MOLECULAR GENETICS

OF INHERITED EYE DISORDERS

M o d e rn G en etics
A series o f boo k s co v erin g new d e v e lo p m en ts across th e e n tire field o f gen etics
E d ite d by H. J. Evans, MRC H u m a n G enetics U n it, W estern G en eral H ospital,
E d in b u rg h , UK

V olum e 1
E m b ry o n a l S tem Cells: In tro d u c in g P la n n e d C h a n g e d in to th e A nim al G erm lin e
M artin L. H o o p e r

V olum e 2
M o lecu lar G en etics o f In h e rite d Eye D isorders
E d ite d by A lan F. W rig h t a n d B arrie Jay

A d itio n al v o lu m es in p re p a ra tio n

V olum e 3
M o lecu lar G en etics o f D ru g R esistance
e d ite d by C. R o lan d W olf a n d J o h n D. Hayes

This book is part o f a series. The publisher will accept continuation orders which may be cancelled at
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tion. Please write for details.
 MOLECULAR GENETICS

OF INHERITED EYE DISORDERS

edited by

Alan F. Wright
M RC Human Genetics Unit, Western General Hospital
Crewe Road, Edinburgh EH4 2XU, UK

Barrie Jay
Emeritus Professor o f Clinical Ophthalmology,
University o f London, UK

informa
healthcare
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 CONTENTS

P reface to th e Series vii

Preface ix
C o n trib u to rs xi

1 B ack g ro u n d to M o lecu lar G en e tic P rin cip les a n d T e ch n iq u es 1

M. B. Gorin and A. F. Wright

(1) INVERTEBRATES

2 R etin al D e g e n e ra tio n M u tan ts o f Drosophila 29

W. L Pak

(2) VERTEBRATES

A. C H O R O ID AND RETINA

3 D iagnosis in I n h e r ite d R etin al D iso rd ers 53

A. C. Bird and B. Jay

4 H u m a n A lbinism a n d M ouse M odels 89

R. A. King, I. J. Jackson and W. S. Oetting

5 In h e rite d R etin al D e g e n e ra tio n s in th e M ouse 123

D. B. Farber and M. Danciger

6 A utosom al D o m in a n t R etinitis P ig m e n to sa 153

P. Humphries, P. Kenna and G. J. Farrar

7 T h e M o lecu lar G en etic A p p ro a c h to M acular D e g e n e ra tio n 173

E. M. Stone and V. C. Sheffield

8 M o lecu lar G en etics o f R e tin o b la sto m a 197

J. K. Cowell

9 C o lo r V ision D efects 217

J. Neitz and M. Neitz

10 X -linked R etinitis P ig m en to sa 259

M. A. Aldred, M. Jay and A. F. Wright

V
vi CONTENTS

11 C o n g e n ita l S ta tio n a ry N ig h t B lindness: A C ritical Review

fo r M o lecu lar A p p ro ach es 277
/'. Heon and M. A. Musarella

12 C h o ro id e re m ia 303
F. P. M. Cremers and H.-H. Ropers

13 N o rrie D isease 321

A. A. B. Bergen, W Berger, Z.-Y. Chen, P. J. Diergaarde,
E. M. Bleek.er-Wagemak.ers, E. M. Battinelli,
M. Warburg, H.-H. Ropers and I. Craig

14 X -linked Ju v e n ile R ed noschisis 339

A. de la Chapelle, T. Alitalo and H. Forsius

15 C linical a n d G en etic H e te ro g e n e ity o f U sh e r S yndrom e 359

W. J. Kimberling, M. Weston and C. Moller

B. V IT R E O U S /L E N S

16 M arfan S y ndrom e 383

P. Tsipouras and. M. W Kilpatrick

17 Lowe O c u lo c e re b ro re n a l S yndrom e 399

I. Okabe and R. L. Nussbaum

18 C rystallin G en es a n d C ataract 413

M. Cartier, L.-C. Tsui, S. P. Ball and N. H. Lubsen

C. A N T E R IO R SEGM ENT

19 A n irid ia 445
I. H anson, T. Jordan and V. van Heyningen

D. M ETABOLIC

20 M ito c h o n d ria l DNA M u tatio n s a n d th e Eye 469

M. D. Brown, M. T. Lott and D. C. Wallace

GLOSSARY 491

IN D EX 515
 PREFACE T O THE SERIES

D ev elo p m en ts in cell a n d m o le c u la r biology have b e e n resp o n sib le fo r e n o rm o u s

advances in o u r u n d e rs ta n d in g o f th e s tru c tu re a n d fu n c tio n s o f g e n e tic factors at
th e m o lecu lar, cell, tissue, org an ism a n d p o p u la tio n levels. T h ese advances are
w ide-ranging, im p in g in g u p o n virtually all fields in th e biological a n d b io m ed ic al
sciences, a n d a re p u b lish e d in a w ide variety o f jo u rn a ls . It see m e d a p p ro p ria te
th e re fo re to p ro v id e a m ean s w h ereb y e x p erts in a field co u ld b e invited to review
re c e n t ad v ances in th e ir own areas o f re sea rch , w hich w ould p rovide th e in te r
e ste d r e a d e r w ith a c o m p reh e n siv e a n d critical app raisal o f p ro g ress a n d p ro s
pects. O u r g e n e ra l aim is to cover new d e v elo p m en ts across th e e n tire field o f
g e n e tic s o f p la n ts a n d anim als, in c lu d in g m an, a n d a t all levels fro m th e m o lecu le
to th e p o p u la tio n . A n im p o rta n t fe a tu re o f th e series will be its flexibility. In d iv id
u al titles will ra n g e fro m s h o rt m o n o g ra p h s o n a n a rro w to p ic to la rg e r m u ltia u
th o r w orks cov erin g several re la te d d e v e lo p m e n ts in a single area.
T h e e d ito rs a n d c o n trib u to rs to th is volum e o n T h e M o lecu lar G enetics o f In
h e rite d Eye D iso rd e rs a re to be c o n g ra tu la te d , fo r this is a lucid, a u th o ritativ e a n d
u p -to -d ate c o m p ila tio n o f pro g ress in an ex citin g a n d im p o rta n t field.

I I .J . Evans
 PREFACE

A b n o rm a litie s o f s tru c tu re re la tin g to th e eye, such as to th e eyelids, lens, iris, etc.

a re r a th e r a p p a re n t o p h th a lm o lo g ic a l featu res as, o f course, a re a b n o rm a litie s o f
fu n c tio n o f this very im p o rta n t sense o rg a n . It is n o t su rp risin g th e re fo re th a t th e
fam ilial n a tu re o f a ra n g e o f eye d efects has b e e n d o c u m e n te d over th e c en tu rie s
a n d a ttra c te d th e a tte n tio n o f th e early h u m a n geneticists. It is w o rth n o tin g th a t
it is alm o st exactly 200 years since D alto n the ch em ist d e sc rib e d his own, a n d his
b ro th e r's , c o lo u r b lin d n e ss (1794) a n d in 1838 C u n iers d e sc rib e d a family, well
know n to m o st stu d en ts, w ith an a u to so m al d o m in a n t fo rm o f c o n g e n ita l statio n
ary n ig h t b lin d n e ss w hich c o u ld b e tra c e d back to a F re n c h m an b o rn in th e early
se v e n te e n th cen tu ry . T h e im p o rta n c e o f in h e rita n c e in a ra n g e o f eye d iso rd ers is
th e re fo re well e sta b lish e d a n d in th e W estern w orld a ro u n d o n e in fo u r o f the
b lin d in th e com m unity, a n d ro u g h ly o n e -h a lf o f individuals w ho are b lin d e d as
c h ild re n , su ffer th e ir disability as a c o n se q u e n c e o f th e in h e rita n c e o f o n e o r m o re
m u ta te d genes.
T h e p a ra m o u n t im p o rta n c e o f th e sense o f vision, th e ra n g e o f variatio n in
stru c tu re a n d fu n c tio n o f h u m a n a n d o th e r anim al eyes, a n d th e w ealth o f in fo r
m a tio n o n th e M e n d e lia n in h e rita n c e o f eye a b n o rm a litie s in h u m an s, has th e re
fo re p ro v id e d b o th a p la tfo rm a n d a s p u r fo r th e ap p lic a tio n o f m o d e rn m o le cu la r
a p p ro a c h e s in th e field o f o p h th a lm o lo g ic a l genetics. T h e p re s e n t volum e b rings
to g e th e r a rich co llectio n o f p a p e rs by in te rn a tio n a lly d istin g u ish e d research ers
d o c u m e n tin g th e m any re m a rk a b le a n d re c e n t advances in o u r u n d e rsta n d in g at
the m o le c u la r level o f th e d e v e lo p m e n t, stru c tu re a n d fu n c tio n o f th e eye in
h e a lth a n d disease. We le a rn th a t in m any cases we are now able to re la te a p a rtic
u la r stru c tu ra l o r fu n c tio n a l d iso rd e r to a p a rtic u la r m u ta tio n in a p a rtic u la r gen e.
We also now have som e u n d e rs ta n d in g o f th e m ech an ism s involved in D alto n 's
c o lo u r b lin d n e ss a n d o f th e ra n g e o f m u ta tio n a l c h an g es resp o n sib le fo r ex am ple
fo r re tin a l d e g e n e ra tio n , c a ta ra c t a n d o th e r im p o rta n t a n d d isab lin g o p h th a lm ic
a b n o rm a litie s. S tu d ies o n th e g en etic s o f re tin o b la sto m a led to th e iso latio n a n d
c h a ra c te risa tio n o f th e first h u m a n tu m o u r su p p re sso r g e n e a n d have essentially
laid th e fo u n d a tio n s fo r o u r u n d e rs ta n d in g o f c ertain in h e rite d c a n c e r p re d isp o
sitions, w hereas th o se o n L e b er's h e re d ita ry o p tic n e u ro p a th y have h ig h lig h te d
th e im p o rta n c e o f m ito c h o n d ria l m u ta tio n s in h u m a n disease. Som e o f th e ad
vances th a t have b e e n m a d e have re lie d o n studies o n o th e r v erteb rates, a n d in
d e e d in v e rte b ra tes, a n d rele v a n t c o n trib u tio n s on th e use o f an im al m o d els in
investigating re tin a l d y stro p h ies a n d a b n o rm a litie s o f eye stru c tu re a n d d evelop
m e n t in h u m a n s a re discussed.

T h e E ditors

IX
H E R E D IT Y

I am th e fam ily face;

Flesh p erish es, I live on,
P ro je c tin g tra it a n d trace
T h ro u g h tim e to tim es a n o n ,
A n d le a p in g fro m place to place
O ver oblivion.

T h e y ears-h eired fe a tu re th a t can

In cu rv e a n d voice a n d eye
D espise th e h u m a n span
O f d u ra n c e - th a t is I;
T h e e te rn a l th in g in m an,
T h a t h e e d s n o call to die.

T h o m a s H ard y (1917)

C h arles D arw in (1859) fro m T h e O rig in o f S p ecies

To su p p o se th a t th e eye, w ith all its in im ita b le con triv an ces fo r ad ju stin g th e focus
to d iffe re n t distan ces, fo r a d m ittin g d iffe re n t a m o u n ts o f light, a n d fo r th e c o rre c
tio n o f sp h erical a n d c h ro m a tic a b e rra tio n , co u ld have b e e n fo rm e d by n a tu ra l se
le c tio n , seem s, I freely confess, a b su rd in th e h ig h est d e g re e. Yet rea so n tells m e,
th a t if n u m e ro u s g ra d a tio n s fro m a p e rfe c t a n d c o m p lex eye to o n e very im p e rfe c t
a n d sim ple, each g ra d e b e in g u seful to its possessor, can be show n to exist; if fu r
ther, th e eye d o e s vary ever so slightly, a n d th e v ariations b e in h e rite d , w hich is cer
tainly th e case; a n d if any v ariatio n o r m o d ific atio n in th e o rg a n be ever useful to
an an im al u n d e r c h a n g in g c o n d itio n s o f life, th e n th e difficulty o f believing th a t a
p e rfe c t a n d c o m p lex eye c o u ld be fo rm e d by n a tu ra l selectio n , th o u g h in su p e ra
ble by o u r im a g in a tio n , can h ard ly be c o n sid e re d real.
 CONTRIBUTORS
M. A. A ld red , MRC H u m a n G e n ed cs
U n it, W estern G e n e ra l U nit,
Crew e R oad, E d in b u rg h E H 4 2XU,
U n ite d K ingdom
T iin a A iitalo, D e p a rtm e n t o f M edical
G enetics, U niversity o f H elsinki,
H a a rtm a n in k a tu 3, 00290 H elsinki,
F in la n d
S arah P. B all, D e p a rtm e n t o f
B iological Sciences, W ash ington
S in g er L aboxatories, U niversity o f
E xeter, E x e te r EX4 4QG,
U n ite d K ingdom
E. M. B attinelli, D e p a rtm e n t o f
B iochem istry, G en etics L aboratory,
U niversity o f O x fo rd , S o u th Parks
R oad, O x fo rd 0 X 1 3Q U ,
U n ite d K ingdom
A. A. B. B erg en , T h e N e th e rla n d s
O p h th a lm ic R esearch In stitu te,
P O Box 12141, NL-1100 AC
A m sterd am , T h e N e th e rla n d s
W. B erger, D e p a rtm e n t o f H u m a n
G enetics, U niversity H ospital
N ijm eg en , P O Box 9101,
6500 HB N ijm eg en , T h e N e th e rla n d s
A lan C. B ird, D e p a rtm e n t o f C linical
O p h th alm o lo g y , M o o rfield s Eye
H ospital, City R oad, L o n d o n
EC1V 2PD, U n ite d K ingdom
E. M. B leeker-W ageinakers, T h e N e th
e rla n d s O p th a lm ic R esearch In stitu te ,
P O Box 12141, N L-1100 AC A m ster
dam , T h e N e th e rla n d s
XI
M ichael D. B row n, D e p a rtm e n t o f
G enetics a n d M o lecu lar M edicine,
E m ory U niversity S chool o f M edicine,
403 D en tal B uilding,
1462 C lifton R oad,
A tlanta, GA 30322,
U n ite d States o f A m erica
M ireille C artier, D e p a rtm e n t o f
G enetics, R esearch In stitu te,
T h e H o sp ital fo r Sick C h ild ren ,
555 U niversity A venue, T o ro n to ,
O n ta rio MSG 1X8, C an ad a
A lb e rt d e la C h ap elle, D e p a rtm e n t o f
M edical G enetics, U niversity o f
H elsinki, H a a rtm a n in k a tu 3,
00290 H elsinki, F in lan d
Z.-Y. C h en , D e p a rtm e n t o f
B iochem istry, G enetics L ab oratory,
U niversity o f O x fo rd , S o u th Parks
R oad, O x fo rd OX1 3Q.U,
U n ite d K ingdom
J o h n K. Cow ell, ICRF O ncology
G ro u p , In stitu te o f C h ild H e alth ,
30 G u ilfo rd S treet, L o n d o n
W C1N 1EH,
U n ite d K ingdom
I. C raig, D e p a rtm e n t o f B iochem istry,
G enetics L aboratory, U niversity o f
O x fo rd , S o u th Parks R oad,
O x fo rd OX1 3Q U,
U n ite d K ingdom
F ran s P. M. C rem e rs, D e p a rtm e n t o f
H u m a n G enetics, U niversity H ospital
N ijm eg en , P O Box 9101, 6500 HB
N ijm eg en , T h e N e th e rla n d s
Xll
M ichael Danciger, L oyola-M arym ount
University, Los A ngeles, CA 90045,
U n ite d States o f A m erica
P. J. Diergaarde, T h e N e th e rla n d s
O p h th a lm ic R esearch In stitu te,
P O Box 12141, N L-1100 AC
A m sterd am , T h e N e th e rla n d s
D ebora B. Farber, J u le s S tein Eye
In stitu te , LTCL\ S chool o f M edicine,
Los A ngeles, CA 90024-7008,
U n ite d States o f A m erica
G. Jane Farrar, D e p a rtm e n t o f
G en etics, T rinity C ollege, D u b lin 2,
Ire la n d
H enrik Forsius, D e p a rtm e n t o f
M edical G enetics, LIniversity o f
H elsin k i, H a a rtm a n in k a tu 3,
00290 H elsinki, F in lan d
M ichael B. Gorin, Eye a n d E ar
In stitu te o f P ittsb u rg h , D e p a rtm e n ts
o f O p h th a lm o lo g y a n d H u m a n
G enetics, U niversity o f P ittsb u rg h ,
PA 15213, U n ite d States o f A m erica
Isabel H anson, MRC H u m a n G enetics
U n it, W estern G e n e ra l H osp ital,
C rew e R oad, E d in b u rg h EH 1 2XLT,
U n ite d K ingdom
E lise H eon , U niversity o f Iowa
H o sp itals a n d C linics, D e p a rtm e n t o f
O p h th alm o lo g y , 200 H aw kins Drive,
Iow a city, Iowa 52242,
U n ite d States o f A m erica
Veronica van H eyningen, MRC H u m a n
G en etics U n it, W estern G e n e ra l
H o sp ital, Crew e R oad, E d in b u rg h
E H 4 2XU, U n ite d K ingdom
CONTRIBUTORS
Peter H um phries, D e p a rtm e n t o f
G enetics, T rinity C ollege, D ublin 2,
Ire la n d
Ian J. Jackson, MRC H u m a n G enetics
U n it, W estern G e n e ral H ospital,
Crew e R oad, E d in b u rg h E H 4 2XU,
U n ite d K ingdom
Barrie Jay, Retired from In stitu te o f
O phthalm ology,
U niversity o f L o n d o n ,
L o n d o n , U n ite d K ingdom
M arcelle Jay, D e p a rtm e n t o f C linical
O p h th alm o lo g y , M oorfields Eye
H ospital, City R oad,
L o n d o n ECIV 2PD,
U n ite d K ingdom
Tim Jordan, MRC H u m a n G enetics
U nit, W estern G en eral H ospital,
C rew e R oad, E d in b u rg h E H 4 2XU,
U n ite d K ingdom
Paul Kenna, D e p a rtm e n t o f G enetics,
T rinity C ollege, D ublin 2, Ire la n d
M ichael W. Kilpatrick, D e p a rtm e n t o f
C linical G enetics, LIniversity o f
B irm in g h am , B irm in g h a m B15 2TT,
U n ite d K ingdom
W. J. Kimberling, Boys Town N atio n a l
R esearch H ospital,
O m a h a , N ebraska, NE 68131,
L inked States o f A m erica
Richard A. King, U niversity o f
M in nesota, Box 485 U M HC,
420 D elaw are S tre et SE, M in neapolis,
M in n eso ta 55455,
U n ite d States o f A m erica
 CONTRIBUTORS
Marie T. Lott, D e p a rtm e n t o f G enetics
a n d M o lecu lar M ed icin e, E m ory
U niversity S chool o f M edicine,
403 D e n ta l B uilding,
1362 C lifto n R oad, A tlan ta,
GA 30322, U n ite d States o f A m erica
N icolette H . Lubsen, D e p a rtm e n t o f
M o lecu lar Biology, U niversity o f
N ijm eg en , T o e rn o o iv e ld , 6525 ED
N ijm eg en , T h e N e th e rla n d s
C. M oller, U niversity o f L in k p in g ,
L in k p in g , Sw eden
Maria A. Musarella, T h e H o spital
fo r Sick C h ild re n , R esearch In stitu te,
D e p a rtm e n ts o f O p h th a lm o lo g y a n d
G enetics, 555 U niversity A venue,
T o ro n to , O n ta rio M 5G 1X8, C an ad a
Jay N eitz, M edical C ollege o f W iscon
sin, D e p a rtm e n t o f C ellu lar Biology
a n d A natom y, a n d D e p a rtm e n t o f
O p h th alm o lo g y , 8701 W atertow n
P lank R oad, M ilw aukee, WI 53226,
U n ite d States o f A m erica
M aureen N eitz, M edical C ollege o f
W isconsin, D e p a rtm e n t o f C ellu lar
Biology a n d A natom y,
8701 W atertow n P lank R oad,
M ilw aukee, WI 53226,
U n ite d States o f A m erica
R obert L. N ussbaum , C hief,
L a b o ra to ry fo r G en etic D isease
R esearch , N a tio n a l C e n te r fo r
H u m a n G e n o m e R esearch,
N IH , B u ild in g 4 9 /4 A 7 2 ,
9000 Rockville Pike, B eth esd a,
M ary lan d 20892,
U n ite d S tates o f A m erica
x i ii
W illiam S. O etting, U niversity
o f M in nesota, Box 485 U M H C ,
420 D elaw are S treet, SE M in neapolis,
M in n e so ta 55455
U n ite d States o f A m erica
I. O kabe, H ow ard H u g h es M edical
In stitu te L ab oratory, U niversity o f
Pennsylvania S chool o f M edicine,
422 C u rie B oulevard, P h ilad elp h ia ,
PA 19104, U n ite d States o f A m erica
William L. Pak, D e p a rtm e n t o f
B iological Sciences, Lilly H all o f Life
Sciences, P u rd u e U niversity, West
L afayette, In d ia n a 47907,
U n ite d States o f A m erica
H ans-H ilger R opers, D e p a rtm e n t o f
H u m a n G enetics, U niversity H o spital
N ijm eg en , P O Box 9101, 6500 HB
N ijm egen, T h e N e th e rla n d s
Val C. Sheffield, D e p a rtm e n t o f
Pediatrics, U niversity o f Iow a C ollege
o f M edicine, Iow a City, Iow a 52242,
U n ite d States o f A m erica
Edwin M. Stone, D e p a rtm e n t o f
O p h th alm o lo g y , U niversity o f Iowa
C ollege o f M edicine, Iow a City, Iowa
52242, U n ite d States o f A m erica
Petros Tsipouras, D e p a rtm e n t o f
Pediatrics, U niversity o f C o n n e c tic u t
H e a lth C enter, F a rm in g to n ,
CT 06030, U n ite d States o f A m erica
Lap-Chee Tsui, D e p a rtm e n t o f
G enetics, R esearch In stitu te ,
T h e H o spital fo r Sick C h ild re n ,
555 U niversity A venue, T o ro n to ,
O n ta rio M 5G 1X8, C an ad a
xiv CONTRIBUTORS

D ouglas C. W allace, D e p a rtm e n t o f M. W eston, Boys Town N atio n al

G en etics a n d M o lecu lar M edicine, R esearch H ospital, O m a h a , N ebraska,
E m o ry U niversity S chool o f M edicine, N E 68131, U n ite d States o f A m erica
403 D e n ta l B uilding, 1462 C lifton
R oad, A tlan ta, GA 30322, A lan F. W right, MRC H u m a n G enetics
U n ite d States o f A m erica U n it, W estern G e n eral H o spital ,
C rew e R oad, E d in b u rg h E H 4 2XU,
M. W arburg, G e n to fte H osp ital, U n ite d K ingdom
D e p a rtm e n t o f O p h th alm o lo g y ,
D ivision o f P aed iatric O p h th a lm o lo g y
a n d H a n d icap s, 40 Sognevej, DK-2820
G en to fte, D en m ark
 1. BACKGROUND T O MOLECULAR GENETIC
PRINCIPLES AND TECHNIQUES

M ICHAEL B. G O R IN 1 a n d ALAN F. W RIG H T2

1The Eye and Ear Institute o f Pittsburgh, Departments of Ophthalmology and H um an

Genetics, University of Pittsburgh, Pittsburgh, PA 15213, USA
2 M RC H um an Genetics Unit, Western General Hospital, Crewe Road,
Edinburgh EH 4 2XU, UK

T h ro u g h o u t this b o o k , th e re a re n u m e ro u s exam p les o f th e g e n e tic a n d physical

m a p p in g o f g en es re la te d to o p h th a lm ic d iso rd e rs a n d o f th e u n ra v ellin g o f th e ir
g e n e tic fu n ctio n s. T h e te c h n iq u e s th a t are u sed cover a ra n g e o f disciplines
in c lu d in g m o le c u la r biology, g en etics, b io c h em istry a n d cytology a n d , to th e nov
ice, th e te rm s a n d m e th o d s can b e o v erw h elm in g . It is th e objective o f this c h a p
te r to p ro v id e a sim p le g u id e fo r th e p e rp le x e d to assist th e reader. T h e
d e sc rip tio n s a n d h isto rical orig in s are k e p t to a m in im u m , so th a t m a terial can be
rapidly accessed. T h e d e fin itio n s a re by n o m ea n s co m p re h en siv e a n d som e
e x c e lle n t a n d c o m p le te m o le c u la r g e n e tic s g u id eb o o k s a re available (Davies a n d
R ead, 1988; King a n d S tansfield, 1990; T h o m p so n e t a!., 1991; Casey, 1992).
T h e re a re th re e sectio n s to this in tro d u c tio n . T h e first covers th e an ato m y o f th e
m am m a lia n g e n o m e a n d o f th e g en e . It is im p o rta n t to re co g n iz e th e e x te n t o f th e
p ro b le m o f id en tify in g a single base p a ir m u ta tio n w ithin a g e n o m e th a t spans
over th re e b illio n base p airs a n d co n ta in s u p to 100,000 g enes. W hile th e basic al
p h a b e t o f th e g e n e tic c o d e - A ,C,G a n d T (U in RNA) - has b e e n re c o g n iz e d since
W atson a n d C rick s classic p a p e r in 1953, it is still n o t clear w hat is th e full signifi
c an ce o f th e 9598% o f DNA th a t d o es n o t d irectly code fo r g enes. T his m a terial
is full o f highly re p e titiv e e le m e n ts th a t m ake th e task o f th e g e n e h u n te r c o n sid
erably m o re d ifficu lt a n d g e n es them selves are sp lit u p in to s h o rt c o d in g stretch es
(ex o n s), ea c h only a few h u n d re d s o f base p airs in le n g th , th a t may b e sc a tte red
over a re g io n th a t covers te n s o r h u n d r e d s o f th o u sa n d s o f base pairs.
T h e se c o n d sectio n ad d resses th e c o n c e p ts o f linkage analysis a n d re la te d g e n e t
ic, as o p p o se d to physical, m a p p in g tec h n iq u es. G e n e tic m a p p in g is c o n c e rn e d
w ith th a t m o st fu n d a m e n ta l o f all g e n e tic ists tec h n iq u e s, p e d ig re e analysis, in
w hich p a re n ta l a n d re c o m b in a n t type o ffsp rin g a re c o u n te d a n d fro m w hich ge
n e tic d istan ces can be c o m p u te d . T h is a re a has d ev e lo p ed very rapidly d u rin g th e
p ast te n years to th e e x te n t th a t a trickle o f g en es m a p p e d u sing re stric tio n frag
m e n t le n g th p o ly m o rp h ism s in th e first h a lf o f th e 1980s has now tu rn e d in to a
flo o d usin g new types o f g e n e tic m a rk e r such as th e m icro satellite re p e a t. T h e n e x t
five years will alm o st certa in ly see th e a u to m a tio n o f such g e n etic m a p p in g p ro c e
d u re s a n d th e in tro d u c tio n o f new m e th o d s, m aking it feasible to e lu c id ate th e ge
n etics o f m o re c o m p lex o p h th a lm ic d iso rd e rs such as g lau c o m a a n d ag e-related
m a c u la r d ystrophies.
2 MICHAEL B. GORIN AND ALAN F. WRIGHT

T h e re is a vast array o f te c h n iq u e s th a t th e m o lec u lar g en e ticist em ploys fo r th e

analysis o f n u cleic acids a n d g e n e ex p ressio n , m any o f w hich are re fe rre d to in the
su c c e e d in g c h a p te rs. Essentially all o f th e se m e th o d s are b u ilt u p o n a fu n d a m e n
tal set o f tools. T h e th ird sectio n provides a b rie f overview o f th ese tools. T h e rev
o lu tio n in g en etics - th e new g e n e tic s - is u n d e rp in n e d by basic advances in
fu n d a m e n ta l m o le c u la r b iological te c h n iq u e s such as th e ability to clo n e DNA in
m icro o rg an ism s, to identify specific n u cleic acids w ithin co m p lex m ix tu res by fil
te r h y b rid isatio n , to e x p o n e n tia lly am plify small segm ents o f DNA by th e rm a l cy
cling, to identify th e se q u e n c e o f n u c le o tid e bases o n a lin e a r stretch o f DNA a n d
so o n . R a th e r th a n review ing th ese essential advances in a histo rical c o n te x t, th e
tools th a t have e m e rg e d fro m th e m will b e sum m arised . T his will give som e in sig h t
in to th e everyday te c h n iq u e s u sed by th e g e n e m ap p er.
In a d d itio n , a glossary is in c lu d e d a t th e e n d o f th e book th a t may h e lp to clarify
b o th th e basic lan g u ag e o f m o le c u la r g e n etics a n d th e m o re te c h n ic a l te rm s a n d
ab b rev iatio n s th a t m ak e th e su bject less accessible th a n it sh o u ld be.

1(a) ANATOMY O F T H E GEN OM E

T h e m a m m a lia n g e n o m e consists o f an e stim a te d 3 b illion base pairs o f DNA

o rg a n iz e d in to 23 p airs o f c h ro m o so m e s in m a n a n d 20 p airs in th e m ouse. T h e
in v e rte b ra te g e n o m e is co n sid erab ly sm aller, so th a t fo r ex am p le th e fru it fly
Drosophila melanogaster h as only fo u r pairs o f c h ro m o so m es a n d 300 m illio n base
p airs o f DNA. T h is sm aller g e n o m e size, very h ig h re so lu tio n cytological a n d
g e n e tic m ap s a n d r a th e r special te c h n iq u e s b o th fo r m u ta tin g a n d re sto rin g th e
fu n c tio n o f g enes, has m a d e D ro so p h ila a pow erful m o d el fo r g e n e tic analysis -
n o t least in th e analysis o f m u ta tio n s affectin g th e eye a n d its d e v e lo p m e n t
(C h a p te r 2). By c o n trast, th e m am m alian g e n o m e has g re a te r com plexity, n o t
only in size b u t also in o rg a n iz a tio n . T h e basic a p p a ra tu s fo r g en etic rep lica tio n
a n d se g re g a tio n a re sim ilar however. E ach c h ro m o so m e co n tain s, firstly, a d o u b le
s tra n d e d lin e a r DNA m o lecu le w ith two a n d p a ra lle l a -h elical stra n d s -th e y are
in o p p o site o rie n ta tio n w ith re sp e c t to th e 5 ' (h e a d ) a n d 3 ' (ta il) c a rb o n
ato m s o f th e d eoxyribose in th e su g a r-p h o s p h a te b a c k b o n e w hich is o rie n te d
as a 5 '- 3 '- 5 '- 3 ' array. H isto n e a n d n o n -h isto n e ch ro m o so m al p ro te in s b in d a n d
c o n trib u te to th e fo ld in g o f DNA such th a t it u n d e rg o e s a lin e a r c o n tra c tio n o f at
least th re e o rd e rs o f m a g n itu d e w ithin th e c h ro m o so m e. C h ro m o so m e s co n ta in a
c e n tro m e re fo r a tta c h m e n t to th e m ito tic o r m eio tic sp in d le, e n su rin g p ro p e r
se g re g a tio n in to d a u g h te r cells, a n d specialised ch ro m o so m a l stru c tu re s called
te lo m e re s th a t facilitate th e re p lic a tio n o f c h ro m o so m al ends. O n e o f th e c h ro
m o so m al p airs is th e sex ch ro m o so m es, w hich in th e h e te ro g a m e tic sex (m ales in
m am m als, w ith an X a n d Y c h ro m o so m e ) sh a re only a sm all re g io n o f hom ology'
(p seu d o -au to so m al re g io n ); th e m ajority o f th e X a n d Y c h ro m o so m es c o n ta in
d iffe re n t g en es. Recessive m u tatio n s o c c u rrin g on th e X ch ro m o so m e are m o re
readily id e n tifie d th a n o n non -sex ch ro m o so m e s (au to so m es) since in th e hem -
 MOLECUIAR GENETIC PRINCIPLES AND TECHNIQUES 3

izygous (XY) state they lack a sec o n d , n o rm a l copy o f the affe cted g en e. T h e
e x p ressio n o f th e X c h ro m o so m e is also d iffe re n t fro m th e au to so m es in the
h o m o g a m e tic sex (X X ), th e fem ale in m am m als, because o f th e process o f ra n
d o m fu n c tio n a l in activ ation o f o n e o f th e two c h ro m o so m e s in each cell in early
em b ry o g en esis (X-inactivation). T his pro cess takes place at a stage o f em b ry o g en -
esis w h ere cell n u m b e rs a re sm all (e.g. <5 day o ld m o u se em bryos) so th at
c h a n c e flu c tu a tio n s in th e d istrib u tio n o f cells w ith o n e o r o th e r X ch ro m o so m e
active can have larg e effects o n th e e x p ressio n o f d e le te rio u s g e n es in heterozy
g o tes - p artic u la rly w h ere th e n u m b e r o f p rim o rd ia l cells giving rise to a tissue,
such as th e re tin a , a re relatively sm all. T his process is resp o n sib le fo r th e variable
a n d o fte n su b tle m a n ife statio n s o f disease in h eterozygous c a rrie rs o f X -linked
d iso rd ers.
A u to so m al g e n e s a re ex p ressed in every cell a lth o u g h it is now know n th a t th e
p a re n ta l so u rce (fa th e r o r m o th e r) o f th e c h ro m o so m e can have a p ro fo u n d effect
o n th e ex p ressio n o f c e rta in genes. T h is process has b e e n te rm e d im printing a n d ,
u n til recently, m ost ex am p les cam e fro m studies o f g en es ex p ressed in early m am
m alian d e v e lo p m e n t. R ecen t studies o n th e p a re n ta l o rig in o f ch ro m o so m e 15 d e
le tio n s associated w ith th e P ra d e r-W illi a n d A n g elm an sy n d ro m es in m an has now
esta b lish e d a sig n ifican t ro le fo r this process n o t only in early d e v e lo p m e n t b u t
also in th e e x p ressio n o f disease.
G e n e tic diversity is ach iev ed by two m a jo r m ech an ism s am o n g d ip lo id species.
T h e first is by m utation, w hich o ccu rs a t a slow b u t finite ra te th ro u g h o u t th e ge
n o m e . T h e e v o lu tio n a ry c o n stra in ts o n a lte rin g th e fu n c tio n o f g e n e s are such
th a t relatively few m u ta tio n a l d iffe re n c es survive in reg io n s th a t co d e fo r p ro te in s
o r c o n tro l g e n e ex p ressio n . T h e s ile n t re g io n s o f th e g e n o m e , on th e o th e r
h a n d , m a in ta in a h ig h level o f diversity. T h e ad vantage o f this h ig h po ly m o rp h ism
c o n te n t b ecam e ev id e n t to g en e m a p p e rs in th e early 1980s, w ho p ro p o se d th e use
o f such se q u e n c e varian ts as m a rk e rs to follow th e se g reg atio n o f g en es in k in
d red s. T h e v arian ts w ere id e n tifie d initially by th e ir ability to a lte r re stric tio n e n
d o n u c le a se cleavage sites (restriction fragm ent length polym orphism s, RFLPs),
w hich c o u ld b e d e te c te d by h y b rid isatio n o f specific ra d io la b e lle d p ro b e s ag ainst
g e n o m ic DNA a fte r d ig e stio n w ith th e a p p ro p ria te e n d o n u c le a se (F igure 1). Such
varian ts w ere fo u n d to o c c u r o n c e every 300 -5 0 0 base p airs in n o n -c o d in g re g io n s
o f th e g e n o m e a n d to b e p re s e n t as fre q u e n t a ltern ativ e fo rm s (alleles) in th e g e n
eral p o p u la tio n . T h ey have b e e n e x p lo ite d to th e full in m a p p in g g e n e tic traits,
w h ere only a lim ite d ra n g e o f b lo o d g ro u p a n d se ru m p ro te in p o ly m o rp h ism s ex
iste d previously fo r this p u rp o se.
In c o n tra st to th e n u c le a r g e n o m e , th e m itochondrial genom e consists o f a
sm all, c irc u la r DNA m o le c u le (16.5 x 10 3 base p airs long) th a t is m a in ta in e d in th e
cytoplasm as h u n d r e d s o r th o u sa n d s o f copies p e r cell. It en c o d e s several su b u n its
o f oxidative p h o sp h o ry la tio n enzym es th a t are vital to th e fu n c tio n o f re sp irin g
cells, th e o th e rs b e in g c o d e d by th e n u c le a r g e n o m e. T h e re are im p o rta n t differ
en ces betw een th e se two g e n o m es (C h a p te r 21), in c lu d in g a significantly h ig h e r
ra te at w hich m u ta tio n s a re in c o rp o ra te d in to th e m ito c h o n d ria l g e n o m e (fixa-
from the presence of a previous mutation within the restriction endonuclease cleavage site (G 'A) on H' thal results in failure to cleave al that p o in t.
The digested fragments are separated electrophoretically, d en atu red , transferred to a m em brane (Southern transfer) and hybridised with a single
stranded ~P labelled probe sequence that specifically anneals to the fragments containing the RFI The segregation of the two alleles are show n
within a small pedigree on the right. The RFI.P alleles do not segregate with the disease phenotype (shaded symbols) in this family.
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 5

tio n ), som e o f w hich give rise to g e n e tic eye d iso rd ers. M u tatio n also arises as a
re su lt o f ch ro m o so m a l a b e rra tio n s su ch as tran slo catio n s, inversions a n d d e le
tions. T h ese a re im p o rta n t b o th in c re a tin g e v o lu tio n ary diversity a n d , in th e c o n
tex t o f g e n e m a p p in g , p ro v id e an im p o rta n t m ea n s o f id en tify in g g e n e s th a t have
b e e n d is ru p te d by tra n slo c a tio n o r d e le tio n events (C h ap te rs 8, 9, 10, 12, 13, 17,
1 9 ,2 1 ).
T h e se c o n d m e c h a n ism fo r g e n e ra tin g diversity occu rs d u rin g m eiosis w hen h o
m o lo g o u s p a re n ta l ch ro m o so m e s pair, e x ch an g e g e n e tic m a te rial by re c o m b in a
tio n a n d seg reg ate in to th e g e rm cells. T h e average n u m b e r o f ex ch an g e s p e r
ch ro m o so m e d u rin g m eiosis in a m am m a lia n cell is only 2 -4 , so th a t th e d a u g h te r
c h ro m a tid s a re c o m p o sites m ad e u p o f relatively lo n g seg m en ts o f e ith e r m a te rn a l
o r p a te rn a l ch ro m o so m es. T h e re c o m b in a tio n process e n su re s th a t th e p ro b a b il
ity o f an in d iv id u al in h e ritin g two g e n e s o r m ark ers fro m th e c h ro m o so m e o rig i
nally c o n trib u te d by a g ra n d p a r e n t decreases rapidly to th e level o f ch an ce,
d e p e n d in g o n how fa r a p a rt they are. T h e p aucity o f re c o m b in a tio n a l crossovers
m akes fo r a relatively efficien t m a p p in g p ro c e d u re in w hich th e ra te at w hich th e
two sites (genes, m ark ers) re c o m b in e is a m easu re o f th e g en e tic d ista n c e betw een
th e m (sectio n II). M istakes th a t o c c u r e ith e r d u rin g ch ro m o so m al re p lic a tio n o r
d u rin g m eio tic p a irin g can give rise firstly to d u p lic a tio n o f g en es a n d la te r to m u l
tip le co p ies o f g en es, p ro v id in g sco p e fo r th e d iv erg en ce o f g en es w ith re la te d
fu n ctio n s, as discu ssed in C h a p te r 9 o n co lo r vision.
T h e n u m b e r o f g e n e s in th e m am m a lian g e n o m e has b e e n e stim a ted to be in
th e re g io n o f 50,000-1 0 0 ,000. T h e average cell expresses o n e -q u a rte r to o n e h a lf
o f this n u m b e r a n d som e g e n e tra n sc rip ts a re very a b u n d a n t, o th e rs m ake only a
few co p ies p e r cell. T h is h ighly skew ed d istrib u tio n o f g e n e exp ressio n can b e an
im p o rta n t c o n sid e ra tio n in id en tify in g c a n d id a te g en es fo r g e n e tic d iso rd ers, p ar
ticularly w ith re g a rd to th e c o n stru c tio n o r sc re e n in g o f ex p re sse d se q u en ce
(cDNA) libraries. If an average m am m alian g e n e p ro d u c t c o n ta in s a b o u t 300 am i
n o acids, th e e q u iv a le n t a m o u n t o f D NA re q u ire d to co d e a n d express such a g en e
is only a b o u t o n e kilobase p a ir (kb; 103 base pairs). W hen e x tra p o la te d to th e size
o f th e w hole g e n o m e , it is readily a p p a r e n t th a t only 2 -4% o f th e g e n o m e actually
co des fo r p ro te in s. N u cleic acid re asso ciatio n studies show th a t as m u c h as 60% o f
m a m m a lia n DNA c o n ta in s u n iq u e o r low copy n u m b e r (<10) seq u en c es, w hile the
re m a in d e r is rep etitiv e. W ithin th e la tte r g ro u p , th re e classes o f re p e titiv e DNA
can b e d istin g u ish e d - h ig h , in te rm e d ia te a n d low a b u n d a n c e classes. T h e first
class o f highly re p e titiv e seq u e n c es in c lu d e s th o se w ith re ite ra tio n fre q u e n c ies
above a b o u t 100,000 copies, su ch as th e tan d em ly rep etitiv e sate llite seq u en c es
n e a r th e c e n tro m e re s o f som e ch ro m o so m e s. A n o th e r im p o rta n t ex am p le o f this
g ro u p is th e A lu rep etitiv e e le m e n t, w hich is 300 base p airs in le n g th a n d in te r
sp ersed th ro u g h o u t th e g e n o m e , o c c u rrin g on average every 3 -4 kb. T h ese a n d
o th e r in te rs p e rs e d re p e a ts a re useful fo r d istin g u ish in g th e DNA o f d iffe re n t m am
m alian species b u t in g e n e ra l are a g en eticists' n ig h tm a re . T h ey m ak e th e task o f
id en tify in g specific se q u e n ce s su ch as g e n e s con sid erab ly m o re difficult, so th a t
ra d io la b e lle d p ro b e se q u en c es o fte n re q u ire co m p e titio n w ith h ig h c o n c e n tra
tio n s o f rep etitiv e D NA in o rd e r to p re v e n t non-specific hy b rid isatio n signals.
6 MICHAEL B. GORIN AND AIAN F. WRIGHT

O th e r re p e titiv e e le m e n ts have b e e n e x p lo ite d to g re a t advantage. T h e m icro

satellite re p e a t class consists o f two to fo u r bases re p e a te d in ta n d e m u p to 50-fold
a n d again widely d isp e rse d th ro u g h o u t th e g e n o m e . M icrosatellites a re th o u g h t to
occur, o n average, every 30 kb a n d are increasingly e x p lo ite d as g e n e tic m arkers.
M ost c a teg o ries o f rep etitiv e DNA a c cu m u la te m u ta tio n s, som e o f w hich beco m e
g en etically fixed in th e p o p u la tio n , b u t th e se q u en ce v ariatio n w ithin m icro satel
lites is p articu larly useful. Tow ards th e e n d o f th e 1980s, it e m e rg e d th a t m icro sat
ellites show a h ig h d e g re e o f v ariatio n in th e number o f re p e a t u n its w ithin d iffe re n t
m e m b e rs o f m ost o u tb re d p o p u la tio n s. T h ese are relatively easily typed after se
q u e n c e am p lificatio n by m e a n s o f th e p o lym erase ch ain re a c tio n (see below ) a n d
now c o n stitu te a m ajo r class o f g e n e tic m a rk e r fo r m a p p in g studies. T h e m ost
w idely u se d m icro satellite is th e CA re p e a t p o ly m o rp h ism , in w hich the CA m o tif
is r e ite ra te d fro m 10 to 50 tim es en bloc.
If m o st o f th e m a m m a lia n g e n o m e d o e s n o t code fo r g enes, w hat is this j u n k
DNA doin g ? T h e rep etitiv e e le m e n ts d o , in c e rta in cases, have th e ability to re p li
cate a n d re -in se rt them selves in new re g io n s o f the g e n o m e , b u t fo r th e m o st p a rt
this ability is lo st o r very ra re . Som e n o n -c o d in g DNA may sim ply p ro vide an evo-
lu tio n arily u seful strategy o f spacing o u t th e regions (exons) c o d in g fo r sm all d o
m ain s w ith in p ro te in s (see below ), p ro v id in g scope fo r c h ro m o so m a l re a rra n g e
m e n ts to c re a te new g e n e tic c o m b in a tio n s a n d fu n ctio n s. Som e DNA is certainly
c o n c e rn e d w ith th e ability to re p lic ate itself, p ro v id in g re c o g n itio n se q u e n c e s fo r
re p lic a tio n p ro te in s a n d c h ro m o so m al scaffold a tta c h m e n t sites. O th e r sites c o n
trib u te to th e re g u la tio n o f g e n e e x p ressio n e ith e r directly, as w ith p ro m o te r a n d
e n h a n c e r se q u e n c e s (see b e lo w ), o r in d irectly by m e a n s o f seq u en ces necessary
fo r th e p ack ag in g o f DNA in to ch ro m o so m es. However, m o st se q u en ces know n to
b e re q u ire d fo r re p lic a tio n o r ex p ressio n a re sh o rt a n d a c c o u n t fo r only a small
p r o p o rtio n o f th e g e n o m e . T h e m ajority o f th e g e n o m e a p p e a rs to re ta in m u ta
tio n s a t a h ig h ra te , w hich itself im p lies a non-specific b u t p e rh a p s ev o lutionarily
im p o rta n t ro le. Specificity may n o t always b e obvious how ever, since th e se co n d a ry
a n d h ig h e r-o rd e r s tru c tu re o f DNA is m u c h less well u n d e rsto o d th an its p rim a ry
s tru c tu re a n d th e re may be c o n sid e rab le co n strain ts o n th e seq u en ce c o m p o sitio n
o f w hat is c u rre n tly re g a rd e d as j u n k DNA.

I (b) ANATOMY O F T H E GENE

T h e eu k ary o tic g e n e can be c o n sid e re d in two parts. T h e first p a rt c o n ta in s the

in fo rm a tio n re q u ire d fo r a rib o n u c le ic acid (RNA) g e n e p ro d u c t. T ra n sfe r a n d
rib o so m al RNA g e n e tra n sc rip ts are re q u ire d fo r th e a d d itio n o f c h a rg e d am in o
acids to th e n a sc e n t p o ly p e p tid e ch ain a n d fo r rib o so m al b in d in g a n d tra n sla tio n
o f th e m e sse n g e r RNA (m RNA) respectively. M essen g er RNA c o n ta in s all th e
se q u e n c e in fo rm a tio n re q u ire d fo r tra n sla tio n in to p ro te in a lth o u g h it first
u n d e rg o e s c o n sid e ra b le m o d ifica tio n w ithin th e n ucleus. U n lik e b a c te ria o r
m ito c h o n d ria , th e p a rts o f eu k ary o tic g e n e s th a t e n c o d e a p ro te in are typically
 MOLECUIAR GENETIC PRINCIPLES AND TECHNIQUES 7

b ro k e n u p in to d isc o n tin u o u s c o d in g seg m en ts c o n ta in e d w ithin exons, sep a

ra te d by in te rv e n in g se q u en c es o r introns, w hich a re rem o v ed fro m th e initial
tra n sc rip t. T h e RNA p o ly m erase tra n s c rip t co m m e n ce s a b o u t 30 base p a irs (th re e
DNA h elix tu rn s) d o w n stream o f a p ro m o te r e le m e n t (e.g. TATA b o x ) , a t th e cap
site, w hich m arks th e 5 ' e n d o f th e tra n sc rip t a n d is m o d ified by th e enzym atic
a d d itio n o f a g u a n o sin e re sid u e in th e o p p o site o rie n ta tio n (5 ' to 5 ') to th e
m RN A ch ain (5 ' to 3 ') : this is th e n m e th y late d to fo rm th e c a p (F igure 2). T h e
cap m ay p ro te c t th e m RN A fro m n u c le a se attack o r assist in rib o so m e b in d in g .
T ra n sc rip tio n p ro c e e d s in a 5 ' to 3 ' d ire c tio n th ro u g h th e 5' untranslated region
o f th e g e n e , p ast th e p ro te in c o d in g re g io n s a n d in tro n s in to th e 3' untranslated
region o f th e g e n e a n d fin ishes at o n e o r m o re stop sequ en ces, w hich are usually
in m a m m a lia n genes. T h e initial tra n sc rip t, w hich is called heterogeneous
nuclear (hn) RNA, is rap idly p ro c essed in th e n u cleu s. T h e 3' te rm in a l RNA c o n
tains a p o ly ad en y latio n signal se q u e n c e (F igure 2), to w hich a re a d d e d 5 0 -200
a d e n o sin e re sid u e s (poly.-Y ta il) , w hich serve to stabilise th e tra n sc rip t a n d to
e ffect its tra n slo c a tio n to th e cytoplasm .
H n RNA is also p ro c e sse d by splicing - enzym atic cleavage a t partially co n se rv e d
re sid u e s flan k in g th e exons. Som e g en es a re spliced a t d iffe re n t p o in ts in d iffe re n t
cell types o r even w ithin th e sam e cell type - d iffe ren tial splicing. T his may resu lt
fro m cell-specific p ro te in s stabilising c e rta in splice co m p lex es at th e ex p en se o f
o th ers. D ifferen tial splicing can re su lt in fu n ctio n ally significant d iffe ren c es in
g en e ex p ressio n .
E xons m ay b e ten s to th o u sa n d s o f base p airs in le n g th a n d a re se p a ra te d by in
tro n s w hich a re typically m u c h lo n g e r a n y th in g u p to h u n d re d s o f kilobases long.
T h e re m ay be fro m o n e to over o n e h u n d r e d ex o n s in a e u k a ry o tic gen e. D u rin g
tra n sla tio n o f th e m a tu re , p ro ce sse d eu k ary o tic mRNA, th e 40S rib o so m a l sub
u n it, w hich c o n ta in s th e in itia to r tRNAmet, b in d s to th e 5 ' e n d a n d tracks alo n g
th e tra n sc rip t u n til it re a c h es th e first initiation codon, AUG (m e th io n in e ), w hen
th e 60S rib o so m al s u b u n it a ttach es a n d tra n sla tio n begins.
M u tatio n s w ith in th e c o d in g o r asso ciated re g io n s re q u ire d fo r pro cessin g th e
tra n s c rip t (e.g. splice sites) a re th e c o m m o n e st cause o f g e n e tic d isorder. T h ese
m ay tak e th e fo rm o f m issense m utations, in w hich the trip le t c o d o n fo r o n e am i
n o acid is c h a n g e d to th a t o f a n o th e r (e.g. CAC TAC ch an g es a h istid in e to a
ty ro sin e). T h e re su lta n t ch a n g e m ay be fu n ctio n ally b e n ig n o r conservative if
th e re is su b stitu tio n o f a sim ilar a m in o acid e.g. b o th re sid u e s have n o n e t ch arg e
a n d sh a re sim ilar biophysical p ro p e rtie s. A lternatively, a c h an g e fro m an acidic to
a basic o r u n c h a rg e d a m in o acid is no n -co n serv ativ e a n d is m o re likely to b e d e l
e terio u s. N o n sen se m utations in tro d u c e a p re m a tu re tra n sla tio n stop c o d o n
(UAA, UAG) in to th e tra n sc rip t, p ro d u c in g a tru n c a tio n o f th e re su lta n t p o ly p ep
tide. Fram eshifts a re sm all in se rtio n s o r d e le tio n s th a t a lte r th e c o d o n re a d in g
fram e a n d so b o th c h a n g e th e su b se q u e n t seq u en ce a n d o fte n lead to tru n c a tio n
o f th e p ro te in d u e to in tro d u c tio n o f a stop co d o n . Splice site m utations resu lt in
a b n o rm a l p ro cessin g o f th e tra n sc rip t le a d in g to an a b n o rm a l p ro te in p ro d u c t.
M u tatio n s can o c c u r w ith in any fu n c tio n ally im p o rta n t p a rt o f th e g en e a n d the
 705 TRANSCRIPTION TERMINATION

2
V .5

y (U r
a *>

ci -G
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 9

relative d is trib u tio n o f m u ta tio n a l types m ay vary w ith th e g e n e s tru c tu re a n d th e

n a tu re o f th e p h e n o ty p e . G enes c o n ta in in g re p e a te d se q u en c es m ay b e u n stab le
e ith e r b ecau se o f a te n d e n c y to e x p a n sio n o f th e re g io n (as in H u n tin g to n 's dis
ease) o r b ecau se they m ay b e m o re p ro n e to d e le tio n as a re su lt o f m isp airin g b e
tw een re p e a te d se q u e n c e s in m eiosis (as in som e LDL re c e p to r m u ta tio n s).
D eletio n s a re a very im p o rta n t m ean s o f id en tify in g disease g e n e s a n d m ay re p r e
se n t a h ig h p ro p o rtio n o f all m u ta tio n s in th e case o f large g en es such as facto r
VIII (10% ) o r d y stro p h in (65% ), w hich cause h a e m o p h ilia A a n d D u c h e n n e m us
cu lar d y stro p h y respectively. In m o st g enes, how ever, d e le tio n s only co m p rise a
sm all p e rc e n ta g e o f m u ta tio n s.
T h e se c o n d p a r t o f th e eu k ary o tic g e n e is c o n c e rn e d w ith th e c o n tro l o f g e n e
ex p ressio n a n d c o n ta in s c o n serv ed seq u en ces re g u la tin g th e level o f tra n sc rip tio n
a n d tissue o r d e v e lo p m e n ta l exp ressio n . T h ese seq u en ces are n o t tra n sc rib e d in to
RNA. T h ey in c lu d e p ro m o te r a n d e n h a n c e r seq u en ces. T h e p ro m o te r re g io n is lo
c a te d u p stre a m o r 5' to th e tra n sc rip tio n sta rt site a n d in clu d es o n e o r m o re o f sev
eral sites w ith relatively s h o rt se q u e n c e m otifs, m any o f th e m b in d in g sites fo r
tra n sc rip tio n activating o r re p re ssin g p ro te in s. M ost g en es c o n ta in a TATA bo x , a
seven base p a ir AT-rich se g m e n t lo c a te d 2 0 -3 0 b p u p stre a m fro m th e tra n sc rip tio n
sta rt site (F ig u re 2), w hich b in d s p a rt o f th e TFTID tra n sc rip tio n fac to r com plex.
T his a p p e a rs to o rie n ta te th e sta rt o f tra n sc rip tio n by RNA polym erase II. Som e
g e n e s c o n ta in a variety o f u p stre a m e le m e n ts lo c ate d 80-2 0 0 b p u p stre a m o f th e
cap site, such as th e CCAAT b o x a n d G C b o x , w hose p recise fu n c tio n s in re g u la t
in g th e level o f tra n sc rip tio n are less clear. T h e se u p stre a m e lem en ts m ay be
p re s e n t in m u ltip le co p ies a n d are o fte n g e n e fam ily o r p ro m o te r - specific. G enes
th a t a re constitutively ex p ressed in all tissues - h o u se k e e p in g g e n e s - te n d to
lack b o th TATA a n d CCAAT boxes b u t have GC boxes, w hich b in d th e S p l tra n
scrip tio n factor. Som e tra n sc rip tio n facto rs are th o u g h t to b in d cooperatively to
th e m o re g e n e ra l p ro m o te r seq u en ces, h e lp in g to p ro d u c e an active tra n sc rip tio n
in itia tio n co m p lex . O th e rs a re m o re tru ly re g u la to ry fo r ex p ressio n o f specific
g en es o r g e n e fam ilies. T h e p ro m o te r re g io n s o f h o u se k e e p in g g e n es also te n d to
have sm all 1 -2 kb isla n d s o f C pG -rich se q u en ce s th a t a re u n d e rm e th y la te d , co m
p a re d w ith C pG se q u e n c e s elsew here in th e v e rte b ra te g e n o m e th a t a re m ethylat
e d a t th e C5 p o sitio n . T h ese C pG islan d s a re fo u n d a ro u n d th e 5 ' e n d o f a
significan t p r o p o rtio n o f all g en es a n d a lth o u g h th e ir ro le is still un clear, they p ro
vide im p o rta n t la n d m a rk s fo r id en tify in g th e lo c atio n o f genes. A n islan d can be
id e n tifie d by a clu ste rin g o f sites fo r m ethylation-sensitive re stric tio n enzym es th a t
cleave u n m e th y la te d C pG -rich sequ en ces.
In a d d itio n to th e m o re g e n e ra l u p stre a m re g u la to ry e le m e n ts discussed above,
th e re a re im p o rta n t e n h a n c e r se q u e n c e s th a t are essential fo r full o r tissue-specific
ex p ressio n a n d act f u r th e r u p stre a m o r even w ithin in tro n s o r do w n stream re
gions. U n lik e p ro m o te r ele m en ts, e n h a n c e rs can act in an o rie n ta tio n a n d
p o sitio n -in d e p e n d e n t m a n n er. T h e se a re o fte n r a th e r s h o rt (1 0 -2 0 b p ) se q u en ces
b u t a re o fte n p re s e n t in several copies, as w ith GC boxes. E n h a n c e rs gen erally ac
tivate a g e n e b u t they m ay also in h ib it it, d e p e n d in g o n th e c ellu la r co n d itio n s.
10 MICHAEL B. GORIN AND ALAN F. WRIGHT

B ecause b o th p ro m o te r a n d e n h a n c e r se q u en c es a re s h o rt a n d , in som e cases,

p re s e n t in m u ltip le copies, they are less v u ln e ra b le to p o in t m u ta tio n th a n ex o n
o r splice seq u en ces, so th a t co m paratively few re g u la to ry m u ta n ts a re fo u n d in
(h e rita b le ) g e n e tic diseases. In th e case o f som atic d iso rd e rs such as cancer,
c h a n g e s in g e n e tic re g u la to ry reg io n s a re o f g re a t im p o rta n c e .

II G EN ETIC MAPPING

T h e c o n c e p t o f g e n e tic linkage was first in tro d u c e d in 1910 by T H M organ w ho

realised th a t th e te n d e n c y o f som e allelic p a irs to seg reg ate to g e th e r (co u p lin g )
a n d o f o th e rs to seg reg ate a p a rt in th e o ffsp rin g (rep u lsio n ) w ere two aspects o f
th e sam e p h e n o m e n o n , in w hich g en es re sid e o n th e sam e c h ro m o so m e (F igure
3).

A
B II X a
b
A
b
A
b
X a a
B
parental
chromosomes

A
BI a
b
A
b
a
B
gametes

coupling repulsion

Figu re 3 C o u p lin g a n d re p u lsio n . Two alleles a t e a ch o f two loci (A,a a n d B,b) a re show n, two
o f w hich a re in c o u p lin g (th e sam e c h ro m o s o m e ), two o f w hich are in re p u lsio n (o n o p p o site
ch ro m o so m es) a n d seg reg ate to g e th e r o r a p a rt in th e gam etes.

M o rg an h y p o th esised th a t th e stre n g th o f th e o b serv ed linkage betw een p a ire d

g e n e s was d e p e n d e n t o n th e s e p a ra tio n o f th e g e n e s o n th e c h ro m o so m e . N on-
p a re n ta l c o m b in a tio n s o f g am etes in th e o ffsp rin g o r re c o m b in a n ts w ere th o u g h t
to re su lt fro m ex c h a n g e (crossing-over) b etw een th e h o m o lo g o u s p a re n ta l c h ro
m o so m es d u rin g m eiosis (F igure 4).
T h is h y p o th esis h as since b e e n fully c o n firm e d . G en etic linkage is th e re fo re
p re s e n t w h en two loci o c c u r o n th e sam e c h ro m o so m e a n d / o r a re in h e rite d to
g e th e r m o re o fte n th a n e x p e c te d fro m in d e p e n d e n t a sso rtm e n t o f th e c h ro m o
som es. G en etic d istan ces a re m e a su re d e ith e r in term s o f th e frac tio n o f re c o m b i
n a n t to to tal o ffsp rin g o r g a m e tes - th e re c o m b in a tio n fra c tio n (usually d e sig n a t
ed by th e sym bol 0) - o r in g e n e tic m ap u n its such as cen tiM o rg an s. T h e early
D ro so p h ila g en eticists so o n o b se rv e d th a t g e n e tic distan ces m e a su re d in te rm s o f
re c o m b in a tio n fra c tio n s w ere ap p ro x im ately additive, w hich led to th e h y p othesis
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 11

bI Os
U x s D D s DOT U U genotypes

A
1
---, I gametes
B
I i| sO 0
L
recombinants

Fig u re 4 R eco m b in a tio n . Two loci on th e sam e c h ro m o so m e are show n, each w ith two alleles
(A,a a n d B,b) w hich seg reg ate in th e g am e tes e ith e r as p a re n ta l (AB, ab) o r re c o m b in a n t (Ab
a n d aB) co m b in atio n s. Loci th a t a re fa r a p a rt o n th e sam e c h ro m o so m e will asso rt in d e p e n
d en tly o f each o th e r in th e g am etes (a n d offsp ring ) as a resu lt o f re c o m b in a tio n b etw een h o
m o lo g o u s c h ro m o so m e s in m eiosis.

o f co lin earity o f g en es a lo n g th e c h ro m o so m e a n d to th e d e v e lo p m e n t o f th e first

g en e tic m ap s o f ch ro m o so m es. Since th e o c c u rre n c e o f d o u b le cross-overs b e
tw een two g e n e tic loci will n o t b e o b se rv e d as re c o m b in a n t o ffsp rin g , the o c cu r
re n c e o f all c a teg o ries o f m u ltip le crossover betw een loci will lead to
u n d e re s tim a tio n o f th e tru e g e n etic d istan ce. T his leads to no n -lin earity o f th e ge
n e tic m ap w h en m ark ers a re s e p a ra te d by lo n g e r distances. T his effe ct is offset to
som e e x te n t by th e w id esp read o c c u rre n c e o f interference, th e te n d e n c y o f o n e
crossover to su p p ress th e o c c u rre n c e o f a d ja c e n t ones. G en etic m aps are th e re fo re
usually ex p ressed in te rm s o f g e n e tic m a p u n its, w hich are additive, even over long
distances. T h e co m m o n ly u se d g e n e tic m ap u n it is th e centiM organ, d e fin e d as
th e le n g th o f c h ro m o so m e th a t show s o n e crossover per chromatid strand in
100 m eioses. T h e re la tio n sh ip b etw een th e re c o m b in a tio n fra ctio n , 0, a n d m ap
u n its is e x p re sse d in te rm s o f m apping functions, such as th a t o f Kosambi:

0 1 ( e 4x - 1)
2 ( e 4 x + 1)

w h ere x is in m a p u n its (M o rg an s). T h e re la tio n sh ip is ap p ro x im ately lin e a r

over s h o rt distan ces, so th a t in m ost m am m als a re c o m b in a tio n fra ctio n o f 0.1 (o r
10% fre q u e n c y o f re c o m b in a tio n ) c o rre sp o n d s to a g e n etic d istan ce o f 10 centi-
M organs, d u e to th e lack o f d o u b le crossovers over this d istan ce. Above a b o u t 20
cen tiM o rg an s (cM) th e two m easu res o f re c o m b in a tio n diverge increasingly u n til
at a re c o m b in a tio n fra c tio n o f 0.5, this c o rre sp o n d s to an in fin ite d istan ce b e
tw een m ark ers. Two m a rk ers se p a ra te d by a 0 value o f 0.5 m ay b e o n d iffe re n t
ch ro m o so m e s a n d th e re fo re assort in d e p e n d e n tly at m eiosis o r may be o n th e
12 MICHAEL B. GORIN AND ALAN F. WRIGHT

sam e c h ro m o so m e b u t s e p a ra te d by a large g e n e tic d istan ce. In th e la tte r case, a

re c o m b in a tio n fra c tio n o f 0.5 is th e m ax im u m value th a t can be ex p e c te d , b ecause
ea c h o f a p a ir o f c h ro m a tid stra n d s has only a 50% c h a n c e o f b e in g involved in any
o n e e x c h a n g e , p ro v id ed th a t each stra n d is equally likely to be involved in a cross
over.
T h e total g en etic m a p le n g th s in m an a n d m o u se are know n q u ite precisely fro m
g e n e tic m a rk e r stu d ies (4000 cM in m a n a n d 1600 cM in m o u se), b u t in b o th cases
th e re is a sig n ifican t sex d iffe re n ce in re c o m b in a tio n fre q u en cies. In m an , fo r ex
a m p le, th e to tal au to so m al le n g th in m ales is a b o u t 2800 cM a n d in fem ales 4800
cM. T h e re a so n fo r th e in c re a se d re c o m b in a tio n in fe m a les1, w hich is g enerally
tru e in m am m als, is u n k n o w n , b u t g e n etic m aps a re th e re fo re b est ex p re sse d as
se p a ra te m ale a n d fem ale m aps. R e c o m b in a tio n rates vary in d iffe re n t p a rts o f th e
g e n o m e , w ith som e h o t sp o ts a n d o th e r c o ld sp o ts, b u t over lo n g e r distan ces
they te n d to equalise. R ates te n d to be in c re a se d tow ards th e e n d s o f ch ro m o so m es
a n d d e c re a se d n e a r th e c e n tro m e res. T h e re is a ro u g h c o rre s p o n d e n c e betw een
th e n u m b e r o f ch iasm ata - th e p re su m e d physical c o u n te rp a rt o f th e crossover -
o b se rv e d in m ale m eioses a n d th e n u m b e r o f crossovers m ea su re d by g e n e tic m a p
p in g studies. Finally, th e re is an a p p ro x im a te re la tio n sh ip betw een g e n e tic a n d
physical d ista n c e such th a t 1 c e n tiM o rg a n in m an c o rre sp o n d s to a b o u t 1 M ega
base (106 base pairs) o f DNA (a b o u t 1.6 M b in m o u s e )2, a lth o u g h this m ay vary
m ark ed ly in d iffe re n t p a rts o f th e g e n o m e d e p e n d in g o n w h e th e r re c o m b in a tio n
is o c c u rrin g above o r below th e average rate.
G en etic lin k ag e is m o st co m m o n ly ev alu ated by m e a n s o f th e lo d score statistic
d evised by M o rto n in 1951. In o rd e r to calculate a lo d sc o re fo r a given set o f data,
first th e p ro b ab ility (lik elih o o d ) o f ob serv in g r re c o m b in a n ts o u t o f n info rm ativ e
m eioses is c a lc u la ted fo r ea c h possible value o f th e re c o m b in a tio n fra c tio n (i.e. o f
th e g e n e tic d ista n c e b etw een two m a rk e rs), u sing b in o m ial th e o re m :

L (e) = ( 9 ) r ( i - 0 ) n ^ r p T (7 7 r 7 )- ! '

F or e x am p le, w h en 1 re c o m b in a n t o u t o f 6 o ffsp rin g are o b serv ed , th e likeli

h o o d fo r 0 = 0.1 is:

L (e = o.ij = (0-1) (Q.9)5^ '

If th e n u m b e r o f re c o m b in a n ts c a n n o t b e in fe rre d u n a m b ig u o u sly (e.g. two

g e n e ra tio n fam ilies) sin ce th e linkage phase o f th e p a re n ts is un k n o w n (i.e. w h e th
e r th e alleles a re in c o u p lin g o r re p u lsio n ), the average lik e lih o o d o f th e two a lter

1There are exceptions to this, particularly towards the ends of some chromosomes.
2The total am ount of DNA per haploid genome is about 3 x 109 base pairs (bp) in mammals, these val
ues are therefore obtained by dividing the genetic length for each species by 3 x lO^bp.
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 13

native p h a se s is c o m p u te d . N ext, th e p ro b a b ility o f th e fam ily d a ta given th e n u ll

h y p o th esis o f ra n d o m a sso rtm e n t (0 = 0.5) is c alcu lated in th e sam e way. In o rd e r
to assess th e overall lik e lih o o d o f linkage, th e lik e lih o o d ra tio (LR) is o b ta in ed :

T h e logjQ o f th e LR is c o n v en tio n ally u sed, w hich is th e lo d sc o re o r log o f th e

o d d s ratio . T h is ca lc u la tio n is th e n re p e a te d over several values o f 0 a n d th e 0 val
ue c o rre s p o n d in g to th e m a x im u m lik e lih o o d v alue o f th e lo d sco re (Zmax) is g e n
erally ta k e n to b e a n a p p r o p r ia te su m m a risin g statistic, since it m in im ise s th e
v arian ce o f th e estim ate. T h e 95% co n fid e n c e lim its o f 0max are m o st con v en ien tly
a p p ro x im a te d by re a d in g th e 0 values c o rre s p o n d in g to Zmax 1.00, p ro v id ed th a t
th e sam ple size is larg e e n o u g h . T h e lo d score is a se q u en tial test, in th a t lods can
be a d d e d to g e th e r fro m d iffe re n t fam ilies a n d if necessary over d iffe re n t studies
u n til th e re q u ire d d e g re e o f statistical significance is o b ta in e d . In h u m a n genetics,
a lo d sco re o f 3 o r g re a te r (LR >1000:1) is c o n v e n tio n ally u sed , fo r M e n d e lia n
traits, to d e m o n s tra te a statistically sig n ifican t linkage, w hile linkage is ex c lu d e d a t
8 values fo r w h ich th e lo d score is - 2 o r less. F o r X -linked loci, the u p p e r critical
lim it is re d u c e d fro m 3 to 2. T h e d eriv a tio n o f th ese lim its takes in to a c c o u n t th e
fact th a t th e p rio r p ro b a b ility o f lin k ag e is a b o u t 1 in 20 fo r au to so m a l loci, a n d
re d u c e s th e p o s te rio r p ro b a b ilitie s b o th th a t lin k ag e is actually p re se n t, given th a t
it has b e e n e x c lu d e d (type 2 e r r o r ) , a n d th a t it is a b sen t, given th a t it has b e en co n
firm e d (type 1 e rro r) to suitably low values. U n d e r the n u ll h y p othesis (0 = 0.5),
th e fu n c tio n 2 In (LR) is d istrib u te d as y j with o n e d e g re e o f fre e d o m , so th a t a
lo d score o f 3 c o rre sp o n d s to a o f 13.8.
L od scores fro m sm all n u c le a r fam ilies can be c h e ck e d m anually b u t fo r m o st
p u rp o se s they a re c a lc u la ted u sin g c o m p u te r p ro g ra m m e s d e sig n e d fo r th e p u r
pose. L in k ag e to an u n m a p p e d disease is usually estab lish ed by sc re e n in g a large
n u m b e r o f m a rk e r loci. A b o u t 200 m ark ers, evenly spaced a lo n g the h u m a n g e n e t
ic m ap , w ould be su ffic ie n t to d e te c t lin k a g e to any locus w ithin 10 cM o n e ith e r
side o f a m ark er. T y p in g a larg e set o f individuals w ith this n u m b e r o f m a rk e rs is
clearly a very larg e u n d e rta k in g b u t th e strategy has pro v ed rem a rk a b ly successful
(C h a p te rs 6, 7, 15, 16). O n c e linkage is estab lish ed betw een a disease g e n e a n d a
m arker, o th e r m a rk e rs fro m th e re g io n are typed a n d a m u ltip o in t linkage analysis
is c a rrie d o u t. H e re th e p o sitio n o f th e disease locus is usually v aried with resp ect
to a fixed series o f m a rk e r loci, w hose o rd e r a n d se p a ra tio n s a re know n. T hese
c o m p u ta tio n s a re c o n sid erab ly m o re d e m a n d in g th a n tw o-point lo d scores a n d
m ay take a lo t o f c o m p u te r tim e. T h e o u tp u t is again usually in th e fo rm o f (m u l
tip o in t) lo d scores c o rre s p o n d in g to d iffe re n t disease g e n e lo catio n s over th e e n
tire m ap. T h e relative lik e lih o o d o f various g e n e o rd e rs can th e n be estim ated.
In h u m a n linkage, th e re are m any p o te n tia l p ro b le m s, m o st n o tab ly sm all sam
ple size, re su ltin g in w ide c o n fid e n c e lim its. G en etic h e te ro g e n e ity is also a m ajor
p ro b le m in g e n e tic eye disease w ith m any loci p ro d u c in g virtually in d istin g u ish
14 MICHAEL B. GORIN AND ALAN F. WRIGHT

ab le p h e n o ty p e s. Lack o f in fo rm a tio n on lin k ag e p hase, th e n e e d to in fe r th e g e n

otypes o f m issing inividuals, p h e n o c o p ie s, re d u c e d p e n e tra n c e a n d n o n -p a te rn ity
all c o m p o u n d th e difficulties. R ep licatio n o f a h u m a n g e n e tic linkage re su lt is
th e re fo re p articu larly im p o rta n t.
In D ro so p h ila, th e o r d e r o f lin k ed loci was first in fe rre d by pairw ise c o m p a ri
sons a n d m in im isatio n o f th e n u m b e r o f d o u b le o r m u ltip le re c o m b in a n ts to p ro
d u c e a lin e a r m a p o r linkage g ro u p . In th e m ouse, lin k ag e m aps a re c o n stru c te d
a lo n g sim ilar lines. T h e m o u se d a ta p rovide f u rth e r ju stific a tio n fo r this sim ple a p
p ro a c h since th e n u m b e r o f d o u b le crossovers has b e e n fo u n d to be even less fre
q u e n t th a n e x p e c te d , as a re su lt o f in te rfe re n c e . T h e pow er o f this a p p ro a c h
in creases as th e d a ta a c c u m u la te u n til th e p o in t is re a c h e d w here all crossover sites
can be in fe rre d accurately. A m ea n s o f calc u latin g th e relative p ro b a b ilitie s o f dif
f e re n t g e n e o rd e rs a n d a b est estim ate o f th e likely lo catio n o f a g e n e m ay still be
re q u ire d however.

G enetic markers

W hat m a rk e rs a re u sed for g en etic m ap p in g ? Initially g e n es w ere m a p p e d in m an

a n d m o u se u sin g e ith e r b lo o d g ro u p v ariants o r p ro te in e le c tro p h o resis p o lym or
phism s. T h e re are co m paratively few such m a rk e rs a n d they a re n o t highly infor
m ative fo r m a p p in g studies - they show low heterozygosity. S ince only the
o ffsp rin g o f d o u b ly o r m u ltip ly h etero zy g o u s individuals provide in fo rm a tio n
a b o u t th e se g re g a tio n o f g a m e te s in th e o ffsp rin g , th ese are said to be in fo rm a
tive fo r linkage. T h e first RFLPs (F u rc 1) w ere u sed in linkage stu d ies in the
early 1980s, w hich m ark ed a rev o lu tio n in g e n e m ap p in g , since it b ec am e clear
th a t th e re was now a lim itless supply o f p o ly m o rp h ic DNA m arkers. Increasingly,
th e la b o u r in ten siv e n a tu re o f RFLP typing a n d th e ir com paratively low h etero zy
gosity c o m p a re d w ith n ew er m arkers, has le d to th e ir d e clin e . T h e m e th o d th a t is
c u rre n tly in favour is to use m icro satellite re p e a t polym orphism s. T h ese m ark ers
can b e selected to have a h ig h heterozygosity (> 0.5), a n d are relatively evenly
sp aced th ro u g h o u t th e g e n o m e s o f m o st m am m a lian species. T hey are easily a n d
rap id ly typed u sin g th e poly m erase ch ain re a c tio n (section 111(3) a n d F ig u re 6)
a n d have a low e n o u g h m u ta tio n rate to b e suitable fo r linkage m ap p in g .
R ecently, h u m a n g e n e tic m ap s c o n ta in in g over 1000 m icrosatellites have b e en
p u b lish e d . S ta n d a rd ise d h u m a n g en etic lin k ag e m aps a re o b ta in e d by typing
m a rk e rs ag ain st a set o f 40 larg e th re e -g e n e ra tio n re fe re n c e (C EPH ) fam ilies.

T he m ouse genetic map

T h e la b o ra to ry m o u se (M ils musculus) is o n e o f th e m ost intensively stu d ie d m am

m alian species, w hich has led to th e id en tific atio n o f a b o u t 500 m u ta n t p h e n o
types, in c lu d in g m any th a t affect th e eye, a n d close to 1000 genetically m a p p e d
loci. T h e c lo n in g o f th e small eye, rd a n d rds loci in th e m ouse led d irectly to the
id e n tific a tio n o f th e c o rre s p o n d in g h u m a n disease g en es (discussed in C h a p te rs
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 15

5, 6, 19). T h e pow erfu l g e n e tic a n d analytical reso u rce s th a t a re available in th e

m ouse to g e th e r w ith th e stro n g ev o lu tio n a ry co n serv atio n o f large g e n o m ic
re g io n s b etw een m o u se a n d m an has le d to the in cre asin g im p o rta n c e o f m ouse
g enetics. M ore recently, this has b e e n fu rth e r e n h a n c e d by th e ability to create
tra n sg e n ic m ice.
T h e p rim a ry re so u rc e fo r g en etic m a p p in g o f an u n k n o w n locus in th e m ouse
u se d to be re c o m b in a n t in b re d (R I) strain s in w hich closely re la te d la b o ra to ry
strains w ere cro ssed a n d b ackcrossed to g en etically fix th e re c o m b in a n t c h ro m o
som es o n an o th erw ise in b r e d b a c k g ro u n d (intraspecific crosses). In this way, th e
seg reg atio n o f m a rk e r alleles th a t a re d istin g u ish a b le betw een th e p a re n ta l strains
can be follow ed by typing th e RI o ffspring. U n fo rtu n a tely , m any m a rk e r alleles are
n o t d istin g u ish a b le b etw een th e p a re n ta l strain s so th a t th e RI o ffsp rin g are u n in
form ative - p a rtic u la rly w ithin m o re in te re stin g ev o lu tio n arily co n se rv e d reg io n s
such as genes. T his p ro b le m has to a larg e e x te n t b ee n o v ercom e by th e in tro d u c
tio n o f in ter-specific crosses b etw een M. musculus a n d M. spretus, o n e o f th e m ost
distantly re la te d M us species th a t still b re e d s w ith M. musculus. T h e se species have
d iv erg ed sufficiently so th a t th e m ajority o f m ark ers d iffer betw een th e m . T h e off
sp rin g o f th e ( .Vi. musculus x M. spretus) FI hybrids can th e re fo re be typed directly
a n d analysed fo r linkage. T h e re a re so m e m in o r draw backs with th e m e th o d , fo r
ex am p le th e FI fem ales b u t n o t th e m ales are fe rtile so th a t only a fem ale g en etic
m ap is p ro d u c e d . In this way, over 600 loci have b e e n g en etically m a p p e d , p ro d u c
in g an average m a p re so lu tio n o f <3 cM.
In m ice, th e to tal g e n e tic le n g th is in th e re g io n o f 1600 cM c o m p a re d with 4000
cM in m an d esp ite th e fact th e th e two species have alm o st id e n tica l a m o u n ts o f
DNA p e r cell (3 x 10y bp p e r h a p lo id g e n o m e ) . T h e co m p arativ e m ap o f m a n a n d
m o u se is in creasin g ly im p o rta n t, sin ce closely lin k e d g en e s in o n e species te n d
to re m a in so in th e o th er. T h e m ea n le n g th o f au to so m al seg m en ts c o n serv ed
betw een m an a n d m o u se is 10.4 2.4 cM a n d a p p ro x im a te ly 90 co n se rv ed seg
m en ts have b e e n id e n tifie d to d ate. Id e n tifica tio n o flin k a g e to a disease g e n e in
m an resu lts in a search b o th fo r c a n d id a te g en e s a n d fo r m ouse m u ta n ts w ith a
re la te d p h e n o ty p e e ith e r o f w hich m a p to th e c o rre sp o n d in g c o n serv e d re g io n o f
th e m o u se g e n o m e . T h is has p ro v e d useful in m oving fro m m ouse to m an a n d vice
versa. Several ex am p les a re discussed in detail in o th e r c h a p te rs (C h a p ters 4, 5, 18,
19).

T ransgenics a n d ES cell technology

T h e ability to c reate tra n sg en ic m ice, in c o rp o ra tin g fo reig n g e n es e ith e r with

m u ta tio n s o r show ing a b n o rm a l ex p re ssio n , has e n o rm o u sly e n ric h e d th e field o f
m o le c u la r g enetics. T h e fu n c tio n o f g en es can be in vestigated by p ro d u c in g m ice
w ith a b e r r a n t ex p ressio n , overex p ressio n o r by m u ta tin g th e n o rm a l g e n e . A
m u ta n t p h e n o ty p e can be re scu ed by in se rtio n o f a c a n d id a te g e n e , p ro v id in g
th e final p r o o f th a t th e c o rre c t g e n e has b e e n id e n tifie d . T h e re a re two m ain
strategies fo r c re a tin g tra n sg en ic m ice th a t are illu stra ted in F igure 5.
16 MICHAEL B. GORIN AND ALAN E WRIGHT

B
ES cells containing Pronuclear injection of
targeted gene transgene
disruption

I
Blastocyst injection

I
Transgenic
mouse

Outcross

Selection of offspring
/
Intercross offspring
with germline transmission to produce homozygous
of transgene transgenic mice

Figu re 5 T ran sg en ic ex p erim e n ts. T ran sg en ic m ice can b e p ro d u c e d in two m ain ways. M ouse
e m b ry o n ic stam (ES) cells may b e ta rg e te d by h o m o lo g o u s re c o m b in a tio n in to a g e n e in or
d e r to in tro d u c e a specific m u ta tio n (e.g. k n o c k o u t). (A) T h e ta rg e te d ES cells are m icro in
je c te d in to a blatocyst, re im p la n te d a n d c h im aeric p ro g e n y id e n tifie d by m ean s o f c o a t c o lo u r
m arkers. T h e se can b e o u tc ro ssed to identify th o se cap ab le o f g e rm lin e tran sm issio n o f th e
tra n sg e n e a n d in tercro ssed to p ro d u c e h om ozygotes. A lternatively th e m ale p ro n u c le u s w ith
in fertilised oocytes is m ic ro in je c ted with th e tra n sg e n e a n d a fter re im p la n ta tio n in to a
p se u d o p re g n a n t fem ale, th e p ro g en y a re o u tc ro ssed to iden tify those show ing hem izygous ex
p ressio n o f th e tra n sg e n e a n d th e n in te rc ro sse d to p ro d u c e h o m ozygous e x p ressio n (B).

T h e first a p p ro a c h involves th e use o f to tip o te n tia l m ouse em b ry o n ic stem cells

(ES cells), w hich can be m o d ified by th e in tro d u c tio n o f g e n e co n stru cts aim ed
a t d is ru p tin g a g e n e e ith e r specifically by h o m o lo g o u s re c o m b in a tio n o r non-spe-
cifically by in sertio n ] in activ atio n (F igure 5 A ). T h e g e n e c o n stru c t can b e in tro
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 17

d u c e d in to ES cells by e le c tro p o ra tio n o r m ic ro in je ctio n . D isru p tio n o f a g e n e by

h o m o lo g o u s re c o m b in a tio n in m itotically dividing cells is a low fre q u e n c y ev en t so
th a t te d io u s selectio n a n d sc re e n in g p ro c e d u re s are o fte n re q u ire d to d e te c t th e
ra re clo n es th a t have in te g ra te d th e g e n e c o n stru c t at th e h o m o lo g o u s site. Spe
cific m u ta tio n s can b e in tro d u c e d in this way o r th e g e n e d is ru p te d by in se rtio n o f
th e c o n stru c t. O n c e th e tra n sg e n e h as b e e n in tro d u c e d in to an ES cell, w hich is
usually re c o g n ise d by th e acq u isitio n o f a d ru g -resistan ce p h e n o ty p e , th e cells are
in tro d u c e d in to m o u se blastocysts by m ic ro in je c tio n a n d after re im p la n ta tio n in to
a fo ste r m o th e r, c h im a e ric o ffsp rin g can b e id e n tifie d by co at c o lo u r o r o th e r g e
n e tic m ark ers. F u rth e r p ro b le m s arise in fin d in g c h im ae ra s th a t express th e tran s
g e n e in th e g e rm lin e a n d so a re ab le to tran sm it it to th e o ffsp rin g a fter
o u tc ro ssin g to an a p p ro p ria te strain . Finally, in te rc ro ssin g o f g e rm lin e ch im aeras
m ay p ro d u c e id e n tifia b le h o m o zygous o ffspring, u n less th e m u ta tio n is a hom ozy
gous leth al.
T h e se c o n d a n d o fte n sim p ler strategy fo r p ro d u c in g tran sg e n ic an im als in
volves m ic ro in je c tio n o f p ro n u c le i w ithin fertilised m ouse oocytes w ith th e g e n e
c o n stru c t, w hich a re re im p la n te d in to p se u d o -p re g n a n t fem ales a n d le a d to the
p ro d u c tio n o f m ice th a t a re hem izygous fo r th e tra n sg e n e (F igure 5B ). T h e re
su lta n t p ro g e n y can ag ain b e o u tc ro sse d a n d in te rc ro sse d to o b ta in hom ozygotes.
In this way, a w ide variety o f investigations can be c a rrie d o u t o n g en e exp ressio n
a n d fu n c tio n in c lu d in g th e "rescue" o f m u ta n t p h e n o ty p e s. T h e hom ozygous rd
and rds m u ta n ts, w hich a re b o th asso ciated w ith re tin a l d e g e n e ra tio n , w ere p artia l
ly re sc u e d u sing p ro n u c le a r in je c tio n o f th e c lo n e d [3-subunit o f cGM P p h o s p h o d
iesterase a n d p e rip h e rin /R D S g en es respectively (C h a p ters 5, 6). T h e te c h n iq u e
can also be u se d to see if a d o m in a n t m u ta tio n in a c lo n e d g en e (e.g. rh o d o p sin ,
C h a p te r 6) co n fers an a b n o rm a l p h e n o ty p e even in th e p re se n c e o f two n o rm a l
copies o f th e g e n e . T h e final p r o o f th a t a disease g e n e has b e e n c lo n e d , is to rec
re a te th e p h e n o ty p e in th e m o u se w ith a m u ta n t c a n d id a te g e n e o r to rescu e th e
n o rm a l p h e n o ty p e in th e m u ta n t m o u se by in tro d u c tio n o f th e c a n d id a te g e n e. In
b o th cases, u p stre a m re g u la to ry re g io n s m u st also be in tro d u c e d in o rd e r to re ta in
a p p ro p ria te ex p ressio n w ithin th e eye.
T h e use o f th e m o u se fo r studying h u m a n g e n e tic d iso rd ers d o e s have lim ita
tions, b u t they a re fa r few er th a n was g en erally a p p re c ia te d te n years ago. T h e vidx
m o u se has a rem ark ab ly m ild p h e n o ty p e c o m p a re d w ith m u ta tio n s in th e h u m a n
h o m o lo g u e o f m o u se d y stro p h in w hich causes th e fatal d iso rd e r o f D u c h e n n e
m u sc u la r dystrophy. Similarly, som e o f th e tran sg e n ic m ice d e v e lo p e d as m odels
o f cystic fibrosis show d iffere n ces fro m th e h u m a n d isorder. In g e n e ra l, th e re is
still so m u c h to b e le a r n t fro m studying m o u se m u ta n ts a n d why they may differ
fro m th e h u m a n d is o rd e r th a t th e ir in c re a sin g use is easily ju stifie d . T h is is p a rtic
ularly tr u e as g e n e th e ra p y starts to co m e o f age in th e 1990s.

Ill T H E T O O L S O F T H E M OLECULA R GENETICIST

T h ese m e th o d s a re n o t u n lik e th e sim ple m ach in es, such as th e lever o r w heel,

th a t u n d e rlie th e w orkings o f co m p le x m ech an ical in stru m e n ts. T h e basic m e th
o d s o r tools o f m o le c u la r g en etics can be c o n sid e re d u n d e r five h eadings:
18 MICHAEL . GORIN AND ALAN E WRIGHT

{1 ) T h e se p a ra tio n o f n u c le ic acids o n th e basis o f size a n d stru c tu re .

(2) T h e m a n ip u la tio n o f n u cleic acids w ith specific enzym es o r chem icals th a t e n
ab le m o lecu les to be m o d ified, cleaved, jo in e d (ligated) o r co p ied .
(3) T h e p ro d u c tio n o f sp ecific n u cleic acids by enzym atic o r ch em ical synthesis o r
by in tro d u c tio n in to m icro o rg an ism s (clo n in g ).
(4) T h e visu alisatio n a n d q u a n tifica tio n o f nu cleic acids by non -co v alen t b in d in g
o f stains o r dyes, by cov alen t a tta c h m e n t o f d e te c to r m o lecules a n d by ultravi
o le t a b so rb an ce.
(5) T h e h y b rid isa tio n o f n u cleic acids to c o m p le m e n ta ry sin g le-stran d ed n u c le
o tid e p ro b e s in o rd e r to d e te c t specific se q u en ces w ithin c o m p lex m ixtures.

U n d e r ea c h o f these h e a d in g s th e re are n u m e ro u s variatio n s a n d m od ificatio n s

th a t allow a b ro a d ra n g e o f tasks to be c a rrie d o u t o n a variety o f n u cleic acid so urc
es. T h e re s u lta n t analyses can involve the d isc rim in atio n o f single n u c le o tid e dif
fe re n c es b etw een two g e n e s b u t a re also cap ab le o f assessing e n o rm o u s DNA
stru c tu re s such as ch ro m o so m es. All ro u tin e la b o ra to ry m e th o d s in m o le c u la r ge
netics use th e se five p rin cip les. A b rie f overview will b e given o f ea ch tool a n d its
p rin c ip a l ap p licatio n s, follow ed by a m o re d e ta ile d table o f specific m e th o d s w ith
in ea c h category.

1 ) S ep aratio n o f nucleic acids o n th e basis o f size an d stru ctu re

N u cleic acids a re ro u tin e ly se p a ra te d by gel e lec tro p h o resis. For relatively sm all,
lin e a r d o u b le -s tra n d e d (ds) DNA m o lecu les (<50 k b ), m ig ratio n w ithin an agar
ose gel in th e p re se n c e o f an electric field is a fu n c tio n o f m o le c u la r size ra th e r
th an c o n fo rm a tio n o r seq u en ce. T h e n u m b e r o f negatively c h a rg e d g ro u p s on
th e p h o s p h a te b a c k b o n e is p ro p o rtio n a l to th e n u m b e r o f base pairs, so th a t th e
ch a rg e density is co n sta n t. T h e d iffe re n t n u c le o tid e resid u es have little effect on
th e c h a rg e b e h a v io u r o f DNA, a lth o u g h this is less so fo r e le c tro p h o resis in poly
acry lam id e. DNA se q u e n c e analysis a n d h ig h re so lu tio n sep a ra tio n o f m o lecu les
u p to a b o u t 1000 n u c le o tid e s in le n g th (e.g. se q u e n c in g o r m icro satellite analy
sis) is g en erally c a rrie d o u t u sin g polyacrylam ide gel e le c tro p h o re sis u n d e r d e n a
tu rin g c o n d itio n s. In th e case o f sin g le-stran d ed (ss) DNA, th e d iffe re n t
n u c le o tid e s can have a m ajo r e ffect on th e c o n fo rm a tio n o f th e m o lecu le, w hich
can a lte r its b eh a v io u r in a gel unless d n a tu ra n ts are u sed to e lim in ate th e sec
o n d a ry stru c tu re . T h is is e x p lo ite d in th e te c h n iq u e o f single stra n d c o n fo rm a
tio n al p o ly m o rp h ism analysis (SSCP) in o rd e r to d e te c t single base ch a n g es fo r
m u ta tio n screen in g .
L arg e m o lecu les o f DNA (>50 kb) w ould n o rm ally be u n a b le to m ig rate w ithin
an ag aro se gel, b u t th e use o f p ulsed-field a n d field-inversion gel e le c tro p h o resis
(PFGE, FIGE) e x p lo it a c h a n g in g electric field to a lte r th e ir se c o n d a ry stru c tu re
so th a t they can m ig ra te at a ra te th a t is inversely p ro p o rtio n a l to size. In c o n trast
to yeast c h ro m o so m e s, h u m a n ch ro m o so m e s are m u c h too large to b e se p a ra te d
by PFGE a n d have first to b e cleaved w ith ra re c u tte r restric tio n e n d o n u c le a se s
in to fra g m e n ts below a b o u t 2 x 106 base pairs (2 Mb) in size. Yeast o r b a cte ria l ar-
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 19

dficial chrom osom es (YACs, BACs) a n d PI vectors have been used to clone h u
m an, m ouse o r D rosophila DNA fragm ents in the size range th at can be separated
and purified by PFGE.
Flow cytom etry or sorting is based on a d ifferent principle than electrophore
sis since it distinguishes chrom osom es (or cells) on the basis of their ability to bind
fluorescent dyes (e.g. bisbenzim ide, DAPI) that are detected after laser excitation.
A dilute solution o f chrom osom es is passed th ro u g h a laser and the em itted fluo
rescent signal is d etected and a m agnetic field is activated to divert the electrically
charged water d ro p let containing the chrom osom e o f interest into a tube. T he
separation o f chrom osom es is th erefo re based on a com bination o f size an d the
structural properties th at allow the bin d in g of dyes.
Table 1 illustrates the d ifferent m ethods o f nucleic acid separation, the size
range an d sources o f the m olecules separated an d special applications of each
technique.

2) M anipulation o f nucleic acids with specific enzymes o r chemicals

DNA can be m anipulated by a wide variety o f specific enzymes, by chemical

reagents, radiation and physical shearing. These m ethods allow nucleic acids to
be specifically cleaved, m odified by methylase, phosphatase or kinase enzymes,
jo in e d an d synthesised. T he following table sum m arises the m ajor m ethods by
m eans of which nucleic acids are altered. Only a small n u m b er o f the large rep er
toire o f enzymes and chem icals is listed bu t table 2 gives some indication o f the
scope o f these tools.

3. Production o f pure nucleic acids - synthesis, cloning

Specific pieces of DNA can be p ro d u ced in large quantities to allow for direct
study o r use as m olecular tools. This am plification process can be carried out by
chemical synthesis (as in the m anufacture o f oligonucleotides), by enzymatic
am plification using the polym erase chain reaction (PCR), or by cloning into vec
tors such as plasmids, cosmids, bacteriophage, PI or into yeast artificial chrom o
somes (YACs) (Table 3).
Chem ical synthesis of DNA is lim ited to m olecules o f less than ab o u t 100 nucle
otides in length an d is widely used for synthesising specific oligonucleotide se
quences (prim ers) for prim ing PCR and sequencing reactions. Small synthetic
oligonucleotides th at act as adaptors o r linkers for m odifying DNA ends are also
useful.
T he pro d u ctio n o f DNA by enzymatic am plification using PCR is central to a
wide range o f m olecular genetic experim ents. It is illustrated an d explained in Fig
ure 6. PCR synthesis o f DNA uses the o th er fundam ental tools, for exam ple an
nealing o f prim ers to the DNA tem plate, replication of DNA using heat-stable
polym erase, visual detection (as p art o f screening and analysis) an d purification
o f the p ro d u c t by electrophoresis but it is o f such fundam ental im portance that it
alm ost am ounts to a basic tool in itself.
20 MICHAEL B. GORIN AND ALAN F. WRIGHT

T ab le 1 F ra c tio n a tio n o f D N A by Size

DNA Type Method Size Range Special Uses

Chromosomes, human Flow sorting 30-200 Mb Chromosome-specific libraries

Metaphase spreads FISH, microdissected libraries

Chromosomes, yeast PFGE 0.1-10 Mb Purification o f YAC DNA

Genomic DNA PFGE 0.1-10 Mb Large-scale genomic mapping

FIGE 25 kb-2 Mb DNA purification, mapping
Agarose 100 b-50 kb Mapping, RFLPs

RNA Agarose, denaturing 1-20 kb Sizing and analysis o f RNA

dsDNA fragments Polyacrylamide 15-1500 b Sizing and analysis o f dsDNA

partial dsDNA fragments Polyacrylamide or SSCP analysis
Hydrolink H eteroduplex analysis
DGGE

ssDNA fragments Polyacrylamide, 1-500 b, single Sequencing, SI nuclease,

denaturing base resolution micro satellite analysis
Gel retardation assays

RNA fragments (small) Polyacrylamide, 50-1500b RNase protection assays -

denaturing to define transcribed regions

T ab le 2 M e th o d s fo r M odifying N u cleic A cids

Process Type Reagents Comments

Cleavage o f DNA Nonspecific

Enzymatic DNasel Use for radio-labelling DNA
by nick translation
Chemical 1,10 phenanthroline-Cu,
EDTA-Fe+2
Radiation UV, Xray Fragm ent size determ ined
by dose
Physical Shearing, ultrasound
Specific for structure
a) Single-stranded
Non-specific SI nuclease, DNA/RNA hybrid analysis
micrococcal nuclease for transcript and intron
mapping
Ends only Mung bean nuclease
b) Double-stranded
Ends only X exonuclease, Bal31
nuclease
Specific for sequence
Enzymatic Restriction endonucleases 4-8 b recognition sequences
Chemical 0 s 0 4, hydroxylaminine + Mismatch cleavage used in
piperidine mutation detection
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 21

T ab le 2 Continued.

Process Type Reagents Comments

Cleavage o f RNA Non-specific RNase A Transcript mapping

Specific for structure
Single-stranded SI nuclease RNase protection
Double-stranded RNaseH
RNA/DNA hybrid RNaseH cDNA synthesis
Specific for sequence RNase M i, T 1,T 2 RNA sequencing

Modification Mthylation Methylase Protects against R E.

Removal o f 5 Phosphatase Prevents ligation
phosphate
Addition o f 5 Polynucleotide kinase Used to label with i2P
phosphate

Ligation (joining) Blunt ends T4 ligase Covalent joining of

Overlapping ends T4 ligase DNA molecules
Single-stranded RNA ligase

Synthesis Chemical Oligonucleotide synthesis Up to 100 bases

Enzymatic
Not sequence-specific
3' Tailing Terminal deoxynucleotide Labelling, cloning
transferase (Tdt)
Sequence-specific
DNA template
DNA product DNA polymerase I, Klenow Labelling, cloning,
enzyme, T4 polymerase, sequencing, thermostable
Taq polymerase enzyme used for PCR
RNA product RNA polymerase RNase protection, in vitro
transcription, cosmid
walking
RNA template
DNA product Reverse transcriptase cDNA cloning, RT/PCR

C lo n in g o f DNA resu lts in th e ability to p ro d u c e large a m o u n ts o f a specific DNA

as well as o f its g e n e p ro d u c ts - RNA a n d p ro te in . T h e essence o f c lo n in g is th a t
DNA can be in tro d u c e d in to fo re ig n cells a n d stably p ro p a g a te d . T h e DNA o f in
te re st is jo in e d to v e c to r se q u en c es th a t allow th e cell to re p lic a te th e fo reig n ge
n e tic m a te ria l a n d th a t co n tain selectab le m ark e rs (e.g. a n tib io tic resistance
gen es) w hich can be u se d to select re c o m b in a n t clones - th o se th a t co n ta in a fo r
eign in sert. T h e p ro d u c tio n o f p u re DNA o f know n o rig in o r se q u e n c e is o f such
fu n d a m e n ta l im p o rta n c e th a t it m u st be c o n sid e re d a p rim ary tool.

4. N ucleic acid visualisation a n d quantification

DNA can b e visualised by a variety o f m e th o d s, b o th specific a n d non-specific,

th a t in c lu d e c o lo rim e tric , rad ioactive, flu o re sc en t, c h e m ilu m in e sc e n t a n d cyto-
22 MICHAEL B. GORIN AND ALAN F. WRIGHT
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Table 3 Production of Nucleic Acids: Synthesis and C lo n in g

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MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 23
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24 MICHAEL B. GORIN AND ALAN F. WRIGHT

logical stains o r dyes. S electio n o f th e m e th o d d e p e n d s on th e re q u ire m e n ts fo r

sensitivity, specificity a n d co n v e n ien c e. E th id iu m b ro m id e sta in in g o f DNA w ithin
gels is ro u tin e ly u sed to m o n ito r th e c o n c e n tra tio n o f specific b a n d s by ex a m in
in g th e flu o re sc e n c e e m itte d a fte r u ltra v io le t (UV) irra d ia tio n . Q u an tific a tio n o f
n u cleic acids can b e c a rrie d o u t m o re accu rately by m easu rin g th e ab so rb a n c e o f
UV lig h t a t 260 n m o r by flu o rim e try a fte r b in d in g o f flu o re sc e n t dye. T able 4 is
n o t co m p re h e n siv e b u t h ig h lig h ts th e ch o ices available fo r d iffe re n t types o f
e x p e rim e n t.

5, Specific se q u en ces o f DNA can be id en tifie d by hybridisation

C o m p le m e n ta ry stra n d s o f n u c le ic acid will a n n e a l o r hybridise to each o th e r a n d

fo rm stable s tru c tu re s even u n d e r u n fa v o u ra b le c o n d itio n s such as elevated tem
p e ra tu re a n d low salt (h ig h strin g en cy ). H yb rid isatio n rea ctio n s in w hich o n e
stra n d (th e p r o b e ) is ra d io la b e lle d a n d p re se n t at h ig h c o n c e n tra tio n can b e
u se d to re c o g n ise specific se q u en c es o f im m o b ilise d DNA w ithin co m p lex m ix
tu re s w ith very h ig h sensitivity. As little as a few pico g ram s o f n u c le ic acid can be
d e te c te d u sin g th ese m e th o d s (T able 5, F igure 1). F or ex am p le 10|Xg o f m a m m a
lian g e n o m ic DNA c o n ta in s only 3pg o f a 1 kb single copy se q u e n c e (1 in 5 x 106)
w hich can b e read ily d e te c te d by S o u th e rn hy b rid isatio n u sing a h ig h specific
activity p ro b e .
W h en d o u b le -s tra n d e d m o le c u les a re fo rm e d , they align so th a t th e h e a d (5 '
e n d ) o f o n e stra n d is m a tc h e d with th e ta il (3 ' en d ) o f th e o th e r stran d . T h e n u
cle o tid e bases a re p a ire d so th a t a d e n in e a n d th ym ine fo rm a stable s tru c tu re , h e ld
to g e th e r by two h y d ro g e n b o n d s, w hile g u a n in e a n d cytosine are m o re tightly
b o u n d by th re e h y d ro g e n b o n d s. Since they d o n o t fo rm co v alent b o n d s, th e two
stra n d s can be relatively easily d e n a tu re d a n d se p a ra te d by h e at, low salt a n d
ch em icals such as alkali, u re a a n d fo rm a m id e . By varying th e h y b rid isatio n o r
w ashing c o n d itio n s, h ybrids can be selec ted th a t are e ith e r very well m a tc h e d o r
c o n ta in a sig n ifican t p ro p o rtio n o f m ism a tc h e d bases. S im ilar c o n sid e ra tio n s a p
ply to th e use o f p rim e r a n n e a lin g re a c tio n s d u rin g a m p lificatio n o f DNA by PCR
(1 g u re 6 ) .
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 25
o
eg
CvJ
U
Pm
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Q
o
C
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in co
[primer annealing (55C)
and extension (72C)]
AMPLIFICATION

d
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c
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26 MICHAEL B. GORIN AND ALAN F. WRIGHT

Table 4 Detection of Nucleic Acids

Type of Specificity/ Reagents Detection Uses

Indicator Incorporation

NONSPECIFIC DETECTION

Electron Can distinguish ss Carbon Electron For DNA

structures microscopy and ds DNA Microscopy rarely used
(shadowing) EM

Stains
N onfluorescent Chromosome banding Giemsa Light
photomicroscopy Cytogenetics
Nonspecific Silver Direct visualisation DNA/RNA on gels
Gold (very sensitive)

Fluorescent Chromosome banding Quinacrine Fluorescence Cytogenetics

DAPI microscopy
Nonspecific (ds > ss) Ethidium Fluorescence
(ds + ss) Acridine photography DNA/RNA on gels
Orange

DETECTING SPECIFIC DNA FRAGMENTS

Direct Detection
Radioisotopes
End-labelled Dephosphorylated 5' end y' P-ATP Autoradiography Sequencing,
3' tailing reaction or*2PdNTP, Hybridisation
K S-dNTP

Incorporation PCR, nick-translation, a 3'7P-33P~, Autoradiography Sequencing,

by synthesis random prim er synthesis, S-dNTP Hybridisation
32p _ 33p
in vitro transcription
S5s -n t p

Fluorescent adducts
Chemically Oligonucleotide Fluorescein, Fluorescence Sequencing
incorp. svnthesis rhodam ine Hybridisation
derivatives

Internal PCR, nick translations, Fluor-dUTP Fluorescence Hybridisation

incorporation random prim er FISH
synthesis

Incorporation PCR Derivatized Fluorescence Sequencing

with labelled oligonuc
prim er leotide
 MOLECULAR GENETIC PRINCIPLES AND TECHNIQUES 21

T ab le 4 Continued.

Type of Specificity/ Reagents Detection Uses

Indicator Incorporation

Secondary Detection

Enzyme adducts to
DNA
Chemically linked glutaraldehyde HRP, Stains, Chemilum Hybridisation
crosslink alkaline
amino-modified oligo phosphatase In situ hybrid

Avidin-conjugates
Biotinylated probe Internal incorporation avidin-FITC Fluorescence FISH
microscopy Hybridization
End-labelling avidin-HRP Stains, Chemilum
Photobiotinylation

An tibody-e nzyme
conjugates
Incorp. by synthesis PCR, nick translation, Alk. ptase-, Chemiluminescence, Hybridization
random prim er synthe HRP- colorimetric stains
sis in vitro transcription -galacto-
sidase-
conjugates
Digoxigenin digoxigenin-dUTP Substrates with,
Fluorescein fluor-dUTP chemilum.
or colored
products

Table 5 Hybridisation of Nucleic Acids

Name of Probe Target Immobilized Separation Support Detection

method Component

Southern Oligo, PCR DNA Target Electro- Membrane Probe

product, Cloned phoresis
DNA

N orthern Oligo, PCR RNA Target Electro- M embrane Probe

product, phoresis
Cloned DNA

DNA or RNA Oligo, PCR DNA Target M embrane Probe

dot blots product, Cloned or RNA
DNA
28 MICHAEL B. GORIN AND ALAN F. WRIGHT

T ab le 5 Continued.

Name of Probe Target Immobilized Separation Support Detection

method Component

In situ Oligo RNA Target Glass slides Probe

hybridisation nucleotides,
of RNA PCR products,
transcripts Cloned DNA,
RNA

PolyA+ mRNA oligo dT RNA Probe Washing Membrane, cDNA

purification column,
magnetic beads
Reverse Primer or RNA None Primer- NA Nonspecific
transcription oligo dT directed o r direct
(RT) synthesis detection

Polymerase Primers DNA Product Amplify Electro Nonspecific

Chain Reaction specific phoresis o r direct
(PCR) fragments detection

RNAse RNA RNA Product Resistance NA Nonspecific

protection to RNAse o r direct
detection

Allele-specific. Oligo DNA Probe Retention Membrane Bound target

oligonucleotide nucleotide of target to
(ASO) bound probe

In situ Cloned DNA Chromo Target Retention Glass slides Bound probe
hybridisation somes of probe

FISH, YACs, PCR

Chromosome products
painting

ACKNOW LEDGEM ENTS

We w o u ld like to th a n k A n d rew C a ro th e rs a n d Ian Jac k so n fo r critically re a d in g

p a rts o f th e m a n u sc rip t. M .B.G is s u p p o rte d in p a rt by u n re s tric te d fu n d s fro m
R esearch to P rev en t B lindness, Inc.

REFERENCES
Casey, D. (1992) Prim er on M olecular G enetics In: Human Genome 19919 2 Program Report. U.S.
D ep a rtm e n t o f Energy, W ashington, D.C.
Davies, K. E. and Read, A. P. (1988) Molecular Basis o f Inherited Disease. IRL Press, O xford, 77 pp.
King, R. C. and Stansfield, W. D, (1992) A Dictionary of Genetics, (Fourth ed. ) O xford University
Press, Inc, New York, 406 pp.
T hom pson, M. W., M clnnes, R. R., W illard, H. F. (1991) Thompson & Thompson Genetics in Medi
cine, (Fifth ed.) W. B. Saunders Co., Philadelphia, 500 pp.
 2. RETINAL DEGENERATION MUTANTS
OF Drosophila

W ILLIAM L. PAR

Department o f Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA

KEY W ORDS: Drosophila, re tin a l d e g e n e ra tio n m u tan ts

ABSTRACT

Six g e n e s w hich, w h en defective, cause p h o to r e c e p to r d e g e n e ra tio n have now

b e e n c lo n e d a n d analyzed in Drosophila. M ost o f th ese g en es w ere id e n tifie d fro m
m u ta n ts g e n e ra te d by ch em ical m u tag e n esis a n d iso lated o n th e basis o f th e ir
defects in th e e le c tro re tin o g ra m o r visual behavior. T h e first g e n e to be clo n e d
was th a t e n c o d in g o p sin in R l-6 p h o to re c e p to rs , w hich ro u g h ly c o rre s p o n d to
ro d p h o to re c e p to rs o f v e rteb rates. T h e first m u ta tio n to b e iso la ted in this g e n e
was a n o n se n se m u ta tio n th a t causes severe p h o to re c e p to r d e g e n e ra tio n .
R ecendy, a h u m a n c o u n te r p a r t o f th is m u ta tio n has b e e n id en tified . M ost o f th e
six g en es e n c o d e p ro te in s th a t a re directly o r indirectly im p lic a te d in p h o to tra n s
d u c tio n in this o rg an ism . T h e p ro te in p ro d u c t o f o n e o f th e m , rdgC, however, has
b e e n im p lic a te d in a rh o d o p sin -a ctiv a ted pathw ay d istin c t fro m p h o sp h o lip a se C-
m e d ia te d p h o to tra n s d u c tio n . W h ere m u ltip le alleles o f a given g e n e have b e e n
stu d ied , stro n g allele d e p e n d e n c e o f re tin a l d e g e n e ra tio n has b e e n fo u n d . T h e
severity a n d th e tim e c o u rse o f re tin a l d e g e n e ra tio n can vary greatly a m o n g dif
fe re n t m u ta n ts in a given g e n e . R etin a l d e g e n e ra tio n in m u ta n ts o f all g en es stu d
ie d e x c e p t th o se in th e rh o d o p sin g e n e show stro n g lig h t d e p e n d e n c e . In these
m u ta n ts, stru c tu ra l d e te rio ra tio n can b e p re v e n te d fro m o c c u rrin g o r co n sid er
ably slow ed dow n by raisin g th e flies in th e d ark . In m o st o f th ese m u ta n ts, how
ever, fu n c tio n a l d efects a re alread y p re s e n t b e fo re th e o n se t o f d e g e n e ra tio n . In
m u ta n ts o f all six g en es, R l-6 p h o to re c e p to rs are m o re su scep tib le to d e g e n e ra
tio n th a n e ith e r ju s t R7 o r b o th R7 a n d R8 p h o to re c e p to rs. Eventually, however,
R7 a n d R8 p h o to re c e p to rs , w hich ro u g h ly c o rre s p o n d to cones, also d e g e n e ra te .
T h is is tru e w h e th e r th e defective g e n e is e x p e c te d to b e e x p re sse d ju s t in R l-6
p h o to re c e p to rs , as in R l-6 o p sin g e n e m u ta n ts, o r in b o th R l-6 a n d R 7 /8 classes
o f p h o to re c e p to rs , as in m u ta n ts o f th e o th e r genes.

I IN T R O D U C T IO N

M any Drosophila m u ta tio n s a re know n to affect th e gross m o rp h o lo g y o f th e com

p o u n d eye th ro u g h th e ir effects o n eye d e v e lo p m e n t (e.g. R en fran z a n d Benzer,
30 WILLIAM L. PAK

1989). T h is review, however, will focus on m u ta n ts c h a ra c te riz e d by age-d ep en -

d e n t d e g e n e ra tio n o f th e re tin a . M oreover, th e discussion will b e largely lim ited
to th o se m u ta n ts fo r w hich th e c o rre s p o n d in g g en es have b e e n id e n tifie d a n d
c lo n e d . By a n d larg e, in th e se m u tan ts, th e gross m o rp h o lo g y o f th e eye is n o r
m al, a n d n early o r co m p letely n o rm a l a d u lt re tin a l stru c tu re s fo rm initially b u t
d e g e n e ra te w ith age.
P revious to th e late 1960s th e only eye m u ta n ts available in Drosophila were those
th a t affect th e eye co lo r o r th e e x te rn a l m o rp h o lo g y o f th e eye. B e g in n in g in th e
la te 1960s, several la b o ra to rie s b e g a n chem ically m u ta g e n iz in g Drosophila a n d iso
la tin g m u ta n ts th a t are defective in visual b eh av io r o r light-evoked e lectrical re
sp o n ses o f th e eye (e le c tro re tin o g ra m , ERG ) (H o tta a n d Benzer, 1969; Pak e t al.,
1969; H e ise n b e rg , 1971; K oenig a n d M erriam , 1977). E ach o f th ese la b o ra to rie s
h a d d iffe re n t e x p e rim e n ta l objectives a n d em p lo y ed d iffe re n t m u ta n t selectio n
schem es. F o r ex am p le, th e B en zer la b o ra to ry was in te re ste d in d issectin g fly b e
hav io r a n d iso lated m u ta n ts defective in p h o to ta x is (B enzer, 1973); th e P ak lab o
ra to ry was lo o k in g fo r m u ta n ts defective in th e ERG fo r use in d issectio n o f th e
p h o to tra n s d u c tio n process (P ak e t al., 1969); a n d M. H e ise n b e rg , th e n w orking in
K. G o tz la b o ra to ry in T b in g e n , G erm any, iso lated m u ta n ts defective in th e opto-
m o to r b e h a v io r fo r use in th e analysis o f th e in te g ra tio n o f visual in fo rm a tio n
(H e ise n b e rg , 1972). T h e in itial m u tag e n esis schem es by th ese th re e la b o ra to rie s
ta rg e te d th e X c h ro m o so m e . M utagenesis o f th e a u to so m es fo r th e iso latio n o f
ERG -defective m u ta n ts was c a rrie d o u t by th e M e rriam la b o ra to ry (K oenig a n d
M erriam , 1977) a n d th e Pak la b o ra to ry (review ed by Pak, 1979). All th e se ap
p ro a c h e s, how ever, y ield ed m any m u ta n ts o f th e sam e g enes, a n d som e o f th e m u
tan ts iso lated w ere fo u n d to display re tin a l d e g e n e ra tio n . In a d d itio n , C osens a n d
M a n n in g (1969) d e sc rib e d a sp o n ta n e o u sly arising ERG -defective m u ta n t, w hich
tu rn e d o u t to be a re tin a l d e g e n e ra tio n m u ta n t (C osens a n d Perry, 1972). M ore
recently, S teele a n d O T ousa (1990) have iso lated re tin a l d e g e n e ra tio n m u ta n ts by
lo o k in g fo r s tru c tu ra l d e te rio ra tio n with age u sin g an o p tical te c h n iq u e (see n e x t
se c tio n ).
D escrib ed in S ection III a re re tin a l d e g e n e ra tio n m u ta n ts o f six g enes, all o f
w hich have now b e e n clo n ed . P re lim in a ry analysis o f o th e r ERG -defective m u ta n ts
suggests th a t th e re are m any m o re g en es w hich, w hen defective, cause re tin a l d e
g e n e ra tio n . A lth o u g h d e ta ile d in fo rm a tio n is available only o n a sm all fra c tio n o f
m u ta n ts th a t display re tin a l d e g e n e ra tio n , th e in fo rm a tio n th a t is b ec o m in g avail
ab le is b e g in n in g to p ro v id e insights in to so m e co m m o n p ro p e rtie s sh a re d by
m any o f th ese m u tan ts.

II STRU CTU RE O F T H E Drosophila C O M PO U N D EYE AND M ETH O D S O F

M O N IT O R IN G RETINAL D EGENERATION

T h e Drosophila c o m p o u n d eye consists o f a p p ro x im ately 800 re p e a tin g u n its

called th e o m m a tid ia . E ach o m m a tid iu m c o n ta in s 20 cells, e ig h t o f w hich are
 RETINAL DEGENERATION IN DROSOPHIIA 31

p h o to re c e p to rs (F ig u re 1). Each p h o to re c e p to r has a m e m b ra n e stru c tu re ,

called th e rh a b d o m e re , co n sistin g o f tightly p a ck e d m icrovilli th a t p ro je c t la te r
ally tow ard th e c e n te r o f th e o m m a tid iu m . T h e se stru c tu re s are specialized fo r
lig h t re c e p tio n a n d p h o to tra n s d u c tio n , co n ta in visual p ig m e n t m o lecu les as in te
g ral m e m b ra n e p ro te in s o f th e m icrovillar m e m b ra n e , a n d th u s c o rre s p o n d to
th e o u te r seg m en ts o f v e rte b ra te p h o to re c e p to rs. Six o f th e p h o to re c e p to rs, R l-6,
have rh a b d o m e re s th a t e x te n d th e full d e p th o f th e re tin a a n d th a t a re a rra n g e d
in a tra p e z o id a l p a tte rn in cross-section (F igure 1). R l-6 p h o to re c e p to rs all c o n
tain th e sam e visual p ig m e n t, R l-6 rh o d o p sin , a n d ro u g h ly c o rre s p o n d to ro d s o f
v e rte b ra tes. T h e re m a in in g two p h o to re c e p to rs , R7 a n d 8, have rh a b d o m e re s
n e a r th e c e n te r o f th e o m m a tid iu m , w ith R7 rh a b d o m e re occu p y in g a p o sitio n
d irectly above th e R8 rh a b d o m e re s. R 7 /8 p h o to re c e p to rs have d iffe re n t visual
p ig m e n ts a n d ro u g h ly c o rre s p o n d to v e rte b ra te cones. P ig m e n t cells, w hich c o n
tain sc re e n in g p ig m e n ts, p te rin s a n d o m m a ch ro m es, s u rro u n d eac h o m m a tid
ium (F ig u re 1), optically iso latin g in dividual om m atid ia. F or m any ap p licatio n s, it
is c o n v e n ie n t to e lim in a te th ese p ig m e n ts genetically. In fact, m an y o f th e studies
su m m arized in S ectio n III w ere d o n e in th e ab sence o f th ese p ig m en ts. A lth o u g h
these p ig m e n ts d o n o t affect th e tra n sd u c tio n process, they d o affect th e a m o u n t
o f lig h t re a c h in g th e rh a b d o m e re s a n d can affect th e severity a n d tim e co u rse o f
d e g e n e ra tio n in lig h t-d e p e n d e n t d e g e n e ra tio n m u tan ts.
R etin al d e g e n e ra tio n in Drosophila can b e m o n ito re d in a n u m b e r o f d iffe re n t
ways. A lth o u g h lig h t a n d e le c tro n m icroscopy o f th e r e tin a c a rrie d o u t as a fu n c
tio n o f age is th e m o st d ire c t a n d re liab le m e th o d o f studying d e g e n e ra tio n , th e
m e th o d is tim e-co n su m in g a n d labor-intensive. It is, th e re fo re , d e sirab le to have
o th er, sim p ler m e a n s o f m o n ito rin g d e g e n e ra tio n . M o n ito rin g th e ERG o r th e
d e e p p se u d o p u p il as a fu n c tio n o f age o fte n yields reliab le in d ic a tio n s o f d e g e n
e ra tio n , a lth o u g h d etails o f stru c tu ra l d e te rio ra tio n a re lacking. In m ost retin a l d e
g e n e ra tio n m u ta n ts, th e ERG is alread y a b n o rm a l at eclosion b e fo re any stru c tu ra l
d e te rio ra tio n is a p p a re n t. As th e re tin a d e g e n e ra tes, th e ERG d e te rio ra te s fu r th e r
w ith age. T h u s, d e te rio ra tio n o f th e ERG w ith age is a reliab le initial in d ica tio n o f
re tin a l d e g e n e ra tio n . T h e d e e p p se u d o p u p il is a m icroscopically o b serv e d su p er
p o sitio n o f v irtu al im ages o f a b o u t 20 n e ig h b o rin g o m m a tid ia (F ran cesch in i a n d
K irschfeld, 1971). As th e re tin a d e g e n e ra te s, th e d e e p p se u d o p u p il b ec o m e s in d is
tin c t a n d d isap p ears. B ecause su p e rp o sitio n o f v irtu al im ages is d e p e n d e n t o n the
c o rre c t stru c tu ra l re la tio n sh ip o f n e ig h b o rin g o m m atid ia, th e te c h n iq u e is sensi
tive to an y s tru c tu ra l m isa lig n m e n t o f o m m atid ia . C onsequently, this te c h n iq u e
te n d s to d e te c t d efects b e fo re they a re seen histologically, a n d th e tim e co urse o f
d e g e n e ra tio n d e te rm in e d by this m e th o d te n d s to be faster th a n th a t o b ta in e d his
tologically.
In m o n ito rin g re tin a l d e g e n e ra tio n , th e life sp an o f Drosophila is m e a su re d in
days o r w eeks p o st-eclosion. W hile it is difficult to d e fin e a Drosophila tim e scale
c o rre s p o n d in g to th a t o f h u m a n , co n v e n ie n t y ard sticks to c o m p a re are th e g e n
e ra tio n tim e a n d th e age a t w hich Drosophila start dying off. At 25 C, th e g e n e ra
tio n tim e o f Drosophila is a p p ro x im a te ly 10 d, a n d th ey sta rt dying o ff at a b o u t 5 wk
post-eclosion u n d e r la b o ra to ry c o n d itio n s.
32 WILLIAM L. PAK

cornea --------

pseudocone

! ---- 3 pigment cell

/ / 2 pigment cell

Rl-6 photoreceptor

Rl-6 rhabdomere -

cone cell
R7 rhabdomere
~ r pigment cell
R4 R3
cone cell process R5 _ R2

R1
R8 rhabdomere----- R7

R8

10 \im

cone cell f o o t --------

I anterior
axons -------------------

equator

Figure 1 S tru c tu re o f Drosophila o m m a tid iu m . A r e p e a tin g u n it in th e c o m p o u n d ey e, th e

o m m a tid iu m , c o n ta in s e ig h t p h o to re c e p to rs , R1 to R 8. E ach p h o to re c e p to r has a rh ab

d o m e r e , c o n s is tin g o f tig h tly p a c k e d m ic r o v illi, p r o je c ti n g la te r a lly i n to th e c e n t e r o f th e o m

m a tid iu m (sh o w n in b la c k ) . E a c h o m m a tid iu m is s u r r o u n d e d b y p r i m a r y ( 1 ) , s e c o n d a r y ( 2 ) ,

a n d t e r t i a r y ( 3 ) p i g m e n t c e ll s . R l -6 p h o t o r e c e p t o r s r o u g h l y c o r r e s p o n d t o r o d s , R 7 / 8 p h o t o

r e c e p to r s to c o n e s , a n d th e r h a b d o m e r e s to th e o u te r s e g m e n ts o f v e rte b ra te p h o to r e c e p to r s .
A d a p te d fro m W o lff a n d R eady (1 9 9 3 ). R e p ro d u c e d w ith p e rm iss io n fro m th e C o ld S p rin g

H a r b o r P re ss.
 RETINAL DEGENERATION IN DROSOPHIIA 33

III D ESCRIPTIO N O F M UTANTS

1 M utants o f th e R h o d o p sin G ene, ninaE

T h e R l -6 o p sin g e n e o f Drosophila, ninaE, w hich c o rre sp o n d s to th e ro d opsin

g e n e o f v e rte b ra tes, was id e n tifie d fro m a class o f m u ta n ts in w hich th e a m o u n t o f
R l -6 rh o d o p s in is g reatly re d u c e d . T h e se m u ta n ts, a n d th e c o rre sp o n d in g g enes,
w ere n a m e d nina ( neither inactivation nor afterpotential) b ecau se o f the c h a ra c te ris
tic electro p h y sio lo g ical p h e n o ty p e d isplayed by th e m u ta n ts (Pak, 1979; S te p h e n
son e t al., 1983). Since th e se m u ta tio n s a p p a re n tly d is ru p t e ith e r rh o d o p sin
synthesis o r its m a in te n a n c e , it was re a so n e d th a t o n e o f th e five nina g en es th e n
id e n tifie d , ninaA, B, C, D, o r E, m ig h t co d e fo r th e R l-6 o p sin p ro te in itself. Two
c rite ria w ere u sed to id en tify th e R l -6 o p sin stru c tu ra l g en e a m o n g th e five nina
genes: cell lin e specificity a n d th e g e n e dosage effect. If d iffe re n t classes o f opsin
are e n c o d e d by d iffe re n t g en es, as se e m e d likely, o n e w ould ex p e c t th a t m u ta
tio n s in th e R l -6 o p sin g e n e w ould affect th e visual p ig m e n t c o n te n t only in R l-6
p h o to re c e p to rs a n d n o t in R 7 /8 p h o to re c e p to rs. T h e g en e do sag e e ffect refers to
th e a p p ro x im a te ly lin e a r re la tio n sh ip fo u n d betw een th e n u m b e r o f copies o f a
g e n e (g e n e dosage) a n d th e a m o u n t o r activity o f th e p ro te in e n c o d e d by th e
g e n e (e.g. review by O B rien a n d M acIntyre, 1978). O f th e five n in a g en e s tested,
only th e ninaE g e n e satisfied b o th th ese crite ria, id en tify in g it as th e p rim e ca n d i
d ate fo r th e R l -6 o p sin g e n e in Drosophila (Scavarda e t al., 1983). T h e ninaE g en e
was m a p p e d u sin g d e le tio n s to th e 92B re g io n o n th e rig h t a rm o f th e th ird c h ro
m oso m e (O T ousa e t al., 1985) a n d was c lo n e d by in te rsp ecie s hybrid izatio n
u sin g bovine rh o d o p s in cDNA o r RNA as p ro b e (O T ousa e t al., 1985; Z uker
e t al., 1985).
C o m p a riso n o f th e d e d u c e d Drosophila p ro te in se q u e n c e with th e v e rte b ra te ro d
o p sin se q u en ces th e n available, bovine (O vchinnikov e t al., 1982; N a th a n s a n d
H ogness, 1983) a n d h u m a n (N a th a n s a n d H ogness, 1984), sh ow ed th a t it h a d all
th e stru c tu ra l fe a tu re s o f opsin. F o r e x am p le , th e p ro te in h a d seven tra n sm e m
b ra n e seg m en ts (F ig u re 2), th e c h ro m o p h o re -b in d in g lysine re sid u e n e a r th e m id
dle o f th e sev en th tra n sm e m b ra n e seg m en t, several C -term inal serin e a n d
th re o n in e re sid u e s th a t c o u ld serve as p h o sp h o ry la tio n sites, a n d several helix-
b re a k in g p ro lin e re sid u e s sc a tte re d th ro u g h o u t th e tra n sm e m b ra n e segm ents.
W h en a lig n e d fo r m a x im u m hom ology, th e Drosophila p ro te in show ed a b o u t 36%
overall se q u e n c e id en tity w ith th e m a m m alia n p ro tein s. M ost o f th e se q u e n c e
id e n tity c o u ld be a c c o u n te d fo r by several clusters o f very stro n g se q u e n c e identity.
T h e w ork, th u s, e sta b lish e d th e basic stru c tu ra l sim ilarity o f Drosophila R l-6 opsin
to m am m a lia n o p sin s a n d id e n tifie d several reg io n s o f stro n g se q u e n c e h o m ology
w hich c o u ld be fu n ctio n ally im p o rta n t.
T h e first in d ic a tio n th a t a m u ta tio n in th e rh o d o p sin g e n e m ig h t cause stru c tu r
al d e te rio ra tio n o f p h o to re c e p to rs was o b ta in e d in Drosophila. In th e early seven
ties, J. K oenig w orking in J. M e rria m s lab a t UCLA, iso lated a m u ta n t, la te r n a m e d
oral (outer rhabdomeres absent), th a t se em ed to lack R l -6 rh a b d o m e re s (H arris
34 WILLIAM L. PAK
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 RETINAL DEGENERATION IN DROSOPHILA 35

e t al.. 1976; K oenig a n d M erriam , 1977). S u b se q u e n t studies show ed th a t sm all

a n d d e fo rm e d b u t rec o g n izab le R l-6 rh a b d o m e re s a re p re s e n t in oraJK m u tan ts
at eclo sio n b u t quickly d isa p p e a r w ith age (Schinz e t al., 1982; S tark a n d Sapp,
1987; O T ousa e t al., 1989). It was soon realized fro m c o m p le m e n ta tio n tests th a t
this m u ta n t c a rrie s a lesio n in th e R l-6 o p sin g e n e , n in aE (Scavarda e t al., 1983).
H ow ever, c o m p le m e n ta tio n tests also rev ea le d th a t th e m u ta n t se em ed to be car
ry in g a n o th e r m u ta tio n . O T ousa et al. (1989) show ed th a t (1) ora/K is, in fact,
a d o u b le m u ta n t c a rry in g a m u ta tio n in th e ninaE g en e a n d a n o th e r o n e in a close
ly lin k e d g e n e a n d (2) th e lesion in th e ninaE g e n e is solely re sp o n sib le fo r th e
s tru c tu ra l d e fe c t in R l -6 p h o to re c e p to rs.
Since th e n , p h o to re c e p to r d e g e n e ra tio n has b e e n fo u n d in all ninaE m u tan ts
e x a m in e d to d a te . L e o n a rd a n d Pak (1984) e x a m in e d , at th e lig h t m icro sco p e lev
el, th e p h o to re c e p to r m o rp h o lo g y o f n in e d iffe re n t ninaE m u ta n ts u p to th e age
o f n in e w eeks post-eclosion. S ubsequently, L e o n a rd e t al. (1992) c a rrie d o u t d e
tailed e le c tro n m icro sco p ic studies o n five o f th ese m u ta n ts fo r w hich th e m o lec
u la r d efects in th e ninaE g e n e h a d b e e n e lu c id a te d (W ash b u rn a n d O Tousa, 1989;
su m m arized in F ig u re 3 in se rt). T h e d efects vary fro m a sm all d e le tio n in th e 5
re g io n re su ltin g in n o tra n sc rip ts (n in a E 011" ) to m issense m u ta tio n s re su ltin g in
single a m in o acid su b stitu tio n s (n in a E 1 1 a n d ni naEp ). T h e n o n se n se m u ta
tion c a rrie d by ninaE p352 is id e n tic al to th a t c a rrie d by ora^K84 in th e ninaE g en e
e x c e p t th a t th e la tte r is a d o u b le m u ta n t ca rry in g a n o th e r m u ta tio n . D e g e n e ra tio n
o f p h o to re c e p to rs was fo u n d in all th ese m u tan ts. It is lig h t-in d e p e n d e n t in th a t
illu m in a tio n cycles have little o r n o effect on th e c o u rse o f d e g e n e ra tio n . T h e tim e
c o u rse o f d e g e n e ra tio n varies strongly a m o n g m u tan ts, w ith m u ta n ts having th e
m o st severe d efects show ing th e fastest d e g e n e ra tio n (F igure 3). In stro n g ninaE
m u ta n ts, R l -6 rh a b d o m e re s alm o st com p letely d isa p p e a r by 3 d post-eclosion,
w hile in w eak er m u ta n ts, m any R l -6 rh a b d o m e re s are still p re se n t at 9 wk post-
eclosion at ro o m te m p e ra tu re . T h e d e g e n e ra tio n first affects R l -6 rh a b d o m e res,
b u t eventually R l -6 cell b o d ies also b e c o m e affected. By th e tim e th e d e g e n e ra tio n
o f R l -6 cells is well u n d e r way, even th e R 7 /8 rh a b d o m e re s, in w hich d iffe re n t
classes o f o p sin a re ex p ressed , also start d e g e n e ra tin g . A n o b se rv atio n o f som e im
p o rt is th e o c c u rre n c e o f m e m b ra n e p ro life ra tio n s at th e base o f R l-6 rh a b d o m e
res in m an y o f th e se m u ta n ts at a b o u t th e tim e th ese rh a b d o m e re s are d e g e n e ra t
ing. E ach o f th e se s tru c tu re s consists o f a closely a p p o se d p a ir o f m e m b ra n e s, o n e
e n d o f w hich is c o n tin u o u s w ith th e p lasm a m e m b ra n e at th e base o f R l-6 rh a b
d o m e re s a n d th e o th e r e n d o f w hich e x te n d s in to th e cytoplasm a n d is closed. As
a w orking hypoth esis, L e o n a rd e t al. (1992) p ro p o se d th a t this a c c u m u la tio n o f
m e m b ra n e s m ig h t be c au sed by th e failu re o f newly in se rte d m e m b ra n e s to b e as
sem b led in to R l -6 rh a b d o m e re s. A sim ilar suggestion was m ad e by S tark a n d
C arlson (1983) to e x p la in th e m o rp h o lo g ic a l d e fe c t o f o n J K84.
A n im p o rta n t re c e n t d e v e lo p m e n t in retin itis p ig m e n to sa (RP) rese a rc h is th e
fin d in g th a t a su rp risin g ly large fractio n o f RP is cau sed by various m u ta tio n s in
th e rh o d o p s in g e n e (D ryja e t al., 1990; 1991; S ung e t al., 1991). In fact, R osenfeld
e t al. (1992) have re c e n tly r e p o rte d o n an au to so m a l recessive p a tie n t w ith a d e
fect alm o st id e n tic a l to th a t c a rrie d by oraJK84 o r n in a E r ^2 su ggesting an in cred-
 36
WT A P334
A P318 O P352
P332 oil 7

W IL L IA M L . PAK
Figure 3 Allele dependence of the time course of photoreceptor degeneration inDrosophila rhodopsin gene {ninaE) mutants and the mo
lecular defects carried by the mutants. Mean number of Rl-6 rhabdomeres per ommatidium, determined in light microscopy, is plotted
as a function of age. Adapted from Figure 1 and Table 1 of Leonard et al. (1992). Reproduced with permission from the Journal of Neu
robiology.
 RETINAL DEGENERATION IN DROSOPHILA. 37

ible d e g re e o f co n se rv a tio n o f d e g e n e ra tio n m ech an ism s across species in at least

som e fo rm s o f p h o to re c e p to r d e g e n e ra tio n . However, m o st o f th e h u m a n RP
c au sed by rh o d o p s in g e n e m u ta tio n s r e p o rte d to d a te is a u to so m a l d o m in a n t,
w hile n o d o m in a n t rh o d o p sin g e n e m u ta tio n cau sin g re tin a l d e g e n e ra tio n has yet
b e e n f o u n d in Drosophila. It is possible th a t m am m a lia n p h o to re c e p to rs are m u ch
m o re sensitive to th e effects o f single copies o f rh o d o p sin g e n e m u ta tio n s th a n
Drosophila p h o to re c e p to rs . It is also likely th a t th e re are m any m o re recessive m u
tatio n s, w hich cause p h o to re c e p to r d e g e n e ra tio n only w hen hom ozygous, in h u
m ans, b u t m ost o f th e m re m a in u n re c o g n iz e d b ecau se hom ozygous exp ressio n o f
recessive m u ta tio n s is n o t a fre q u e n t ev en t in a n a tu ra l p o p u la tio n .

2 M utants o f th e P h o sp h o lip ase C G ene, norpA

Iso latio n o f norpA (no receptor potential A) m u ta n ts was first r e p o rte d in d e p e n

d en tly by H o tta a n d B en zer (1970) a n d Pak e t al. (1970). B ecause stro n g m u ta
tions in this g e n e ab o lish th e re c e p to r p o te n tia l, it h a d lo n g b e e n felt th a t th e
p ro te in p ro d u c t o f this g e n e plays a critical ro le in p h o to tra n s d u c tio n in Droso
phila. T h e first p ieces o f evidence th a t norpA m u ta n ts m ig h t be defective in inosi
tol lip id m etab o lism w ere o b ta in e d by In o u e e t al. (1985) a n d Yoshioka et al.
(1984), w ho show ed th a t b o th p h o sp h o lip a se C (PLC) a n d diacylglycerol kinase
(DGK) activities a re g reatly re d u c e d in th e se m u tan ts. S elin g er a n d M inke (1988)
h e lp e d focus o n PLC as a p o te n tia l ta rg e t o f norpA m u ta tio n by show ing that, in a
tem p eratu re-sen sitiv e norpA m u ta n t, th e PLC, b u t n o t DGK, activity is re d u c e d
only w h en th e m u ta n t is ex p o sed to re stric te d te m p e ra tu re s. In th e m ean tim e, a
body o f ev id en ce sta rte d e m e rg in g to suggest th a t th e in v e rte b ra te p h o to tra n s
d u c tio n cascade m ay involve PLC activation (Figure 4). F or e x am p le , in je ctio n o f
in o sito l 1 ,4 ,5 -trisp h o sp h ate (IP3) in to th e v e n tra l p h o to re c e p to rs o f th e h o rse
sh o e c ra b Lim ulus was show n to p ro d u c e electrical activities re se m b lin g those
g e n e ra te d by lig h t (B row n e t al., 1984; Fein e t al., 1984), a n d lig h t-d e p e n d e n t
activation o f PLC was d e m o n s tra te d in a n u m b e r o f in v e rte b ra te eye p re p a ra tio n s
(B row n e t al., 1984, 1987; Szuts e t al., 1986; D evary e t al., 1987; B aer a n d Saibil,
1988; W ood e t al., 1989).
M o lecu lar iso latio n a n d analysis o f th e norpA g e n e p ro v id e d th e m o st c o m p el
lin g ev id en ce fo r th e in v o lv em en t o f PLC in in v e rte b ra te p h o to tra n sd u c tio n . T h e
norpA g e n e was m a p p e d to th e 4B6-4C1 re g io n o f th e X c h ro m o so m e (B anga
et al., 1986) a n d was c lo n e d by a c o m b in a tio n o f c h ro m o so m al w alking a n d trans-
p o so n m u ta g e n e sis (B lo o m q u ist e t al., 1988). S eq u e n ce analysis o f c o g n a te cDNAs
show ed th a t th e g e n e e n c o d e s a p o ly p e p tid e o f 1095 a m in o acids (F igure 2) w ith
stro n g se q u e n c e sim ilarity w ith m a m m a lia n PLC seq u en ces th a t b e cam e know n
fro m cDNA c lo n in g a t a b o u t the sam e tim e (K atan e t al., 1988; Stahl e t al., 1988;
S uh e t al., 1988a, b ). T h ese PLCs fall in to th re e stru ctu rally d istin c t classes, PL C a,
p, a n d y, a n d a re c h a ra c teriz e d by th e p re se n c e o f two, 120-200 a m in o acid d o
m ain s o f s tro n g se q u e n c e sim ilarity, B oxes X a n d Y (R h ee et al., 1989; F igure 2)
C o m p a riso n o f th e d e d u c e d norpA p ro te in seq u en ce w ith m am m alian se q u en ces
38 WILLIAM L. PAR

Figure 4 H y p o th e tic a l p h o to tr a n s d u c tio n s c h e m e in Drosophila p h o t o r e c e p t o r s . L ig h t-a c tiv a t-

e d rh o d o p sin ( R h ) , lo c a te d in th e r h a b d o m e r i c m e m b r a n e , a c tiv a te s G p r o te i n (G ) b y c a ta

ly z in g t h e e x c h a n g e o f G T P f o r G D P . T h e G p r o te i n , in t u r n , a c tiv a te s p h o s p h o li p a s e C (P L C )

to c a ta ly z e t h e h y d ro ly s is o f p h o s p h a tid y lin o s ito l 4 ,5 - b is p h o s p h a te ( P lP o ) i n t o d ia c y lg ly c e r o l

(D G ) a n d in o s ito l 1 ,4 ,5 - tr is p h o s p h a te ( I P 3 ) . IP > b i n d s t o I P 3 r e c e p t o r ( I P 3R ) o n t h e s u b r h a b -
d o m e r i c c i s t e r n a e ( S R C ) m e m b r a n e t o r e l e a s e C a 2+ f r o m th e in tra c e llu la r c o m p a rtm e n ts . T h e

r e l e a s e d C a 2*, t h r o u g h a n u m b e r o f u n k n o w n s te p s , o p e n s a c la s s o f c a t i o n c h a n n e l s w ith r e l
a t i v e l y l o w C a 2+ p e r m e a b i l i t y . T h i s c l a s s o f c h a n n e l s is r e s p o n s i b l e f o r t h e tra n s ie n t p h a s e o f
th e r e c e p t o r p o t e n t i a l . I n th e m e a n t im e , t h e a c tiv a te d IP 3R a ls o o p e n s a d i f f e r e n t c la s s o f io n

c h a n n e l s w i t h h i g h C a 2+ p e r m e a b i l i t y t h r o u g h d i r e c t i n t e r a c t i o n . T h i s c l a s s o f c h a n n e l s i s r e
s p o n s ib le fo r th e s te a d y - s ta te phase o f th e re c e p to r p o te n tia l, a n d th e C a 2+ i n f l u x th ro u g h

t h e s e c h a n n e l s h e l p s r e p l e n i s h C a 2+ i n th e in tr a c e llu la r c o m p a r tm e n ts . P h o s p h a tid y lin o s ito l

tra n s fe r p ro te in (P I-T P ) e n c o d e d b y th e rdgB g e n e lo c a te d in th e S R C m e m b r a n e tra n s p o rts

P I in to th e rh a b d o m e ric m e m b ra n e to re p le n is h it f o r u s e in p h o to tr a n s d u c tio n . E x c e p t fo r

th e firs t fe w s te p s , m u c h o f t h e s c h e m e is s till la r g e ly h y p o th e tic a l. A d a p t e d f r o m M in k e a n d

S e lin g e r (1 9 9 1 ). R e p r o d u c e d w ith p e rm is s io n f ro m th e P e rg a m o n P re ss.

show ed th a t th e fo rm e r is stru c tu ra lly sim ilar to m am m a lian PLCjis a n d has 49 a n d

53% se q u e n c e id e n tity w ith m am m alian PLCps w ithin Boxes X a n d Y, respectively;
35 a n d 44% id en tity w ithin E x te n d e d B oxes X a n d Y, respectively; la rg e r reg io n s
o f se q u e n c e h o m o lo g y w ere rev ealed in a su b se q u e n t analysis (R. S h o rtrid g e a n d
Pak, u n p u b lis h e d ), all stro n g ly in d ic a tin g th a t th e norpA g e n e p ro d u c t is a PLCp.
T oyoshim a et al. (1990) p ro v id ed p r o o f th a t id en tifica tio n o f th e norpA p ro te in
as a PLC is c o rre c t. T h ey p u rifie d th e m ajo r PLC c o m p o n e n t p re se n t in wild-type
 RETINAL DEGENERATION IN DROSOPJIILA 39

b u t a b s e n t in norpA h e a d s a n d show ed th a t seq u en ce s o f p e p tid e fra g m e n ts d e

rived fro m th e p ro te in m a tc h e d th e d e d u c e d norpA p ro te in se q u e n c e (B lo o m q u ist
e t al., 1988). U ltra s tru c tu ra l lo calizatio n o f th e norpA p ro te in by im m u n o -g o ld la
b e lin g show ed th a t th e p ro te in is lo calized in th e lu m e n o f th e m icrovilli o f th e
rh a b d o m e re s in close association w ith th e m icrovillar m e m b ra n e (Schneuw ly
e t al., 1991). It has, th u s, b e e n estab lish e d unequivocally th a t th e norpA g e n e e n
co des a p h o sp h o lip a se C. Since g e n e tic d isru p tio n o f th e norpA g e n e alm ost com
pletely ab o lish es th e re c e p to r p o te n tia l, PLC is an essential c o m p o n e n t o f th e
p h o to tra n s d u c tio n m a c h in e ry in Drosophila (F igure 4 ) . T his c o n c lu sio n is strongly
s u p p o rte d by th e lo calization o f th e norpA p ro te in in th e rh a b d o m e re , th e stru c
tu re specialized fo r p h o to re c e p tio n a n d p h o to tra n sd u c tio n .
M e y erth o len e t al. (1987) stu d ie d th e rh o d o p sin c o n te n t, d e e p p se u d o p u p il,
a n d rh a b d o m e re s tru c tu re in th re e d iffe re n t norpA allelic m u ta n ts u p to th e age
o f 3 wk a n d first r e p o r te d th a t norpA rh a b d o m e re s d e g e n e ra te w ith age. Stark
e t al. (1989) a n d Z inkl e t al., (1990) also stu d ie d a g e -d e p e n d e n t d e te rio ra tio n o f
rh a b d o m e re s in norpA m u ta n ts by e le c tro n m icroscopy a n d sp e c tro p h o to m e try o f
rh o d o p s in c o n te n t u p to th e age o f 10 d. A t eclosion, rh a b d o m e re s are largely n o r
m al even in th e stro n g e st m u ta n ts stu d ie d , even th o u g h a severe electrophysiolog-
ical d e fe c t is p re se n t. If grow n o n a 12 h r l i g h t / 12 h r d a rk illu m in a tio n cycle, th e
rh a b d o m e re s b e c o m e sm aller a n d d is to rte d (Z inkl e t al., 1990) a n d finally disap
p e a r (M e y e rth o le n e t al., 1987), w ith th e rh o d o p sin c o n te n t d e c re a sin g ro u g h ly in
p arallel. T h e tim e c o u rse o f d e g e n e ra tio n is a lle le -d e p en d e n t, te m p e ra tu re -d e -
p e n d e n t, a n d lig h t-d e p e n d e n t (M ey e rth o le n e t al., 1987). T h e m u ta n ts c arry in g
th e stro n g e st alleles display th e fastest d e g e n e ra tio n , with R l -6 rh a b d o m e re s dis
a p p e a rin g w ith in a b o u t 10 d a t 24 C (M e y erth o len e t al., 1987). T h e tim e course
is slow er fo r m u ta n ts c a rry in g w eaker alleles a n d at low er te m p e ra tu res. T h e rh a b
d o m e re s o f R l-6 p h o to re c e p to rs are m u c h m o re susceptible to d e g e n e ra tio n th a n
those o f R 7 /8 p h o to re c e p to rs (M e y erth o len e t al., 1987), even th o u g h th e norpA
p ro d u c t is ex p ressed in all a d u lt p h o to re c e p to rs (Schneuw ly e t al., 1991). B ecause
rh a b d o m e re d e g e n e ra tio n is strongly lig h t-d e p e n d e n t, n o d e g e n e ra tio n is a p p a r
e n t even in severely affe c ted m u ta n ts if grow n in c o m p le te d ark n ess (M ey erth o len
e t al., 1987; Z inkl e t al., 1990). M ost o f th e above stu d ies w ere c a rrie d o u t o n m u
tan ts fro m w hich th e sc re e n in g p ig m e n ts in th e eye w ere genetically elim in a te d . If
th e s c re e n in g p ig m e n ts a re left in ta c t a n d th u s th e a m o u n t o f lig h t re a c h in g the
rh o d o p sin m o lecu les is re d u c e d , rh a b d o m e re d e g e n e ra tio n is n o t a p p a r e n t u p to
10 d post-eclo sio n (S tark e t al., 1991).
T h e p h o to tra n s d u c tio n cascade in m a m m a lia n p h o to re c e p to rs d o e s n o t utilize
PLC. T h u s, a q u e stio n o f som e im p o rta n c e is w h e th e r p h o to re c e p to r d e g e n e ra
tio n in norpA m u ta n ts h as any relev an ce to m am m alia n p h o to re c e p to rs. T h e q u es
tio n m ay be an sw ered a lo n g two lines. T h e first o f th ese is to n o te th at, in
v e rte b ra te p h o to tra n s d u c tio n , cGM P p h o sp h o d ie ste ra se (PDE) o ccupies a posi
tio n an a lo g o u s to th a t o c c u p ie d by PLC in in v e rte b ra te p h o to tra n sd u c tio n in th a t
it is th e ta rg e t o f tra n sd u c in in te ra c tio n . T h u s, by analogy, o n e m ig h t e x p e c t to
fin d th a t a d e fe c t in th e g en es e n c o d in g cGM P PDE su b u n its m ig h t cause p h o to
40 WILLIAM L. PAR

re c e p to r d e g e n e ra tio n in m am m als. In fact, th e m ouse re tin a l d e g e n e ra tio n m u

ta n t rd has b e e n show n to c a rry a m u ta tio n in th e g e n e e n c o d in g th e PDE 3
s u b u n it (Bowles et a t , 1990; P ittle r a n d B aehr, 1991).
A n o th e r way to answ er th e q u estio n is to ask if th e re are any m a m m alia n PLCs
h o m o lo g o u s to th e norpA p ro te in e x p ressed p re d o m in a n tly o r exclusively in th e
re tin a . A lth o u g h PLC has n o ro le in m am m a lian p h o to tra n sd u c tio n , it c o u ld have
a ro le in o th e r signal tra n sd u c tio n c a rrie d o u t by p h o to re c e p to rs. PLCs involved
in retina-specific fu n c tio n s c o u ld b e highly h o m o lo g o u s to the norpA p ro te in in
b o th s tru c tu re a n d fu n c tio n , a n d th u s a d e fe c t in th e g en e s e n c o d in g th ese p ro
tein s c o u ld have c o n se q u e n c e s sim ilar to th o se c au sed by norpA m u ta tio n s. S earch
fo r m a m m a lia n h o m o lo g s o f th e norpA p ro te in has le d to th e isolation o f bovine
cDNAs e n c o d in g PLCP isoform s, ex p ressed alm ost exclusively in th e re tin a a n d
hav in g se q u e n c e id e n tity to th e norpA p ro te in a t least 10% h ig h e r th a n any o th e r
PLCps id e n tifie d to d a te (F e rre ira et al., 1991).

3 M utants o f th e trp G ene

T h e first m u ta n t to be iso lated in th e trp (transient receptor potential) g e n e was a

sp o n ta n e o u sly arisin g o n e th a t b eh av ed as th o u g h b lin d in b rig h t a m b ie n t lig h t
(C osens a n d M an n in g , 1969; C osens, 1971). O th e r m u ta n t alleles o f this g en e
w ere iso lated by ch em ical m u ta g en e sis (review by Pak, 1979). M u tatio n s in this
g e n e cause th e re c e p to r p o te n tia l to decay to th e baselin e d u rin g a b rig h t a n d / o r
p ro lo n g e d lig h t stim ulus, th e re b y causing blin d n ess, w hile in wild type a steady-
state re sp o n se is p re s e n t th r o u g h o u t th e d u ra tio n o f stim ulus (C osens a n d M an
n in g , 1969; C osens, 1971; M inke et al., 1975). T h e re c e p to r p o te n tia l is g e n e ra te d
by a su m m a tio n o f a larg e n u m b e r o f sm all, d iscrete p o te n tia ls know n as b u m p s,
e a c h o f w hich arises in re sp o n se to a single p h o to n (R u sh to n , 1961; F u o rte s a n d
Y eandle, 1964; D odge e t al., 1968). F rom noise analysis, M inke e t al. (1975) a n d
M inke (1982) c o n c lu d e d th a t th e decay o f re c e p to r p o te n tia l d u rin g su stain ed
stim u lu s in trp m u ta n ts is c au sed by a severe re d u c tio n in th e rate o f b u m p s, i.e.
th e a b so rb e d p h o to n s b e c o m e ineffective in g e n e ra tin g bum ps.
W ong e t al. (1989) m a p p e d th e g e n e to th e 99C5-6 re g io n o f th e th ird c h ro m o
som e, a n d M on tell a n d R u b in (1989) a n d W ong e t al. (1989) c lo n e d th e g e n e by
a s h o rt c h ro m o so m a l walk u sin g a head-specific g en o m ic clo n e th a t m aps to th e
99C re g io n , iso lated by Levy e t al. (1982). A g en o m ic DNA fra g m e n t o b ta in e d in
th e walk was u se d to rescu e a trp m u ta n t by P -elem en t-m ed ia ted g e rm lin e tra n s
fo rm a tio n (M o n tell e t al., 1985), p ro v id in g definitive p r o o f th a t th e DNA frag
m e n t c o n ta in e d th e e n tire trp g en e . T h is is p ro b ab ly th e first ex a m p le o f g en e
th e ra p y a p p lie d to a re tin a l d e g e n e ra tio n m u ta n t in any o rganism . T h e g e n e e n
cod es a novel p ro te in o f 1275 a m in o acid resid u es w ith a t least four, a n d possibly
as m any as eig h t, p o te n tia l tra n s m e m b ra n e d o m a in s (F igure 2). H ow ever, b ecause
th e p ro te in has n o h o m o lo g y to any k now n p ro te in , g e n e c lo n in g p ro v id e d little
clue to th e fu n c tio n o f th e p ro te in .
T h e followdng two sets o f o b se rv a tio n s le d M inke a n d S elin g er (1991) to suggest
th a t th e trp p ro te in is a C a+~ tra n s p o rte r lo calized in th e p lasm a m e m b ra n e n e a r
 RETINAL DEGENERATION IN DROSOPHIIA 41

th e base o f th e rh a b d o m e re : (1) T h e p h e n o ty p e s o f Irp a n d an an a lo g o u s m u ta n t

nss (n o steady state) o f th e sh eep blowfly Lucilia are closely m im ick ed by th e ap p li
catio n o f L a+3, a know n in h ib ito r o f C a+2-b in d in g p ro te in s in c lu d in g Ca+2 tra n s
p o rte rs, to th e e x tra c e llu la r space o f w ild-type p h o to re c e p to rs (H o c h stra te , 1989),
a n d (2) several lines o f in d ire c t e v id en ce exist to suggest th a t th e Irp a n d nss m u
ta n t p h o to re c e p to rs d o n o t display an in crease in in tra c e llu la r [Ca+2] th a t n o rm a l
ly o ccu rs d u rin g illu m in a tio n (su m m a rized by M inke a n d Selinger, 1991).
A cc o rd in g to this hypoth esis, IP3 g e n e ra te d by light-activated PLC b in d s to th e IP3
re c e p to rs o n th e in tra c e llu la r C a+2 sto rag e c o m p a rtm e n t to re lease C a+2, an d , at
th e sam e tim e, th e IP 3-b o u n d IP 3 re c e p to r d irectly in te ra c ts w ith th e ^ -e n c o d e d
C a+2 tra n s p o rte r in th e p lasm a m e m b ra n e to cause tra n s p o rt o f C a+2 in to th e cy
to p lasm a n d th u s to re p le n ish Ca4" in th e sto rag e c o m p a rtm e n t (F igure 4). T h e
C a+" p o o l in th e sto rag e c o m p a rtm e n t is su fficien t to g e n e ra te only th e tra n sie n t
p h ase o f th e re c e p to r p o te n tia l d u rin g a p ro lo n g e d stim ulus, a n d th e re p le n ish
m e n t o f th e p o o l th ro u g h th e a ctio n o f th e C a+2 tra n s p o rte r is n e e d e d to m a in ta in
th e steady-state re sp o n se . M ore recently, w hole-cell rec o rd in g s fro m dissociated
p h o to re c e p to rs o f b o th w ild type a n d trp m u ta n ts le d H a rd ie a n d M inke (1992) to
p ro p o se th a t th e re c e p to r p o te n tia l in Drosophila is m e d ia te d by two classes o f light-
activated ch a n n e ls: o n e co n sistin g o f th o se w ith h ig h C a+2 p e rm ea b ility m e d iatin g
th e steady-state p h ase o f th e re c e p to r p o te n tia l a n d th e o th e r co n sisting o f those
w ith m u c h low er C a"2 p e rm e ab ility m e d ia tin g th e tra n sie n t p h ase (see Figure 4).
T h ey fu r th e r (1) su g g ested th a t th e trp g e n e e n c o d e s e ith e r th e su b set o f c h an n els
w ith h ig h C a+2 p e rm e a b ility o r a p ro te in p ro d u c t essential fo r th e ir fu n c tio n a n d
(2) id e n tifie d this su b set o f h ig h C a+~ p e rm e ab ility c h a n n e ls with th e C a+2 tra n s
p o r te r p ro p o s e d e a rlie r by M inke a n d S elin g er (1991), unifying th e two h y p o th e
ses, Since th e h y p o th esis re q u ire s th a t th e C a+2 c h a n n e l be lo c a te d in th e plasm a
m e m b ra n e n e a r th e base o f the rh a b d o m e ra l m icrovilli (F igure 4 ), im m u n o lo cal-
ization o f th e trp p ro te in to th e base o f th e rh a b d o m e re by J. A. Pollock (cited by
H a rd ie a n d M inke, 1992) was tak en as stro n g s u p p o rt fo r th e ir hypothesis.
C osens a n d P e rry (1972) c a rrie d o u t EM studies o f trp p h o to re c e p to rs a n d re
p o r te d th a t m any o f th e R l -6 rh a b d o m e re s show d e g e n e ra tiv e c h an g e s with age.
T h ese rh a b d o m e re s a re stru c tu ra lly n o rm a l at eclosion, b u t by 2 to 4 wk post-eclo-
sion, m any o f th e m a re re d u c e d in cross-sectional a re a a n d are d is ru p te d by vesic-
u la tio n . R8 rh a b d o m e re s also u n d e rg o sim ilar d e g en era tiv e chan g es, b u t R7
rh a b d o m e re s a p p e a r to b e largely fre e o f th ese c h a n g e s a t the ages tested. Al
th o u g h n o o th e r p u b lish e d stu d ies o f rh a b d o m e re d e g e n e ra tio n in trp a re avail
able, o u r ow n u n p u b lis h e d o b serv a tio n s (K. Isono a n d D. L e o n a rd ) are c o n sisten t
w ith C osens a n d P e rry s early re p o rt. As in th e case o f norpA, th e d e g e n e ra tio n is
lig h t-d e p e n d e n t. It d o e s n o t m a n ife st itself if th e flies are m a in ta in e d in co m p lete
d a rk n e ss u p to th e age o f 3 1 /2 wk post-eclosion. T h e p re se n c e o f sc re e n in g pig
m e n ts seem s to c o n sid erab ly slow dow n th e d e g e n e ra tio n tim e course, a n d R l-6
p h o to re c e p to rs a re m o re su scep tib le to d e g e n e ra tio n th a n R7 cells.
42 WILLIAM L. PAK

4 Mutants o f the rdgB Gene

H o tta a n d B en zer (1970) first re p o r te d th a t m u ta tio n s in two g en es, la te r n a m e d

rdgA a n d B ( retinal degeneration A a n d B), cause a g e -d e p e n d e n t d e g e n e ra tio n o f
rh a b d o m e re s. In d e p e n d e n tly , o th e rs have also iso lated m u ta tio n s in th e se g enes
(H e ise n b e rg , 1971; review by Pak, 1975). H a rris a n d S tark (1977) re p o r te d th a t
d e g e n e ra tio n c au sed by rdgA m u ta tio n s p ro c e e d s in d e p e n d e n tly o f lig h t while
th a t c au sed by rdgB m u ta tio n s is lig h t d e p e n d e n t. Since th e rdgA g e n e has n o t yet
b e e n c lo n e d , n o fu r th e r m e n tio n will be m a d e o f this g en e in this review.
T h e first d e ta ile d stu d ies o f rdgB m u ta n ts w ere c a rrie d o u t by H arris a n d Stark
(1977). T h e y show ed th a t rh a b d o m e re d e g e n e ra tio n is n o t only lig h t-d e p e n d e n t
b u t also te m p e ra tu re - a n d a lle le -d e p e n d e n t. T hus, if ex p o se d to a 12 h r lig h t/1 2
h r d a rk cycle a t 25 C, th e R l-6 rh a b d o m e re s, w hich are histologically n o rm a l at
eclo sio n , d isa p p e a r w ithin a b o u t a w eek. T h e tim e co u rse is faster if th e flies are
raised at h ig h e r te m p e ra tu re s o r o n c o n tin u o u s illu m in atio n . R 7 /8 rh a b d o m e re s
a re m u c h less sensitive to d e g e n e ra tio n th a n R l-6 rh a b d o m e re s, th e e x te n t to
w hich R 7 /8 rh a b d o m e re s d e g e n e ra te b e in g a lle le -d e p e n d e n t. Som e alleles cause
virtually n o d e g e n e ra tio n o f R 7 /8 rh a b d o m e re s, w hile o th e rs cause alm o st co m
p le te d e g e n e ra tio n o f R 7 /8 rh a b d o m e re s w ith in a week. T h e allele d e p e n d e n c e o f
R 7 /8 d e g e n e ra tio n , however, was d isp u te d late r by S tark et al. (1983).
D e g e n e ra tio n in rdgB m u ta n ts can be largely p re v e n te d if th e m u ta n ts a re raised
in th e d a rk o r d e p riv e d o f vitam in A (H a rris a n d Stark, 1977). Since vitam in A d e p
rivation drastically re d u c e s th e rh o d o p sin c o n te n t, it w ould be e q u iv alen t to d a rk
re a rin g . Sim ilarly, norpA m u ta tio n s, if c o m b in e d w ith an rdgB m u ta tio n in d o u b le
m u ta n ts, in h ib it rdgB-ca u sed d e g e n e ra tio n (H arris a n d Stark, 1977; S tark e t al.,
1983), p resu m ab ly b ecau se th e norpA m u ta tio n p rev e n ts th e g e n e ra tio n o f th e
light-evoked p o te n tia l. (rdgiJ-caused d e g e n e ra tio n can be d istin g u ish e d fro m
norpA-cau sed d e g e n e ra tio n b ecause o f th e ir d iffe re n t tim e courses. ) H a rris a n d
S tark (1977), however, iso lated a norpA allele w hich d o es n o t abolish th e re c e p to r
p o te n tia l a n d y et p rev en ts re tin a l d e g e n e ra tio n cau sed by a specific rdgB allele.
T h e allele-specific rescu e le d th em to p ro p o se th a t th e p ro te in p ro d u c t o f norpA
in te ra c ts w ith th a t o f rdgB.
S tark a n d C arlson (1982) show ed by e le c tro n m icroscopy th a t rdgiJ-induced d e
g e n e ra tio n is n o t c o n fin e d to th e rh a b d o m e re s. In fact, R l-6 p h o to re c e p to r axons
a re a m o n g th e first s tru c tu re s to be affected . In ad d itio n , by 7 d o n a 12 h r lig h t/
12 h r d a rk cycle, th e p h o to re c e p to r cytoplasm beco m es e le c tro n d en se a n d b egins
to show various s tru c tu re s asso ciated w ith d e g e n e ra tio n , such as vacuoles, e le c tro n
d e n se re tic u lu m , a n d d e g e n e ra te m ito c h o n d ria . M oreover, p h o to re c e p to r ax o n
te rm in a ls lack synaptic vesicles a n d p resy n ap tic stru c tu re s.
M inke, S elin g er a n d co lleag u es (review ed by M inke a n d Selinger, 1991) show ed
th a t a n u m b e r o f ch em icals a p p lie d to th e re tin a o f rdgB m u ta n ts cause p h o to re
c e p to r d e g e n e ra tio n in th e d ark , w hile th e sam e tre a tm e n t has n o effect o n wild
type flies. In p articu lar, p h o rb o l e ster was f o u n d to cause p h o to re c e p to r d e g e n e r
a tio n in d istin g u ish a b le fro m th a t cau sed by lig h t (M inke et al., 1990). S ince p h or-
 RETINAL DEGENERATION IN DROSOPHIIA 43

b o l e ste r is a pow erfu l activator o f p ro te in kinase C (N ishizuka, 1988), th e above

re su lt su g g ested th a t p h o to re c e p to r d e g e n e ra tio n in rdgB m ig h t b e c au sed by ex
cessive p h o sp h o ry la tio n o f p h o to r e c e p to r p ro te in s. In fact, th ese investigators
show ed th a t, in p u lse la b e lin g e x p e rim e n ts w ith ra d io la b e lle d p h o sp h a te , m u ch
g re a te r in c o rp o ra tio n o f label is fo u n d in th e eye p ro te in s o f rdgB m u ta n ts th a n
wild type. M ore d ire c t ev idence fo r possible in v olvem ent o f p ro te in kinase C
(PKC) was p ro v id e d by S m ith e t al. (1991), w ho show ed th a t rdgB-caused p h o to re
c e p to r d e g e n e ra tio n is su p p ressed by a m u ta tio n in th e s tru c tu ra l g e n e fo r eye-spe
cific PKC.
O n e o f th e early electro p h y sio lo g ical m an ife statio n s o f p h o to re c e p to r d e g e n e r
atio n in rdgB is th e a p p e a ra n c e o f spikes su p e rim p o se d o n th e ERG (R u b in stein et
al., 1989). T h e spikes have re g e n e ra tiv e p ro p e rtie s a n d arise fro m th e norm ally
no n -sp ik in g ax o n s a n d ax o n te rm in a ls o f p h o to re c e p to rs. T h e v o lta g e -d e p e n d e n t
C a '+ c h a n n e l b lo ck ers, Cd~+, verap im il c h lo rid e , a n d d iltiazem , elim in a te th e
spikes w ith o u t affectin g th e lig h t-in d u c e d resp o n se. In fact, a p p lic a tio n o f diltiaz
em to th e eyes o f rdgB m u ta n ts over a 7-day p e rio d largely p re v en ts lig h t-in d u c ed
p h o to re c e p to r d e g e n e ra tio n . C o m b in in g these results, M inke a n d S elin g er
(1991) a n d Sahly e t al. (1992) p ro p o s e d th a t p h o to re c e p to r d e g e n e ra tio n in rdgB
m u ta n ts is cau sed by a toxic in crease in in tra c e llu la r C a c o n c e n tra tio n th ro u g h
voltag e-g ated C a + c h a n n e ls, possibly as a resu lt o f excessive p h o sp h o ry la tio n .
T h e rdgB g e n e h a d b e e n cytogenetically m a p p e d to th e 12A-E re g io n o f th e X
c h ro m o so m e (H a rris a n d Stark, 1977). V ihtelic e t al. (1991a) re fin e d th e m a p p in g
to 12C1 u sin g X-ray in d u c e d rdgB alleles c o n ta in in g d e te c ta b le cytological lesions
a n d iso lated g e n o m ic DNA c o rre s p o n d in g to th e re g io n by a c h ro m o so m a l walk.
T h ey fo u n d a single tra n sc rip tio n u n it affe cted by ea ch o f th e new rdgB alleles w ith
in th e c lo n e d DNA fra g m e n t a n d th u s id e n tifie d it as th e rdgB g en e . Im m u n o lo cal-
izatio n o f th e rdgB p ro te in show ed th a t, in p h o to re c e p to rs, it is fo u n d in the
cytosolic base o f th e rh a b d o m e re s o f R l-6 p h o to re c e p to rs, n e a r th e su b rh a b d o -
m eric c iste rn a e (SRC) (F ig u re 4) a n d th e axons o f all p h o to re c e p to rs (V ihtelic
e t al., 1991b). T h e SRC a re a n etw o rk o f sm o o th e n d o p la sm ic re tic u lu m lo c ate d
just b e n e a th th e rh a b d o m e re s o f rh a b d o m e re -b e a rin g p h o to re c e p to rs (e.g. Walz,
1982; M atsum oto-S uzuki et al., 1989). T h e p ro te in is also fo u n d in th e ocelli, o p tic
lobes, a n te n n a e , a n te n n a l lobes, a n d m u sh ro o m b o dies o f th e c e n tra l b rain.
S e q u e n c e analysis o f th e tra n sc rip tio n u n it rev ealed an o p e n re a d in g fram e ca
p ab le o f e n c o d in g a p ro te in o f 1054 a m in o acids (V ihtelic et al., 1991a), th e am in o
te rm in a l 280 resid u es o f w hich are m o re th a n 40% id en tic al to th e e n tire le n g th
o f th e ra t b ra in p h o sp h a tid y lin o sito l tra n sfe r p ro te in (PI-TP; D ickeson e t al.,
1989) (F ig u re 2). T h e PI-TP-like d o m a in is follow ed by six p o te n tia l tra n sm e m
b ra n e d o m ain s. T h u s, it has b e e n p ro p o s e d th a t the rdgB p ro te in is a novel, m em
b ra n e -b o u n d m e m b e r o f th e PI-TP fam ily localized in o r n e a r th e SRC, involved
in sh u ttlin g PI b etw een th e SRC a n d th e rh a b d o m e re s fo r use in p h o to tra n s d u c
tio n (V ihtelic e t al., 1991b) (F igure 4 ). T h e re la tio n sh ip betw een this fin d in g a n d
th e p revious p ro p o sals o f excessive p h o sp h o ry la tio n a n d C a "' c h a n n e l d e fe c t has
n o t yet b e e n clarified.
44 WILLIAM L. PAK

5 Mutants o f the ninaC Gene

A long w ith th e ninaE g e n e (S ection III 1), th e ninaC g e n e was o n e o f th e o rig in al

five nina g e n e s id e n tifie d fro m m u ta tio n s th a t re d u c e th e rh o d o p sin c o n te n t
(Pak, 1979; S te p h e n so n e t al., 1983). F ro m th e ir studies o n several alleles o f
ninaC, M atsu m o to et al. (1987) su g gested (1) th a t th e ninaC g e n e likely en c o d e s
two retina-specific p ro te in s o f 130 an d 170 kD id e n tifie d in SDS polyacrylam ide
gel e le c tro p h o re s is a n d (2 ) th a t th ese p ro te in s are p ro b ab ly cytoskeletal c o m p o
n e n ts o f rh a b d o m e ra l m icrovilli. T h e first suggestion was b ased o n stro n g ninaC
g e n e d o sag e d e p e n d e n c e d isplayed by th e two p ro te in s, a n d th e se co n d sugges
tio n was b ased o n e le c tro n m ic ro g ra p h s show ing re d u c e d o r a b se n t ce n tra l
cytoskeletal co res o f rh a b d o m e ra l m icrovilli in ninaC m u tan ts. N o tin g th a t th e
rh a b d o m e re size is re d u c e d in ninaC m u ta n ts, they f u rth e r su ggested th a t th e
re d u c tio n in rh o d o p sin c o n te n t is d u e to th e re d u c e d size o f rh a b d o m e re s re su lt
ing from d efective m icrovillar cytoskeleton.
M o n tell a n d R u b in (1988) iso lated th e ninaC g e n e u sin g a h ead -sp ecific g e
n o m ic clo n e (Levy e t al., 1982) m a p p in g to th e 28A re g io n o f th e sec o n d c h ro m o
som e to w hich th e n in a C g e n e h a d also b e e n m a p p e d (M atsum oto e t al., 1987). To
p ro v e th a t a DNA fra g m e n t d eriv ed fro m th e Levy clo n e c o n ta in s th e ninaC g e n e,
M o n tell a n d R u b in (1988) re sc u e d a ninaC m u ta n t by in tro d u c in g th e DNA fra g
m e n t in to th e m u ta n t by P -e le m e n t-m e d iated g e rm lin e tra n sfo rm a tio n . M olecu
lar analysis o f th e g e n e by M on tell a n d R u b in (1988) show ed th a t it is ex p ressed
as two highly o v erlap p in g , altern ativ ely sp liced mRNAs w hich e n c o d e two very
closely re la te d , p h o to re c e p to r-sp ecific p ro te in s o f 1135 ( p l3 2 ) a n d 1501 (p l7 4 )
a m in o acids (F igure 3 ) . E ach isozyme consists o f two a d ja c e n t d o m ain s h o m o lo
g o u s to p ro te in kinases a n d th e h e a d re g io n o f th e m yosin heavy c h ain , respective
ly (F igure 2). T h u s, th e ninaC p ro te in p ro d u c ts a p p e a r to b e novel p h o to re c e p to r-
specific cytoskeletal p ro te in s c o n ta in in g a p ro te in kinase d o m ain .
P o rte r e t al. (1992) show ed th a t a stro n g ninaC m u ta tio n causes lig h t-d e p e n d e n t
a n d a g e -d e p e n d e n t d e g e n e ra tio n o f rh a b d o m e re s. W hen m a in ta in e d o n a 12 h r
lig h t/ 12 h r d a rk cycle, th e rh a b d o m e re s, w hich a re essentially n o rm a l at eclosion,
a lm o st d isa p p e a r by 3 wk post-eclosion. R7 rh a b d o m e re s a re som ew hat less affect
e d th a n e ith e r R l -6 o r R8 rh a b d o m e re s. If grow n in th e d ark , o n th e o th e r h a n d ,
d e g e n e ra tiv e ch an g es, if p re s e n t a t all, are only slight at 3 wk post-eclosion.
To study th e effects o f e lim in a tin g o n e o r th e o th e r o f th e two isozymes e n c o d e d
by ninaC, P o rte r e t al. (1992) g e n e ra te d two ninaC m u ta n ts expressing, resp ectiv e
ly, only p i 74 a n d only p i 32 by a p p ro p ria te o lig o n u c le o tid e -d ire c te d m u ta g en e sis
o f th e ninaC g e n e a n d in tro d u c tio n o f th e a lte re d g en e s in to a ninaC null m u ta n t.
T h ey f o u n d th a t e lim in a tio n o f p i 74 alo n e is su fficien t to cause b o th m o rp h o lo g
ical a n d physiological p h e n o ty p e s in d istin g u ish a b le fro m those o f ninaC n u ll m u
tan ts, w hile e lim in a tio n o f p l3 2 has little o r n o effect. EM im m u n o lo caliz atio n o f
th e two isozym es show ed th a t th e p i 74 a n d p i 32 p ro te in s a re lo c a te d prim arily in
th e rh a b d o m e re s a n d cytoplasm , respectively. F rom th ese observ atio n s, th ese a u
th o rs have su g g ested th a t th e p i 74 p ro te in associates with actin in th e ce n tra l
 RETINAL DEGENERATION IN DROSOPHIIJ l 45

co res o f rh a b d o m e ra l m icrovilli th ro u g h its myosin-I-like tail a n d is involved in

p h o sp h o ry la tin g rh a b d o m e ric p ro te in s im p o rta n t in p h o to tra n sd u c tio n .

6 M utants o f th e rdgC G en e

M u tatio n s in th e rdgC g e n e w ere iso lated fro m chem ical m u tag en e sis by lo o k in g
fo r th e ir d e g e n e ra tiv e effects o n th e d e e p p se u d o p u p il w ith age (Steele a n d
O T ousa, 1990). R h a b d o m e re s o f rdgC m u ta n ts a re n o rm a l w hen y o u n g b u t
u n d e rg o age- a n d lig h t-d e p e n d e n t d e g e n e ra tio n . As m o n ito re d by ex a m in in g th e
d e e p p se u d o p u p il, d e g e n e ra tio n is essentially c o m p lete by 8 d post-eclosion w hen
re a re d o n a 12 h r l i g h t / 12 h r d a rk cycle. R7 a n d R8 rh a b d o m e re s are m u c h m o re
re sista n t to d e g e n e ra tio n th a n R l-6 rh a b d o m e re s a n d ju s t b e g in show ing signs o f
d e g e n e ra tio n w h en th e la tte r have a lm o st com p letely d e g e n e ra te d . D e g e n e ra tio n
is n o t o b se rv e d if th e flies a re re a re d in c o m p lete dark n ess. U n lik e m o st o th e r
know n re tin a l d e g e n e ra tio n m u tan ts, p rio r to th e o n se t o f d e g e n e ra tio n , n o obvi
o us d e fe c t in th e re c e p to r p o te n tia l is ev id en t in rdgC m u tan ts. D e g e n e ra tio n
re q u ire s p h o to a c tiv a tio n o f rh o d o p sin b ec au se it d o e s n o t o c c u r if th e rh o d o p sin
level is re d u c e d by e ith e r vitam in A d e p riv a tio n o r th e use o f a rh o d o p sin ( ninaE)
g e n e m u ta tio n . H ow ever, u n like rdgB -induced d e g e n e ra tio n , rdgC-induced
d e g e n e ra tio n d o es n o t re q u ire th e norpA-en c o d e d PLC, su ggesting th a t it is m e d i
a te d by a rh o d o p sin activated pathw ay d iffe re n t fro m th e P L C -m ediated p h o
to tra n s d u c tio n pathway.
T h e g e n e was m a p p e d to 77B1 o f th e th ird ch ro m o so m e a n d was isolated by
ch ro m o so m a l w alking (S teele e t al., 1992). A DNA fra g m e n t th o u g h t to co n ta in
th e g e n e , iso lated fro m th e walk, was u se d to rescu e rdgC m u ta n t flies by g e rm line
tra n sfo rm a tio n , p ro v id in g p r o o f th a t th e g e n e h a d b e e n clo n e d in th e fra g m en t.
T h e g e n e e n c o d e s a p ro te in o f 661 a m in o acids w hich c o n ta in s a d o m a in h o m o l
o gous to th e catalytic d o m a in s o f types 1, 2A, a n d 2B s e r in e /th r e o n in e p ro te in
p h o sp h a ta se s (F igure 2). T h e p u tativ e catalytic d o m a in is follow ed by a d o m a in
c o n ta in in g several EF h a n d m otifs, o r p o te n tia l C a2+-b in d in g sites (F igure 2).
S teele e t al. (1992), th u s, su g g ested th a t th e rdgC p ro te in m ig h t be a novel type o f
s e r in e /th r e o n in e p h o sp h a ta se re g u la te d d irectly by C a2 r levels. T h e p ro te in is ex
p ressed in th e re tin a , ocelli, a n d m u sh ro o m b o d ie s o f th e b ra in . T h e fu n c tio n o f
th e p ro te in h as n o t yet b e e n e lu cid ate d .

IV DISCUSSION

A lth o u g h only a few g e n e s w hich, w h en defective, cause p h o to re c e p to r d e g e n e ra

tio n have b e e n iso lated a n d analyzed in Drosophila to d a te , som e ten tativ e co n c lu
sions m ay be ad v a n c e d re g a rd in g re tin a l d e g e n e ra tio n in this org an ism . First o f
all, as we have seen in th e previous sec tio n , th e re is a stro n g c o rre la tio n betw een
re tin a l d e g e n e ra tio n a n d defective p h o to tra n sd u c tio n . A lm ost all m u ta tio n s th a t
affect p h o to tra n s d u c tio n m ay also cause re tin a l d e g e n e ra tio n (see also M inke
46 WILLIAM L. PAR

a n d Selinger, 1991). H ow ever, m u ta tio n s th a t cause re tin a l d e g e n e ra tio n d o n o t

necessarily all affect p h o to tra n sd u c tio n . F or ex am p le, retin al d e g e n e ra tio n
cau sed by rdgC m u ta tio n s p ro c e e d s in th e ab se n ce o f norpA-en c o d e d PLC (Steele
a n d O T ousa, 1990), sug g estin g th a t this m o d e o f d e g e n e ra tio n is n o t c o n se q u e n t
o n a d e fe c t in P L C -m ediated p h o to tra n s d u c tio n . R ather, it a p p e a rs to be a co nse
q u e n c e o f a d e fe c t in a n o th e r light-activated, rh o d o p sin -m e d ia te d pathw ay
(S teele a n d O T ousa, 1990). T h e im p lica tio n is th a t d efects n o t only in p h o
to tra n s d u c tio n b u t also in any o th e r light-activated sig n alin g pathw ay o f p h o to re
c e p to rs m ay cause p h o to re c e p to r d e g e n e ra tio n .
A llele d e p e n d e n c e o f d e g e n e ra tio n has b e e n stu d ie d in detail only fo r ninaE
m u ta n ts to d a te (Section III 1; L e o n a rd et al.. 1992). As show n in F igure 3, th e tim e
c o u rse o f d e g e n e ra tio n is strongly a lle le -d e p e n d e n t. T h e results, th o u g h striking,
a re n o t u n e x p e c te d , since d iffe re n t m u ta n t alleles can have very d iffe re n t effects
o n th e fu n c tio n o f th e p ro te in e n c o d e d by th e gen e. T h u s, a lth o u g h allele d e p e n
d e n c e has b e e n r e p o rte d only fo r ninaE (L e o n a rd e t al., 1992), rdgB (H a rris a n d
Stark, 1977), a n d norpA (M ey erth o len et al., 1987) m u ta n ts to date, it is e x p e c te d
to be a p ro p e rty c o m m o n to all d e g e n e ra tio n m u tan ts.
R etin al d e g e n e ra tio n in m ost m u ta n ts stu d ie d to d a te is strongly lig h t-d e p e n
d e n t (S ection III). T h e only e x c e p tio n a p p e a rs to be ninaE m u ta n ts (S ection III 1;
L e o n a rd et al., 1992). M u tan ts o f a n o th e r g e n e , rdgA, w hich are n o t discussed in
this review, have also b e e n r e p o rte d to u n d e rg o lig h t-in d e p e n d e n t re tin a l d e g e n
e ra tio n (H a rris a n d Stark, 1977). In d e e d , d e g e n e ra tio n p ro c e e d s in th e d a rk in
this m u ta n t. H ow ever, o u r own u n p u b lis h e d o b serv atio n s in d ic ate th a t d e g e n e ra
tio n h as a su b stan tial lig h t-d e p e n d e n t c o m p o n e n t in rdgA in th a t it is con sid erab ly
a c c e le ra te d by illu m in a tio n (D. L e o n a rd a n d V. Bow m an, u n p u b lis h e d d a ta ).
T h u s, in m o st m u tan ts, re tin a l d e g e n e ra tio n is e ith e r com p letely b lo ck ed o r su b
stantially re ta rd e d by raisin g th e m u ta n ts in th e dark. H owever, d a rk re a rin g does
n o t re su lt in fu n c tio n a l re sc u e in m ost lig h t-d e p e n d e n t d e g e n e ra tio n m u ta n ts b e
cause fu n c tio n a l defects a re already p re s e n t b e fo re any evidence o f d e g e n e ra tio n
is a p p a re n t. As th e flies age a n d d e g e n e ra tio n p ro cee d s, electro p h y sio lo g ical re
sp o n ses d e te rio ra te fu rth e r. T h u s, in th ese m u tan ts, stru c tu ra l d e g e n e ra tio n does
n o t in itia te a fu n c tio n a l d efect, b u t ra th e r it fu rth e r e x ac erb ates a d e fe c t th a t is
alread y p re se n t. T h e re are, how ever, e x cep tio n s. In rdg'C m utants, fo r ex am p le, the
ERG is initially n o rm a l (S teele a n d O T ousa, 1990) b u t d e te rio ra te s w ith age a p
p a re n tly as a c o n se q u e n c e o f re tin a l d e g e n e ra tio n . T h e re fo re , d a rk re a rin g w ould
re su lt in b o th fu n c tio n a l a n d s tru c tu ra l re sc u e o f rdgC m u tan ts.
In all re tin a l d e g e n e ra tio n m u ta n ts stu d ie d to date, R l-6 p h o to re c e p to rs te n d
to d e g e n e ra te b e fo re R7 o r R8 p h o to re c e p to rs , a lth o u g h eventually th e la tte r d e
g e n e ra te also. In th e case o f ninaE m u ta n ts, th e g re a te r susceptibility o f R l-6 p h o
to re c e p to rs to d e g e n e ra tio n is u n d e rs ta n d a b le , since th e ninaE g e n e e n c o d e s R l-
6 o p sin . In fact, th e q u e stio n in this case is why R7 a n d R8 p h o to re c e p to rs d e g e n
e ra te a t all. However, R7 a n d R8 p h o to re c e p to rs d o d e g e n e ra te o n c e d e g e n e ra tio n
o f R l -6 p h o to re c e p to rs is well u n d e r way (L e o n a rd e t al., 1992). A p a rt o f th e ex
p la n a tio n m ig h t b e th a t R 7 /8 p h o to re c e p to rs a re tro p b ically d e p e n d e n t o n th e
 RETINAL DEGENERATION IN DROSOPHILA 47

p re se n c e o f R l -6 p h o to re c e p to rs. T h u s, w hen R-6 p h o to re c e p to rs d e g e n e ra te ,

R 7 /8 p h o to re c e p to rs can n o lo n g e r m a in ta in them selves a n d d e g e n e ra te also.
Even in m u ta n ts in w hich th e g e n e p ro d u c ts are ex p ressed in b o th R l -6 a n d R 7 /
8 cells, R l -6 p h o to re c e p to rs te n d to d e g e n e ra te first. Since d e g e n e ra tio n in these
m u ta n ts is lig h t-d e p e n d e n t, g re a te r susceptibility o f R l -6 p h o to re c e p to rs to d e
g e n e ra tio n m ay be d u e , in p a rt, to g re a te r sensitivity o f R l-6 p h o to re c e p to rs to
lig h t b ecau se o f th e ir la rg e r rh a b d o m e re s. However, it has b e e n re p o rte d th at in
trp a n d ninaC, R8 p h o to re c e p to rs d e g e n e ra te as fast as R l -6 p h o to re c e p to rs, w hile
R7 p h o to re c e p to rs a re m o re re sista n t to d e g e n e ra tio n (C osens a n d Perry, 1972;
P o rte r e t al., 1992). P o rte r e t al. (1992) have su g gested th a t R7 p h o to re c e p to rs are
re sista n t to d e g e n e ra tio n b ecau se they a re UV re c e p to rs a n d th e a m b ie n t lig h t
c o n ta in s relatively little UV. In o th e r lig h t-d e p e n d e n t re tin a l d e g e n e ra tio n m u
tants, o n th e o th e r h a n d , d iffe re n tia l susceptibility to d e g e n e ra tio n betw een R7
a n d R8 cells h as n o t b e e n re p o rte d . T h u s, n e ith e r o f th e above ex p la n a tio n s is a d
e q u a te to e x p la in b o th sets o f ob serv atio n s: th a t only R7 p h o to re c e p to rs a re resis
ta n t to d e g e n e ra tio n in som e classes o f m u ta n ts a n d th a t b o th R7 a n d R8
p h o to re c e p to rs are resistan t to d e g e n e ra tio n in o th e r classes o f m u tan ts. T hus,
w hile th e se e x p la n a tio n s m ay play a p a rt in th e o b serv ed p h e n o m e n a , th e w hole
e x p la n a tio n is still to b e o b ta in e d in fu tu re e x p erim en ts.
Finally, m u ta tio n s in a r a th e r large n u m b e r o f g en es can cause re tin a l d e g e n e r
a tio n in Drosophila. E stim ating fro m p re lim in a ry analysis o f a c o llectio n o f ERG-de-
fective m u ta n ts, m o re th a n a d o ze n o th e r g enes, in a d d itio n to th o se d e sc rib e d in
this review, a re likely to cause re tin a l d e g e n e ra tio n w hen defective. T h u s, th e
g en es d e sc rib e d in S ection III very likely re p re s e n t only a fra c tio n o f re tin a l d e
g e n e ra tio n g e n e s in this org an ism . M oreover, analyses o f d e g e n e ra tio n p h e n o
types are still largely in c o m p le te even fo r those d e sc rib e d in S ection III. Iso lation
a n d analysis o f th e se o th e r g en es a n d m o re c o m p lete know ledge o f th e know n d e
g e n e ra tio n m u ta n ts a re likely to b e im p o rta n t in th e u n d e rs ta n d in g o f re tin a l d e
g e n e ra tio n n o t only in this o rg an ism b u t also in m am m als as well.

V ACKNOW LEDGM ENTS

I th a n k M artin B urg, P aulo F e rre ira , L ydia L. R andall, a n d B h arath Srinivasan fo r

th e ir c o m m e n ts o n th e m a n u sc rip t, B a rb ara Petty a n d Lydia L. R andall fo r ed ito
rial a n d s te n o g ra p h ic assistance, D o n a ld R eady fo r h e lp w ith F igure 1. T h e p re p a
ra tio n o f th e m a n u sc rip t was s u p p o rte d in p a rt by a g ra n t fro m th e N a tio n al Eye
In stitu te (EY 00033). I g ratefully ack now ledge th e w ork o f m any colleag u es w hose
c o n trib u tio n s are review ed in th e m a n u sc rip t a n d apologize fo r any in a d v e rte n t
om issions.
48 WILLIAM L. PAK

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 3. DIAGNOSIS IN INHERITED RETINAL DISORDERS

ALAN C. BIRD a n d BARRIE JAY

Department o f Clinical Ophthalmology, Moorfields Eye Hospital, City Road,

London EC1V 2PD, UK

IN T R O D U C T IO N

R ed n al re c e p to r d y stro p h ies co m p rise a variety o f d isp a ra te genetically d e te r

m in e d c o n d itio n s th a t d iffer fro m o n e to a n o th e r in th e ir m o d e o f in h e rita n c e ,
th e ir p a tte rn o f visual loss a n d th e ir o p h th a lm o sc o p ic a p p e a ra n c e s. O ver th e past
few years an in c re a sin g n u m b e r o f d istin c t d iso rd e rs have b e e n rec o g n ised w ithin
this h e te ro g e n e o u s g ro u p , a n d in som e a clue to th e ir p a th o g e n e sis h as sta rte d to
em e rg e . It is p ossible to subdivide p a tie n ts with re c e p to r dystro p h ies in to several
g ro u p s d e p e n d in g o n th e ir clinical fea tu res. Som e p a tie n ts have sym ptom s early
in th e ir disease, in d ic a tin g p rim a ry loss o f ro d fu n c tio n , a n d ex a m in a tio n reveals
defective vision in th e m id zo n e o f th e visual field a n d m o rp h o lo g ic a l ch an g es in
th e p o ste q u a to ria l fu n d u s; th e diseases in such cases are know n collectively as
re tin itis p ig m en to sa. O th e r d iso rd ers, w ith loss o f co n e fu n c tio n a n d m o rp h o lo g
ical c h a n g e s in th e c e n tra l fu n d u s, a re know n as m a c u la r d e g e n e ra tio n s o r co n e
dystrophies.
T his subdivision in to p e rip h e ra l d e g e n e ra tio n s in w hich th e ro d s may be th e
p rim a ry ta rg e t o f disease a n d c e n tra l d e g e n e ra tio n s in w hich th e co nes may be
th e cells initially a ffe c ted is superficially attractive. It is te m p tin g to assum e th a t
th e p a th o g e n e sis o f c o n d itio n s w ithin th e retin itis p ig m e n to sa g ro u p is re la te d to
a d is o rd e r o f a m e ta b o lic fu n c tio n o r stru c tu ra l p ro te in p e c u lia r to ro d s a n d c o n
versely th a t m a c u la r d y stro p h ies are d u e to sim ilar defects o f cones. H owever,
m any p a tie n ts have c o m b in e d d iso rd e rs o f b o th ro d a n d co n e systems, even in th e
early stages o f th e ir disease, a n d all p a tie n ts w ith re tin itis p ig m e n to sa have som e
d e g re e o f co n e loss. T h e re c e p to r d y stro p h ies, th e re fo re , c o m p rise a sp e c tru m o f
d iso rd e rs ra n g in g fro m p u re ro d d y stro p h ies, th ro u g h r o d /c o n e a n d c o n e /r o d
dystro p h ies, to p u re c o n e d ystrophies. It is p ro b a b le th a t d iffe re n t fo rm s o f disease
usually re su lt fro m th e effect o f d iffe re n t genes b u t in som e in stan ces th e d iffe re n c
es m ay be d u e to o b se rv a tio n o f th e sam e d iso rd e r a t d iffe re n t stages o f evolution,
o f d iffe re n t p h e n o ty p ic e x p ressio n o f th e sam e g e n e tic d iso rd e r o r even o f differ
e n t m u ta tio n s w ithin th e sam e g e n e. D espite th ese reserv atio n s, re c e p to r dystro
p h ies will be c o n sid e re d in two b ro a d categories: p e rip h e ra l re c e p to r dystro p h ies
typified by re tin itis p ig m en to sa; a n d c e n tra l d y strophies. In a d d itio n , re tin a l dys
tro p h ie s m ay also o c c u r as p a r t o f m ultisystem disease in a variety o f w ell-described
sy ndrom es, two o f w hich, R efsum disease a n d a b e ta lip o p ro te in a e m ia , will also be
d iscussed h ere.
54 ALAN C. BIRD AND BARRIE JAY

PERIPHERAL RECEPTO R DYSTROPHIES

Retinitis pigm entosa

R etin itis p ig m e n to sa (RP) is a solitary m a n ifestatio n o f several genetically d e te r

m in e d d iso rd e rs c h a ra c te rise d by loss o f d a rk a d a p ta tio n a n d progressive re d u c
tio n o f p e rip h e ra l visual fields early in th e disease, le a d in g eventually to
im p a irm e n t o f c e n tra l vision. It is b e c o m in g increasingly obvious th a t th e re are
m any d iffe re n t g e n e tic d e fe c ts w ithin this fam ily of diso rd ers.

Gmetic studies

R etinitis p ig m e n to sa m ay be in h e rite d as a n au to so m al d o m in a n t, an au to so m a l
recessive o r an X -linked trait. W h en transm ission has o c c u rre d th ro u g h several
g e n e ra tio n s th e re c o g n itio n o f X -linked o r au to so m al d o m in a n t in h e rita n c e is
usually sim ple. As w ith o th e r d iso rd ers, p a re n ta l co n san g u in ity may in d ica te th e
possibility o f au to so m a l recessive in h e rita n c e . W h en faced with a sp o ra d ic case,
how ever, th e p ro b le m o f id en tifying th e g e n e tic fo rm o f th e disease may be m o re
difficult. In th e larg e series o f cases a t M o o rfield s Eye H ospital, th e fre q u e n c y o f
sim p lex (sp o rad ic) cases in fam ilies w ith re tin itis p ig m e n to sa is at p re se n t 52 p e r
c e n t (Jay M, p e rso n a l c o m m u n ic a tio n ), a figure a little la rg e r th a n th a t p u b
lish ed 10 years ago (42% - Jay, 1982). O th e r a u th o rs have fo u n d a sim ilar h ig h
fre q u e n c y o f sim plex cases (63% - P e a rlm a n , 1979; 48% - H u , 1982; 50% -
B o u g h m a n 8c F ishm an, 1983; 42% - H eckenlively, 1987). U ntil fairly recen tly it
was co m m o n ly assu m ed th a t p a tie n ts w ith sim plex RP h a d au to so m a l recessive
disease b u t th e re is in c re a sin g clinical e v id en ce th a t m any o f th e se p a tie n ts do
n o t ex p ress th e disease in th e sam e m a n n e r as p a tie n ts with pro v en au to so m al
recessive RP (H eckenlively, 1988). T h e re is also g e n e tic evidence th a t som e cases
o f sim p lex RP c a rry a m u ta tio n o f th e rh o d o p sin g e n e (D ryja e t al, 1991). Sim
p lex cases m ay be X -linked if h etero zy g o u s fem ales are asym ptom atic o r only very
m ildly a ffected a n d affe c ted m ale relatives are n o t know n to th e p ro b a n d , o r th e
disease m ay b e au to so m a l d o m in a n t as a re su lt o f a new m u ta tio n .
T h e fre q u e n c y o f th e d iffe re n t g e n e tic fo rm s o f retin itis p ig m e n to sa varies in
d iffe re n t series a n d in d iffe re n t co m m u n itie s (Table 1).
F u rth e r g e n e tic in fo rm a tio n may b e o b ta in e d by e x a m in in g asym ptom atic re la
tives. In p articu lar, it is u sefu l to ex am in e th e m o th e rs o f severely affe cted m ales
b e c a u se they fre q u e n tly show m ild retin al involvem ent, in d ic a tin g th e h etero zy
g o u s state o f X -linked disease (B ird, 1975).
T h e severity o f th e disease m ay b e u sed as a g u id e to th e likely in h e rita n c e (Jay
& Bird, 1973). In g e n e ra l X -linked a n d a u to so m a l recessive retin itis p ig m e n to sa
cause severe d isease o f early o n set, w hile d o m in a n t disease is m ilder. Severe dis
ease in a fem ale usually in d ic a tes a u to so m a l recessive a n d m ild disease in a m ale
au to so m a l d o m in a n t disease. M ild disease in a fem ale is c o m p atib le w ith e ith e r th e
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 55

T a b le 1

Author No of AI) AR XL Sp
Gases % % * %

Ammann et al., 1961 153 9 90 1

Franois & Verriest, 1962 153 19 35 4.1 41.3
Imaizumi, 1971 1091 2.1 40.1 43.2
Fishman, 1978 173 26 19 16 38
Pearlman, 1979 250 11 21 5 63
Boughman et al.., 1980 670 10 69 6 15
Hu, 1982 151 11 33.1 7.7 48.3
Jay, 1982 426 24.4 15.5 18 42.1
Boughman & Fishman, 1983 300 21.7 16 9 50
Heckenlively 1987 609 16.9 30.8 10.1 42.2

(AD = autosomal dominant; AR = autosomal recessive; XL = X-linked; Sp - sporadic

h etero zy g o u s state o f X -linked disease o r a u to so m a l d o m in a n t disease, w hereas se

vere disease in a m ale suggests X -linked o r au to so m al recessive disease.

Functional studies

By carefu l stu d ies o f visual fu n c tio n , co n sid e ra b le success has b e e n achieved in

estab lish in g a subdivision o f RP. In som e diso rd ers, th e ta rg e t cells have b e en
id e n tifie d , a n d th e ch a ra c teristic s o f fu n c tio n a l loss in d ica te th e n a tu re o f th e dis
ease m ech an ism .
Two b ro a d categ o ries o f a u to so m a l d o m in a n t RP have b e e n id e n tifie d w hich
are d e sig n a te d as type I o r d iffu se a n d type II o r re g io n a l fo rm s (M assof &
F inkelstein, 1981; A rd e n e t al., 1983; Lyness et al., 1985). T h e fu n c tio n a l ch a rac
teristics a re c o n siste n t w ith in fam ilies in d ic a tin g g e n e tic h e tero g en e ity . In fam ilies
w ith diffuse RP, a ffected m e m b e rs show w id esp read loss o f ro d fu n c tio n w ith rela
tively well p re se rv e d c o n e fu n c tio n at som e stage in th e ev o lu tio n o f th e ir disease.
T h o se w ith re g io n a l RP show v aria tio n in th e state o f ro d a n d c o n e fu n c tio n w ith
severe losses o f b o th system s in som e re g io n s a n d n e a r n o rm a l fu n c tio n in o th ers.
In re g io n a l RP, loss o f rh o d o p sin a c c o u n ts fo r th e re d u c tio n o f sensitivity, b u t in
diffuse RP th e re is m o re rh o d o p sin th a n w ould be p re d ic te d if re d u c e d lig h t ab
so rp tio n by visual p ig m e n t was resp o n sib le fo r sensitivity loss (K em p et al., 1988).
T h ese fin d in g s suggest th a t in re g io n a l RP loss o f ro d fu n c tio n is d u e to p h o to re
c e p to r cell d e a th , s h o rt o u te r seg m en ts o r a c o m b in a tio n o f th e two, w hile in dif
fuse RP this c a n n o t be th e case.
A d d itio n a l varian ts have b e e n id e n tifie d in au to so m al d o m in a n t disease. S ector
RP (B ietti, 1937; H aase & H e lln er, 1965) is c h a ra c te rise d by re tin a l a tro p h y re
stric te d to p a rt o f th e fu n d u s, a n d gross field loss is c o n fin e d to th e a re a o f visual
field c o rre s p o n d in g to th e involved re tin a . W h en this occurs, retin itis p ig m e n to sa
u sually affects th e low er h a lf o f th e fu n d u s w ith loss o f visual fu n c tio n in th e u p p e r
56 ALAN C. BIRD AND BARRIE JAY

field (F led eliu s & S im o n sen , 1970), b u t rarely th e disease affects only th e su p e rio r
(R ag n etti, 1962), nasal (V ukovich, 1959) o r te m p o ral fu n d u s (A lezzandrini,
1965). In m o st re p o r te d cases w ith lim ited secto r involvem ent th e in h e rita n c e was
au to so m al d o m in a n t a n d th e re g io n a l d istrib u tio n o f disease was c o m m o n to af
fe c te d m e m b e rs o f th e fam ily (H om m er, 1959; K uper, 1960; Lisch, 1960; H aase &
H e lln e r, 1965; F led eliu s & S im o nsen, 1970). However, sim ilar cases also a p p e a r in
fam ilies w ith relatives w ho have inv o lv em en t o f th e w hole fu n d u s (Krill e t al.,
1970) a n d som e a re h e tero zy g o u s fo r X -linked re tin itis p ig m e n to sa (Krill e t al.,
1970; B ird, 1975). By flu o re sc e in angiography, m in o r ch a n g e s are o fte n fo u n d to
be m o re w id esp read th a n m ig h t b e a p p re c ia te d by o p h th a lm o sc o p y with w hite
light, a n d th e secto r o f retin itis p ig m e n to sa may b e an a rea o f m ax im u m disease
r a th e r th a n o f exclusive in v o lv em en t (Krill et al., 1970; B ird, 1975). In typical sec
to r RP, ro d a n d c o n e ERGs show m ild re d u c tio n in a m p litu d e w ith n o rm a l co n e
im p licit tim es (B erson 8c H o w ard, 1971; M assof 8c F inkelstein, 1979; F u lto n &
H a n se n , 1988). T h is p a tte rn o f disease is co m m o n to all a ffected m e m b e rs w ithin
a fam ily irresp ectiv e o f age, su g gesting th a t th e disease, in c o n tra st to o th e r fo rm s
o f au to so m a l d o m in a n t RP, is n o n -p rogressive o r p ro g resses very slowly.
In som e fam ilies th e re is slow recovery fro m b leach (A lex an d er & F ishm an,
1984). C o n e a d a p ta tio n a n d th e early p a rt o f th e reco v ery o f ro d sensitivity follow
th e n o rm a l tim e co u rse, b u t th e la te r p h a se o f ro d a d a p ta tio n is m ark ed ly p ro
lo n g ed .
U n ila te ra l re tin itis p ig m e n to sa was r e p o rte d as early as 1865 by P e d ra lg ia a n d
th e o p h th a lm o sc o p ic o b serv atio n s w ere c o rro b o ra te d by histological e x a m in a tio n
25 years later. In 1952, F ran o is a n d V erriest review ed th e 56 u n ila te ra l cases re
p o r te d u p to th a t tim e a n d c o n c lu d e d th a t only 10 cases h a d th e typical o p h th a l
m o sco p ic a p p e a ra n c e o f g en etically d e te r m in e d retin itis p ig m e n to sa a n d w ere
strictly u n ila te ra l. S u b se q u e n t re p o rts (K olb & Galloway, 1964; C a rr & Siegel,
1973) have show n th a t, in som e cases, m in o r c h an g es can be d e m o n stra te d in th e
u n a ffe c te d fellow eye by so p h istica te d e x a m in a tio n . F u rth e rm o re , C a rr a n d Siegel
(1973) th o u g h t th a t th e lesions in cases d e sc rib e d by th e m a n d by som e prev io u s
a u th o rs w ere d u e to vascular disease a n d w ere n o t h e ritab le . To illu strate th e dif
ficulty in assessing th e sig n ificance o f such cases, P e arlm an a n d colleagues (1976)
d e sc rib e d a p a tie n t w ith m ild b u t w id e sp rea d u n ila te ra l o u te r re tin a l disease, b u t
they p re s e n te d n o ev idence as to th e in h e rita n c e o f th e disease. It sh o u ld b e e m
p h a sise d th a t in n o case o f u n ila te ra l retin itis p ig m e n to sa has a fam ily h isto ry o f
eye disease b e e n id e n tifie d a lth o u g h , in o n e p a tie n t, th e p a re n ts w ere c o n sa n g u in
e o u s (C o rd ie r e t al., 1966). M ost w orkers take th e view th a t th e re is n o g o o d evi
d e n c e th a t u n ila te ra l retin itis p ig m e n to sa re p re se n ts a d istin c t h e rita b le disease.
T h e expressivity in som e fam ilies is hig h ly variable su ch th a t a b o u t 70% o f
m e m b e rs w ith th e a b n o rm a l g en o ty p e have m o d e ra te to severe disease w hile
th e re m a in d e r a re asym ptom atic, b u t w ith m ild fu n d u s a b n o rm a litie s a n d electro-
physiological c h a n g e s in d ic a tin g th e p re se n c e o f th e a b n o rm a l g e n e (E rn st &
M o o re, 1988). In th ese fam ilies, th e re a p p e a rs to b e b im o d a l e x p ressio n o f disease
a lth o u g h th e factors th a t m o d u la te g e n e ex p re ssio n are un k n o w n .
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 51

Specific clinical subtypes o f RP have also b e e n re c o g n ise d with au to so m a l re ce s

sive disease. RP has b e e n d e sc rib e d with p re se rv a tio n o f the re tin a l p ig m e n t e p i
th e liu m a d ja c e n t to th e re tin a l a rte rio le s, a n d u n lik e m any p a tie n ts w ith RP they
a re co n sisten tly h y p e rm e tro p ic (H eckenlively, 1982; P o rta e t al., 1992).
A f u r th e r v a ria n t co m p rises n ig h t b lin d n e ss a n d variably re d u c e d visual acuity
associated w ith re stric te d p ig m e n ta tio n in th e o c u la r fu n d u s, cystoid m ac u lar
c h an g es re se m b lin g m a c u la r schisis, a n d distinctive e le c tro re tin o g ra p h ic a b n o r
m alities (M a rm o r e t al., 1990). T h e ro d ERG is u n re c o rd a b le , m axim al stim ulus
evokes a larg e re sp o n se w ith very p ro lo n g e d im p licit tim e o f th e b-wave, p h o to p ic
a n d sco to p ic re sp o n se s a re o f sim ilar m a g n itu d e , a n d th e re sp o n se is m u c h la rg e r
w hen p ro d u c e d by s h o rt th a n by lo n g w avelengths. T h e last a ttrib u te is know n as
th e e n h a n c e d S c o n e re sp o n se.
As early as 1914, D iem re p o r te d a b n o rm a l fu n d u s reflexes in fem ales h etero zy
gous fo r X -linked retin itis p ig m e n to sa , b u t d u rin g su b se q u e n t years sim ilar p h e
n o m e n a w ere re p o r te d in o th e r g e n e tic fo rm s o f retin itis p ig m e n to sa a n d w ere
re fe rre d to as ta p e ta l re flex es (M an n , 1937). An a b n o rm a l ta p e tal reflex has
b e e n re p o r te d to b e th e m o st co m m o n ex p re ssio n o f th e hetero zy g o u s state in X-
lin k e d disease (Falls & C o tte rm a n , 1948; W ie n e r & Falls, 1955; Francois, 1962;
Ricci e ta l., 1963; H ussels, 1967; W a rb u rg & S im o n sen 1968; Krill 1969). Ricci a n d
his co lleag u es (1963) fo u n d th a t this reflex c o u ld n o t be seen a fte r e x p o su re o f
th e fu n d u s to lig h t b u t re a p p e a re d a fte r a p e rio d o f darkness; th ey called this the
p h n o m n e d e M izuo in v e rse. H ow ever, S chappert-K im m ijser (1963) co u ld
identify this a b n o rm a l reflex in fem ales o f only o n e fam ily o u t o f eig h t a n d c o n
c lu d e d th a t a lth o u g h this sign is useful w hen p re se n t, its ab se n ce d o e s n o t in d ica te
a n o rm a l g en o ty p e.
A ttem p ts have b e e n m ad e to subdivide X -linked retin itis p ig m e n to sa on th e ba
sis o f d iffe re n tia l affectio n o f h eterozygotes. O nly a few o f th e early re p o rts o f fam
ilies w ith X -linked disease d e sc rib e d re tin a l d e g e n e ra tio n in fem ales (M cQ u arrie,
1935; Ja n sse n , 1938) b u t p ro fo u n d visual loss in h etero zy g o tes was re p o rte d by
M cK enzie (1951) in N ew Z ealand. In 1960 K obayashi d esc rib e d a fam ily with even
m o re severe in v o lv em en t o f th e fem ale m em b e rs, five hetero zy g o u s fem ales hav
in g re tin itis p ig m e n to sa , 11 having so m e stigm ata o f th e disease a n d at least fo u r
b e in g n o rm a l. N o a c c u ra te co m p arativ e analysis was m a d e o f th e severity o f th e
disease b etw een affe c ted m e n a n d w om en, a lth o u g h it was sta te d th a t th e fem ales
w ere m o re m ildly affe c ted th a n th e m ales. F ro m this in fo rm a tio n , it was c o n c lu d e d
th a t th e re w ere th re e se p a ra te X 4 in k e d con d itio n s: X -linked recessive, X -linked
in te rm e d ia te a n d X -linked d o m in a n t retin itis pig m en to sa. H ow ever, su b se q u e n t
e x p e rie n c e in d ic a te s th a t th e re is co n sid e ra b le intrafam ilial v ariatio n in th e sever
ity o f th e disease in h etero zy g o tes, a n d th a t th e re is n o ju stific a tio n fo r se p a ra tin g
X -linked re tin itis p ig m e n to sa in to d iffe re n t c ateg o ries on th e basis o f th e severity
o f th e disease in w o m en (B erson e t al., 1969; B ird, 1975).
It d o e s a p p e a r th a t th e p re se n c e o f a ta p etal reflex in w om en hetero zy g o u s fo r
th e a b n o rm a l g e n e c h a ra c terises som e fam ilies b u t n o t o th e rs (Schappert-K im -
mijser, 1963), such th a t this sign a p p e a rs to d iffe re n tia te X lin k ed RP in to two cat
58 ALAN C. BIRD AND BARRIE JAY

eg o ries o n clinical g ro u n d s, an o b serv atio n in k eep in g with re c e n t g e n e tic studies

th a t in d ic a te th e ex isten ce o f two loci fo r X -linked RP.

Molecular genetic studies

T h e first RP g e n e to be localised (RP2) was o n e fo r X -linked RP using a p ro b e

L I .28 (B h a tta c h a ry a e t al., 1984) w hich m aps to th e p ro x im al X ch ro m o so m e
sh o rt a rm in b a n d X p l l . S u b se q u e n t stu d ies have s u p p o rte d th e o rig in al o b ser
v ation (F rie d ric h et al., 1985; M ukai e t al., 1985; W right et al., 1987). T h e first
in d ic a tio n o f a se c o n d locus re su lte d fro m th e id e n tifica tio n o f a d e le tio n at
Xp21 in a ch ild with re tin itis p ig m en to sa, D u c h e n n e m u sc u la r dystrophy a n d X-
lin k e d c h ro n ic g ra n u lo m a to u s disease (F ran ck e et al., 1985); this locus is distal
(te lo m e ric ) to th a t o f RP2 a n d is d e sig n a te d RP3. L ocalisation o f an X -linked
retin itis p ig m e n to sa g e n e to this distal site was s u p p o rte d by o th e r linkage studies
(N u ssb au m e t al., 1985a; D e n to n e t al., 1988). T h e re is n o w ide a g re e m e n t o n the
clinical d iffe re n c es b etw een diseases tra n sm itte d at th e two loci, a lth o u g h all h e t
erozygotes in w hom a ta p e ta l reflex is seen a p p e a r to b e lo n g to fam ilies in w hich
m u ta tio n is a t th e RP3 locus. T h e possibility o f m o re th a n o n e a b n o rm a l allele at
each locus has n o t b e e n ex c lu d e d ; th a t n o t all fam ilies w ith disease tra n sm itte d at
th e RP3 site have a ta p e ta l reflex w ould s u p p o rt this view.
In 1989, a locus fo r au to so m al d o m in a n t retin itis p ig m en to sa (adR P) was id e n
tified o n th e lo n g a rm o f c h ro m o so m e 3 (M cW illiam et al., 1989). T h re e c a n d id a te
g e n e s exist close to th e p ro p o s e d site, a n d w ithin a s h o rt tim e a p ro lin e /h is tid in e
m u ta tio n was d e te c te d at c o d o n 23 o f th e rh o d o p sin m o lecu le (D ryja e t al.,
1990a). Since th e n , m o re th a n 30 d iffe re n t m u ta tio n s have b e e n d e te c te d in th e
rh o d o p sin g e n e a n d it a p p e a rs th a t this g e n e acco u n ts fo r a b o u t 25% o f au to so m al
d o m in a n t RP (D ryja et al., 1990b; Sung e t al., 1991; K een et al., 1991; In g le h e a rn
et al., 1992). M ost o f th e su b stitu tio n s a p p e a r to be p e c u lia r to a single family, al
th o u g h som e, n o tab ly th e m u ta tio n at c o d o n 347, have b e e n fo u n d in m any p a rts
o f th e w orld. T h e p r o lin e /h is tid in e m u ta tio n a t co d o n 23 was id e n tifie d in a p ro
p o rtio n o f subjects in th e U n ite d States b u t n o t in o th e r co u n tries. T h e re is now
som e ev id en ce th a t th e RP p a tie n ts w ith this m u ta tio n may fo rm p a rt o f o n e large
p e d ig re e .
In several fam ilies with au to so m al d o m in a n t RP, th e re is n o linkage with m ark ers
in th e lo n g a rm o f c h ro m o so m e 3 (L ester e t al., 1990), c o n firm in g h e te ro g e n e ity
o f d o m in a n t disease, a n d it has now b e e n show n th a t g en es o th e r th a n th a t fo r
rh o d o p sin may be re sp o n sib le fo r a u to so m al d o m in a n t disease (B lanton e t al.,
1990, 1991; F a rra r et al., 1991a,b; Kajiwara et al., 1991). O n e o f th ese is th e g e n e
fo r rd s p e rip h e rin th a t is o f p a rtic u la r in te re st since it is a m u ta tio n in this g e n e
th a t is re sp o n sib le fo r th e re tin a l d e g e n e ra tio n in th e rds m ouse (Travis e t al.,
1989; C o n n e ll e t aL, 1991). P e rip h e rin is localised to th e o u te r se g m e n t disc m em
b ra n e s o f b o th ro d s a n d cones, a n d is th o u g h t to be essential fo r th e assembly, o ri
e n ta tio n a n d physical stability o f th e o u te r se g m e n t discs o f th e re tin a l
p h o to re c e p to rs (C o n n e ll & Molday, 1990; Travis e t al., 1991a; A rokaw a et al.,
1992).
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 59

O f g re a t in te re st is th e id en tifica tio n th a t m a c u la r dystrophy may also b e d u e to

m u ta tio n s in th e RDS g e n e . Two fam ilies w ith a fo rm o f m ac u la r dystrophy have
b e e n show n to have a try p to p h a n su b stitu tio n fo r a rg in in e a t c o d o n 172, a n d a n
o th e r fam ily w ith a lm o st id en tica l disease has a g lu ta m in e su b stitu tio n fo r a rg in in e
a t th e sam e c o d o n . A m u ta tio n re su ltin g in a sto p c o d o n a t p o sitio n 258 has b e e n
id e n tifie d in a n o th e r fam ily w ith a re tin a l d e g e n e ra tio n sim ilar in a p p e a ra n c e to
a d u lt vitellifo rm m a c u la r dystrophy. T h ese fin d in g s d e m o n stra te th a t som e form s
o f au to so m al d o m in a n t re tin itis p ig m e n to sa a n d d o m in a n tly in h e rite d m ac u lar
d y stro p h ies a re c au sed by m u ta tio n s in th e sam e (RDS p e rip h e rin ) g e n e. T h e vari
ability o f p h e n o ty p e c au sed by m u ta tio n s at d iffe re n t c o d o n s suggests th a t th e
fu n c tio n a l significance o f ce rta in am ino-acids to c o n es a n d ro d s m ay b e d iffe re n t
(Wells e t al 1993).

Functional consequences o f mutations in adRP genes

In th e fam ilies w ith au to so m a l d o m in a n t RP show ing m u ta tio n s o f th e rh o d o p sin

g e n e o n c h ro m o so m e 3, d iffe re n t p a tte rn s o f re tin a l d y sfunction have b e e n d e m
o n stra te d w ith d iffe re n t m u ta tio n s (F ishm an e t al., 1991; H eckenlively e t al.,
1991; Ja c o b so n e t al., 1991; K em p e t al., 1992; M oore e t al., 1992).
F u n ctio n al loss co m p a tib le w ith diffuse (type I) au to so m al d o m in a n t RP was
fo u n d in p a tie n ts w ith th re e types o f rh o d o p sin m u ta tio n in w hom ro d a n d co n e
fu n c tio n was tested; p ro lin e to le u c in e a t c o d o n 347, lysine to glu tam ic acid at
c o d o n 296 (w hich is th e b in d in g site fo r r e tin a l) , a n d an iso leu cin e d e le tio n at
c o d o n 255 o r 256. W ith all th re e m u ta tio n s, p o o r n ig h t vision was consistently
id e n tifie d in early life a n d ro d ERGs w ere severely re d u c e d o r u n m e a su ra b le . In
th o se w ith m easu rab le visual fu n c tio n , psychophysical testing show ed ro d fu n c tio n
to be severely affe c ted th ro u g h o u t th e re tin a (with th re sh o ld elevations o f m o re
th a n 3 log u n its), even in y o u n g e r individuals, w hile loss o f co n e fu n c tio n varied
widely b etw een fam ilies, a n d was less w id esp read a n d less severe th a n loss o f ro d
fu n c tio n . In th e m o st severely a ffe c te d fam ily (lys296glu), th e re was little visual
fu n c tio n a fte r th e age o f 30 years in m o st m em b ers (K een e t al., 1991). W ith th e
iso leu cin e d e le tio n a t c o d o n 256, c o n e fu n c tio n was lim ite d to th e c e n tra l 10 by
th e age o f 25 years b u t little f u r th e r loss o c c u rre d th e re after. C o n e fu n c tio n was
re ta in e d over m o st o f th e visual field u n til m id d le life in th e c o d o n 347 m u ta tio n
d e sp ite w id e sp re a d a n d severe early loss o f ro d fu n c tio n .
T h e fu n c tio n a l a b n o rm a lity in fam ilies with m u ta tio n s th re o n in e to a rg in in e at
c o d o n 17, p ro lin e to h istid in e at c o d o n 23, th re o n in e to a rg in in e a t co d o n 58, gly
cin e to a rg in in e at c o d o n 106 a n d g u a n in e to a d e n in e at c o d o n 182 is qualitatively
sim ilar w ith a ltitu d in a l o r sectorial d istrib u tio n o f disease a n d this a ttrib u te ap
p ears to be c o n sta n t w ith in th e fam ilies (F ish m an e t al., 1991, 1992; H eckenlively
et al., 1991; R ich ard s e t al., 1991; S to n e e t al., 1991; K em p e t al., 1992; M oore et
al., 1992). T h is c h a ra c teristic o f disease is in m a rk e d c o n tra st w ith th a t seen in p a
tien ts w ith o th e r rh o d o p s in m u ta tio n s e x a m in e d to d ate. R od fu n c tio n is severely
60 ALAN C. BIRD AND BARRIE JAY

d e p re sse d in th e s u p e rio r field b u t is n early n o rm a l in th e in fe rio r field. Loss o f

co n e sensitivity closely follow s th a t o f th e rods. In this re sp e c t th e p a tte rn o f dis
ease resem b les re g io n a l (type II) RP.
A n a d d itio n a l strik in g fin d in g in th ese fam ilies with sectorial type RP is a c h a r
acteristic ab n o rm a lity in o n e c o m p o n e n t o f th e kinetics o f d a rk a d a p ta tio n follow
in g e x p o su re to a b rig h t light. P rio r to b le ac h in g , m e a su re m e n ts m ad e in the
relatively in ta c t p o rtio n o f th e visual field show m ild th re sh o ld elevations o f a p
p ro x im a te ly o n e log u n it. F ollow ing lig h t a d a p ta tio n th e re is a m a rk e d delay in th e
reco v ery o f sensitivity. T h e in itial p o rtio n o f recovery m e d ia te d by co n es a n d rods
is n o rm a l. H ow ever by o n e h o u r, w hen in th e n o rm a l subjects recovery o f sensitiv
ity is co m p lete, th ese p a tie n ts show re sid u al th re sh o ld elevations o f 1-2 log u n its
fro m th e p re b le a c h values. Even a fte r n early two h o u rs, th re sh o ld s are still elevat
e d by m o re th a n 1.0 log u n it. In two subjects, it was fo u n d th a t th e tim e co u rse o f
th is slow reco v ery o f sensitivity was o f th e o r d e r a t least 8 0 -1 2 0 h o u rs (M oore e t
al., 1992).
U sin g a m o d e l based u p o n p rim a te d a ta o f ro d o u te r se g m e n t le n g th a n d tu r n
over, it has b e e n calcu lated th a t th e delayed phase o f th e recovery o f ro d sensitivity
follow ing stro n g lig h t a d a p ta tio n co u ld be d u e in p a rt to th e fo rm a tio n o f new disc
m e m b ra n e s w ith a n o rm a l c o n c e n tra tio n o f rh o d o p sin ra th e r th a n in situ re g e n e r
a tio n o f p h o to p ig m e n t (M o o re e t al., 1992). T h e m o d el re q u ire s th a t th e o u te r
seg m en ts a re s h o rt as a re su lt o f RP, a n d th a t a m ajo r p o rtio n o f th e o u te r se g m e n t
is sh e d follow ing stro n g lig h t a d a p ta tio n .
T h ese o b se rv a tio n s show th a t th e re a re b o th q u an titativ e a n d qualitative differ
en ces in th e RP c o n s e q u e n t u p o n d iffe re n t m u ta tio n s in th e rh o d o p sin g e n e . Also
im p o rta n t is th a t it has b e e n show n fo r th e first tim e th a t som e fo rm s o f RP a re
d u e to d efects o f m etab o lic system s th a t a re lim ited to ro d p h o to re c e p to rs, a n d yet
it is ev id e n t th a t loss o f p h o to p ic fu n c tio n is consistently fo u n d in th ese d iso rd ers.
N o e x p la n a tio n exists to a c c o u n t fo r c o n e cell d e a th . It is possible th a t c o n e loss
o ccu rs as a re su lt o f e ith e r a release o f toxic p ro d u c ts by dying ro d s o r th e co nes
b e in g m etabolically d e p e n d e n t u p o n th e p re se n c e o f rods. It follows th a t loss o f
c o n e fu n c tio n , a lth o u g h im p o rta n t to th e p a tie n t, is a sec o n d ary effect. It is also
c le a r th a t th ese stu d ies a re in c o m p le te a n d th a t f u rth e r w ork will p ro d u c e m o re
in fo rm a tio n .
T h e c u r r e n t k now ledge o f th e putative fu n c tio n o f rd s p e rip h e rin provides an
e x p la n a tio n fo r d iffe re n t p h e n o ty p e s b e in g associated with d iffe re n t m u ta tio n s in
th e RD S g e n e . T h e p h o to re c e p to r o u te r se g m e n t p ro te in , rds p e rip h e rin , has 346
a m in o acids w ith fo u r h y d ro p h o b ic tra n sm e m b ra n e d o m a in s a n d two p u tativ e N-
lin k e d glycosylation sites, o n e o f w hich is co n serv e d across fo u r species a n d is
th o u g h t to b e im p o rta n t to th e p ro te in 's fu n c tio n in stabilising th e p ara llel array
o f p h o to re c e p to r o u te r se g m e n t m e m b ra n e s (Travis e t al., 1991a; b ). Im m u n o h is-
to c h e m ic a l stu d ies have show n th a t th e p ro te in is lim ited to th e m e m b ra n e s o f o u t
e r seg m en ts o f b o th ro d s a n d co n es a lth o u g h th e re is d isa g re e m e n t over its p recise
localisatio n . O n e study u sing a polyvalent an tib o d y to a sh o rt p e p tid e se q u e n c e
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 61

n e a r th e carboxyl te rm in u s im p lie d th a t the p ro te in was d istrib u te d over the e n

tire le n g th o f th e o u te r se g m e n t disc m e m b ra n e (Travis e t al., 1991a). It was sug
g e ste d th a t co v alen t b o n d in g betw een rd s p e rip h e rin m o lecu les was resp o n sib le
fo r th e m a in te n a n c e o f th e p arallel a rra n g e m e n t o f th e o u te r se g m en t m e m
b ra n e s. By c o n tra st, a se p a ra te study u sing a m o n o c lo n a l a n tib o d y to p u rifie d p h o
to re c e p to r discs show ed th a t lab ellin g was c o n fin e d to th e rim s o f th e o u te r
seg m en t, im plying th a t th e p rim a ry fu n c tio n o f th e p ro te in was to stabilise the
th erm o d y n am ically u n fa v o u rab le b e n d a t th e disc rim (Kajiwara e t al., 1991). To
e x p lain th e se d iffe re n t fin d in g s it was h y p o th e sise d th a t th e e p ito p e s available fo r
a n tib o d y b in d in g may d iffe r at th e disc rim w hen c o m p a re d with o th e r p a rts o f the
disc m e m b ra n e . R egardless o f th e p recise m ec h an ism , th e g e n e ra l b e lie f is th a t rds
p e rip h e rin is im p o rta n t to th e stru c tu ra l stability o f th e o u te r se g m e n t disc m e m
b ra n e . S tu d ies o f th e rds m o u se s u p p o rt this belief. In the hom ozygous rds/rds g e n
otype, o u te r se g m e n t discs a re n o t fo rm e d , th e disc m e m b ra n e b e in g d isc h arg ed
as sm all vesicles in to th e su b re tin a l space (Sanyal & Ja n se n , 1981; C o h e n , 1983;
J a n s e n & Sanyal, 1984; U su k u ra & Bok, 1987). In th e hetero zy g o u s r d s /+ m ouse,
o u te r se g m e n t disc m e m b ra n e s a re lo n g a n d d isto rte d (H aw kins e t al., 1985; San
yal & H aw kins, 1988).
R ecently it h as b e e n show n th a t rd s p e rip h e rin m ay b e non-covalently associated
w ith ROM 1, a p ro te in stru ctu rally re la te d to p e rip h e rin . ROM1 has b e e n localised
to th e disc rim s o f ro d o u te r segm ents b u t has n o t b e e n id e n tifie d in co nes (Bas-
com et al., 1992). It has b e e n p ro p o se d th a t th e fo rm a tio n a n d stability o f th e b e n d
in th e disc m e m b ra n e in ro d s are d e p e n d e n t u p o n th e association betw een rds p e
rip h e rin a n d ROM 1. H ow ever, th e ab sen c e o f RO M ! in con es im plies th a t differ
en ces exist b etw een th e p recise m ec h an ism s by w hich rd s p e rip h e rin stabilises
o u te r se g m e n t m e m b ra n e s in th e two classes o f p h o to re c e p to r. In ro d s, th e associ
a tio n b etw een rd s p e rip h e rin a n d ROM1 m ay be im p o rta n t, w h ereas in cones, rds
p e rip h e rin m ay b in d to a d iffe re n t m e m b ra n e p ro te in o r act a lo n e. If th e b in d in g
sites a re d iffe re n t in ro d s a n d cones, c o n se rv a tio n o f o n e a m in o acid in rds p e r ip h
e rin m ay b e im p o rta n t to ro d s only, a n d a m u ta tio n at this site c o u ld cause a dys
tro p h y falling w ithin th e c ate g o ry o f re tin itis p ig m en to sa , in w hich ro d s w ere th e
ta rg e t cell o f disease w ith relative p re se rv a tio n o f cones. Conversely, a d iffe re n t
m u ta tio n in th e RDS g e n e m ay d is ru p t th e m etab o lism o r stru c tu re o f e ith e r con es
a lo n e cau sin g m a c u la r d y strophy o r o f b o th ro d s a n d cones. F ro m o u r data, it a p
p ears th a t th e p re se n c e o f a rg in in e a t p o sitio n 172 is im p o rta n t to th e stru c tu re
a n d fu n c tio n o f co n es b u t n o t ro d s (Wells e t al., 1993).

C h o ro id erem ia

C h o ro id e re m ia , a diffuse p h o to re c e p to r dystrophy th a t may be d ifficu lt to distin

guish fro m RP, p a rtic u la rly in y o u n g m ales, was re c o rd e d as a d istin ct c o n d itio n
by M a u th n e r (1871) a n d th e in h e rita n c e was re co g n ised as X -linked in 1942
(G o e d b lo e d , 1942; W a a rd en b u rg , 1942). A p a rt fro m its in h e rita n c e , th e d istin c
62 ALAN C. BIRD AND BARRIE JAY

tive fe a tu re o f this d is o rd e r is diffuse progressive atro p h y o f th e re tin a l p ig m e n t

e p ith e liu m a n d ch o rio cap illaris.
T h e sym ptom s in affe c ted m e n are id e n tic al to retin itis p ig m en to sa, w ith loss o f
d a rk a d a p ta tio n a n d p e rip h e ra l fields early, w hich p ro g resses to leave a sm all ce n
tral field. V isual acuity is m a in ta in e d at a g o o d level u n til late in th e d iso rd er, o fte n
well in to th e 50s. E lectro p h y siological re sp o n ses are severely d e p re sse d early in
th e disease, as they are in RP.
In m ales, th e fu n d u s shows a g e n e ra lise d loss o f th e re tin a l p ig m e n t e p ith e liu m ,
so m etim es re su ltin g in a b lo n d fu n d u s as early as two years o f age, follow ed by th e
a p p e a ra n c e o f fin e g ra n u la r su b retin al p ig m e n t dep o sits in th e m id e q u a to ria l re
gio n . By th e th ird d e c a d e o f life, th e re a re m u ltifo cal areas o f loss o f th e re tin a l
p ig m e n t e p ith e liu m with c o n c u rr e n t u n d e rly in g ch o rio cap illaris a tro p h y in a typ
ically scallo p ed p a tte rn , b e c o m in g m o re w id esp read a n d slowly p ro g re ssin g to a
g e n e ra lise d c o n flu e n t loss o f re tin a l p ig m e n t e p ith e liu m a n d chorio cap illaris. T h e
disease p ro g resses u n til a sm all a re a o f ch o rio ca p illaris a n d p ig m e n t e p ith e liu m
survives at th e fovea (M cC ulloch & M cC ulloch, 1948) , a n d even this is eventually
lost. F lu o rescein a n g io g ra p h ic studies d e m o n stra te well th e loss o f p ig m e n t in the
p ig m e n t e p ith e liu m a n d th e associated loss o f ch o rio cap illaris.
H isto p a th o lo g ic a l e x a m in a tio n c o n firm e d th e extensive c h o rio re tin a l atro p h y
(R afuse & M cC ulloch, 1968; M cC ulloch, 1969; C a m ero n e t al., 1987), as well as
e p ire tin a l m e m b ra n e fo rm a tio n a n d vascular e n d o th e lia l cell ab n o rm a litie s
(C a m e ro n e t al., 1987).
T h e h etero zy g o u s fem ales alm o st always have a b n o rm a l fu n d i with ch an g es in
th e p ig m e n t e p ith e liu m , b u t fu n c tio n a l loss is u n u su a l in th e y o u n g a n d is m ild
la te r in life. T h e m o st c o m m o n a p p e a ra n c e is o f m id p e rip h e ra l p ig m e n ta tio n lo
calised d e e p in th e re tin a a n d asso ciated w ith spotty areas o f p ig m e n t ep ith e lia l
atrophy. T h e re m ay be p ig m e n t clu m p in g th a t m ay be lin e a r in d istrib u tio n . O c
casionally, focal areas o f c h o ro id a l atro p h y occur, a ro u n d th e o p tic disc o r in the
m id p e rip h e ry . T h e se fu n d u s ch an g es are usually stationary, b u t p ro g re ssio n has
b e e n n o te d (Krill, 1977). M icroscopic e x a m in a tio n d e m o n stra te d a b n o rm a litie s
o f th e p ig m e n t e p ith e liu m only (Krill, 1977), w hile o th e r re p o rts a re o f w ide
s p re a d m a lfo rm a tio n o f th e o u te r re c e p to r seg m en ts a n d p atch es o f re tin a l a tro
phy (G h o sh e t al., 1988), o r o f s h o rt o r a b se n t p h o to re c e p to r o u te r seg m en ts in
m u c h o f th e e q u a to ria l reg io n , irre g u la r th ickness a n d p ig m e n ta tio n o f th e re tin a l
p ig m e n t e p ith e liu m , a n d areas o f p ro fo u n d atro p h y in th e e q u a to ria l re g io n
(F la n n e ry e t al., 1990).
C h o ro id e re m ia is a d istin c t en tity w ithin th e g ro u p o f o u te r retin al d y strophies,
in w hich it is th e re tin a l p ig m e n t e p ith e liu m th a t is p rim arily affec ted by th e dis
o rd e r, r a th e r th a n th e c h o ro id as th e n a m e im plies. T h e c h o ro id e re m ia lo cu s has
b e e n m a p p e d to b a n d q21 o n th e X c h ro m o so m e (N ussbaum et al., 1985b) a n d
DNA clo n es th a t span X q21 d e le tio n s in p a tie n ts w ith th e disease have b e e n isolat
ed (C rem ers e t al., 1990). In som e o f th e p a tie n ts w ith m ic ro d e le tio n s, m e n ta l re
ta rd a tio n a n d / o r d eafn ess have b een re p o r te d (H o d g so n e t al., 1987; R o sen b erg
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 63

e t al., 1987; C re m e rs et al., 1989; M erry e t al., 1989), b u t d esp ite th e size o f th e
d e le tio n , th e c h o ro id e re m ia rem ain s rem a rk ab ly c o n sta n t in clinical exp ressio n .
It is likely th a t th e p ro m in e n t c h o ro id a l a tro p h y is a se c o n d a ry resp o n se to p ig
m e n t e p ith e lia l cell loss. C h o ro id a l a tro p h y is n o t u n iq u e to c h o ro id e re m ia , since
it h as b e e n id e n tifie d in a b o u t h a lf th e eyes w ith retin itis p ig m e n to sa su b ject to his-
to p a th o lo g ic a l study, b u t p ro fo u n d a tro p h y early is u n lik e re tin itis p ig m e n to sa a n d
signifies a p a th o g e n ic p ro cess d iffe re n t fro m th o se in o th e r o u te r re tin a l diseases.

R e fs u m d ise a se

T his ra re au to so m a l recessive d iso rd e r, c h a ra c terise d by re tin itis p ig m en to sa,

c h ro n ic p o ly n eu ritis a n d c e re b e lla r signs, was first d esc rib ed in 1945 (R efsum ,
1945) a n d in 1963 it was show n th a t a ffec ted individuals h a d elev ated levels o f
se ru m p h y tan ic acid (K lenk & K ahlke, 1963). T his fatty acid is ex o g en o u s, b e in g
d eriv ed fro m th e b reak d o w n o f c h lo ro p h y ll. C u ltu re d fibroblasts fro m skin have
defective alp h a-h y d ro x y latio n o f p h y ta n a te a n d in hetero zy g o tes th e level o f activ
ity is a b o u t h a lf th e n o rm a l level a lth o u g h they have n o sym ptom s (S tein b erg
et al., 1967; H e r n d o n e t al., 1969; H u tto n & S te in b erg , 1973).
Loss o f n ig h t vision is usually th e first sym ptom o c c u rrin g b efo re th e age o f 30
years. T h e p ro g re ssio n a n d fu n d u s c h a n g e s are sim ilar to th o se o f m o re classical
fo rm s o f retin itis p ig m e n to sa , a lth o u g h loss o f ce n tra l vision a n d m a c u la r chan g es
o c c u r e a rlie r th a n is usual. M iosis (sm all po o rly re a c tin g pupils) is co m m o n an d
c o n trib u te s to th e re d u c e d vision (G ib b e rd e ta l., 1985).
H y p e rtro p h ic in te rstitia l p e rip h e ra l n e u ro p a th y is a c o n sta n t fe a tu re o f th e dis
o rd e r a lth o u g h it is usually n o t clinically a p p a re n t u n til a fte r th e o n se t o f o cu lar
disease, w hile an o sm ia is usually a p p a re n t fro m an early age. Progressive n eu ro -
sen so ry h e a rin g loss, a b n o rm a l v estib u lar resp onses, ichthyosis a n d card iac c o n
d u c tio n d efects are co m m o n ly fo u n d .
H isto p a th o lo g ic a l c h a n g e s o f th e eye show a cc u m u la tio n o f an a b n o rm a l d e p o
sition, p re su m e d to be p h y tan ic acid in th e p ig m e n t e p ith e liu m o f iris a n d re tin a
(Levy, 1970; T o u ssain t & D anis, 1971; D ick e t al., 1990).
T re a tm e n t consists o f re d u c tio n o f in tak e o f all fo o d s c o n ta in in g ch lo ro p h y ll,
w hich in c lu d e s all fat p ro d u c ts o f herb iv o res. T h e p rin cip al fo o d s allow ed are fish,
lean m eat, p o u ltry , m a rg a rin e a n d g ra in p ro d u c ts. P lasm ap h eresis p e rfo rm e d
o n c e o r twice a m o n th effectively rem oves p h y tan ic acid fro m th e body a n d re d u c
es th e d ie ta ry re stric tio n (G ib b e rd e t al., 1979; M oser e t al., 1980). R e d u ctio n in
p lasm a p h y tan ic acid p ro d u c e s a co n sid e ra b le clinical im p ro v e m e n t in th e n e u r
o p a th y b u t n o t in th e re tin itis p ig m e n to sa (G ib b erd e t al., 1985).

A b e ta lip o p ro te in a e m ia

T his ra re au to so m a l recessive d iso rd er, w hich was first d e sc rib e d in 1950 (Bassen
& K ornzw eig, 1950), p re se n ts initially w ith m a la b so rp tio n sy ndrom e a n d fat in to l
e ra n c e , a n d has a p ig m e n ta ry re tin o p ath y , progressive ataxic n e u ro p a th y an d
64 ALAN C. BIRD AND BARRIE JAY

acan th o cy to sis as o th e r c o m m o n featu res. T h e re tin o p a th y is in d istin g u ish a b le

fro m th a t in a se p a ra te g e n e tic d iso rd er, h y p o b e ta lip o p ro te in a em ia . It has
b e c o m e cle a r th a t th e m a jo r ab n o rm a lity is defective fat a b so rp tio n causing defi
ciency o f vitam ins A a n d E, u n d e te c ta b le b eta -lip o p ro te in s (Salt et al.. 1960) a n d
low c h o le ste ro l in seru m , re d u c e d p lasm a p h o sp h o lip id s, total lipids, triglycer
ides a n d a lp h a -l-lip o p ro te in s. T h e in testin al m ucosa shows c h ara c teristic
ch an g es, in c lu d in g p a c k in g o f th e villa w ith lip id d ro p le ts. Som e ery th ro cy tes
have a c u rio u s c re n a te d a p p e a ra n c e , te rm e d acanthocytosis (Isselb ach er e t al.,
1964).
F u n d u s a b n o rm a litie s m ay b e co m e a p p a re n t in th e first year o f life with w hite
d o ts at th e level o f th e p ig m e n t e p ith e liu m th a t is re m in isc e n t o f ch an g e s seen in
d ie ta ry vitam in A deficiency. W ith tim e, th e ch a ra cteristic fu n d u s a p p e a ra n c e s a n d
h isto p a th o lo g ic a l ch an g es o f retin itis p ig m e n to sa a p p e a r (Ja m p e l & Falls, 1958;
Von S allm an e t al., 1969; C o gan et al., 1984).
P rogressive n e u ro lo g ic a l disease develops w hich is usually m an ifest initially as
a b n o rm a l gait. A b n o rm a litie s in c lu d e c e re b e lla r ataxia, m uscle w asting a n d senso
ry loss.
T re a tm e n t by restrictio n o f d ie ta ry fats a n d su p p le m e n ta tio n w ith large doses o f
vitam ins A a n d E a p p e a rs to b e effective in p re v e n tin g th e advance o f th e disease
(W olff e t al., 1964; M u ller e t al., 1977; B ishara e t al., 1982) a n d reversal o f th e fu n
d u s c h a n g e s has b e e n re p o r te d (G ouras et al., 1971). P rophylactic vitam ins in the
first 12 to 18 years o f life a p p e a r to p re v e n t o r m odify th e o c c u rre n c e o f re tin o p a
thy (R u n g e e ta l ., 1986).

CENTRAL R EC EPTO R DYSTROPHIES

A n u m b e r o f g en etically d e te rm in e d d iso rd e rs cause progressive loss o f visual

fu n c tio n s asso ciated w ith cones: losses o f visual acuity, c o lo u r vision a n d c e n tra l
visual field, a n d diffuse, p o o r vision in b rig h t light. T h ese p a tie n ts also re m a rk
u p o n difficulty w ith n ig h t vision, b u t o n clo ser q u e stio n in g this o fte n rela te s to a
s h o rt tra n s ie n t p e rio d o n first e n te rin g a d a rk e n v iro n m e n t.
Specific d iso rd e rs, such as Best's disease, c an be id e n tifie d b u t th e re m a in d e r in
c lu d e a g ro u p o f e n titie s th a t c a n n o t b e clearly d istin g u ish e d o n e fro m a n o th e r.
T h e se in c lu d e th e re c e n tly d e sc rib e d m u ta tio n s in th e RDS g e n e causing m ac u lar
d y stro p h y (Wells e t al., 1993).

V ite llifo rm m a c u la r d y stro p h y (B ests d is e a s e )

T h e typical lesion o f B ests disease is a r o u n d yellow d e p o sit at th e m a cu la th a t

m ay b e id e n tifie d w ithin a s h o rt tim e o f b irth (B arkm an, 1961) o r w hich m ay
d ev elo p la te r in a previously n o rm a l fu n d u s (D eu tm a n , 1971; B arricks, 1977). O n
ra re occasions, th e m a c u la r lesion is n o t seen u n til la te r life (D eu tm a n , 1971);
th e lesio n m ay b e ex tra m a c u lar, th e disease m ay b e asym m etrical (M aloney et al.,
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 65

1977) o r th e fu n d u s m a y b e en tirely n o rm a l (D e u tm an , 1971; Krill, 1977; M o h ler

& Fine, 1981; G o d el, e t al., 1986). G odel a n d colleagues (1986) state th a t a b o u t
h a lf o f th o se w ith th e a b n o rm a l g e n o ty p e have a n o rm a l o r n e a r n o rm a l fu n d u s
a p p e a ra n c e a n d n o rm a l vision. T h e a b n o rm a l m a terial a p p e a rs to a c c u m u la te at
th e level o f th e p ig m e n t e p ith e liu m , h isto p a th o lo g ic a l evidence in d ic a tin g th a t it
is p ro b ab ly b etw een B ru c h 's m e m b ra n e a n d th e p ig m e n t e p ith e liu m (O 'G o rm a n
et al., 1988). A lth o u g h flicker fu sio n studies in d ic ate co n e d y sfunction m u c h ear
lie r (M assof e t al., 1977), c e n tra l c o n e fu n c tio n re m a in s relatively g o o d in m ost
p a tie n ts u n til th e se c o n d d e c a d e o f life, a t w hich tim e th e re is progressive loss o f
c e n tra l vision, asso ciated w ith d isin te g ra tio n o f th e c e n tra l yellow area. T h e evolu
tio n o f th e lesio n d u rin g this p e rio d is d e te rm in e d by w h e th e r o r n o t new b lo o d
vessels invade th e a b n o rm a l d e p o sit fro m th e c h o ro id . If new vessels invade th e
lesion, th e re m ay b e su b retin a l h a e m o rrh a g e s a n d fibrosis (disciform re sp o n se ),
a n d th e visual o u tc o m e is p o o r (M iller et al., 1976). In o th e r p a tien ts, th e yellow
m aterial m ay b eco m e p artially liq u efied , fo rm in g flu id levels w ithin th e su b re ti
nal space a n d even tu ally th e m a te ria l reso rb s, leaving c o n flu e n t a tro p h y o f th e
c e n tra l p ig m e n t e p ith e liu m a n d ch o rio c ap illaris. T h e a p p e a ra n c e s o f p atie n ts
w ith n o rm a l m acu lae, m in im al p ig m e n t e p ith e lia l changes, a tro p h ic m a cu lae o r
fib ro u s m a c u la r scars te n d e d n o t to ch an g e; th o se w ith v itelliform o r p seu d o h y
po p y o n lesions p ro g re sse d to la te r a tro p h ic m a c u la r stages (M o h le r & Fine,
1981).
T h e au to so m al d o m in a n t m o d e o f in h e rita n c e o f this d iso rd e r was first suggest
e d by Best (1905) a n d was su b sta n tia te d la te r by fu r th e r o b se rv atio n s o n th e sam e
fam ily (Vossius, 1921; W eisel, 1922; Ju n g , 1936).
T h e rise in o c u la r p o te n tia l in d u c e d by light,as seen in th e electro -o cu lo g ram ,
is always re d u c e d in p a tie n ts w ith th e a b n o rm a l g e n o ty p e fo r B est's disease, w hat
ever th e ir clinical status, b u t ERG p o te n tia ls are n o rm a l (D eu tm an , 1969, 1971).
T h e universal re d u c tio n o f th e lig h t-in d u c ed rise in o c u la r p o te n tia l even in those
p a tie n ts w ith n o rm a l fu n d i, in d ic a te s a w id e sp rea d d y sfunction o f th e re c e p to r p ig
m e n t e p ith e lia l c o m p le x th a t has possibly b e e n p re se n t fro m b irth .
In p a tie n ts w ith ad v a n c ed disease, h isto p ath o lo g y show ed extensive atro p h y o f
p h o to re c e p to rs in th e m a c u la r are a (M cF arland, 1955; A n d e rso n , 1977). In slight
ly less ad v a n c e d cases, th e re was also w id esp read a b n o rm ality o f th e re tin a l pig
m e n t e p ith e lia l cells, in w hich an excessive a m o u n t o f lipofuscin h a d ac c u m u la te d
(F ra n g ie h e t al., 1982; W eingeist e t al., 1982; O 'G o rm a n e ta l., 1988). In o n e case,
an a c c u m u la tio n o f h e te ro g e n e o u s m a terial betw een B ru c h 's m e m b ra n e a n d th e
p ig m e n t e p ith e liu m a t th e fovea was b eliev ed to re p re s e n t th e lo c atio n o f a previ-
tellifo rm lesion. T h is m a te ria l a p p e a re d to b e d eriv ed fro m d e g e n e ra tin g p ig m e n t
e p ith e lia l cells (O 'G o rm a n e t al., 1988).

A d u lt v ite llifo rm m a c u la r d y stro p h y

A d u lt v itelliform m a c u la r dystrophy was first d e sc rib ed by Gass (1974) as a p e c u

liar fo v eo m acu lar dystrophy. It is c h a ra c terise d by a focal, r o u n d o r oval sh a p ed ,
66 AI AN C. BiRD AND BARRIF. JAY

su b re tin a l yellow ish foveal lesion, o ften w ith o n e o r m o re p ig m e n t spots on the

a n te rio r su rface a t th e level o f th e p ig m e n t e p ith e liu m . T h e lesions may vary in
size b u t are typically o n e th ird to o n e h a lf o f a disc d iam eter, a n d are usually b ilat
eral a n d sym m etrical. P a tie n ts usually p re s e n t in th e fo u rth o r fifth d e c a d e o f life
a n d te n d to have m in im al visual sym ptom s (B re c h e r & B ird, 1990).
T h e d is o rd e r differs fro m vitelliform m a c u la r dystrophy (Best's disease) in th a t
th e foveal lesions a re sm aller, it p rese n ts at a la te r age, it do es n o t d e m o n stra te evo
lu tio n a ry c h a n g e s o f th e foveal lesion, a n d the lig h t in d u c e d rise in o c u la r p o te n
tial is rarely ab sen t.
A lth o u g h series have b e e n re p o rte d w h ere th e re was n o evid en ce o f fam ilial in
v o lv em en t (K ingham & L o c h e n , 1977), this c o n d itio n is now gen erally re g a rd e d
as b e in g tra n s m itte d as an au to so m a l d o m in a n t trait (Gass, 1974; V ine & Schatz,
1980; B re c h e r & Bird, 1990). It is in te re stin g th a t a m u ta tio n in th e p e rip h e rin
RDS g e n e , in a sto p c o d o n a t p o sitio n 258, has b e e n fo u n d in o n e p a tie n t w ith a
re tin a l d e g e n e ra tio n sim ilar in a p p e a ra n c e to a d u lt vitelliform m ac u lar dystrophy
(Wells e ta l ., 1993).

B u tte rfly -s h a p e d d y stro p h y

In 1970, five m e m b e rs o f a fam ily w ere d e sc rib e d fro m H o lla n d with a u n iq u e

fu n d u s a p p e a ra n c e co n sistin g o f p a tte rn e d h y p e rp ig m e n ta tio n a t th e m acula
(D e u tm a n e t al., 1970). T h e p ig m e n t a p p e a re d to be in th e p ig m e n t e p ith e liu m ,
a n d flu o re sc e in a n g io g ra p h y show ed g ra n u la r h y p e rp ig m e n ta tio n o f th e re m a in
in g p ig m e n t e p ith e liu m a t th e p o ste rio r p o le. T h e d iso rd e r caused few sym ptom s
since th e w orst visual acuity was 0.8. T h e m o st striking fe a tu re was th e universal
re d u c tio n o f th e lig h t-in d u c e d rise in o c u la r p o te n tia l to 130% o r low er in th e
p re se n c e o f g o o d visual fu n c tio n .
D e u tm a n a n d co lleag u es (1970) c o n c lu d e d th a t th e re was diffuse d y sfunction
o f th e re tin a l p ig m e n t e p ith e liu m w ith little associated visual deficit since th e re
was n o in d ic a tio n o f ro d d y sfu n ction as g a u g e d by th e e le c tro re tin o g ra m a n d no
a b n o rm a lity o f c o lo u r vision. In this re sp e c t b u tterfly -sh ap ed dystrophy is sim ilar
to B est's disease. T h e o rig in al p e d ig re e is suggestive o f au to so m a l d o m in a n t in h e r
itan ce, a lth o u g h th e disease was id e n tifie d in only two g e n e ra tio n s.

B ie ttis c ry sta llin e d y stro p h y

A u n iq u e fu n d u s a p p e a ra n c e was r e p o rte d by B ietti in th re e p atie n ts, two o f

w hom w ere b ro th e rs, w ho h a d crystals in th e re tin a a n d in th e p e rip h e ra l c o rn e a
(B ietti, 1937). T h e re is progressive a tro p h y o f th e re tin a with p ro m in e n t a tro p h y
o f th e p ig m e n t e p ith e liu m a n d c h o ro id . As th e a tro p h y su p erv en e s, th e in trare ti-
n al crystals d isap p ear. T h e p a tte rn o f re tin a l fu n c tio n a l loss is sim ilar to th a t o f
retin itis p ig m e n to sa w ith th e initial sym ptom s usually a p p e a rin g in th e th ird o r
fo u rth d e c a d e o f life. A u to so m al recessive in h e rita n c e was e sta b lish ed in this c o n
d itio n by H u (1983). P a tie n ts w ith a sim ilar fu n d u s a p p e a ra n c e b u t w ith o u t cor
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 67

n eal c h a n g e s have b e e n d e sc rib e d a n d c o n sid e re d to re p re s e n t th e sam e d iso rd e r

(F ranois a n d D e Laey 1970; W elsh 1977; H ayasaka & O koyum a 1984). N o sys
tem ic b io c h e m ic a l d is o rd e r has b e e n id e n tifie d , a lth o u g h th e c o rn e a l crystals
re se m b le c h o le ste ro l o r c h o le ste ro l ester, suggesting th a t B iettis crystalline dys
tro p h y m ay be d u e to a system ic a b n o rm a lity o f lip id m etab o lism (W ilson e t al.,
1989).

O th e r c e n tra l d y stro p h ie s

T h e re m a in in g c o n e d y stro p h ies have b e en su bdivided with re sp e c t to in h e rit

an ce a n d th e a p p e a ra n c e o f th e fu n d u s, b u t no satisfactory c a te g o risatio n o f
th ese c o n d itio n s has yet b e e n devised, a n d it is likely th a t each subdivision c o n
tains m o re th a n o n e c o n d itio n . Two b ro a d subdivisions, fu n d u s flavim aculatus
a n d b u ll's eye m acu lo p ath y , can be id e n tifie d o n th e basis o f fu n d u s m orphology,
a lth o u g h , th e d istin c tio n may n o t be ab so lu te.

F u n d u s flav im ac u latu s

T h is b ro a d c a te g o ry co m p rises diseases in w hich th e re is d e p o sitio n o f w hite

m a te ria l a t th e level o f th e p ig m e n t e p ith e liu m ; S ta rg a rd ts disease (S targ ard t,
1909, 1913) a n d fu n d u s flavim aculatus (F ran cesch etti, 1963; F ran cesch etti &
F rancois, 1965) have b e e n used to d e n o te these d iso rd ers, b u t th e re is n o evi
d e n c e th a t these two te rm s d escrib e se p a ra te c o n d itio n s. M ost cases show a u to so
m al recessive in h e rita n c e a lth o u g h a u to so m al d o m in a n t fo rm s are occasionally
seen (Cibis e t al., 1980). T h e se c o n d itio n s usually cause ra p id loss o f c e n tra l
vision d u rin g a 6 m o n th p e rio d in th e first 15 years o f life, a lth o u g h in som e cases
g o o d visual acuity is m a in ta in e d u n til th e age o f 40 years.
At th e tim e o f visual loss, c o n flu e n t atro p h y o f th e o u te r re tin a , p ig m e n t e p ith e
lium a n d c h o rio c a p illa ris o ccurs a t th e fovea, a n d this a rea grow s slowly d u rin g the
re st o f th e p a tie n t's life. T h e w hite fish ta il flecks occupy th e re m a in in g p a rt o f
th e p o s te rio r p o le w ith c h a ra c teristic sp a rin g o f th e p e rip a p illa ry re g io n . T hese le
sions can b e id e n tifie d at th e tim e o f th e initial visual loss a n d resolve as a d d itio n al
o n es a p p e a r elsew here (H a d d e n & Gass, 1976).
O n flu o re sc e in an g io g raphy, th e c h o ro id is n o t seen in th e m ajority o f p atien ts
a lth o u g h it a p p e a rs n o rm a l in som e (B o n in , 1971; Fish e ta l., 1981; F ishm an e ta l.,
1987). It seem s likely th a t th e n o n -a p p e a ra n c e o f th e c h o ro id signifies an even
d e p o sitio n o f a b n o rm a l m a terial at th e level o f th e p ig m e n t e p ith e liu m th a t ab
sorbs b lu e -g re e n light. T h is has b e e n c o n firm e d by h isto p ath o lo g ic al studies in
w hich it has b e e n d e m o n s tra te d th a t th e re tin a l p ig m e n t e p ith e lia l cells w ere
p a c k e d w ith lipofu scin a n d m ela n o lip o fu sc in (Eagle e t al., 1980; L opez e t al.,
1990); th ese c h an g es sta rt to d ev elo p in c h ild h o o d (S tein m etz e t al., 1991). A t the
site o f p revious w hite lesions, a tro p h y o f th e p ig m e n t e p ith e liu m o ccurs a n d flu o
rescein a n g io g ra p h y shows m u ltifo cal h y p e rflu o re sc en c e , c o rre sp o n d in g to th e ar
eas o f p ig m e n t e p ith e lia l atrophy.
68 MAX C. BIRD AND BARRIE JAY

O n e le c tro re tin o g ra p h y , all p a tie n ts have som e significant a b n o rm a lity o f co n e

fu n c tio n w hile m any have a b n o rm a l ro d fu n c tio n (M oloney e t al., 1983). T h e
E O G is initially n o rm a l b u t fre q u e n tly b ec o m e s su b n o rm a l late in th e disease (N o
b le & C arr, 1979; M oloney et al., 1983).

B u lls eye d y s tro p h ie s (in c lu d in g c o n e a n d c o n e - r o d d y s tro p h ie s )

T h e typical fu n d u s c h a n g e s in this g ro u p o f co n e a n d c o n e -ro d d y stro p h ies c o n

sist o f o n e o r m o re c o n c e n tric rings o f p ig m e n t e p ith e lial ch a n g e a ro u n d th e
fovea, w hich gives rise to a c h a rac teristic a p p e a ra n c e o n flu o rescein angiography.
Visual loss m ay o c c u r at any tim e d u rin g th e se co n d to th e fifth d e ca d e s o f life
a n d o n c e sta rte d p ro g resses slowly (G rey e t al., 1977). T h e re m ay o r m ay n o t be
w hite d ep o sits at th e level o f th e p ig m e n t e p ith e liu m . T h e p h e n o m e n o n o f d ark
c h o ro id is also seen in som e fam ilies w ith b u lls eye dystrophy (Fish e t al., 1981).
T h e h e te ro g e n e ity o f this g ro u p is in d ic a te d by th e in h e rita n c e in d iffe re n t fam
ilies, w hich may b e au to so m al d o m in a n t, au to so m a l recessive o r X -linked. W ith in
this g ro u p a re th e progressive c o n e d y stro p h ies th a t usually p re se n t in ad o le sc en c e
o r early a d u lt life w ith re d u c e d vision, p h o to p h o b ia , a n d defects o f c o lo u r vision
a n d w hich usually p re c e d e th e m ac u la r ch an g es. T h ese m ac u lar c h an g e s re su lt
fro m a tro p h y o f th e re tin a l p ig m e n t e p ith e liu m , giving rise to a b u lls ey e a p
p e a ra n c e .
T h e c o n e d y stro p h ies a re a t p re s e n t classified by th e ir m o d e o f in h e rita n c e ,
w hich m ay be au to so m a l d o m in a n t (G o o d m a n et al., 1963; B erson e t al., 1968;
Krill 8c D e u tm a n , 1972; P e a rlm a n et al., 1974; van S ch o o n ev eld et al., 1991; W ent
et al., 1992), au to so m a l recessive (G ouras e t al., 1983; Yagasaki & Ja c o b so n , 1989)
o r X - lin k e d (H eckenlively & W eleber, 1986; Ja c o b so n e t al., 1989; R eichel et al.,
1989; van E v e rd in g e n e t al., 1992). Even w ithin th ese d iffe re n t g e n e tic fo rm s o f
c o n e d ystrophy th e re is h etero g en eity . In th e g ro u p o f d o m in a n t c o n e dystro p h ies
th e re is o n e re c e n tly d e sc rib e d fo rm w ith early a n d n e a r c o m p lete ab sen c e o f blu e
c o n e fu n c tio n , b u t w ith a blue-sensitive visual p ig m e n t g e n e in d istin g u ish ab le
fro m n o rm a l (W ent e t al., 1992). In a fam ily w ith X -linked c o n e dystrophy a cco m
p a n ie d by loss o f re d c o n e fu n c tio n , a 6.5 kilobase d e le tio n w ithin th e re d co n e
p ig m e n t g e n e was fo u n d (R eichel et al., 1989).
H e te ro g e n e ity is fu r th e r in d ic a te d by th e p re se n c e o f p e rip h e ra l re tin a l d e g e n
e ra tio n in som e cases, a n d th ese c o n e -ro d d y stro p h ies, w ith ro d involvem ent, m ust
b e d istin g u ish e d fro m c o n e d y strophies, w h e re ro d fu n c tio n is n o rm a l, by m e a n s
o f electro p h y sio lo g ical tests o f ro d fu n c tio n . It m u st also b e re m e m b e re d th a t
m any p a tie n ts w ith each o f th e g e n e tic fo rm s o f retin itis p ig m e n to sa (ro d -co n e
d y stro p h ies) also have a b u lls-eye a p p e a ra n c e at th e ir m acu lae (P ru e tt, 1983).
D ru g in d u c e d p h e n o c o p ie s o c c u r (K earns & H o lle n h o rst, 1966; Krill e t al., 1971)
a n d c o m p a ra b le d y stro p h ies have also b e e n d esc rib ed in th e P ierre-M arie type o f
h e re d ita ry a tax ia (Bjork e t al., 1956), in fucosidosis (Snodgrass, 1976) a n d in
a m e la n o g e n esis im p e rfe c ta (Jalili & S m ith, 1988).
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 69

T h e re is c o n sid e ra b le d o u b t as to w h e th e r th e fu n d u s ch an g es o f fu n d u s flavi-
rn acu laiu s a n d b u lls eye d ystrophy in d ic a te th a t th e d iso rd e rs a re clearly se p a ra t
e d in to two b ro a d g ro u p s. S tudies o f fu n d u s a p p e a ra n c e show th a t in som e fam ilies
th e ch an g es are c o n sta n t, w hilst in o th e rs b u lls eye dystrophy m ay b e seen in early
disease a n d flavim aculatus lesions seen later.

S o rsb y 's f u n d u s d y stro p h y (p s e u d o in fla m m a to ry m a c u la r d y stro p h y )

M acular d y stro p h ies have b e e n d e sc rib e d in w hich th e re are fe a tu re s o th e r th a n

a tro p h y o f th e o u te r re tin a l layers. Sorsby a n d his colleag u es (1949) first re p o rte d
an au to so m a l d o m in a n t d iso rd e r in w hich th e re is b ila teral c e n tra l visual loss in
th e fifth d e c a d e o f life fro m su b re tin a l n eo v ascu larisatio n , a n d progressive a tro
p hy o f th e p e rip h e ra l re tin a a n d c h o ro id le a d in g to loss o f a m b u la to ry vision by
th e sev en th d e c a d e in m o st cases.
T h re e o f th e o rig in al fam ilies re p o rte d by Sorsby (Sorsby e t al., 1949) have b een
extensively stu d ie d m o re rec en tly a n d have c o n firm e d th e th re e key fe a tu re s o f the
dystrophy d e sc rib e d by Sorsby: a u to so m al d o m in a n t in h e rita n c e , loss o f c e n tra l vi
sion o c c u rrin g in th e fifth d e c a d e o f life, a n d loss o f a m b u la to ry vision o c c u rrin g
d u rin g th e e ig h th d e c a d e . In o n e o f th ese fam ilies (E fam ily), p rio r to any re d u c
tio n o f ce n tra l vision, th e a u th o rs f o u n d fine d ru s e n in som e p a tie n ts, a n d in o th
ers a c o n flu e n t d e p o sit o f yellow su b re tin a l m a terial at th e m acu la (H oskin e t al.,
1981). In a n o th e r fam ily (K fam ily), p e rip h e ra l re tin a l d y sfunction o c c u rre d , with
a d e p o s it o f a yellow su b re tin a l m a teria l th ro u g h o u t th e fu n d u s a n d a trita n c o lo u r
d efect, all p rio r to th e loss o f c en tra l vision. In som e o f th ese p a tie n ts th e re was
loss o f c e n tra l vision as a re su lt o f a tro p h ic disease, r a th e r th a n fro m in grow th o f
su b re tin a l new vessels (C ap o n e t al., 1988). In a th ird fam ily (C fam ily), as in th e
o th e r two, yellow m a te ria l was p re s e n t at th e level o f B ru c h s m e m b ra n e early in
th e co u rse o f th e disease, a lth o u g h th e ea rlie st p h en o ty p ic m a rk e r was delayed fill
ing o f th e c h o rio cap illaris. T h e loss o f c e n tra l vision was co m m o n ly d u e to atro p h y
o f th e o u te r re tin a a n d c h o ro id ; su b re tin a l neov ascu larisatio n was a ra re o c cu r
re n c e (P o lk in g h o rn e e t al., 1989). In a n o th e r fam ily (n o t o n e o f Sorsbys), th re e
v ariatio n s in th e fu n d u s a p p e a ra n c e w ere seen: firstly a n d m o st com m only, w hite
to yellow fu n d u s spots a c c o m p a n ie d a d isciform m ac u lar d e g e n e ra tio n ; in th e sec
o n d , th e fu n d u s spots w ere a b sen t; in th e th ird , th e yellow dep o sits w ere associated
w ith a tro p h ic m a c u la r d e g e n e ra tio n (H a m ilto n e t al., 1989).
E lectro p h y sio lo g ical resp o n ses w ere originally th o u g h t to be usually w ithin n o r
m al lim its, sugg estin g th a t th e d is o rd e r was localised to th e m ac u la r re g io n (C arr
et al . 1975), a lth o u g h a re d u c e d lig h t rise o n e le ctro -o cu lo g rap h y has m o re re
cently b e e n re p o r te d (C ap o n et al., 1988; D reyer & H idayat, 1988).
A lig h t a n d e le c tro n m icro sco p ic study o f th e eyes o f o n e p a tie n t show ed a 30
m ic ro n th ick d e p o sit w ithin B ru c h s m e m b ra n e th a t stain e d positive fo r lipids. In
a d d itio n , th e re was gross loss o f th e o u te r re tin a , a d isc o n tin u o u s re tin a l p ig m e n t
e p ith e liu m a n d a tro p h y o f th e c h o rio ca p illa ris (C ap o n e t al., 1989).
70 ALAN C. BIRD AND BARRIE JAY

It has to b e a p p re c ia te d th a t a tro p h y o f th e c h o rio ca p illa ris o r o f th e w hole

th ick n ess o f th e c h o ro id a p p e a rs to be a c o m m o n p h e n o m e n o n at th e te rm in a l
p h a se in all o u te r re tin a l dystro phies. It has b e e n d esc rib e d histologically in h a lf
th e cases o f retin itis p ig m e n to sa stu d ie d a n d is a co m m o n late fe a tu re o f m ost, if
n o t all, c e n tra l dystro p h ies, such as S ta rg a rd ts disease a n d b u ll's eye dystrophy.
T h e se o b serv atio n s suggest th a t th e c h o ro id a l a tro p h y is a resp o n se to ab se n ce o f
th e re tin a l p ig m e n t e p ith e liu m a n d re tin a l p h o to re c e p to rs. Initially, th e c h o rio
capillaris was th o u g h t to b e p rim arily a ffe cted in c e n tra l a re o la r c h o ro id a l a tro p h y
(Sorsby, 1939; Sorsby & C rick, 1953; N o b le, 1977) b ecau se atro p h y o f th e re tin a l
p ig m e n t e p ith e liu m a n d c h o ro id at th e m a cu la was a p p a re n tly th e initial o r a t least
th e m o st p ro m in e n t o p h th a lm o sc o p ic c h a n g e w hen th e p a tie n t was first seen.
H ow ever, A sh to n (1953) d e m o n s tra te d th a t th e m a jo r c h o ro id a l b lo o d vessels
w ere n o rm a l in a case o f c e n tra l a re o la r c h o ro id a l sclerosis by h isto p ath o lo g ic al
e x a m in a tio n . It is also e v id e n t th a t N oble (1977) co u ld n o t have b e e n able to co m
m e n t o n th e state o f th e c h o rio c ap illaris h a d th e p ig m e n t ep ith e lia l p ig m e n t c o n
te n t b e e n n o rm a l. Clearly, th e r e is d o u b t c o n c e rn in g th e p rim a ry site o f disease in
so-called c h o ro id a l d y stro p h ies. T h e two possibilities a re p rim a ry a tro p h y o f th e
c h o ro id o r p rim a ry atro p h y o f th e p ig m e n t e p ith e liu m , each cau sing se c o n d a ry
a tro p h y o f th e n e ig h b o u rin g tissues.
It is possible th a t early ch o rio ca p illaris clo su re d istin g u ish es d iso rd e rs with early
p ig m e n t e p ith e lia l cell d e a th fro m those in w hich th e disease affects p rim arily the
re tin a l re c e p to rs.

CO N G EN ITA L REC EPTO R DEFECTS

A series o f c o n d itio n s has b e e n re c o g n ise d in w hich th e re is a genetically d e te r

m in e d n o n p ro g ressiv e visual d e fect th a t m ay be re la te d to e ith e r th e c o n e o r the
ro d systems.

C o n e d e f e c ts

Colour vision defects

D efects in c o lo u r vision have b e e n classified o n th e basis o f th e c o n c e p t th a t

c o lo u r vision is d e te rm in e d by th re e classes o f cones: red-sensitive, green-sensitive
a n d blue-sensitive. T h e basis fo r this classification has b e e n c o n firm e d by the
se q u e n c in g o f th e th re e c o n e p ig m e n t g en es (N ath an s e t al., 1986a,b; V ollrath et
al., 1988). If c o lo u r m a tc h in g tests d e m o n stra te th a t o n e o f th e th re e systems is
d efective b u t p re s e n t (tric h ro m a ts), th e term s p ro tan o m aly (re d ), d e u te ra n o m -
aly (g re e n ) a n d tritan o m aly (blue) are u se d a n d if o n e is fu n c tio n ally a b se n t
(d ic h ro m a ts ), th e suffix -an o p ia re p la ce s th e suffix -anom aly.
D eu teran o m aly , d e u te ra n o p ia , p ro ta n o m a ly a n d p ro ta n o p ia are X -linked c o n
ditions: d e u te ra n o m a ly is fo u n d in a b o u t 5% o f th e p o p u la tio n o f W estern E u ro p e
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 71

a n d N o rth A m erica; d e u te ra n o p ia , p ro ta n o m a ly a n d p ro ta n o p ia are each fo u n d

in a b o u t 1% (W a a rd en b u rg e t al., 1963). T ritan o m aly a n d trita n o p ia , w hich a re in
h e rite d as a u to so m a l d o m in a n t d iso rd ers, a re m u ch less co m m o n affectin g b e
tw een 0.002 a n d 0.007% o f th e p o p u la tio n (V erriest, 1974). In dividuals w ith these
a b n o rm a litie s o f c o lo u r vision have n o rm a l visual acuities, a n d a n o m alo u s tric h ro
m ats a re o fte n u n a w a re o f this c o n d itio n . A p a rt fro m th e c o lo u r d efect, th e eye is
o th erw ise n o rm a l. T h e m o le c u la r g e n e tic basis o f r e d /g r e e n c o lo u r vision defects
is discu ssed in d e ta il in C h a p te r 9.

Mono chromatism

P a tie n ts su fferin g fro m m o n o c h ro m a tism have a b se n t o r m ark ed ly im p a ire d

c o lo u r vision. In c o m p le te ro d m o n o c h ro m a tism th e re is little evid en ce o f c o n e
fu n c tio n , so th a t visual acuity is poor, th e p a tie n t has nystagm us a n d e x tre m e p h o
to p h o b ia is ch a ra c teristic . T h e fu n d i show n o gross abnorm ality, a lth o u g h , th e
foveola m ay a p p e a r a b n o rm a l a n d c h a n g e s in th e c e n tra l p ig m e n t e p ith e liu m are
so m etim es id e n tifie d (W a a rd en b u rg e t al., 1963). T h e d a rk a d a p ta tio n curve is
typically m o n o p h a sic , fu sio n fre q u e n c y is very low a n d th e p h o to p ic (co n e ) ERG
is a b se n t b u t th e sco to p ic (ro d ) ERG is n o rm al. R od m o n o c h ro m a tism is an a u to
som al recessive trait. A n in c o m p le te fo rm o f ro d m o n o c h ro m a tism has b e e n
d e sc rib e d w h e re th e sym ptom s are less severe, visual acuity is b e tte r a n d p h o to
p h o b ia a n d nystagm us may b e a b se n t (Sloan & N ew hall, 1942; Siegel e t al., 1966).
In a n o th e r fo rm o f m o n o c h ro m a tism th e re is ab se n ce o f c e n tra l co nes (Krill,
1977).
B lue c o n e m o n o c h ro m a tism p re se n ts in a sim ilar fashion to c o m p le te ro d
m o n o c h ro m a tism , e x c e p t th a t th e re a p p e a rs to be an in ta c t b lu e co n e system a n d
th e in h e rita n c e is X -linked (A lp ern e t al., 1965). T h e g e n e fo r this fo rm o f m o n o
c h ro m a tism m aps to X q28, a n d this ra re d iso rd e r results fro m alte ra tio n s o f th e
re d a n d g re e n p ig m e n t g en es, o r fro m th e ir d e le tio n (Lewis e t al., 1987; N ath an s
e t al., 1989).
F o u r eyes fro m in d iv id uals w ith m o n o c h ro m a tism have b e e n e x a m in e d histo-
p ath o lo g ically (L arsen , 1921; H a rris o n e t al., 1960; Falls et al., 1965; G lickstein &
H e a th , 1975). T h ese show ed a c h a n g e a t th e fovea w ith a re d u c e d n u m b e r o f
co n es a n d an a b n o rm a lity o f th o se w hich re m a in e d .

R o d d e fe c ts

Congenital stationary night blindness

C o n g e n ita l sta tio n a ry n ig h t b lin d n e ss (CSNB) is c h a ra c terise d by n ig h t b lin d

ness, n o rm a l fu n d u s a p p e a ra n c e , th e ab sen c e o f ro d d a rk a d a p ta tio n (m o n o p h a
sic d a rk a d a p ta tio n cu rv e) a n d lack o f p ro g ressio n . It may be in h e rite d as an
au to so m al d o m in a n t, an a u to so m al recessive o r an X -linked trait.
72 AIAN C. BIRD AND BARRIE JAY

T h e m ost widely r e p o rte d fo rm o f CSNB is an au to so m al d o m in a n t d iso rd e r

(D ejean & G assenc, 1949). T h e se p atien ts a p p e a r to have n o rm a l c o n e fu n c tio n
b u t little ro d fu n c tio n . Visual acuity, c o lo u r vision a n d p h o to p ic visual fields are
n o rm a l o r a re a t m o st m ildly a b n o rm a l a n d d a rk a d a p ta tio n shows only a c o n e seg
m e n t th a t m ay be a b n o rm a lly p ro lo n g e d (Krill & M artin, 1971); th e re is n o shift
fro m c o n e to ro d c h aracteristics in th e d a rk a d a p ta tio n curve, a n d th e ERG shows
n o p ro lo n g a tio n o f th e b wave im p licit tim e betw een th e p h o to p ic a n d scotopic
reco rd s. H ow ever, fu n d u s re fle c to m e try in d ic ate s a n o rm a l c o n c e n tra tio n o f
b le a c h a b le rh o d o p s in in o n e case (C arr e t al., 1966) a n d histological stu d ies have
show n n o stru c tu ra l a b n o rm a litie s e ith e r in th e re tin a as a w hole o r in th e ro d s in
p a rtic u la r (Vaghefi e t al., 1978).
T h e m ajo rity o f p a tie n ts w ith au to so m a l recessive a n d X -linked CSNB, a n d oc
casional p a tie n ts w ith au to so m a l d o m in a n t CSNB, have an ERG w ith a n e a r n o r
m al a wave a n d a substantially re d u c e d b wave o n testin g u n d e r scotopic
c o n d itio n s (negative ERG) (N oble e t al., 1990). W ith in cre asin g in ten sity o f the
test stim ulus, th e a m p litu d e o f th e a wave in creases b u t th a t o f th e b wave is u n
c h a n g e d (S c h u b e rt & B o rn sc h e in , 1952). T h ese p a tie n ts co u ld be f u rth e r divided
in to two g ro u p s: o n e g ro u p lacked ro d fu n c tio n (co m p lete ty p e ), th e o th e r re
ta in e d so m e ro d fu n c tio n (in c o m p le te type) (M iyake e t al., 1986). P a tien ts with
th e c o m p le te type are m yopic; th o se w ith th e in c o m p le te type m ay b e h y p ero p ic
o r m yopic (K liouri e t al., 1988). A lth o u g h c o m p le te a n d in c o m p le te CSNB d id n o t
co ex ist in any o f th e fam ilies r e p o rte d by M iyake e t al. (1986), o th e rs have fo u n d
p a tie n ts w ith b o th c o m p le te a n d in c o m p le te CSNB w ithin th e sam e X -linked p e d
ig rees (K h o u ri e t al., 1988; P earce e t al., 1990). T his led P earce e t al. (1990) to p ro
p o se th a t X -linked CSNB is a single clinical entity m an ifestin g a wide v a ria tio n in
clinical ex p ressio n . It has also b ee n su g g ested th a t in c o m p le te CSNB a n d A land
Isla n d eye disease m ay be th e sam e c o n d itio n (W eleber e t al., 1989; A litalo e t al.,
1991).
M yopia is alm o st always associated w ith th e X -linked fo rm o f CSNB in w hich vi
sual acuity is re d u c e d a n d nystagm us m ay b e p re se n t. C a rriers o f X -linked CSNB
a re n o t n ig h t b lin d b u t m ay show a b n o rm a l oscillatory p o te n tia ls o n ERG (M iyake
& Kawase, 1984; Y oung e t al., 1989). M yopia also o ccu rs in those cases o f a u to so
m al recessive CSNB th a t have a b n o rm a l vision an d , again, th ese p a tie n ts may have
n ystagm us (Krill, 1977). T h e se fo rm s o f CSNB m ay p re se n t in infancy with b lin d
n ess (W eleber & T o n g u e , 1987).
T h e c o m p le te X -linked CSNB g e n e has b een assigned to X p l l .2 - p l l .3
(M usarella e t al., 1989; Gal e t al., 1989; B ech -H an sen et al., 1990).
L ig h t a n d e le c tro n m icro sco pic studies o f o n e eye w ith CSNB a n d a negative
ERG show ed a n o rm a l a rra n g e m e n t o f ro d o u te r se g m e n t discs, n o rm a l synaptic
e n d s o f th e p h o to re c e p to rs a n d c o m p le te loss o f g a n g lio n cells. It was su ggested
th a t th e cause o f CSNB in th is case may b e re la te d to m ec h an ism s in h ib ito ry to
cells o f th e b ip o la r layer (W atanabe et al., 1986). T h e ab sen ce o f r o d - c o n e in te r
a c tio n , to g e th e r w ith an a b se n t scotopic b wave, also im plies th a t th e d efect is in
th e m id re tin a l layers (Siegel e t a l., 1987).
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 73

Prolonged Dark Adaptation

T h re e d istin c t c o n d itio n s have b e e n d e sc rib e d in w hich th e final th re sh o ld o f

d a rk a d a p ta tio n is n o rm a l b u t in w hich th e ro d p h ase is a b n o rm ally p ro lo n g e d ;
th e d e fe c t in each a p p e a rs to b e static.

Oguchi's disease

T h is au to so m a l recessively in h e rite d d iso rd e r w ith p ro lo n g e d d a rk a d a p ta tio n

was first re p o r te d in J a p a n in 1907 by O g u c h i a n d m ost su b se q u e n t re p o rte d
cases have co m e fro m th e sam e co u n try , a lth o u g h som e n o n -Jap an ese p a tie n ts
have b e e n d e sc rib e d (K lein, 1959; W in n e t al., 1939). In m ost p a tien ts, defective
n ig h t vision is th e only co m p lain t, visual acuity b e in g n o rm a l. T h e c h arac teristic
fe a tu re o f this c o n d itio n is th e a b n o rm a l c o lo ra tio n o f th e lig h t-ad a p ted fu n d u s,
th e a b n o rm a l w hite o r c re a m -co lo u re d a p p e a ra n c e b e in g deriv ed fro m th e in n e r
lim itin g m e m b ra n e a n d presu m ab ly fro m th e fo o t plates o f M uller cells. T h e
a b n o rm a l c o lo u r resolves over a p e rio d o f 0.5 - 8 h o u rs in dark n ess a n d has b e e n
te rm e d th e M iz u o -N a k a m u ra p h e n o m e n o n a fte r th e a u th o rs w ho p ro v id e d the
first d e sc rip tio n (M izuo, 1915; N ak a m u ra . 1920). A sim ilar p h e n o m e n o n has
b e e n d e sc rib e d in X -linked co n e dy stro p h y (H eckenlively & W eleber, 1986) a n d
in X -linked retin o sch isis (d e J o n g e t al., 1991). D ark a d a p ta tio n is ch a ra c teristi
cally slow a n d a final ro d th re sh o ld m ay b e a tta in e d only a fte r several h o u rs, a n d
even th e n m ay b e slightly elevated (F ran o is & V erriest, 1954).
H isto p a th o lo g ic a l e x a m in a tio n has b e e n u n d e rta k e n o n th re e eyes. A n excess
o f co n es c o m p a re d w ith ro d s to g e th e r w ith an a b n o rm a l layer o f m a teria l betw een
th e re c e p to rs a n d p ig m e n t e p ith e liu m was fo u n d by O g u c h i (1925). P arallel his
to logical stu d ie s o n th e o th e r h a lf o f th e sam e eye by a n o th e r investigator (Ya-
m an ak a, 1924) failed to c o n firm th is a d d itio n a l layer. H e c o n sid e re d th e
a b u n d a n c e o f r o u n d fuscin g ra n u le s c o n fin e d to th e apical p o rtio n o f th e p ig m e n t
cells as th e c h a ra c te ristic fe a tu re , H isto p ath o lo g ical a n d e le c tro n m icroscopic
study o f a n o th e r eye m o re re ce n tly (K uw abara e t al., 1963) c o n firm e d th e exist
en ce o f an a b n o rm a l layer betw een th e o u te r seg m en ts o f th e p h o to re c e p to rs a n d
th e p ig m e n t e p ith e lia l cells. H ow ever, th e c o n stitu e n ts w ere n o rm a l c o m p o n e n ts
o f th e re tin a co n sistin g o f fuscin g ra n u le s a n d p ro tru sio n s o f th e p ig m e n t e p ith e
lium w ith c o m p lex in te rd ig ita tio n s o f th e o u te r segm ents. T h e re was n o a b n o rm a l
c o n e d istrib u tio n . A th ird h isto lo g ical study (Y am anaka, 1969) is o p e n to q u e stio n
as th e p a tie n t h a d re d u c e d vision w ith re tin a l p ig m e n ta ry ch an g es a n d b o th p a r
en ts h a d retin itis p ig m e n to sa. O n electrophysiology, b o th th e a-wave o f th e ERG
a n d th e lig h t rise in th e sta n d in g p o te n tia l o f th e eye are n o rm a l, b u t th e scotopic
b-wave o f th e ERG is severely d e p re sse d even in th e fully d a rk a d a p te d eye (C arr
& R ipps, 1967).
T h e p a th o g e n e sis o f O g u c h i's disease is n o t u n d e rs to o d a n d a lth o u g h it is
te m p tin g to sp e c u la te th a t it is re la te d to a slow re g e n e ra tio n o f rh o d o p sin , this is
n o t substan tia te d by a single study o f rh o d o p sin kinetics in this d iso rd er, in w hich
74 ALAN C. BIRD AND BARRIE JAY

rh o d o p s in re g e n e ra tio n was n o rm a l (C arr & R ipps, 1967). T h ese o b se rv a tio n s im

ply th a t th e p rim a ry ab n o rm a lity is u n re la te d to lig h t catch a n d rh o d o p sin b le a c h
in g b u t is re la te d to o th e r systems o f tra n sd u c tio n . F rom th e electrophysiology
results, th e re g io n o f b ip o la r cells a p p e a rs to be th e e a rlie st stage in th e visual p a th
way e x h ib itin g signs o f d efective fu n c tio n . H owever, th e p re se n c e o f an a b n o rm a l
layer e ith e r o n th e vitreal side o f th e p ig m e n t e p ith e lia l cells o r w ithin th e apical
cytoplasm in cites som e a b n o rm a lity o f o u te r-se g m e n t sh e d d in g o r o f th e p ig m e n t
e p ith e liu m , w h e th e r d u e to slow phagocytosis o r in a d eq u a cy o f th e lysosom al sys
tem . F rom th e ir o b se rv a tio n in X -linked retinoschisis, d e J o n g a n d his colleagues
(1991) sugg ested th a t th e M iz u o -N a k a m u ra p h e n o m e n o n is c au sed by an excess
o f e x tra c e llu la r p o tassiu m in th e re tin a as a re su lt o f a d e c re a se d p o tassiu m scav
e n g in g capacity o f re tin a l M u ller cells.

Fundus albipunctatus

F u n d u s a lb ip u n c ta tu s is a static au to so m al recessive c o n d itio n in w hich th e only

sym ptom s a re re la te d to defective d a rk a d a p ta tio n (L auber, 1924). T h e c o n d itio n
sh o u ld n o t be co n fu sed with a lb ip u n c ta te dystrophy (re tin itis p u n c ta ta albes
c e n s), w hich is progressive a n d w hich re p re se n ts a v aria n t o f retin itis pig m en to sa.
T h e fu n d u s shows w id esp read d istrib u tio n o f u n ifo rm -sized alm o st w hite d o ts at
th e level o f th e p ig m e n t e p ith e liu m , w hich are m o st d e n se in th e p o st-eq u ato ria l
fu n d u s; th e m acu la m ay o r m ay n o t be involved. C han g es in th e d istrib u tio n o f th e
w hite d o ts have b een d e sc rib e d (M arm or, 1977), as have th e ir ch an g e fro m flecks
in c h ild h o o d to relatively p e rm a n e n t p u n c ta te dots th a t in crease in n u m b e r over
th e years (M arm or, 1990). D iffuse p ig m e n ta ry ch an g es in th e p ig m e n t e p ith e liu m
a re u n u su a l. F lu o rescein a n g io g ra p h y shows p u n c ta te h y p e rflu o re sc en c e th a t
d o e s n o t, how ever, c o rre s p o n d w ith th e p u n c ta te w hite d o ts (Gass, 1970).
A lth o u g h fu n d u s a lb ip u n c ta tu s usually involves b o th eyes, u n ila te ra l disease has
b e e n re p o r te d (H en k es, 1963). Typically, th e visual acuity a n d visual fields a re n o r
m al, b u t m in o r loss o f field h as b e e n d e sc rib e d (M arm or, 1977). In a m ajority o f
cases, th e d a rk a d a p ta tio n o f b o th cones a n d ro d s is m arkedly p ro lo n g e d (Krill &
M artin , 1971; C a rr & Siegel, 1973; M arm or, 1977; M argolis et al., 1987) a n d th e re
is delay in th e a cq u isitio n o f scotopic ERG th re sh o ld s (Sm ith e t al., 1959; Krill &
M artin , 1 9 7 1 ;M arm o r, 1977; M argolis e t al., 1987). V ariation fro m this p a tte rn has
b e e n d e sc rib e d in w hich th e d a rk a d a p ta tio n a n d ERG are n o rm a l (F ranois et al.,
1956; F ra n c e sc h e tti e t al., 1963) o r d a rk a d a p ta tio n shows a co n e se g m e n t only
(M a n d e lb a u m , 1941). It is n o t cle ar w h e th e r this variatio n im plies various d eg re es
o f severity o f a single disease o r th a t several d iso rd ers have b e e n d e sc rib e d w hich
sh a re this fu n d u s abnorm ality.
S tu d ies o f rh o d o p s in k in etics show ed slow rh o d o p sin re g e n e ra tio n th a t parallels
d a rk a d a p ta tio n (C a rr e t al., 1974). T his im plies th at, by c o n tra st w ith O g u c h i's dis
ease, th e sen so ry d e fe c t is d u e to a b n o rm a l p h o to p ig m e n t kinetics in fu n d u s albi
p u n c ta tu s.
 DIAGNOSIS IN INHERITED RETINAL DISORDERS 75

Fleck retina o f Kandori

T h is ra re c o n d itio n in w hich th e re is p ro lo n g e d d a rk a d a p ta tio n giving rise to dif

ficulty w ith n ig h t vision b u t n o o th e r sym ptom s, has b e e n d esc rib e d only in J a p a n
(K an d o ri e t al., 1972). D ark a d a p ta tio n shows a p ro lo n g e d ro d p h ase re a c h in g
n o rm a l th re sh o ld s w ithin 40 m in u te s. T h e fu n d u s p rese n ts large irre g u la r w hite
lesions a t th e level o f th e p ig m e n t e p ith e liu m th a t are m o st c o n c e n tra te d in th e
e q u a to ria l reg io n . T h e p h o to p ic ERG is n o rm a l a n d a p ro lo n g e d in terv al o f d a rk
a d a p ta tio n is n e e d e d to re a c h sco to p ic p o ten tia ls.

C O N C LU SIO N

T h e re c e n t advances in m o le c u la r biology w hereby specific g e n e tic d efects have

b e e n id e n tifie d , a n d in cell biology d e m o n stra tin g th e in flu e n c e o f g e n etic
defects o n cell fu n c tio n , r e n d e r th e fin d in g s o f clinical in v estigation m u c h m o re
significant. In th e past, th e fu n c tio n a l deficits in retin itis p ig m e n to sa w ere o f
value in d istin g u ish in g o n e d iso rd e r fro m a n o th e r o r a t least iden tify in g categ o
ries o f disease. It is now possible to re la te th ese ch aracteristics to putative disease
m ech an ism s a n d to g e n e ra te h y p o th eses th a t a re a m en ab le to testin g in th e lab o
ratory. It is also th e case th a t o b serv a tio n s are possible in m an, p a rticu la rly w ith
re sp e c t to d e ta ile d re c o rd in g o f fu n c tio n a l loss in th o se w ith re tin a l dystrophies,
w hich c a n n o t b e m ad e in anim als. T h u s th e results o f clinical a n d la b o ra to ry
studies a re re le v a n t o n e to th e o th er.
T h e in itial subdivision o f retin itis p ig m e n to sa was achieved o n th e basis o f its in
h e rita n c e ; fu n c tio n a l stu d ies a d d e d to o u r u n d e rs ta n d in g o f this c o m p lex g ro u p
o f d iso rd ers. T h e re c e n t id e n tific a tio n o f a n u m b e r o f d iffe re n t m u ta tio n s in the
rh o d o p sin a n d p e rip h e rin RDS g en es are at p re s e n t b e in g c o rre la te d w ith clinical
a n d fu n c tio n a l a b n o rm a litie s, a n d we can ex pect, in th e n e a r fu tu re , to fin d o th e r
g e n e s re sp o n sib le fo r f u r th e r m e m b e rs o f this g ro u p o f disorders.
T h e m e c h a n ism by w hich a lte ra tio n o f th e a m in o acid se q u e n c e in rh o d o p sin
o r rd s p e rip h e rin m o lecu les may in flu e n c e p h o to re c e p to r fu n c tio n is u nclear, b u t
m u c h m ay d e p e n d u p o n th e site o f ex p re ssio n o f th e a b n o rm a l p ro te in . F u rth e r
stu d ies sh o u ld clarify this a sp ec t o f th e p ro b lem .
A lth o u g h th e re has b e e n a very ra p id advance in th e u n d e rs ta n d in g o f disease
m ec h a n ism s in retin itis p ig m en to sa, th e re is every p ro sp e c t th a t th e ex p a n sio n o f
k now ledge will a c c e lera te in th e n e x t few years, p articu larly if th e re is c o n tin u e d
an d , if possible, in c re a sin g c o lla b o ra tio n betw een clinicians a n d basic scientists.
T h e c o n sid e ra b le ad v ances so far ac h iev e d have n o t yet b ro u g h t any g re a t advance
in therapy, a lth o u g h som e effect o n clinical m a n a g e m e n t is alread y evident. G e
n e tic c o u n se llin g in a fam ily is u n d o u b te d ly sim plified if th e g e n e tic a b n o rm ality
is know n, a n d advice o n visual p ro g n o sis m ay be m ad e on a firm e r basis if single
noso lo g ical e n titie s a re id e n tifie d by g e n e tic studies. In a d d itio n , it is to be h o p e d
th a t w ith th e kno w led g e th a t will re su lt fro m ex te n sio n o f c u rre n t studies, it may
76 Al AN C. BIRD AND BARRI E JAY

even tu ally b e possible to o ffe r effective tre a tm e n t to th e p a tie n t with re tin itis pig
m en to sa.

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 4. HUMAN ALBINISM AND MOUSE MODELS

RICHARD A. KING, IAN J. JACKSON* a n d WILLIAM S. O E T T IN G

University o f Minnesota, BO X 485 UMHC 4 2 0 Delaware Street SE, Minneapolis, Minnesota

55455, USA
*M R C H um an Genetics Unit., Western General Hospital, Crewe Road, Edinburgh, EH 4 2XU,
UK

ABBREVIATIONS USED

AS A n g elm an S y n d ro m e
c m o u se albino locus
CH S C h e d ia k H ig ash i S yndrom e
H PS H erm an sk y P u d la k S yndrom e
OA O c u la r A lbinism
OA1 X -L inked OA
O CA O c u lo c u ta n e o u s A lbinism
OCA1 T y ro sin ase-related OCA
OCA2 T yrosinase-positive OCA
p m o u se pink-eyed dilution locus
P H u m a n P g e n e locus w hich is h o m o lo g o u s to m o u se pink-eyed
dilution locus
PWS Prader-W illi S y n d rom e
TRP1 T yrosinase re la te d p ro te in 1; gp75; brown locus
TRP2 T yrosinase re la te d p ro te in 2; d o p a c h ro m e ta u to m e rase ; slaty locus
TYR H u m a n tyrosinase g e n e locus

IN T R O D U C T IO N

In h e rite d a b n o rm a litie s o f m e la n o g en esis th a t p re se n t w ith a c o n g e n ita l re d u c

tio n in m e la n in synthesis in all m elan o cy tes o f th e skin, h a ir a n d eyes are te rm e d
o c u lo c u ta n e o u s albinism (O C A ), a n d those in w hich th e h y p o p ig m e n ta tio n p ri
m arily involves th e m elan o cy tes o f th e eye are te rm e d o c u la r albinism (O A ). T h e
te rm a lb in o is d e riv e d fro m th e L atin albus fo r w hite, a n d is a ttrib u te d to Bat-
h azar Tellez w ith his 1660 d e sc rip tio n o f A frican individuals w ith w hite skin
(P earso n et a l.,1911). Sir A rch ib a ld G a rro d in c lu d e d OCA in his o rig in al d e sc rip
tio n o f in b o rn e rro rs o f m etab o lism (G a rro d , 1908). O CA is universal in d istrib u
tio n a n d has b e e n d e sc rib e d in all h u m a n e th n ic groups.
A sim ilar g e n e tic v arian t, involving w hite skin a n d h a ir a n d p in k eyes is seen in
m any o th e r m am m alian species, in c lu d in g cats, dogs, cattle, m ice, rats a n d rab b its
(S earle, 1990). P ig m e n ta tio n d efects have b e e n p a rtic u larly well stu d ie d in th e
m ouse, w h ere th e re a re m o re th a n 80 d iffe re n t g en etic loci at w hich m u ta tio n s af-
90 RICHARD A. KING ET AL.

feet h a ir o r eye p ig m e n ta tio n . Som e o f th e se m u tatio n s, in a d d itio n to p ro v id in g

usefu l tools by w hich th e d e v e lo p m e n t o f m elan o cy tes can be dissected, a re also
m o d e ls fo r th e u n d e rs ta n d in g o f h o m o lo g o u s h u m a n diseases. T hey are p a rtic u
larly in fo rm ativ e fo r e x a m in in g th e effect o f d iffe re n t m u ta tio n s o f th e sam e g en e.
T h e te rm a lb in o in m o u se is u se d fo r an im als m u ta n t at o n e p a rtic u la r locus, th e
e-locus o n c h ro m o so m e 7 a n d th e m o d el fo r h u m a n OCA1. O th e r form s o f h u m a n
a lb in ism c o rre s p o n d to o th e r m ouse m u ta tio n s.

M ELANIN SYNTHESIS

B io c h e m ic a l P ro c e s s

M elan in biosynthesis has b e e n stu d ied fo r over seventy years (R aper, 1928;
M ason, 1948; L e rn e r a n d F itzpatrick, 1950), a n d fo r m o re th a n 50 years was
th o u g h t to be re g u la te d solely by th e enzym e tyrosinase w ith all su b se q u e n t reac
tio n s o c c u rrin g non-enzym atically (C o lem an , 1962). In 1978, L ogan a n d W eath-
e rh e a d re p o r te d th a t h a ir follicle tyrosinase activity o f th e C h in ese h a m ste r was
h ig h w ith th e w in te r a n d su m m e r m olt, w hile p ig m e n te d h airs w ere p ro d u c e d
only w ith th e su m m e r m o lt (L ogan a n d W ea th e rh ea d , 1978). T his show ed th a t
tyrosinase activity in itself wras n o t su fficien t fo r p ig m e n ta tio n a n d o th e r m e c h a
n ism s fo r c o n tro l o f th e p ro cess m u st exist. T his o b serv a tio n led to a se arch fo r
o th e r enzym es a n d facto rs th a t re g u la te m e la n in synthesis, a n d several have now
b e e n id e n tifie d , in c lu d in g tyrosinase re la te d p ro te in 1 o r gp75 (TRP1) (Shiba-
h a ra e t al., 1986; Jack so n , 1988; Jac k so n e t al.,1990), tyrosinase re la te d p ro te in 2
o r d o p a c h ro m e ta u to m e ra se (TRP2) (Ja c k so n , 1988; Jac k so n et al.,1992; Tsuka-
m o to e t al., 1992) a n d Pm el 17-1 (Kwon e t al., 1987). T h e specific ro le o f th ese
newly d e sc rib e d facto rs in n o rm a l m e la n o g e n esis is u n k n o w n b u t a g re a t d e a l o f
w ork is b e in g d e v o te d to this p ro b le m , a n d h u m a n a n d m ouse m u ta n ts are p ro
v idin g im p o rta n t insights.
T h e biosynthesis o f m e la n in starts w ith th e o x id a tio n o f tyrosine a n d e ith e r red-
yellow p h e o m e la n in o r black-brow n e u m e la n in can fo rm (P rota, 1988a; P ro ta a n d
T h o m so n , 1976; P ro ta, 1986; P ro ta, 1988b; P ro ta, 1980). T h e c o m m o n steps to
b o th types o f m e la n in are th e o x id a tio n o f tyrosine to d o p a a n d th e d e h y d ro g e n a
tio n o f d o p a to d o p a q u in o n e , th e two steps b e in g catalyzed by tyrosinase (L e rn e r
a n d F itzpatrick, 1950; P o m e ra n tz a n d W arner, 1967). If sulfhydryl c o m p o u n d s are
p re se n t, they re a c t w ith d o p a q u in o n e a n d c o m m it th e process to p h ae o m elan iza -
tio n th ro u g h a series o f cysteinyldopa a n d b en z o th ia z in e in te rm e d ia te s th a t poly
m erize to acid-soluble p h e o m e la n in (Je rg il e t al., 1983; A g ru p et al., 1979, 1982;
H a n sso n e t al., 1980; R o rsm an e t al., 1 9 7 3 ;J a ra e t al., 1988). T h e p ro d u c tio n o f
p h e o m e la n in a p p e a rs to be c o n tro lle d by th e availability o f sulfhydryl c o m p o u n d s
in th e m e la n o so m e ( J a r a et al.,1988). In th e ab sen ce o f sulfhydryl c o m p o u n d s,
d o p a is c o n v e rte d to d o p a c h ro m e (C ab an es et al., 1987) w hich is th e n c o n v e rte d
to 5,6-dihydroxy indole-2-carboxylic acid by th e catalytic actio n o f d o p a c h ro m e
 HUMAN ALBINISM AND MOUSE MODELS 91

ta u to m e ra se (L e o n a rd e t al., 1988; H e a rin g e t al., 1982; K rn e r a n d Pawelek,

1980; Paw elek, 1985, 1990, 1991; Paw elek et al., 1980; C h ak rab o rty e t al. 1992) o r
to 5,6-dihyd ro x y in d o le by sp o n ta n e o u s re a c tio n (L e o n a rd et al., 1988). Tyrosinase
m ay also play a ro le in c o n tro llin g th e o x id a tio n o f dih y d ro x y in d o le-2-carboxylic
acid to th e c o rre s p o n d in g in d o le q u in o n e (K rn e r a n d Pawelek, 1982). T h e steps
b e y o n d th e d ih y d ro x y in d o le c o m p o u n d s a re n o t well e lu c id a te d , b u t gen erally in
volve th e fo rm a tio n o f in d o le q u in o n e s w hich th e n polym erize to acid-insoluble
e u m e la n in (P ro ta, 1988b; P ro ta , 1980; Ito a n d W akam atsu, 1989). In a sense, th e
fo rm a tio n o f m e la n in can be view ed, in p a rt, as a d e to x ifica tio n m e ch an ism o f th e
cell by c o n v e rtin g relatively toxic in te rm e d ia te c o m p o u n d s such as q u in o n e s in to
in e rt m e la n in th a t c a n n o t diffuse o u t o f th e cell (P rota, 1980).
T yrosinase is a m u ltifu n c tio n a l c o p p e r c o n ta in in g g ly co p ro tein with a de novo
m o le c u la r w eight o f a b o u t 65kD a a n d a glycosylated w eight o f 75kD a (N ishioka,
1978; M iyazaki a n d O h ta k i, 1976; H e a rin g et al., 1978). T h e tyrosinase p o ly p ep
tide c o n ta in s 528 a m in o acids in c lu d in g an 18 a m in o acid le a d e r se q u e n c e th a t is
m issing in th e m a tu re enzym e (W ittb jer e t al., 1990). T yrosinase c o n ta in s two c o p
p e r atom s as a b in u c le a r co m plex. T h e c o p p e r c o m p lex fo rm s a p e ro x id e w ith di
oxygen w hich th e n in te ra c ts w ith tyrosine as th e first step in m e la n in fo rm a tio n
(J a c k m a n e t al., 1992; Ross a n d S o lo m o n , 1991). T h e a m in o acid seq u e n ce o f th e
c o p p e r b in d in g sites in h u m a n tyrosinase a re sim ilar to th e a m in o acid seq u en ce
o f h em o cy an in (V olbeda a n d H oi, 1989a; Ross a n d S o lom on, 1991), a c o m p o u n d
fo r w hich X-ray d e fra c tio n d a ta are available, a n d this has p e rm itte d d e ta ile d ter
tiary s tru c tu ra l analysis o f th e c o p p e r b in d in g site o f h u m a n tyrosinase (O e ttin g
a n d King, 1992b). T yrosinase has a h y d ro p h o b ic tra n sm e m b ra n e re g io n th a t a n
ch o rs th e p ro te in to th e m e m b ra n e a n d a cytoplasm ic tail, b o th at th e carboxy e n d
o f th e p ro te in .
D o p a c h ro m e ta u to m e ra se catalyzes th e co n version o f d o p a c h ro m e to 5,6-dihy
droxy indole-2-carboxylic acid (L e o n a rd et al., 1988; H e a rin g et al.,1982; K rn er
a n d Paw elek, 1980; Paw elek, 1990, 1991). T h e m u rin e o r h u m a n enzym e has n o t
b e e n iso lated a n d p u rifie d , b u t th e g e n e has b e e n c lo n e d as cDNA. T h e p re d ic te d
a m in o acid se q u e n c e o f d o p a c h ro m e ta u to m e ra se has a m o d e ra te d e g re e o f sim i
larity to th a t o f tyrosinase (40% ) a n d in c lu d e s areas th a t m ay b e a tra n sm e m b ra n e
re g io n a n d two c o p p e r b in d in g sites as well as having two co n serv ed cysteine-rich
d o m ain s. T h e fu n c tio n o f th ese cysteine-rich d o m a in s is u n k n o w n , b u t o n e o f
th e m is h o m o lo g o u s to th e EGF m o tif fam ily o f a m in o acid seq u e n ces w hich are
fo u n d in m an y m e m b ra n e -b o u n d o r se c re te d p ro te in s fro m n e m a to d e a n d Droso
phila d e v e lo p m e n ta l g en es, m a m m alia n grow th factors a n d e x tra c e llu lar m atrix
p ro te in s. (Ja c k so n e t al., 1992; Davis, 1990) T h e m o tif m ay fu n c tio n to m e d iate
p r o te in - p r o te in in te ra c tio n betw een th e m ela n o g e n ic enzym es a n d o th e r p ro
tein s in th e m e la n o so m e . A lth o u g h d o p a c h ro m e ta u to m e ra se has a specific actio n
in th e co n v ersio n o f d o p a c h ro m e a n d is e n c o d e d by a re co g n iz ed coat c o lo r locus
in th e m o u se (slaty lo c u s ), th e p re cise ro le o f this enzym e in n o rm a l m e la n o g e n e-
sis re m a in s largely u n k n o w n .
92 RICHARD A. KING ET AL.

M o le c u la r B iology

Tyrosinase

T h e h u m a n tyrosinase g e n e (TYR) is lo c a te d o n c h ro m o so m e 11 q 14- q 2 1 (B a rto n

et al.,1988). T h e r e is also a tyrosinase p se u d o g e n e (TYRL) th a t is a tru n c a te d
g e n e o n c h ro m o so m e l l p l l . 2 - cen (B a rto n e t al 1988; T akeda e t al., 1989,
1991). T h e r a g e n e is ap p ro x im ately 50 kb in le n g th a n d th e c o d in g re g io n is
d iv id ed in to 5 ex o n s, w ith alm o st h a lf th e c o d in g re g io n in ex o n 1 (G iebel e t al.
1991b). T h e m o u se g e n e , o n c h ro m o so m e 7, has a n id en tical ex o n stru c tu re to
th e h u m a n , b u t a t 70kb is a little lo n g e r (R u p p e rt e t a l , 1988). E x p ressio n studies
u sin g th e m o u se tyrosinase g e n e have show n th a t only th e first 270 b p o f th e p ro
m o te r re g io n a re n ecessary fo r b o th m elan o cy te specific exp ressio n a n d th e cor
re c t te m p o ra l e x p ressio n in tran sg e n ic m ice (K luppel e t al.,1991), a lth o u g h
e x p ressio n stu d ies u sin g th e e n tire g en e in c lu d in g 50 kb o f th e p ro m o te r re g io n
is n ecessary to co m p letely m im ic wild type exp ressio n o f the g en e (S chedl et al..
1992, 1993).

TRP1

A seco n d p ig m e n t cDNA was id e n tifie d th a t e n c o d e s a p ro te in with a b o u t 40%

a m in o acid id e n tity w ith tyrosinase a n d is c u rre n tly c alled tyrosinase re la te d p ro
tein-1 o r TR P1, initially n a m e d TRP (S h ib a h a ra e t al., 1986, C o h e n e t al., 1990).
T h e e n c o d e d p ro te in has th e ch ara cteristic c o p p e r-b in d in g sites, m e m b ra n e
s p a n n in g d o m a in a n d cysteine-rich fe a tu re s seen in tyrosinase a n d d o p a c h ro m e
ta u to m e ra se . T h e m o u se g e n e m aps to th e brown ( b) locus o n c h ro m o so m e 4
(Jackson, 1988). M u tatio n s at this locus th a t affect th e p ro te in se q u e n c e o r
e x p ressio n levels a lte r th e type o f e u m e la n in m a d e fro m black to brow n (Ja ck so n
e t al., 1990). Interestingly, d e sp ite th e cle a r c o m m o n ev o lu tio n ary history, TRP1
has an ex o n s tru c tu re q u ite d iffe re n t fro m tyrosinase (Ja c k so n e t al., 1991),
w hich is a c h a ra c teristic o f g e n e fam ilies se p a ra te d by a very lo n g history. T h e
im p lic a tio n is th a t th e fam ily o f re la te d p ro te in s sh o u ld be fo u n d in a w ide ra n g e
o f species.
T h e h u m a n TRP1 sh ares a b o u t 93% a m in o acid id en tity with m ouse TR P1, b e
in g a p o ly p e p tid e o f 527 a m in o acids w ith a m o le c u la r w eight o f 60,000. T h e h u
m an g e n e has b e e n m a p p e d to h u m a n ch ro m o so m e 9p23 u sin g in situ
h y b rid izatio n (M urty e t al., 1992). T h e TRP1 locus has b e e n show n to be th e g en e
fo r th e h u m a n m e la n o m a a u to a n tig e n ic g ly co p ro tein 75 (gp75) (H a la b an a n d
M o ellm an n , 1990; V ijayasaradhi a n d H o u g h to n , 1991). T h e g ly co p ro tein gp75 is
th e m o st a b u n d a n t g ly co p ro tein in m e la n o m a cell lines, b u t a b se n t in n o n p ig -
m e n te d m e la n o m a cell lin es (Tai e ta l., 1983; T h o m so n e t al., 1985).
B ioch em ical analysis o f m o u se TRP1, p u rifie d by im m u n e affinity c h ro m a to g ra
phy, has show n th a t th e p ro te in ex h ib its low levels o f tyrosine hydroxylase activity
( J im n e z e t al., 1991) b u t c o lu m n p u rifie d h u m a n gp75 fro m m e la n o m a has b e e n
 HUMAN ALBINISM AND MOUSE MODELS 93

show n n o t to c o n ta in any tyrosine hydroxylase activity (V ijayasaradhi a n d

H o u g h to n , 1991). T h is d iscrep an cy m ay be d u e to th e h ig h sim ilarity o f tyrosinase
to gp75 a n d th e im m u n o p re c ip ita te d brow n p ro te in m ay have b e e n c o n ta m in a te d
w ith tyrosinase. O th e rs have ascrib ed catalase activity to this p ro te in a n d have
given it th e locus n a m e CAS2 fo r th e h u m a n g e n e b u t this activity m ay n o t be the
p rim a ry catalytic fu n c tio n o f this p ro te in (H a lab a n a n d M o ellm an n , 1990; C hinta-
m a n e n i e t al., 1991). It has b e e n show n th a t b o th tyrosinase a n d gp75 c o n ta in cat-
alase-like activity in d ic a tin g th a t this activity may be artifactual.

TRP2

A th ird m e m b e r o f th e ty ro sin ase-related family, TRP2, was initially c lo n e d as a

c a n d id a te fo r m o u se tyrosinase, a n d has a b o u t 40% a m in o acid id e n tity to b o th
tyrosinase a n d TR P1, in c lu d in g th e v ario u s c h aracteristic m otifs (Ja c k so n e t al.,
1992). Im m u n o p u rific a tio n has su b seq u e n tly show n th a t TR P2 is d o p a c h ro m e
ta u to m e ra se (T su k am o to e t al., 1992). T h e h u m a n cDNA has recen tly b een
c lo n e d (B. B o u c h a rd , p e rso n a l c o m m u n ic a tio n ) a n d is 80% id en tic al to its
m o u se h o m o lo g u e . T h e m o u se g e n e m ap s to a n d is m u ta te d at th e slaty locus on
c h ro m o so m e 14, (Ja c k so n et al., 1992) w hilst th e h u m a n g e n e is a t 13q32 (B.
B o u c h a rd , p e rso n a l c o m m u n ic a tio n ).

D EFIN ITIO N O F HUM AN ALBINISM

O u r c u rre n t u n d e rs ta n d in g o f th e path o p h y sio lo g y o f h y p o p ig m e n ta tio n allows a

precise d e fin itio n o f albinism . First, th e re a re n o c h a rac teristic c h a n g e s associ
a te d w ith th e skin h y p o p ig m e n ta tio n o th e r th a n a g e n e ra liz e d re d u c tio n o f m ela
n in in OCA. T h e h y p o p ig m e n ta tio n o f th e skin is associated with sensitivity to
u ltra v io le t ra d ia tio n a n d a p re d isp o sitio n to skin cancer, b u t th e d e g re e o f the
sensitivity in alb in ism is th e sam e as in n o rm a lly p ig m e n te d individuals w ith lig h t
o r fair p ig m e n ta tio n . T h e specific a n d d e fin in g c h an g es in albinism o c c u r in the
o p tic system , a n d a re p a rt o f th e d ia g n o stic c rite ria (C reel e t al., 1974, 1990).
M elan in is involved in th e n o rm a l d e v e lo p m e n t o f th e eye a n d th e o p tic system
th ro u g h u n k n o w n m ech an ism s, a n d re d u c e d m e la n in in th e eye d u rin g d evelop
m e n t is asso ciated w ith lifelo n g nystagm us, re d u c e d re tin a l p ig m e n t, foveal hypo
plasia w ith re d u c e d visual acuity, a n d a b n o rm a l ro u tin g o f th e n e u ra l fibers from
th e re tin a to th e b ra in re su ltin g in strabism us a n d th e loss o f b in o c u la r vision.
T h e o p tic system c h a n g e s a re fo u n d in all types o f albinism , su ggesting th a t it is
th e re d u c tio n in m e la n in in th e involved tissue r a th e r th a n a p le io tro p ic effect o f
th e re sp o n sib le g en e.

N y stag m u s

A lm ost all in d iv id u als w ith albinism have nystagm us (Collew ijn e t al., 1985; van
D o rp , 1987; C reel et al., 1990). Several recen tly re p o rte d fam ilies co n ta in individ
94 RICHARD A. KING ET AL.

uals w ho a p p e a re d to have albinism w ith o u t nystagm us, b u t this is q u ite rare

(S u m m ers e t al.,1991; C h e o n g et al.,1992; C astro n u o v o e t al.,1991). N ystagm us is
p re s e n t at b irth o r d evelops w ithin th e first few m o n th s o f life, a n d th e a m p litu d e
is usually g re a te r early in life a n d b ec o m e s less m a rk e d w ith tim e, to th e p o in t
w h ere it can be m in im al in an a d u lt. T h e re can be an associated h e a d tilt o r tu rn
th a t a p p e a rs to d a m p e n th e a m p litu d e o f th e nystagm us.

P h o to p h o b ia

M ost individuals w ith alb in ism have som e d e g re e o f p h o to p h o b ia , th e d e g re e

re la te d to th e a m o u n t o f p ig m e n t p re se n t in th e iris a n d re tin a . P h o to p h o b ia is
usually m o st m a rk e d at a y o u n g age, a n d babies w ith albinism , p artic u larly the
tyrosinase-negative type o f OCA1 will n o t w ant to go o u tsid e a n d will cry w hen
they a re in th e sun. Som e in d iv iduals w ith alb in ism m ay have n o p h o to p h o b ia .

V isu al A cuity

A ffected individuals usually have d im in ish e d visual acuity, typically in th e ra n g e

o f 2 0 /1 0 0 to 2 0 /2 0 0 , w ith a ra n g e o f 2 0 /4 0 -5 0 to 2 0 /4 0 0 + (E d m u n d s, 1949;
F o n d a , 1962; S u m m ers e t al., 1988). Several re c e n t re p o rts have id e n tifie d in d i
viduals w ith albinism w ho have n o rm a l o r n ea r-n o rm a l visual acuity, b u t this is
th o u g h t to b e ra re (S u m m ers e t al., 1991). V isual d e v e lo p m e n t in an affected
in fa n t is slow er th a n n o rm a l, w ith a tta in m e n t o f visual acuity delayed in th e first
years o f life. R efractive e rro rs are co m m o n , a n d m any affected individuals have
im p ro v e m e n t o f th e ir acuity w ith p ro p e r c o rre c tio n (Taylor, 1978; F o n d a, 1962).
C o lo r vision is essentially n o rm a l.

Iris P ig m e n t

Iris p ig m e n t is n o rm ally fo u n d in th e stro m a a n d in th e p o ste rio r iris e p ith e liu m ,

a n d b o th c o m p a rtm e n ts have re d u c e d a m o u n ts in albinism . Strom al p ig m e n t
ch an g es th e iris from a b lu e c o lo r to o n e th a t is partially o r com p letely brow n,
w hile p ig m e n t in th e p o s te rio r iris e p ith e liu m re d u c e s th e tran slu cen cy o f th e
iris. S tro m al p ig m e n t in albinism can b e a b se n t, m in im al (lig h t brow n co lo r to
c e n tra l p o rtio n s o f iris), o r m o d e ra te (lig h t brow n iris), a n d o fte n in creases w ith
age (S u m m ers et al., 1988). W h en th e p o ste rio r e p ith e lia l p ig m e n t is a b sen t, th e
iris is fully tra n s lu c e n t o n g lo b e tra n sillu m in a tio n . In crea sin g a m o u n ts o f strom al
a n d e p ith e lia l p ig m e n t re d u c e tran slu cen cy to a c a rtw h e e l o r w agon-w heel,
se g m e n ta l o r spotty p a tte r n o n globe tra n sillu m in a tio n (S um m ers et al., 1988).

R e tin a l P ig m e n t a n d F o v eal D e v e lo p m e n t

T h e a m o u n t o f p ig m e n t in th e p ig m e n t e p ith e liu m o f th e re tin a is re d u c e d o r

a b se n t, allow ing th e c h o ro id a l vessels to b e seen b e n e a th th e re tin a . T h e n o rm a l
 HUMAN ALBINISM AND MOUSE MODELS 95

foveal d e p re ssio n a n d lig h t reflex in th e c e n te r o f th e fovea are a b se n t (U sher,

1920; S u m m ers et al., 1988; A badi a n d D ickinson, 1983; A dler, 1910), a n d this
d e v e lo p m e n ta l a b n o rm a lity is largely re sp o n sib le fo r th e re d u c e d acuity. T h e
m a c u la r a re a can be tra n s lu c e n t o r o p a q u e , th e la tte r p ro d u c e d by lu tea l ra th e r
th a t m e la n in p ig m e n t (S u m m ers et al., 1988).

O p tic T ra c t D e v e lo p m e n t

All individuals w ith alb in ism have excessive decu ssatio n o f th e o p tic fibers at the
chiasm (C reel e t al., 1974, 1990; G uillery e t al., 1975). T h e ipsilateral p ro je ctio n s
fro m th e re tin a to th e la te ra l g e n ic u la te are re d u c e d a n d the s tru c tu re o f th e lat
eral g e n ic u la te is a b n o rm a l (G u illery e t al., 1975). T h e d e m o n stra tio n o f optic
tra c t m is ro u d n g by visual evoked p o te n tia l studies is th e critical diag n o stic p ro c e
d u re fo r q u e stio n a b le cases (C reel e t al., 1974, 1978, 1981, 1990; A p k arian e t al.,
1990). M isro u tin g o f th e o p tic fib ers can be d e m o n s tra te d w ith visual evoked
p o te n tia ls re c o rd e d w ith m o n o c u la r stim u latio n (L ev en th al a n d C reel, 1985;
C reel e t al., 1974, 1978, 1981; A p k arian e t al., 1990). T h e a lte re d d e v e lo p m e n t o f
th e o p tic tracts c o n trib u te s to th e strab ism u s a n d d im in ish e d stereo acu ity th a t is
usually p re se n t.

O C U LO C U TA N EO U S ALBINISM: PRIMARY DEFECT SPECIFIC FOR

M E IA N IN SYNTHETIC PATHWAY

O v erv iew

O c u lo c u ta n e o u s alb in ism can b e d iv id ed in to th o se types in w hich th e p rim a ry

d e fe c t is in th e m e la n in sy nthetic system in th e m elanocyte a n d th o se p re se n tin g
w ith albinism a n d asso ciated d efects in w hich th e p rim a ry d e fe c t is n o t specific
fo r th e m e la n in sy n th etic system (T able 1). All types are a u to so m a l recessive in
in h e rita n c e , a n d o b lig ate h etero zy g o te s d o n o t regularly m an ifest c u ta n e o u s o r
o c u la r h y p o p ig m e n ta tio n . T h e m elan o cy te stru c tu re a n d n u m b e r in th e skin a n d
h a ir follicle a re n o rm a l in all types o f OCA, a lth o u g h th e m ela n o so m a l stru c tu re
may b e a lte re d , p a rtic u la rly in those types in w hich th e p rim a ry d efec t is n o t spe
cific fo r th e m e la n in syn thetic pathw ay (i.e. C H S). P rim ary OCA can be divided
in to OCA1 o r ty ro sin ase-related OCA, OCA2 o r tyrosinase-positive OCA (using
th e m o st re c e n t te rm in o lo g y fro m M cK usick (M cKusick, 1992)) a n d several types
th a t a re u n classified b ecau se o f in c o m p le te clinical a n d b io ch e m ic al data.
96 RICHARD A. KING ET AL.

T able 1 C lassification O f A lbinism

O C U L O C U T A N E O U S A L B IN IS M (O C A )

Primary Defect Specific for Melanin Synthetic Pathway

OCA1 Tyrosinase related OCA
Mutations associated with no residual enzyme activity
Tyrosinase negative OCA (OCA1A)
Mutations associated with residual enzyme activity
Yellow OCA (OCA1B)
Minimal Pigm ent OCA (OCA1MP)
Mutation associated with unusual enzyme activity
Tem perature sensitive OCA (OCAITS)

OCA2 Tyrosinase positive OCA

Prader-Willi Syndrom e/Angelm an Syndrome

Unclassified types
Brown OCA
R ufous/red OCA
Primary Defect not Specific for Melanin Synthetic Pathway

Hermansky-Pudlak Syndrome
Chediak-Higashi Syndrome

OCULAR ALBINISM
OA1 X-Linked OA
OA2 Autosomal Recessive OA

T y ro sin a se R e la te d OCA (O CA1)

Clinical phenotype

T yrosinase re la te d O CA is p ro d u c e d by m u ta tio n s o f th e stru c tu ra l g e n e fo r th e

tyrosinase enzym e. A ffected in dividuals have re d u c e d o r a b se n t h a irb u lb tyrosi
nase activity, a n d h etero zy g o u s individuals g en erally have very low activity (King
a n d O lds, 1985; K ing e t al., 1978; King a n d W itkop, 1977). T h e p h e n o ty p e o f
ty ro sin ase-related OCA varies fro m having n o m elan in p ig m e n t in th e skin a n d
eyes to hav in g an u n u su a l p a tte rn o f c u ta n e o u s p ig m e n t. An im p o rta n t c h a ra c te r
istic o f OCA1 is th e m a rk e d h y p o p ig m e n ta tio n at b irth , with m ost affec ted in d i
viduals having w hite hair, milky w hite skin (in co m p a riso n to th e cream y w hite
skin o fte n p re se n t in individuals w ith tyrosinase-positive O C A ), a n d b lu e eyes a t
b irth . T h e irid es can be very lig h t b lu e a n d tra n slu c e n t su ch th a t th e w hole iris
has a p in k o r re d co lo r in a m b ie n t light. W ith tim e, th e irid es o fte n b e c o m e a
d a rk e r b lu e a n d re m a in tra n s lu c e n t o r b e c o m e lightly p ig m e n te d with re d u c e d
translucency. T h e skin re m a in s w hite b u t may a p p e a r to have m o re co lo r with
tim e. S un e x p o su re p ro d u c e s e ry th e m a a n d a b u rn if th e skin is u n p ro te c te d , a n d
a g e n e ra liz e d tan is a b s e n t o r m in im al in m ost, a lth o u g h an occasional individual
w ith OCA1 can tan well, p a rtic u la rly if th e h a ir a n d eye a re p ig m e n te d . Few pig
 HUMAN ALBINISM AND MOUSE MODELS 97

m e n te d lesions (nevi, freckles, len tig in e s) d ev elop in th e skin. T h e clinical p h e

n o ty p e is d e p e n d e n t o n th e type o f m u ta tio n p re se n t, a n d th e v ariatio n in
p h e n o ty p e is m o re e x tre m e th a n previously reco g n ized . It is e x p e c te d th a t fu tu re
stu d ies will b ro a d e n th is g ro u p to in c lu d e individuals w ho have n early n o rm a l
p ig m e n t asso ciated w ith m u ta tio n s o f th e tyrosinase g en e th a t only m inim ally
re d u c e activity o f th e enzym e.
Ind iv id u als w ith tyrosinase negative O CA are b o rn w ith w hite h a ir a n d skin a n d
b lu e eyes, a n d th e re is n o m e la n in synthesis in th ese tissues th r o u g h o u t life (Wit-
k o p e t al., 1989; K ing a n d O lds, 1985; K ing a n d W itkop, 1976). T h e p h e n o ty p e is
th e sam e in all e th n ic g ro u p s. W ith tim e, th e h a ir m ay d evelop a d a rk e r w hite o r a
slight yellow tin t th a t varies w ith d iffe re n t sham poos. T h e irid es are tra n slu c e n t
a n d a p p e a r p in k early in life a n d o fte n tu rn a gray-blue co lo r with tim e. N o pig
m e n te d lesio n s d ev elo p in th e skin, a lth o u g h a m e lan o tic nevi can b e p re se n t. In
th e U.S., th e fre q u e n c y o f tyrosinase negative O CA is e stim a ted to b e 1 in 39,000
in th e C aucasian a n d 1 in 28,000 in th e A frican-A m erican p o p u la tio n (W itkop
e ta l., 1989).
Yellow O CA was o rig in ally th o u g h t to re p re s e n t a d iscrete type o f O CA w ith a
c h a ra c teristic p h e n o ty p e b u t m o le c u la r stu d ies now show th a t this is p a rt o f O C A 1.
T h e te rm yellow O C A re fe rs to th o se types o f tyrosinase re la te d O CA p ro d u c e d by
m u ta tio n s asso ciated w ith resid u a l tyrosinase fu n c tio n (leaky m u ta tio n s). T h e
a m o u n t o f re sid u a l enzym e fu n c tio n is sufficient to p ro d u c e sm all to m o d e ra te
a m o u n ts o f iris, h a ir a n d p e rh a p s skin p ig m e n t.
Yellow O CA was first re c o g n iz e d in th e Am ish (N an ce et al., 1970) a n d subse
q u en tly id e n tifie d in o th e r g ro u p s. Fam ily stu d ies suggested th e resp o n sib le g e n e
was allelic to th a t fo r tyrosinase-negative OCA (H u et al., 1980) a n d m o le c u la r
stu d ies eventually d e m o n s tra te d several d iffe re n t m u ta tio n s o f th e tyrosinase g e n e
in this type o f OCA. T h e p h e n o ty p e o f yellow OCA varies, b u t all affected individ
uals have w hite h a ir a n d skin a n d blu e eyes at b irth . H a ir a n d iris p ig m e n t develop
in th e first few years o f life. T h e h a ir c o lo r ch an g es to lig h t yellow, lig h t b lo n d o r
g o ld e n b lo n d first, a n d eventually can tu rn d a rk b lo n d in th e ad u lt (G iebel e t al.,
1991 d; N an ce e t al., 1970). T h e irid e s can d evelop lig h t tan p ig m e n t, p articu larly
in th e in n e r th ird o f th e iris, a n d th e skin m ay tan w ith sun ex p o su re. E th n ic a n d
fam ily p ig m e n t p a tte rn s can in flu e n c e th e p h e n o ty p e , a n d h a ir co lo r can tu rn
lig h t re d o r brow n. T h e fre q u en cy o f this type o f m u ta tio n is u n k n o w n , b u t does
n o t a p p e a r to be ra re (G iebel e t al., 199Id ; W itkop et al., 1989).
A n o th e r OCA1 type asso ciated w ith som e m e la n in fo rm a tio n is m in im al p ig
m e n t O CA (K ing et al., 1986). T h e o rig in a l d e sc rip tio n o f m in im al p ig m e n t OCA
in c lu d e d a p h e n o ty p e sim ilar to tyrosinase negative O CA ex cep t fo r th e d ev elo p
m e n t o f m in im al a m o u n ts o f iris p ig m e n t in th e first d ec ad e o f life, a n d b io c h e m
ical ev alu atio n su g g ested th a t this was a tyrosinase re la te d OCA. M o lecular analysis
has id e n tifie d m u ta tio n s o f th e tyrosinase g e n e in individuals w ith this type (O et-
tin g a n d King, 1993). It is h y p o th esize d th a t th e resp o n sib le m u ta n t allele is asso
c iated w ith less re sid u a l enzym e activity th a n a yellow allele, a n d th e p ig m e n t is
seen prim arily in th e iris b ecau se this is a stable r a th e r th a n a d e c id u o u s p ig m e n t
98 RICHARD A. KING ET AL.

tissue (i.e. th e p ig m e n t can a c c u m u la te w ith o u t b e in g sh ed w ith tu rn o v e r o f th e

tissue). It m ay be m o re a p p ro p ria te to in c lu d e m in im al p ig m e n t O CA w ithin the
b ro a d ra n g e o f yellow O CA
A ffected individuals have w hite skin a n d h a ir a n d b lu e eyes at b irth . Iris p ig m e n t
is p re s e n t a t b irth o r d evelops in th e first d ecad e. T h e h a ir re m a in s w hite o r devel
o ps a very slight yellow tin t. T h e skin rem ain s milky w hite a n d ta n n in g has n o t
b e e n o b serv ed . To d ate, all in d iv iduals rec o g n iz e d with this type o f OCA have b e e n
C au casian , a n d th e p h e n o ty p e in o th e r e th n ic g ro u p s is u n k n o w n . T h e freq u en cy
is n o t know n. P la tin u m O CA (W itkop et a l , 1989) m ost likely re p re se n ts this sam e
p h e n o ty p e .
A very in te re s tin g a n d u n u su a l tyrosinase re la te d OCA has recen tly b e e n d e
sc rib e d (K ing e t al., 1991b). T h e p ro b a n d o f th e o rig in al fam ily a p p e a re d to have
tyrosinase negative O CA th ro u g h th e first years o f h e r life, w ith w hite h a ir a n d
skin, a n d b lu e eyes. W ith p u b e rty h e r body h a ir c o lo r c h a n g e d . T h e axillary h a ir
re m a in e d w hite, th e scalp h a ir also re m a in e d w hite b u t w ith a slig h t yellow tin t, the
a rm h a ir tu r n e d lig h t re d d ish brow n, a n d th e leg h a ir tu r n e d d a rk brow n. T h e
eyes re m a in e d b lu e a n d th e skin re m a in e d w hite. Analysis o f tyrosinase fro m scalp
a n d leg h a irb u lb s show ed th a t th e enzym e was te m p e ra tu re sensitive, losing activ
ity above 35C. As a resu lt, p ig m e n t c o u ld fo rm in th e c o o le r b u t n o t th e w arm er
p a rts o f th e body. M o lecu lar analysis show ed th a t th e p ro b a n d was a c o m p o u n d
h etero zy g o te a t th e tyrosinase locus, having two d iffe re n t m issense m u ta tio n s th a t
in c lu d e d th e te m p e ra tu re sensitive allele a n d a tyrosinase-negative allele (G iebel
e t al.,1991c). T h e re w ere th re e sim ilarly affec ted siblings in th e fam ily a n d n o o th
e r fam ily o f this type has b e e n p u b lish e d to d ate. T his type o f tyrosinase re la te d
O CA is an a lo g o u s to th e Siam ese cat a n d th e H im alayan m ouse.

Molecular phenotype

Analysis has show n th a t m u ta tio n s in th e tyrosinase g en e a re resp o n sib le fo r

OCA1. DNA se q u e n c e analyses o f th e g en o m ic DNA fro m individuals w ith OCA1
h as rev ealed th a t m any d iffe re n t m u ta tio n s are re sp o n sib le fo r this single g e n e
d is o rd e r (O e ttin g a n d King, 1993). A t p re s e n t 25 m issense, 4 n o n se n se a n d 9
fra m e sh ift m u ta tio n s have b e e n re p o rte d fo r a to tal o f 38 d iffe re n t m u tatio n s.
T h is analysis has show n th a t m ost individuals with OCA1 a re c o m p o u n d hetero zy
g o tes w ith d iffe re n t m u ta n t m a te rn a l a n d p a te rn a l alleles. T h ese individuals have
all c o m b in a tio n s o f m issense, n o n se n se a n d fram esh ift m u ta tio n s. A pp ro x im ately
15% o f all in d iv id u als in w hich th e e n tire c o d in g re g io n a n d flan k in g in tro n
se q u e n c e s have b e e n se q u e n c e d have show n only o n e m u ta tio n a n d in these
cases th e c o rre c t n u c le o tid e is displayed o n th e h o m o lo g o u s allele to th a t o f th e
m u ta tio n show ing th a t they a re c o m p o u n d heterozygotes. T h e se c o n d m u ta tio n
is th o u g h t to be w ithin a re g u la to ry reg io n th a t is o u tsid e o f th e reg io n
se q u e n c e d o r in th e in tro n se q u en c es p ro d u c in g a cryptic splice site o r affectin g
an in tro n ic tra n sc rip tio n a l reg u lato r. M ost o f th e se m u tatio n s have b e e n fo u n d in
in div id u als w ith th e classic tyrosinase negative type o f OCA1.
 HUMAN ALBINISM AND MOUSE MODELS 99

T h e m ajo rity o f tyrosinase m u ta tio n s have b e e n fo u n d in C aucasian individuals

b u t analysis o f in d iv id u als w ith d iffe re n t racial b a c k g ro u n d s are revealing m u ta
tions th a t seem to be u n iq u e to th a t g ro u p o f p eo p le. Two d iffe re n t m u ta tio n s
have b e e n fo u n d in J a p a n e s e individuals, a m issense m u ta tio n at c o d o n 77 (R77Q)
re su ltin g in an a rg in in e to g lu ta m in e su b stitu tio n a n d a single base in se rtio n o f a
cytosine a t c o d o n 310 (+AC310) re su ltin g in a p re m a tu re te rm in a tio n at co d o n
316 (T om ita e t al., 1989; K ikuchi e t al., 1990; T ak ed a et al., 1990). T h e re have also
b e e n re p o rte d a m issense m u ta tio n in an A m erican black a t c o d o n 89 (cysteine to
a rg in in e , C 8 9 R ), a n o n se n se m u ta tio n a t c o d o n 178 in an A fghan in dividual (try p
to p h a n to te rm in a tio n , T178X ) a n d a d in u c le o tid e d e le tio n in a P ak astani individ
u al (-AGA115) (G iebel e t al., 1991a; Spritz et al., 1991; O e ttin g et al., 1993a).
M ap p in g th e lo c a tio n o f th e m issense m u ta tio n s shows th a t they clu ster in to
th re e d istin c t re g io n s o f th e tyrosinase c o d in g re g io n (F igure 1). Two o f th ese clus
ters a re w ith in th e p u tativ e c o p p e r A o r c o p p e r B b in d in g re g io n s w hile th e th ird
is lo c a te d at th e 5 e n d o f th e c o d in g se q u e n c e . It is th o u g h t th a t th ese clusters d e
fine fu n c tio n a l areas o f th e tyrosinase enzym e (K ing et al., 1991a; O e ttin g a n d
King, 1 9 9 2 a). T h e re are several possibilities as to how th ese m u ta tio n s c o u ld cause
th e ir e ffect o n tyrosinase activity. F o r th e p u tativ e c o p p e r A a n d c o p p e r B b in d in g
re g io n th e m o st obvious e x p la n a tio n is th a t the m u ta tio n s a lte r th e p ro te in stru c
tu re in su ch a way th a t th e c o p p e r a to m s a re n o t co rrectly c o o rd in a te d w ith th e
h istid in e lig an d s th a t c o m p lex w ith th e c o p p e r atom s (O e ttin g a n d King, 1992b).
In th e c o p p e r A b in d in g re g io n , two o f th e se m u ta tio n s are w ithin c o n serv ed am i
n o acid m otifs. T h e m u ta tio n F176I alters a p h e n y la la n in e in th e m o tif Phe-X-X-
X-His th a t is c o n se rv e d in th e c o p p e r b in d in g c e n te rs fo u n d in tyrosinase a n d
h e m o c y a n in (V olbeda a n d H oi, 1 9 8 9 a,b ). A seco n d m u ta tio n , A206T, is w ithin th e
m o tif Pro-X-Phe-X-X-X-His th a t is c o n se rv e d in all tyrosinase p o ly p ep tid es in c lu d
ing fu n g al tyrosinase m o lecu les (H u b e r e t al., 1985). F o u r o f th e m issense m u ta
tions, a t c o d o n s 371, 373, 382 a n d 383, are in a lo o p stru c tu re o f th e c o p p e r B
b in d in g re g io n b etw een th e a-h e lice s re sp o n sib le fo r th e c o rre c t p la c e m e n t o f the
th re e h istid in e c o p p e r ligands. T h e se m issense m u ta tio n s in th e c o p p e r A a n d
c o p p e r B b in d in g re g io n s are n e x t to h istid in e re sid u e s w hich a re resp o n sib le fo r
th e c o rre c t o rie n ta tio n o f th e two c o p p e r ato m s in th e catalytic site o f th e enzym e.
C o m p u te r m o d e lin g o f th e c o p p e r B b in d in g re g io n shows p e rtu rb a tio n s in the
se c o n d a ry s tru c tu re w h en th ese m u ta tio n s are su b stitu ted fo r th e n o rm a l se
q u e n c e . T h e ju x ta p o s itio n o f th e two c o p p e r ato m s is critical b ecau se o f th e n e e d
to fo rm a p e ro x id e w ith d ioxygen w hich is n ecessary fo r catalytic fu n c tio n a n d any
a lte ra tio n in th e c o p p e r to c o p p e r d istan ce w ould in te rfe re w ith th e p e ro x id e
b rid g e a n d r e n d e r th e enzym e inactive. T h ese m u ta tio n s may e lim in a te enzym atic
activity by a lte rin g th e lo c a tio n o f th e h istid in e lig an d s a n d a lte rin g co pper-pro-
tein b in d in g (O e ttin g a n d King, 1992b). A se c o n d possible m ec h an ism is th a t
th ese d o m a in s also c o n ta in a su b strate b in d in g reg io n o r a n o th e r catalytic site af
fe c te d by th ese m u ta tio n s. T h e b in d in g o f tyrosine o r DOPA sh o u ld o c cu r n e x t to
th e c o p p e r ato m s a n d if th ese a m in o acids are involved in this fu n c tio n , th e n even
a slig h t c h a n g e in th e s tru c tu re o f th is re g io n o r th e ch em ical n a tu re o f an a m in o
acid side c h a in m ig h t a ffect enzym atic activity. T h e th ird cluster, at th e a m in o te r
100 RICHARD A. KING ET AL.

m in al o f th e enzym e (c o d o n s 42-89), c o u ld re p re s e n t a stru c tu ra l d o m a in th a t in

volves a catalytic site o f th e enzym e. O n e m u ta tio n in this cluster, R77Q , resu lts in
th e rem oval o f a positively c h a rg e d a m in o acid (arg in in e) a n d is su b stitu te d by a
u n c h a rg e d a m in o acid (g lu ta m in e) (S h ib a h a ra e t al., 1990; S h ib a h ara, 1992). T h e
ro le o f a rg in in e in enzym e active sites as an a n io n ic b in d in g site has b e e n re p o rte d
in over o n e h u n d r e d enzym es a n d chem ical m o d ific atio n o f this resid u e resu lts in
in activ atio n o f th e enzym e. T h e a rg in in e a t c o d o n 77 may play a ro le in th e cata
lytic site o f tyrosinase a n d rem oval o f this a m in o acid c o u ld d is ru p t th e in te ra c tio n
o f th e tyrosine su b stra te o r d o p a co-factor w ith th e active site o f this enzym e (Ri-
o rd a n , 1979). T h e clu ster at th e c o p p e r B b in d in g site e x ten d s 3 o f this re g io n
in to ex o n 4. T h is a re a o f th e c lu ste r in clu d e s a yellow OCA1 m u ta tio n (P406L) a n d
th e te m p e ra tu re sensitive OCA1 m u ta tio n . T his re g io n is rem o v ed fro m th e co p
p e r B b in d in g site a n d m ay b e a sep arate fu n c tio n a l d o m a in , possibly involved in
th e su b strate b in d in g re g io n o f th e catalytic site.

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O Z O O 3 3 o O > > ^ - l q - ro ) ro O cow w w u h & ft H O
r o -* cn '"J co co > - T ^ <> o o *nI 'J -i co <x> o j g o o - 1^
r o - v jc n _ .c d c o - * - ^ 03 { o i\> ct> 05 cn -* -w c o ro c o & M co o ?5 c o c n c o co <
co o2
cn O Z < D r H r a c n S x iS W - i S S D x l -n T o D ia m O m m z x CO .

rim r h i m M n 5\
2 1 3 H 4 -CD i

u
Cu (A)
u
Cu (B)

Figure 1 L ocation ol th e M utations in th e T yrosinase G en e A ssociated with ()( A 1

T h e boxes re p re s e n t th e a ex o n s o f th e tyrosinase co d in g re g io n a n d th e C u(A ) a n d C u(B )
labels re p re s e n t th e c o p p e r b in d in g regions. A rrow s show th e lo catio n o f th e m issense, n o n
sense a n d fra m e sh ift m u tatio n s asso ciated w ith OCA1. T h e o u tlin e d tex t re p re se n ts no n -
p a th o g e n ic poly m o rp h ism s. T h e black b ars show th e fo u r re g io n s w h e re th e m issense
m u ta tio n te n d to cluster.

T h e re a re m u ta tio n s o f th e tyrosinase g e n e th a t re d u c e b u t d o n o t com p letely

e lim in a te th e level o f tyrosinase activity, a n d m u ta tio n s th a t c o n fe r te m p e ra tu re
sensitivity to th e enzym atic activity. T h ese m u ta tio n s w ere fo u n d in individuals
w ith yellow OCA1, w ho have p artia l p ig m e n ta tio n in th e ir h a ir a n d eyes (G iebel
e t al., 1991d). A nalysis o f th e A nush fam ilies in w hich this was originally
d e sc rib e d has show n th a t th e affec ted individuals are hom ozygous fo r a c o d o n
406 m issense m u ta tio n (P406L) re su ltin g in a p ro lin e to le u cin e su b stitu tio n
(G ieb el et al., 1991d; N an ce e t al., 1970). E x p ressio n studies in H eL a cells show
th a t this m u ta tio n re d u c e s enzym atic activity to 7% o f th e wild type levels. A
c o d o n 275 m issense m u ta tio n (valine to p h en y la la n in e ; V275F) has b e e n fo u n d
in in d iv id u als w ith this p h e n o ty p e w ho are c o m p o u n d h eterozygotes, w ith the
m u ta tio n o n th e h o m o lo g o u s allele (co d o n 81 p ro lin e to le u cin e ; P81L) fo u n d in
 HUMAN ALBINISM AND MOUSE MODELS 101

tyrosinase neg ativ e OCA1 (su p p o sed ly an allele associated with n o tyrosinase
fu n c tio n ). S ince th e yellow QCA1 p h e n o ty p e varies in fam ilies c o n ta in in g individ
uals hom ozy g o u s fo r th e P406L m u ta tio n as well as in individuals w ho are co m
p o u n d h etero zy g o tes, it a p p e a rs th a t o th e r fam ilial p ig m e n t g en es can in flu e n c e
this p h e n o -ty p e (G iebel e t al., 1991d). In th e case o f re d u c e d tyrosinase activity,
re d /y e llo w p h e o m e la n in b iosynthesis is favored over b la c k /b ro w n e u m e la n in
biosynthesis.
In o n e in d iv id u al, a te m p e ra tu re-sen sitiv e fo rm o f tyrosinase w7as o b se rv ed (see
above) (K ing et al., 1 9 9 1 b ). T his in d iv id u al was fo u n d to have a m u ta tio n at c o d o n
422 (R 422Q ) re su ltin g in an a rg in in e to g lu ta m in e su b stitu tio n w ith a tyrosinase
n egative OCA1 m u ta tio n o n th e h o m o lo g o u s allele (G iebel et al., 1991c). E xpres
sion o f th e 422 c o d o n m u ta tio n p ro d u c e d a tyrosinase th a t was in activ ated above
35C. It is in te re s tin g th a t th e m o u se H im alayan m u ta tio n , also a te m p e ra tu re sen
sitive tyrosinase m u ta tio n , is lo c a te d in th e sam e reg io n o f th e p e p tid e , c o d o n 420
(h istid in e to a rg in in e ) (Kwon e t al., 1989). T h e re has also b e e n re p o rte d a tem
p e ra tu re sensitive m u ta tio n a t c o d o n 402 (R 402Q ) (T rip ath i et al., 1991). It is now
obvious th a t th e re is a c o n tin u o u s sp e c tru m o f re d u c e d tyrosinase activities re su lt
in g fro m m u ta tio n s o f th e tyrosinase g e n e (K ing e t al., 1988; K ing a n d O e ttin g ,
1992). T h ese a lte re d tyrosinase m o lecu les p ro d u c e p h e n o ty p ic v ariatio n fro m a
co m p le te ab se n c e o f p ig m e n t to a p ig m e n t p h e n o ty p e in d istin g u ish a b le fro m n o r
m al. In all th ese cases o f OCA1 th e re is o c u la r involvem ent. T his sp e c tru m o f pig
m e n t has also b e e n o b se rv e d in th e c-Iocus m u ta tio n s o f th e m o u se (Silvers, 1979).
Several n o n p a th o g e n ic p o ly m o rp h ism s have also b e e n d e te c te d . Two o f these
re su lt in a m in o acid su b stitu tio n s. A p o ly m o rp h ism a t c o d o n 192 c o n tain s e ith e r
a serin e (TAT) o r tyrosine (TC T) a n d affects a Mbo I re stric tio n e n d o n u c le a se site
(G iebel a n d Spritz, 1990). It is in te re stin g th a t this m u ta tio n at c o d o n 192 is w ithin
th e c o p p e r A b in d in g site b u t d o es n o t a p p e a r to affect th e p h e n o ty p e o f th e in d i
vidual. T h e d istrib u tio n o f this p o ly m o rp h ism is eq u al in C aucasian p o p u la tio n s,
b u t only th e T C T c o d o n is fo u n d in th e O rie n ta l p o p u la tio n . T h e seco n d polym or
p h ism is at c o d o n 402 a n d is e ith e r a n a rg in in e o r g lu tam in e. T h is poly m o rp h ism
d o es affect tyrosinase activity by re d u c in g enzym atic activity by 75% w hen ex
p ressed in c u ltu re d cells b u t th e clinical p h e n o ty p e is n o t a lte re d (T rip ath i e t al.,
1991). A th ird p o ly m o rp h ism is a t th e C C A \T T bo x o f th e p ro m o te r a n d affects a
Taq I restric tio n e n d o n u c le a se site (O e ttin g et al., 1991). A n o th e r po ly m o rp h ism
lo c a te d in th e p ro m o te r reg io n is a G A -cluster 300 bp u p stre a m o f th e sta rt site;
fo u r alleles have b e e n d e te c te d u sing PC R am p lificatio n (M orris e t al., 1991). Two
o th e r p o ly m o rp h ism s are fo u n d by m e a n s o f R estriction F ra g m e n t L e n g th Poly
m o rp h ism s (RFLP) analysis w ith e ith e r Taq I o r Bgl II a n d u sin g th e tyrosinase
cDNA as a p ro b e (Spritz e t al., 1988; Spritz a n d S tru n k , 1990). H a p lo ty p e analysis
can be p e rfo rm e d w ith analysis o f th e se p o ly m o rp h ic sites. T h e m issense m u tatio n
a t c o d o n 47 (G47D) has b e e n o b se rv e d in th re e d iffe re n t p o p u la tio n s, U n ite d
States, P u e rto Rico a n d th e C a n ary Islands (O e ttin g e t al., 1993b). All individuals
h a d th e G 47D m u ta tio n asso ciated w ith h a p lo ty p e 1 show ing a c o m m o n fo u n d e r
fo r this m u ta tio n . O n th e o th e r h a n d , th e m u ta tio n a t c o d o n 81 (P81L) was fo u n d
102 RICHARD A. KING ET AL.

in in d iv id u als fro m th e U n ite d States a n d th e C an ary Islands, b u t th e two reg io n s

h a d two d iffe re n t hap lo ty p es, h a p lo ty p e 1 b e in g associated w ith th e P81L m u ta
tio n in individuals fro m th e C an ary Islands a n d h ap lo ty p e 4 associated with th e
U n ite d States, show ing th e possibility o f two d iffe re n t fo u n d e rs fo r th e sam e m u
tatio n .

M ouse T yrosinase M utations

T h e recessive m o u se albino m u ta tio n was o n e o f th e first g en es to b e stu d ie d a fter

th e red isco v ery o f M e n d e ls Laws (B ateson, 1903). T h e p h e n o ty p e o f th e m u ta n t
an im al is a cle a r h o m o lo g u e o f h u m a n OCA1; th e re is n o hair, skin o r eye p ig
m e n t synthesised, a n d n o tyrosinase activity is d e tec tab le. T h e iris is n o t n o rm ally
e x te rn a lly visible in th e m o u se, so th e d a rk p ig m e n t seen in th e eye is th e p ig
m e n te d re tin a l ep ith e liu m . Lack o r re d u c tio n o f eye p ig m e n t results in th e eye
a p p e a rin g p in k , re d o r ru b y -co lo u red , d e p e n d in g o n severity. M utants with
a b se n t o r m u c h re d u c e d eye p ig m e n t also have, like th e ir h u m a n c o u n te rp a rts,
d efective vision d u e to a re d u c e d n u m b e r o f ip silateral o p tic n erv e c o n n e c tio n s
to th e b ra in . (G uillery, 1974; LaVail e t al., 1978)
T h e tyrosinase g e n e fro m th e classical albino m u ta tio n has b e e n se q u e n c e d , a n d
in fact e n c o d e s a p ro te in w ith two a m in o acid d iffere n ces fro m wild-type (Shiba-
h a ra e t al., 1990). T h ese a re C103S a n d R405H (n u m b e rin g is the sam e as th e h u
m an e n z y m e ). Several lines o f evidence in d ica tes th a t only o n e o f th ese changes,
C103S is causative o f th e albino m u ta tio n . Firstly, a tyrosinase g e n e with only this
c h a n g e is inactive in ex p ressio n in c u ltu re d cells (S h ib ah ara, 1990) o r in tra n sg e n
ic m ice (Yokoyama e t al., 1990). S eco n d , a p ig m e n te d clone, a p p a re n tly a rever-
ta n t to w ild-type, was id e n tifie d in a c u ltu re d albino m elanocyte line. S e q u e n c in g
o f th e tyrosinase g e n e show ed th a t th e re v e rta n t cells h a d c h a n g e d th e SI 03 back
to th e w ild-type cysteine, b u t only o n o n e c h ro m o so m e (Ja c k so n a n d B e n n e tt,
1990). All co m m o n ly u sed albino m ouse lin es have this sam e m u ta tio n , in d ic a tin g
th a t they all d erive fro m a c o m m o n a n c e sto r (Ja c k so n a n d B en n ett, 1990; Yokoya
m a e t al., 1990). Cysteine-103 is an essential p a rt o f th e E G F-m otif a n d is c o n serv ed
in h u m a n tyrosinase, b u t has n o t b e e n seen to be m u ta n t in any OCAI p a tie n ts so
far. O th e r m u ta tio n s have b e e n seen in th e EGF m otif; cysteine-89 is p a rt o f th e
m otif, w hich is m u ta te d to a rg in in e in h u m a n tyrosinase causing OCAI (Spritz
e t al., 1991) a n d th e C111Y c h a n g e w hich causes th e brown m u ta tio n in m ouse
TRJP-1 is also in th e m o tif (Z darsky et al., 1990).
T h e re a re n u m e ro u s alleles o f th e classic albino m u ta tio n . Som e have th e sam e
lack o f fu n c tio n p h e n o ty p e , a n d are d u e to o th e r (as yet u n c h a ra c te rise d ) p o in t
m u ta tio n s o r to d e le tio n s o f th e g e n e (Yokoyama et al., 1990; R inchik e t al.,
1993a). M ore in te re stin g fro m a co m p arativ e g e n e tic p o in t o f view are th e alleles
giving p h e n o ty p e s in te rm e d ia te b etw een a lb in o a n d full p ig m e n ta tio n . A n u m b e r,
such as chinchilla, ruby-eyed dilute, extreme dilution, a n d platinum fo rm a series, with
 HUMAN ALBINISM AND MOUSE MODELS 103

d e c re a sin g a m o u n ts o f p ig m e n t, re su ltin g in progressively p a le r coats g o in g alo n g

th e series, a n d eyes ra n g in g fro m alm o st black to p in k (Silvers, 1979). T h e p h e n o
typic series c o rre s p o n d s to d e c re a sin g levels o f d e te c ta b le tyrosinase activity, a n d
th ese m u ta n ts are likely to be m ouse m o d els o f yellow OCA. A n o th er, C-44H has
alm o st n o c o a t p ig m e n ta tio n , b u t eyes w hich d a rk e n a fte r b irth to b ec o m e nearly
b lack a t a few m o n th s o f age. Possibly this allele is a m o d el o f m inim al p ig m e n t
O CA o r p la tin u m OCA.
T h e m o le c u la r n a tu re o f th e ch a n g e s in th e tyrosinase g e n e o f th ese p artia l o r
leaky m u ta tio n s is likely to be very info rm ativ e, b u t w ith th e e x c e p tio n o f chin
chilla th e se have n o t b e e n stu d ie d in d etail. T h e chinchilla tyrosinase g e n e c o n tain s
a p o in t m u ta tio n A 482T (B e erm an e t al., 1990), lo ca ted in th e tran sm em -b ran e
re g io n o f th e e n c o d e d enzym e, w hich may b e th e cause o f d e c re a sed activity, al
th o u g h a n e ffect o f th e m u ta tio n o n m RN A levels has n o t b een rig o u ro u sly ru le d
o u t. T h e tem p eratu re-sen sitiv e tyrosinase o f th e Himalayan m u ta tio n was re fe rre d
to above. T h e an im als a re largely very lightly p ig m e n te d , b u t have d a rk ears, nose,
tail a n d sco tu m a n d re d eyes. T h e te m p e ra tu re senstivity o f th e enzym e results
fro m an H 420R c h a n g e (Kwon e t al., 1989). O n e o th e r m u ta tio n has b e en su bject
to m o le c u la r study, chinchilla-mottled, an im als w hich have p a tc h e s o f c h in ch illa fu r
a n d p a tc h e s o f lig h te r fur. T h e chinchilla-mottled tyrosinase stru c tu ra l g e n e seem s
n o rm a l, b u t th e re is a DNA re a rra n g e m e n t, in se rtio n o r d e le tio n lo c a te d several
kilobases u p stre a m . (P o rte r e ta l., 1991) T h e re a rra n g e m e n t affects tra n sc rip tio n
o f th e g e n e , b u t to a v ariab le e x te n t in d iffe re n t clones. T ran sg en ic anim als m ad e
w ith a tyrosinase m in ig e n e , a n d with 5k b o f u p stre a m DNA do n o t always have n o r
m al levels o f g e n e e x p ressio n ( B e e rm a n e t al., 1990). T ran sg e n es c a rry in g th e full
tyrosinase g e n e , p lu s !5 0 k b u p stre a m re su lt in p o s itio n -in d e p e n d e n t ex p ression
(S chedl et al., 1993), in d ic a tin g a re q u ire m e n t fo r seq u en ce e le m e n ts betw een
-5kb a n d -150kb fo r full-level exp ressio n .

T yrosinase Positive O C A (O C A 2)

Overview

A n u m b e r o f o b se rv a tio n s have b e e n m ad e th a t b roadly d efin e this type o f OCA.

First, fresh scalp a n a g e n h a irb u lb s fro m an affected in dividual fo rm visible pig
m e n t w h en in c u b a te d in tyrosine o r d o p a (W itkop e t al., 1970) a n d th e m e lan in
in these h a irb u lb s is synthesized in th e m elan o so m es w hen th e in c u b a te d h air
b u lb s a re o b serv ed by e le c tro n m icroscopy (W itkop et al., 1970). H istorically, this
o b se rv a tio n was th e first to se p a ra te h u m a n OCA in to two types, as h a irb u lb s
fro m an in d iv id u al w ith tyrosinase negative OCA d o n o t fo rm p ig m e n t w hen in c u
b a te d in tyrosine o r d o p a u n d e r id e n tic a l co n d itio n s (W itkop et al., 1970). Sec
o n d , th e activity o f tyrosinase in h a irb u lb s fro m affected individuals is n o m a l o r
n early n o rm a l w h en m e a su re d by a q u a n titativ e assay o r ev alu ated by e le c tro
p h o re sis (K ing a n d O lds, 1985; K ing e t al., 1978; K ing a n d W itkop, 1976). T h ird ,
104 RICHARD A. KING ET AL.

Table 2 Mouse Mutations as Models for Human Albinism

OCA1 albino (c) locus mutations

OCA1A albino
OCA1B, OCA IMP chinchilla
ruby-eyed dilute
extreme dilution
platinum
OCA1TS Himalayan
OCA2 pink-eye dilution locus
multiple mutations

Hermansky-Pudlak Syndrome Mutations at 12 loci:

Chediak-Higashi Syndrome beige, cocoa, pale ear, light ear,
mocha, muted, pallid, pearl,
reduced pigmentation, ruby-eye,
ruby-eye-2, sandy

th e u ltra s tru c tu re o f th e m elan ocyte a n d th e m e lan o so m e a p p e a r to b e n o rm a l

(W itkop e t al., 1973). F o u rth , th e g e n e (o r g enes) resp o n sib le fo r this type o f
O C A is n o t g en etically lin k e d to th e tyrosinase g e n e (H eim et al., 1988).
T h e g e n e ra l p h e n o ty p ic fe a tu re s o f this type o f OCA in c lu d e th e p re se n c e o f
h a ir a n d iris p ig m e n t at b irth o r early in life, a n d th e d e v e lo p m e n t o f localized
(nevi, freckles, a n d len tig in es) skin p ig m e n t, o fte n in sun ex p o se d re g io n s o f th e
skin (W itkop e t al., 1989). T h e e th n ic a n d fam iliar p ig m e n t b a c k g ro u n d o f th e af
fe c te d in d iv id u al has a m o re p ro fo u n d effect o n tyrosinase positive O C A th a n o n
tyrosinase re la te d O C A (W itkop e t al., 1989). M ost a ffec ted individuals a c c u m u
late p ig m e n t in th e ir h a ir a n d eyes d u rin g th e ir lifetim e, a n d this is m o re m a rk e d
in in d iv id u als from d a rk e r e th n ic g roups.
B roadly sp eak in g , any o r all o f th e m any m o u se coat-co lo u r m u ta tio n s c o u ld be
c a n d id a te s fo r m ouse h o m o lo g u e s o f th e h u m a n diso rd ers. How ever, only a frac
tio n o f th e m u ta tio n s affect eye p ig m e n ta tio n to a significant d e g re e . L e ad in g
am o n g st th ese is th e pink-eyed dilution (p) m u ta tio n , o n c h ro m o so m e 7, w hich has
n u m e ro u s alleles w hich p ro d u c e d ilu te d h a ir p ig m e n ta tio n a n d p in k eyes (Silvers,
1979).

Clinical phenotype

In C aucasian individuals, th e a m o u n t o f p ig m e n t p re s e n t a t b irth o r d ev elo p in g

w ith tim e varies from m in im al in n o r th e r n E u ro p e a n (p artic u larly S candinavian)
to m o d e ra te in s o u th e rn E u ro p e a n o r M e d ite rra n e a n individuals. T h e h a ir can
b e very lightly p ig m e n te d at b irth , having a lig h t yellow o r b lo n d color, o r m o re
p ig m e n te d w ith a d e fin ite b lo n d , g o ld e n b lo n d o r even re d color. T h e skin is
cream y w hite a n d p ig m e n te d spots may be p re se n t. T h e iris c o lo r is blue-gray o r
lightly p ig m e n te d , a n d th e d e g re e o f tra n slu cen cy c o rre la te s w ith th e a m o u n t o f
iris p ig m e n t p re se n t. W ith tim e, p ig m e n te d nevi a n d le n tig in e s may d e v elo p a n d
 HUMAN ALBINISM AND MOUSE MODELS 105

p ig m e n te d freckles a re seen in e x p o se d areas with re p e a te d su n ex p o su re. T h e

skin d o e s n o t tan. T h e h a ir slowly tu rn s d a rk e r th ro u g h th e first two o r m o re
d e c a d e s o f life.
In m o st A frican-A m erican in d iv id u als a n d those fro m west A frica, th e p h e n o
type o f tyrosinase-positive O CA is d istin c t (K ing et al. 1980). T h e h a ir is yellow at
b irth a n d re m a in s yellow with tim e, a lth o u g h th e c o lo r may tu rn darker. T h e skin
is cream y w hite a t b irth a n d ch an g e s little with tim e. P ig m e n te d nevi, len tig in es
a n d freckles d evelop, b u t th e re is n o tan. T h e irides are b lu e /g r a y o r lightly p ig
m e n te d . T h is p h e n o ty p e is p e rh a p s th e m ost c o m m o n in th e w orld because o f th e
h ig h fre q u e n c y o f this type in w est A frica (K ing et al., 1980; O k o ro , 1975). In the
A frican-A m erican p o p u la tio n , th e re is som e p h e n o ty p ic v ariatio n w ithin this type,
p artic u la rly in th e h a ir c o lo r w hich m ay be brow n, a u b u rn , o r re d r a th e r th a n yel
low at b irth o r w ith tim e.
A newly re c o g n iz e d asso ciation w ith OCA2 is the h y p o p ig m e n ta tio n fo u n d w ith
P rader-W illi sy n d ro m e (PWS) a n d A n g elm a n S yndrom e (AS) (K ing e t a l.,1993;
W iesn er e t al., 1987; B utler, 1989; B u tler e t al., 1986). PWS is a d e v elo p m en ta l syn
d ro m e th a t in c lu d e s n e o n a ta l h y p o to n ia , h y p e rp h a g ia a n d obesity, hyp o g o n ad ism ,
sm all h a n d s a n d feet, a n d m e n ta l r e ta rd a tio n associated w ith c h a rac teristic behav
io r (Cassidy, 1984; Bray e t al., 1983). A p p ro x im ately 70% o f individuals w ith PWS
have a d e le tio n o n th e lo n g a rm o f th e p a te rn a l c h ro m o so m e 15 (B utler
et a l.,1986; L e d b e tte r et al., 1982; M u tira n g u ra e t al., 1993), a n d m o st o f those
w ith o u t a d e le tio n have u n ip a re n ta l disom y fo r th e m a te rn a l c h ro m o so m e 15 (Ma-
g en is et al., 1990; M ascari et al., 1992).
A p p ro x im ately 50% o f individuals w ith PWS are h y p o p ig m e n te d b u t are w ith o u t
all o f th e typical fe a tu re s o f alb in ism (W iesner e t al., 1987; B utler, 1989; C reel
e t al., 1986; B u tle r e t al., 1986). Interestingly, a n u m b e r o f individuals w ith PWS
a n d th e typical fe a tu re s o f O CA have b e e n id e n tifie d (R inchik et al., 1993b). T h e
h y p o p ig m e n ta tio n in PWS was originally d e sc rib e d as o c u lo c u ta n e o u s alb inoidism
to signify c u ta n e o u s w ith o u t o c u la r inv o lv em en t (H ittn e r e t al., 1982), b u t subse
q u e n t stu d ies have d e m o n s tra te d h y p o p ig m e n ta tio n o f skin, hair, a n d eyes, in d i
catin g th a t th is is o c u lo c u ta n e o u s h y p o p ig m e n ta tio n . F o r th o se w ith o u t obvious
OCA, h a ir a n d skin a re lig h te r th a n u n a ffe c te d fam ily m em b ers, a n d c h ild h o o d
n ystagm us a n d strab ism u s a re c o m m o n . T h e irides are p ig m e n te d b u t show som e
tran slu cen cy o n g lo b e tra n sillu m in a tio n , a n d th e a m o u n t o f re tin a l p ig m e n t is re
d u c e d . A lth o u g h foveal h ypoplasia is n o t usually p re se n t, th e fovea d o es n o t a p
p e a r en tire ly n o rm a l (C reel e t al., 1986). V isual evoked p o te n tia l studies have
rev ealed o p tic tra c t m isro u tin g in a alb in ism p a tte rn in som e individuals w ith PWS
a n d h y p o p ig m e n ta tio n (C reel e t al., 1986); th e p re se n c e o f a b n o rm a litie s o f th e
o p tic tra c t im plies p a th o lo g ic h y p o p ig m e n ta tio n d u rin g d ev elo p m en t, in d ic a tin g
th a t th e h y p o p ig m e n ta tio n in PWS is best d e sc rib e d as a type o f albinism . T h e in
dividuals w ith PWS a n d O CA have n o t b e e n fully d e scrib e d , b u t th e fea tu re s o f th e
albinism are typical fo r OCA2.
Som e individuals w ith AS are also h y p o p ig m e n te d , b u t th e p e rc e n ta g e show ing
this p h e n o ty p ic c h a ra c teristic is u n k n o w n (K ing e t al., 1992; F ry b u rg et al., 1991).
106 RICHARD A. KING ET AL.

AS is a c o m p lex d e v e lo p m e n ta l d iso rd e r th a t in clu d e s d e v e lo p m e n tal delay a n d se

vere m e n ta l re ta rd a tio n , m icrocephaly, n e o n a ta l h y p o to n ia, ataxic m ovem ents,
a n d in a p p ro p ria te la u g h te r (F ry b u rg et al., 1991; W illiam s a n d Frias, 1982; Angel-
m an , 1965). T h o se w ith h y p o p ig m e n ta tio n have lig h t skin a n d h a ir a n d m ay have
a h isto ry o f nystagm us o r strab ism us (K ing e t al., 1992). Iris tran slu cen cy a n d re
d u c e d re tin a l p ig m e n t m ay be p re se n t. N o analysis o f th e o p tic tra c t o rg a n iza tio n
is available.

Molecular Phenotype

Recently, th e h u m a n h o m o lo g u e to th e m o u se pink-eyed-dilution (p) g e n e has

lin k e d to a type o f tyrosinase positive OCA, OCA2 (G a rd n e r e t al., 1992; Ram say
e t al., 1992; R in ch ik e t al., 1993b). T his lin k ag e was fo re sh ad o w ed by th e id e n tifi
c a tio n o f D N 10, a g e n e asso ciated w ith th e PWS c h ro m o so m a l re g io n (N akatsu et
a l.,1992), as th e p ro d u c t o f th e m ouse p g e n e . T his m a d e P, th e h u m a n h o m o
lo g u e o f th e m u rin e p g e n e , a stro n g c a n d id a te fo r OCA2. T h e m ouse p locus is
lo c a te d o n c h ro m o so m e 7 in a re g io n th a t has co n serv ed synteny w ith h u m a n
c h ro m o so m e 15q 11.2-q 12, th e lo ca tio n o f th e critical reg io n fo r PWS (B rilliant,
1992). T h e 3.4 kb m RN A tra n sc rip t is ex p ressed only in m elanocytes (R inchik e t
al., 1993b). T h e P p o ly -p ep tid e co n ta in s 12 tra n sm e m b ra n e d o m a in s w ith several
Af-glycosylation sites a n d p o te n tia l p ro te in kinase C p h o sp h o ry la tio n sites (G a rd
n e r e t al., 1992; R in ch ik e t al., 1993b). It has b e e n h y p o th esized th a t th e P
p o ly p e p tid e is a tyrosine tra n s p o rt p ro te in b o u n d to th e m e m b ra n e o f th e mel-
an o so m e . S om e s u p p o rt is le n t to the id ea by th e o b se rv a tio n th a t in c u b a tin g th e
m elan o cy tes fro m p m u ta n t m ice, o r fro m OCA2 p a tien ts, in excess tyrosine
in creases th e ir p ig m e n ta tio n (S idm an a n d P e rlstein , 1965; W itkop e t al., 1989)
L in k ag e analysis u sing th e p locus cDNA has show n th a t this g en e , w hich c o rre
sp o n d s to th e D15S12 m ark er, is w ithin th e c h ro m o so m al re g io n at 15q 11 -2-q 13
th a t is d e le te d in p a tie n ts w ith PWS a n d AS (G a rd n e r e t al., 1992; R in ch ik e t al.,
1993b). L in k ag e was also e sta b lish ed betw een tyrosinase positive O CA a n d m a rk
ers D15S10 a n d D15S13 w hich also m ap to 1 5 q ll.2 - q l2 (Ram say e t al., 1992). Ev
id e n c e th a t th e P locus is synonym ous w ith th e OCA2 g e n e has b e e n s tre n g th e n e d
by th e fin d in g o f an in d iv id u al w ith tyrosinase positive OCA w ith two d iffe re n t d e
le tio n s o f th e P locus; a d e le tio n o f th e e n tire P locus o n th e p a tern ally deriv ed
c h ro m o so m e 15 a n d a p a rtia l d e le tio n o f th e P locus o n th e m atern a lly d eriv ed al
lele (R in ch ik el al.. 1993b).
T h e m e c h a n ism fo r th e h y p o p ig m e n ta tio n in PWS in th o se individuals w ith o u t
obvious O C A re m a in s an en ig m a. T h e m u rin e p locus is n o t im p rin te d a n d it is
likely th a t th e h u m a n locus is n o t (G a rd n e r et al.,1992). T h e d e le tio n o n th e p a
te rn a l c h ro m o so m e 15 in PWS a n d th e m a te rn a l c h ro m o so m e 15 in AS suggests
th a t th e h y p o p ig m e n ta tio n arises fro m a m e ch an ism o th e r th a n g e n e dose effect,
since h e tero zy g o tes fo r tyrosinase positive O CA are n o rm ally p ig m e n te d . F u rth e r
w ork will b e n ecessary a fte r th e fu n c tio n o f the P locus g e n e p ro d u c t is d e te r
m in ed .
 HUMAN ALBINISM AND MOUSE MODELS 107

T h e m u ltip le alleles o f th e m ouse p m u ta tio n , as at albino, p ro d u c e a ra n g e o f

p h e n o ty p e s fro m only slig ht d ilu tio n o f p ig m e n t o f h a ir a n d eyes, to th e full d ilu
tio n seen in p anim als. Som e alleles, su ch as p-6 Harwell a n d p-cleft palate are co m
p le te d e le tio n s o f th e locus, o th e rs, fo r ex am p le p-Jackson have p artia l d e le tio n s
w hilst p-25 Harwell a n d black-eyed sterile have DNA re a rra n g e m e n ts u p stre a m o f the
g en e. M ost o f th e se m u ta tio n s, as well as th e o rig in al p, p ro d u c e n o mRNA, b u t the
p-Jackson m u ta n t g e n e m akes a sm aller m RN A (presu m ab ly b ecau se it is an in te r
n a l d e le tio n ) a n d black-eyed sterile, as well as o th e rs, have n o rm a l m RN A (w hich p re
sum ably e n c o d e s fo r a m u ta n t p ro te in ) (G a rd n e r e t al., 1992). O n e fu rth e r
m u ta tio n , p-unstable, is p a rticu la rly in te re stin g as it is a larg e (70kb) in te rn a l d u p li
catio n o f p a rt o f th e g e n e . T h e re su lt is th a t th e m RNA p ro d u c e d is la rg e r by 1.5kb,
re su ltin g fro m a d d itio n o f th e d u p lic a te d ex o n s in to th e m a tu re mRNA, w hich
d o es n o t m ak e a fu n c tio n a l p ro te in (G a rd n e r e t al., 1992; R inchik e t al., 1993b).
T h e DNA d u p lic a tio n causes th e m u ta tio n to be u n stab le; it is able to re v e rt a t hig h
fre q u e n c y to wild-type by re c o m b in a tio n , causing elim in a tio n o f th e d u p lic a te d
se g m e n t (G o n d o e t al., 1993). T h e p h e n o ty p e o f p-unstable is n o rm ally c h a ra c ter
ised by sm all d a rk p a tc h e s o n a d ilu te d p ig m e n t coat. O ccasio n al anim als have
la rg e r d a rk p a tc h e s (in d ica tin g a rev ersio n ev en t e a rlie r in d e v e lo p m e n t), a n d
th ese m ice o fte n tra n sm it th e re v e rte d g e n e to th e ir offspring.
T h e e x p e c ta tio n is th a t th e re will b e g re a t allelic h e te ro g e n e ity o f this g en e in
h u m a n s re su ltin g in a sp e c tru m o f effects fro m severe to m ild fo rm s o f albinism ,
as seen in th e m o u se a n d in th e OCA1 locus in h u m a n s.

U nclassified Types

Brown O CA has b e e n r e p o r te d only in individuals in A frica o r in A frican-A m eri-

can individuals, a n d th e p h e n o ty p e in C aucasian individuals is u n k n o w n (K ing
e t al., 1980, 1985). At b irth , th e h a ir a n d skin co lo r are lig h t brow n, a n d th e irides
a re gray to tan . W ith tim e th e re is little c h a n g e in skin color, b u t th e h a ir may
tu rn d a rk e r a n d th e irid es may a c c u m u la te m o re tan p ig m en t. T h e skin is resis
ta n t to th e a c u te effects o f sun ex p o su re a n d d o e s n o t b u rn . T h e iris has p u n c ta te
a n d rad ial translucency, a n d m o d e ra te re tin a l p ig m e n t is p re se n t. T h e skin may
d a rk e n w ith sun e x p o su re . V isual acuity ran g es fro m 2 0 /6 0 to 2 0 /1 5 0 . T h e u ltra
s tru c tu re o f th e m elan o cy te a n d m e la n o so m e a re n o rm a l, a n d h a irb u lb tyrosi
n ase activity is n o rm a l (K ing e t al., 1980, 1985). T h e fre q u en cy o f B row n O CA is
u n k n o w n ; in o n e study, a p p ro x im a te ly 29% o f indivi-duals w ith alb in ism in N ige
ria h a d this type (K ing e t a l., 1980).
In d iv id u als w ith ru fo u s (R ed) O CA having re d h a ir a n d red d ish -b ro w n p ig m e n t
e d skin have b e e n re p o r te d in A frica (P earso n e t al., 1911; S tan n u s, 1913; Krom-
b e rg e t al., 1990) a n d in N ew G u in ea (H o rn a b ro o k e t al., 1980; W alsh, 1971), b u t
clinical d e sc rip tio n s a re in c o m p le te , n o b io ch em ical o r u ltra s tru c tu re d a ta are
available, a n d sim ilar p h e n o ty p e s in th e US a n d E u ro p e a n p o p u la tio n s have n o t
b e e n reco g n iz e d . In d iv id u als with re d h a ir w ho have e ith e r tyrosinase re la te d OCA
o r tyrosinase positive O CA are re co g n ize d , b u t the red d ish -b ro w n skin p ig m e n t is
usually n o t p re se n t. In N ew G u inea, th e d esc rib e d p h e n o ty p e in clu d es re d d ish -
108 RICHARD A. KING ET AL.

brow n skin, d e e p m a h o g a n y hair, red d ish -b ro w n to brow n irides, a n d m o d e ra te

a m o u n ts o f re tin a l p ig m en t.

O CU L O C U T A N E O U S ALBINISM: PRIMARY DEFECT N O T SPECIFIC

FO R MEL SYNTHETIC PATHWAY

Several ra re types o f O CA a re associated w ith clinical fe a tu re s th a t involve tissues

o u tsid e o f th e p ig m e n t system , a n d th e re d u c tio n in m e la n in synthesis in these
c o n d itio n s a p p e a rs to be se c o n d ary to a d e fe c t th a t is n o t specific fo r th e m e la n o
cyte o r th e m e la n in syn th etic pathway. T h e p h e n o ty p e o f th ese c o n d itio n s varies
fro m obvious O CA to m o d e ra te h y p o p ig m e n ta tio n , a n d th e associated m edical
p ro b le m s can b e severe a n d re p re s e n t th e m ajo r m edical p ro b le m fo r the
affe c ted in dividual.

H erm ansky-P udlak Syndrom e

H erm an sk y -P u d lak S y n d ro m e (HPS) is an a u to so m a l recessive c o n d itio n th a t p re

sen ts with OCA, a m ild b le e d in g diathesis, a n d a c e ro id storage disease a ffectin g
p rim arily th e lungs a n d th e g u t. H erm an sk y a n d P u d lak first d e sc rib e d this c o n d i
tio n in two C zechoslovakian individuals in 1959 (H erm an sk y a n d P u d lak , 1959)
a n d it h as su b seq u en tly b e e n re c o g n iz e d th r o u g h o u t th e w orld, w ith th e m ajority
o f affe c ted individuals in th e P u e rto R ican p o p u la tio n (W itkop e t al., 1989). H PS
is au to so m a l recessive in in h e rita n c e .
T h e alb in ism has a v ariable p h e n o ty p e , ra n g in g fro m m a rk e d h y p o p ig m e n ta
tio n th a t is sim ilar to tyrosinase-negative O C A to only m o d e ra te h y p o p ig m e n ta tio n
w ith p ig m e n t p re s e n t in th e skin, h a ir a n d eyes. In th e P u e rto R ican p o p u la tio n ,
th e h a ir c o lo r varies fro m w hite to yellow to brow n. T h e skin is cream y w hite a n d
freckles a re o fte n p re s e n t in th e sun e x p o se d reg io n s (face, neck, a rm s a n d
h a n d s ), o fte n co alescin g in to large are as th a t lo o k like n o rm a l d a rk skin p ig m en t.
P ig m e n te d nevi a re c o m m o n . T h e iris c o lo r varies fro m b lu e to brow n, a n d all o f
th e o c u la r fe a tu re s o f albinism a re p re se n t. V isual acuity ran g e s fro m 2 0 /6 0 to 2 0 /
400 (Sim on e t al., 1982; S u m m ers et al., 1988). T h e p re se n c e o f O CA m ay n o t be
obvious in a P u e rto R ican ind iv idual w ho h a s brow n hair, p ig m e n t in th e ex p o sed
areas o f th e skin, a n d brow n eyes w ith o u t c o m p a riso n o f th e p ig m e n t p a tte rn to
u n a ffe c te d fam ily m e m b e rs (w ho a re g en erally d a rk e r in p ig m e n t) o r w ith o u t rec
o g n iz in g th e o c u la r fe a tu re s o f albinism .
T h e m ost severe clinical m a n ifesta tio n s o f HPS a re re la te d to th e p u lm o n a ry
a n d in te stin a l chan g es. In te rstitia l p u lm o n a ry fibrosis has b e e n d e sc rib e d in m any
individuals with H PS, a lth o u g h th e actual p rev alen ce is u n k n o w n (H a rm o n e t al.,
1994; W hite e t al., 1984). T h e fibrosis resu lts in m o d e ra te to severe restrictive lu n g
disease (H a rm o n et al., 1994). T h e d e v e lo p m e n t o f g ra n u lo m a to u s colitis, p re
s e n tin g w ith a b d o m in a l p ain a n d bloody d ia rrh e a in a ch ild o r an a d u lt, has also
b e e n d e sc rib e d in m any individuals with H PS (M ah ad eo et al., 1991; S ch in ella
 HUMAN ALBINISM AND MOUSE MODELS 109

et al., 1980). T h e etiology is u n k n o w n , a n d im m u n o lo g ic stu d ies do n o t show an

a b n o rm a lity (S h a n a h a n e t al., 1988). T h e p re se n c e o f c e ro id m a teria l in th e e p i
th elial cells o f th e g u t suggests th a t this m aterial may b e involved in th e d ev elo p
m e n t o f th e colitis, b u t th is has n o t b e e n proven.
T h e b le e d in g d iath esis in H PS is re la te d to a deficiency o f sto rag e g ran u les in
th e p la te le ts (i.e. sto rag e p o o l-d efic ie n t p late lets). T h e sto rag e g ra n u le s o r d en se
b o d ies are re d u c e d in n u m b e r o r are a b se n t, a n d this is associated with a d e fic ie n
cy o f s e ro to n in , a d e n in e n u cleo tid e s, a n d calcium in th e p la te le t (W itkop et al.,
1989). As a resu lt, p la te le ts in HPS d o n o t show th e irreversible sec o n d ary ag g re
g a tio n w hen stim u lated . T h is d eficiency p ro d u c e s m ild h e m o rrh a g ic episo d es in
m any a ffe c ted individuals, in c lu d in g easy brusibility, epistaxis, hem optysis, g in g i
val b le e d in g w ith b ru s h in g o r d e n ta l e x tra c tio n , a n d p o stp a rtu m b le ed in g . O cca
sional severe b le e d in g is o bserv ed . T h e basic d e fe ct in H PS is u n k n o w n . H a irb u lb
tyrosinase activity is p re s e n t b u t th e level is low (K ing a n d O lds, 1985). T h e stru c
tu re s o f th e m elan o cy te a n d m e la n o so m e s tru c tu re a re n o rm a l (W itkop et al.,
1989).

C hediak-H igashi S yndrom e (CH S)

T h e C hediak -H ig ash i S y n d ro m e is a ra re au to so m al recessive sy n d ro m e th a t c o n

sists o f in c re a se d susceptibility to b a c te ria l in fectio n s, h y p o p ig m e n ta tio n , a n d the
p re se n c e o f g ia n t pero x idase-positive lysosom al g ra n u les in p e rip h e ra l b lo o d
g ran u lo cy tes (B lum e a n d Wolff, 1972). T h e skin, hair, a n d eye p ig m e n t is d ilu te d
in C H S b u t th e affe c ted individuals d o n o t always have obvious albinism a n d th e
h y p o p ig m e n ta tio n m ay only be obvious in re la tio n sh ip to o th e r fam ily m em bers.
H a ir c o lo r is lig h t brow n to b lo n d , a n d th e h a ir has a m etallic silver-gray sh een .
T h e skin is cream y w hite to slate gray. Iris p ig m e n t is p re s e n t a n d nystagm us a n d
p h o to p h o b ia m ay be p re s e n t o r a b se n t (B enezra e t al.,1980). V isual evoked
p o te n tia l stu d ies show m isro u tin g o f th e o p tic fibers in a p a tte rn th a t is sim ilar to
indiv id u als OCA. T h e p rim a ry d e fe c t in CHS is un k n o w n . T h e susceptibility to
b acterial in fectio n s a p p e a rs to be th e re su lt o f th e a b n o rm a l g ra n u le s in th e n e u
tro p h ils a n d o th e r cells (W hite a n d Claw son, 1980; T anaka, 1980). T h e h y p o p ig
m e n ta tio n also arises fro m th e fo rm a tio n o f a b n o rm a l g ra n u les. G iant
m e la n o so m e s fo rm in th e m elan o cy te a n d are u n a b le to be tra n sfe rre d to th e sur
ro u n d in g k eratin o cy tes, le a d in g to a b n o rm a l m elan o so m e d istrib u tio n a n d
h y p o p ig m e n ta tio n .

M ouse M odels f o r P la te le t Storage P o o l D eficiencies

T h e c o m b in a tio n o f p ig m e n ta tio n d efects with a p la te le t sto rag e p o o l deficiency

has b e e n fo u n d in a n u m b e r o f m o u se m u tan ts. T h e m u ta n ts (beige, cocoa, pale ear,
light ear, mocha, muted, pallid, pearl, reduced pigmentation, ruby eye, ruby eye-2 and
sandy) all have a c o m b in a tio n o f d efects o f lysosom al enzym e sec retio n in kidney
a n d o th e r tissues, a deficiency o f p la te le t d en se g ran u le s, a n d a p ig m e n ta tio n
110 RICHARD A. KING ET AL.

d e fe c t c a u se d by m e la n o so m e ab n o rm a litie s. T h e re a re d ifferen c es b etw een the

12 m u ta n ts in th e ra n g e o f p ig m e n ta tio n d efects a n d th e effects o n th e eye, b u t in
g e n e ra l th e eyes a re lig h te r at b irth th a n in adults. T h re e o f th e m u tan ts, pale ear,
pearl, a n d ruby-eye have b e e n show n to have re d u c e d ipsilateral p ro je c tio n s fro m
th e re tin a to th e la te ra l g e n ic u la te n u cleu s, as in o c u la r p ig m e n t-d efic ien t
h u m a n s. Any o r all o f th ese may be m o d e ls fo r H PS o r fo r CH S (Silvers, 1979;
G reen 1989).
T h is series o f d iffe re n t m u ta n t loci wfiich all affect th e sam e th re e p rocesses sug
gests a co m m o n pathw ay lin k in g th e p rocesses, in w hich th e p ro d u c ts o f th e 12 loci
p a rtic ip a te o r in te ra c t. M elan o som es a n d lysosom es have a n u m b e r o f p ro te in s in
c o m m o n a n d may sh a re a c o m m o n o rig in (O rlow e t al., 1993; Z h o u e t al., 1993).
T h e re has b e e n p ro m isin g progress tow ards e lu c id a tin g the m o le c u la r basis o f
pallid. T h e g e n e e n c o d in g th e m ouse e ry th ro cy te p ro te in b a n d 4.2 (E pb4.2) m aps
a t o r n e a r th e pallid locus, a n d e x p ressio n o f th e E pb4.2 mRNA is a b n o rm a l in kid
n ey (w hich is a ffected by th e m u ta tio n ) b u t n o t in liver o r b ra in (w hich a re n o t
affected ) (W hite e t al., 1992). T h e p ro te in 4.2 m u st be c o n sid e re d a stro n g c a n d i
d a te fo r th e pallid locus, b u t th e d a ta is n o t, as yet, definitive. O n e u n re so lv e d issue
is th a t h u m a n p a tie n ts w ho lack p ro te in 4.2 suffer fro m h e re d ita ry spherocy-tosis,
b u t have n o n e o f th e fe a tu re s o f th e p allid m ice, w hilst p allid m ice d o n o t have
spherocytosis. T h e re m ay b e tissue-specific, o r even organelle-specific, fo rm s o f
th e p ro te in , a n d d iffe re n t diseases m ig h t m an ifest fro m m u ta tio n s in th e sam e
g e n e . T h e id e n tific a tio n o f th e p ro d u c t o f o n e o f th e p la te le t sto rag e p o o l defi
ciency m u ta tio n s in m ouse will greatly a id th e id e n tific atio n o f o th e rs, by lo catin g
th e c e llu la r processes affe c ted a n d fin d in g o th e r in te ra c tin g p ro te in s w ithin the
p rocess, w hich m ig h t be e n c o d e d at th e o th e r m u ta n t loci.

O CU LA R ALBINISM

X -Linked R ecessive OA (OA1)

OA1 has an X -linked recessive p a tte rn o f in h e rita n c e . A ffected m ales have n o r

m al skin a n d h a ir p ig m e n t. T h e irides a re b lu e to brow n, a n d th e o c u la r ch an g es
a re sim ilar to O CA (F reed et al., 1989). In A frican-A m erican m ales, iris co lo r is
o fte n brow n a n d th e re is little iris tran slu cen cy (C harles e t al., 1992). T h e skin o f
C aucasian in d iv id u als w ith OA1 a p p e a rs n o rm a lly p ig m e n te d , w hile m any A fri
can-A m erican individuals have sc a tte re d h y p o p ig m e n te d m acules. H eterozygous
fem ales can b e clinically d e te c te d b ecau se o f o c u la r p ig m e n t ch an g es th a t resu lt
fro m X -inactivation. A v arig ated p a tte rn o f re tin a l p ig m e n t a n d p u n c ta te areas o f
iris tran slu cen cy a re seen in a p p ro x im a te ly 80% o f o b lig ate h etero zy g o tes fem ales
(K in o sh ita e t al., 1989; F re e d e t al., 1989; S m ith e t al., 1988; C h arles e t al., 1992).
A sm all n u m b e r o f h etero zy g o u s fem ales have o cu la r ch a n g es o f alb in ism in c lu d
 HUMAN ALBINISM AND MOUSE MODELS 111

in g nystagm us a n d re d u c e d acuity th a t is th o u g h t to b e th e resu lt o f n o n ra n d o m

p a tte rn s o f x-inactivation ( J a e g e r a n d Jay 1981).
M elanocytes a re n o rm a l in OA1 b u t th e re are ch a n g e s in th e m elan o so m es. M el
anocytes in th e skin a n d h a ir follicles as well as th o se o f th e iris a n d re tin a c o n tain
larg e m ela n o so m e s called g ia n t m e lan o so m e s, m a cro m ela n o so m es, o r m ela n in
m a c ro g lo b u les, a lo n g w ith n o rm a l m elan o so m es. T h e g ia n t m e lan o so m es a re also
fo u n d in th e tissues o f o b lig ate h e tero zy g o u s fem ales. T h e systemic n a tu re o f the
m e la n o so m e d e fe c t in OA1 suggests th a t this is really a type o f OCA in w hich th e
m a jo r m a n ife sta tio n s are in th e eye. T h e re m ay n o t be any type o f albinism with
c h an g es lim ite d to th e eye, a n d all types p ro b ab ly r e p re s e n t variable ex p ressio n o f
o c u lo c u ta n e o u s albinism .
T h e g e n e fo r OA1 h as b e e n lo calized to th e s h o rt a rm o f th e X ch ro m o so m e.
Fam ily stu d ies p la c e d th e g e n e o n th e X ch ro m o so m e. Initial linkage studies
show ed OA1 lin k e d to th e Xg b lo o d g ro u p , jnow localized to X p22.32 (Fialkow
et al., 1967; P earce e t al., 1968). T h e g e n e has now b e e n m a p p e d m o re precisely
to th e X p22.3-X p22.2 re g io n , (S c h n u r e t al., 1991) a n d is th o u g h t to be betw een
th e m ark ers DXS237 a n d DXS143 (B erg en e t al., 1990, 1991). T h e g en e has n o t
b een iso lated a n d th e fu n c tio n o f its p ro d u c t is un k n o w n .
C o n se rv a tio n o f lin k ag e b etw een m o u se a n d h u m a n X ch ro m o so m es readily al
lows id e n tific a tio n o f th e re g io n w h e re th e m ouse h o m o lo g u e o f OA1 sh o u ld be.
C uriously, th e re a re n o m u ta tio n s w hich affect eye o r h a ir m elanocytes in this p a rt
o f th e c h ro m o so m e in th e m ouse. It is possible th a t a m u ta tio n w hich has only a
sub tle effect o n eye p ig m e n ta tio n w o u ld n o t have b e e n id e n tifie d in m o u se stocks
w ith o u t a sc re e n fo cu sed p articu la rly o n eye defects.

A utosom al R ecessive O c u la r Albinism (OA2)

T his type o f OA affects m ales a n d fem ales equally (K ulkarni a n d N a th a n so n ,

1989). T h e o c u la r fe a tu re s o f th e alb in ism are sim ilar to th o se o f O CA a n d OA1,
e x cep t th a t m o st a ffe c ted in d iv id u als have som e re tin a l p ig m e n t in th e p o ste rio r
p o le. M ost p u b lis h e d cases o f OA2 have b e e n in C aucasians, a n d they have fre
q u e n tly h a d lig h t p ig m e n t at b irth w ith a g rad u a l in crease in skin a n d h a ir pig
m e n t w ith tim e. Skin p ig m e n t a p p e a rs to b e n o rm a l in OA2 b u t affec ted A frican-
A m erican in d iv id u als have n o t b e e n d escrib ed .
T h e g e n e re sp o n sib le o f OA2 has n o t b e e n id e n tifie d o r m a p p e d . A re c e n t re
p o r t o f a d y sm o rp h ic ch ild having th e fe a tu re s o f OA a n d an ab se n ce o f g ia n t m el
a n o so m es in asso ciatio n w ith a d e le tio n o f c h ro m o so m e 6 ( q l3 -q l5 ) suggests th a t
this may be th e locus fo r OA2 (R ose e t al., 1992). A n o th e r real possibility is th a t
OA2 d o es n o t exist as a se p a ra te entity, a n d th a t th e previously d e sc rib e d cases ac
tually have tyrosinase g e n e o r P g e n e m u ta tio n s associated with re sid u al enzym e
activity a n d relatively n o rm a l c u ta n e o u s p ig m e n ta tio n . In dividuals having tyrosi
nase g e n e m u ta tio n s asso ciated w ith n early n o rm a l cu ta n e o u s p ig m e n ta tio n have
b e e n d escrib ed .
112 RICHARD A. KING ET AL.

T h e re a re very few m ouse m u ta tio n s w h ere eye p ig m e n ta tio n is affe cted w ith o u t
h a ir a n d skin involvem ent. O n e m u ta n t allele o f ruby eye-2, maroon, has b e e n d e
scrib ed in w hich som e m u ta n t anim als can have lig h te n e d eyes b u t a n o rm a l coat
co lo r; a lth o u g h affe c ted litte rm a te s m ig h t have m u c h lig h te n e d coats. In su p p o rt
o f th e id ea th a t OA2 h u m a n s m ig h t have tyrosinase m u tatio n s, th e d iffe re n t m u
ta n t alleles o f m o u se albino, th e tyrosinase g en e , can have d iffe ren tial effects on
h a ir versus eye p ig m e n ta tio n . T h e extreme dilution m u ta tio n has a very pale coat,
b u t d a rk eyes, w hereas th e ruby-eyed dilute allele gives a d a rk e r coat w ith ru b y eyes.
O n th e w hole, however, th e reverse is tru e ; m u ta tio n s w hich have lig h te n e d h a ir
b u t d a rk eyes o c c u r m u ch m o re frequently.

CLINICAL APPLICATION

D iagnosis o f A lbinism

M ost indiv id u als w ith O CA have an obvious p h e n o ty p e , a n d extensive la b o ra to ry

te stin g is unn ecessary . T h e o c u la r ex a m in a tio n is m o st im p o rta n t, a n d a diagnosis
o f O CA sh o u ld n o t b e m ad e w ith o u t th e o c u la r fe atu res o f albinism , p artic u larly
w hen c u ta n e o u s a n d iris p ig m e n t is p re se n t. It can be d ifficu lt to se p a ra te OCA1
a n d OCA2 w ith th e clinical e x a m in a tio n . T h e m o st reliab le c h a rac teristic fo r this
is th e p re se n c e (OCA2) o r ab sen ce (OCA 1) o f h a ir p ig m e n t at b irth , b u t delayed
h a ir gro w th in m any c h ild re n may m ake this difficult in the first six m o n th s o f
life.
T h e critical d iag n o stic issue is th e p re se n c e o r a b sen ce o f albinism r a th e r th e
specific type o f albinism . K now ledge o f th e specific type can h e lp in fam ily c o u n
seling, b u t d o es n o t accu rately p re d ic t th e e x p e c te d visual o u tc o m e. In g e n e ra l,
individuals w ith O CA w ho fo rm m ela n in in th e ir h a ir a n d eyes will have a b e tte r
visual o u tc o m e th a n th o se w ho fo rm little o r n o p ig m e n t, b u t ex c e p tio n s to this
a re c o m m o n .
F o r OCA, th e p recise d iag n o sis o f OCA1 o r OCA2 re q u ire s m o le c u la r analysis
o f th e tyrosinase o r th e P g e n e . T h e q u a n tita tiv e h a irb u lb tyrosinase assay has b e e n
u se d b u t is im p recise. In d iv id u als w ith OCA1 w ho have alleles c o d in g fo r an e n
zym e w ith m o d e ra te resid u al activity may d e m o n stra te h a irb u lb tyrosinase activity
on assay, a n d th e in c o rre c t in te rp re ta tio n m ay b e OCA2 ra th e r th a n OCA1. H e t
erozygote d e te c tio n can only b e p e rfo rm e d w ith m o le c u la r analysis. At th e p re se n t
tim e, m o le c u la r analysis o f th e OA1 g e n e is n o t possible, a n d a skin o r h a ir follicle
biopsy to evaluate fo r g ia n t m e lan o so m e s re m a in s th e m o st precise m e th o d o f di
agnosis. T h e OA1 g e n e is well localized o n th e X c h ro m o so m e , however, a n d fam
ily c o u n se lin g w ith linkage analysis may b e possible. It is e x p e c te d th a t th e OA1
g e n e will be iso lated in th e very n e a r fu tu re , a n d d ire c t m o le c u la r analysis will th e n
be possible.
 HUMAN ALBINISM AND MOUSE MODELS 113

T re a tm e n t

T h e two c o m p o n e n ts to th e tre a tm e n t o f OCA are skin p ro te c tio n a n d vision su p

p o rt. M ost individuals w ith O CA d o n o t tan a n d a re susceptible to th e ultraviolet
rays o f th e sun. T h e use o f h ats w ith a b rim , long-sleeve shirts a n d lo n g pants,
a lo n g w ith an av o id an ce o f th e sun a t m idday (10:00a.m . to 2:00p.m .) will o fte n
b e e n o u g h , p a rtic u la rly in m o re n o r th e r n clim es. S u n sc re en is necessary w hen
a d d itio n a l p ro te c tio n is n ecessary o r th e above m ea su res a re im practical.
O n g o in g o p h th a lm o lo g ic care is c ritical to s u p p o rt th e n o rm a l d e v e lo p m e n t o f
an indiv id u al with alb in ism . T his sh o u ld in c lu d e yearly eye e x am in a tio n s, full cor
re c tio n fo r m yopia o r h y p ero p ia, a n d c o n su lta tio n with th e a p p ro p ria te local low
vision p ro g ra m . E x cep t fo r special circu m stan ces, c h ild re n w ith alb in ism sh o u ld
a tte n d re g u la r school classes a n d s h o u ld be e n c o u ra g e d to p a rtic ip a te in all as
p ects o f school.

FUTURE

T h e re a re th re e ex citin g areas o f fu tu re re se a rc h in p ig m e n t biology a n d th e eye.

First, th e g e n e s re sp o n sib le fo r all types o f h u m a n albinism will b e id e n tified ,
allow ing c o rre c t d iag n o sis a n d fam ily co u n selin g . P h en o ty p ic v ariatio n o f each
type a n d c o rre la tio n b etw een g en o ty p e a n d p h e n o ty p e , p articu la rly th e visual
d e v e lo p m e n t, will be d e fin ed . S eco n d , th e isolation o f a sig n ifican t n u m b e r o f th e
g en es involved in c u ta n e o u s a n d o c u la r m e lan o g en esis, fro m b o th h u m a n an d
m o u se, will allow th e d e v e lo p m e n t a n d re g u la tio n o f p ig m e n t in th ese tissues to
be stu d ied . F o r ex am p le, an analysis o f th e ro le o f m ela n in in n o rm a l eye devel
o p m e n t will prov id e in sig h t in to th e ch a n g e s th a t o c c u r in albinism , a n d m ay sug
gest m e th o d s fo r a lte rin g th ese ch an g es. T h e m ouse, as an e x p e rim e n ta l system,
sh o u ld play a m a jo r ro le in th ese analyses. T h ird , it m ay be possible to develop
m e th o d s o f g e n e th e ra p y fo r th e c u ta n e o u s a n d th e o c u la r ch an g es in albinism .
T h is is already b e in g d iscussed in several ce n te rs a n d th e m o u se m o d e ls m ig h t be
u seful fo r te stin g g e n e th e ra p y p ro to co ls.

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 HUMAN ALBINISM AND MOUSE MODELS 119

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 5. INHERITED RETINAL DEGENERATIONS
IN TH E MOUSE

DEBORA B. FARBER 1 and MICHAEL DANCIGER 12

Jules Stein Eye Institute and Department of Ophthalmology, UCI-A School of Medicine, Los
Angeles, California 900241; and Loyola-Marymount University, Los Angeles,
California 90045, USA2

U ntil very recently, none o f the genes responsible for the several in h erited
m urine retinal deg en eratio n s1 had been identified. However, in 1989 and 1990
the defective genes responsible for two o f these diseases were isolated and charac
terized. In each case, a candidate gene was cloned by subtractive an d differential
hybridization and m apped to a particular m ouse chrom osom e by somatic cell
hybrid analysis. B oth candidate genes were localized to the m urine chrom osom e
known to contain the respective disease locus. O ne o f the genes identified is the
retinal degeneration or rd, which causes a rapidly developing, early retinal degener
ation m apping to m ouse chrom osom e 5 (Sidm an an d G reen, 1965) an d encodes
the P subunit of the rod-specific enzyme cyclic GM P-phosphodiesterase (Bowes
et al., 1989; 1990). T h e o th er gene isolated is responsible for the retinal degenera
tion slow or rds m utation, which m aps to m ouse chrom osom e 17 (D em ant et al.,
1979; Travis et al., 1989). T he rds gene encodes a rod p h o to rec ep to r o u ter seg
m en t m em brane p rotein known as rd s /p e rip h e rin (C onnell et al., 1991). Fur
ther, the abortive developm ent o f the ro d o uter segm ents in rds is understandable
in term s o f a defective disc m em brane p rotein. Similarly, the degenerative p ro
cess in rd m ouse photoreceptors closely correlates with the finding of increased
levels o f cyclic GMP d u e to a defective cyclic G M P-phosphodiesterase.
In this chapter, the studies that led to the identification o f the rd gene will be
discussed in detail; the work involved in the identification o f the rds gene will be
sum m arized and the use o f the m ouse as a m eans of identifying an d characterizing
hum an disease genes will be discussed.

'T h e following m ouse m utants show signs o f retinal d eg en eratio n (th eir symbols a n d chrom osom al lo
cations, w here known, are shown in parentheses: retinal d eg en eratio n (rd, 5; rd-3, l:/M-4.4), retinal de
generation slow {rds, 17). p urkinje cell d e g en e ratio n (pal, 13), cribriform d eg en e ratio n (cn, 4),
nervous (nr, 8), vitiligo (vit, 6) (G reen, 1989; Heckenlively et al., 1992; C hang et al., 1993).
124 DEBORA B. FARBER AND MICHAEL DANCIGER

THE rd MOUSE

Retinal degeneration in the m ouse (rd m utation) was first described in wild ani
mals caught in different areas of E urope and is p resen t in several inbred labora
tory strains (Sidm an an d G reen, 1965). Over the years, these m ice have been the
object of many genetic, m orphological, electrophysiological, biochem ical, im m u
nological, m olecular biological and behavioral studies.
T he rd disease is in h erited in an autosom al recessive fashion and results in the
rapid and selective degeneration o f the retinal p h o to rec ep to r cells (Sidm an and
G reen, 1965; LaVail and Sidm an, 1974). T he m orphology of the norm al m ouse
retina is shown in Figure 1. T he rd visual cells appear to form norm ally during the
prenatal an d early postnatal period and to achieve some degree o f differentiation,
as indicated by the developm ent of ru d im en tary rod o u ter segm ents and a synap
tic ribbon com plex (LaVail and Sidm an, 1974; Blanks et al., 1974). T he m orphol
ogy o f a rod p h o to rec ep to r is shown in Figure 2. By 10 days of age, the
p h otoreceptors of both the rd/rd m ouse a n d the rd /+ heterozygote (which has a
m orphologically n orm al retina) look alike at the light m icroscope level, b u t by day
20, virtually all the rd/rd rod visual cells have deg en erated (Noell, 1965). T he sin
gle row o f nuclei left from the o u ter nuclear layer at this time in the rd retin a cor
responds to cone nuclei (Carter-Dawson et al., 1978). Most o f these nuclei
disappear in the following few m onths (Figure 3). At the electron m icroscope lev
el, the first signs of ultrastructural pathology in the rd p hotoreceptors are ob
served on the 7th to 8th postnatal day as 1) fewer ribosom es and swelling o f the
m itochondria o f the in n er segm ents, followed by 2) slower developm ent a n d dis
organization o f the o u ter segm ent m em branes and 3) absence of triad configura
tions at the p h o to recep to r term inals (S onohara and Shiose, 1968; Caley, Jo h n so n
an d Liebelt, 1972; Sanyal and Bal, 1973; Blanks, Adinolfi and Lolley, 1974).
T he retinal pigm ent epithelium as well as the cells o f the in n er re tin a rem ain
m orphologically and functionally norm al d u rin g the course of the dissolution of
the o u ter nuclear layer (LaVail an d Sidm an, 1974). However, subtle an d slow
changes occur in the ganglion cells and in the in n er nuclear and in n er plexiform
layers after several m onths o f life (Grafstein, M urray an d Ingoglia, 1972).
T he rd retina is responsive to high intensity illum ination and betw een 12 and 18
postnatal days it displays an atten u ated electroretinogram which becom es com
pletely flat by 28 postnatal days (Noell, 1965). T he rate o f visual cell degeneration
is n o t accelerated by the level o f environm ental light in which the mice are reared.

Biochem ical and Molecular Biological Studies

T he first clues regarding the cause o f the rd m utation cam e from studies o f cyclic
nucleotide m etabolism . Cyclic GMP levels becom e elevated in the rd retin a when
co m pared to control retina by the 6th postnatal day, at least two days p rio r to the
onset o f p h o to rec ep to r cell degeneration, an d they are several-fold h ig h er than
norm al by day 14 (Farber an d Lolley, 1974). Cyclic GMP levels decline thereafter,
correlating with the loss o f visual cells from the rd retina. Analysis o f microdis-
 I N H E R I T E D R E T I N A I . DEC. E N E R A T K ) N S IN T H E M O l SI- 115

Choriocapillaris
RPE
Outer segments
Inner segments
Photoreceptor
Outer nuclear layer
layer

Outer plexiform
layer

Inner nuclear
layer

Inner retinal
layers
Inner plexiform
layer

Ganglion cell
layer

F igure 1 L ig h t m ic r o g r a p h o i th e r e tin a l i o m a n o r m a l a d u lt m o u se , s h o w in g th e m a jo r cell

lay ers (c o u rte s y o f Dr. J o h n F la n n e ry ) . T h e r e tin a l p ig m e n t e p ith e liu m (R P E ) lie s a t th e to p
a n d in te r d ig ita te s w ith th e o u t e r s e g m e n ts o f th e p h o t o r e c e p t o r cells. T h e n u c le i o f th e s e
cells a r e o r g a n iz e d in se v e ra l row s, f o r m in g th e o u t e r n u c le a r layer. B e tw ee n th is lay er a n d
th e in n e r m o s t r e tin a l layer, f o r m e d by th e g a n g lio n c ells a n d th e ir a x o n s, th e r e is a c e llu la r
s tra tu m , th e in n e r n u c le a r layer, f o r m e d by h o riz o n ta l, b ip o la r, in te r p le x if o r m a n d a m a c rin e
n e u r o n s , a n d th e g lia l lik e M u lle r cells. S y n a p tic c o n ta c ts a lso o c c u r in d e f in e d lay e rs o f th e
re tin a . T h e o u t e r p le x ifo r m lay e r c o n ta in s t h e sy n a p se s b e tw e e n th e p h o t o r e c e p t o r s a n d h o r
iz o n ta l a n d b ip o la r cells; a n d t h e i n n e r p le x if o rm lay e r is f o r m e d w ith th e sy n a p tic c o n ta c ts
b e tw e e n g a n g lio n a n d b ip o la r cells, g a n g lio n a n d a m a c r in e cells, a n d a m a c rin e cells in te r a c t
in g w ith e a c h o th e r. I n te r p le x if o r m cells h a v e p ro c e s s e s e x te n d in g i n to th e i n n e r p le x ifo rm
a n d in n e r n u c le a r layers. T h e la tte r p ro c e s s e s m a k e sy n a p tic c o n ta c ts w ith h o r iz o n ta l a n d b i
p o la r cells. M u lle r cells s p a n th e r e ti n a fr o m th e v itre a l b o r d e r th r o u g h th e o u t e r n u c le a r
layer.
 126 DEBORA B. FARBER AND MICHAEL DANCIGER

Pigment Epithelium

F ig u re 2 D ia g ra m o f a v e r te b r a te r o d p h o t o r e c e p t o r cell. T h e o u t e r s e g m e n t c o n ta in s sta ck s
o f m e m b r a n o u s disks f o r m e d b y lip id b ila y e rs w h ic h p ro v id e a m a trix fo r p r o te in m o le c u le s.
M o st o f th e s e d isks a r e n o t c o n tin u o u s w ith th e p la s m a m e m b r a n e o f th e p h o t o r e c e p t o r a l
th o u g h th e y a r e c o n n e c te d by f ila m e n to u s s tru c tu r e s . T h e visual p ig m e n t rh o d o p s in , th e m a
j o r p r o t e i n o f t h e r o d o u t e r s e g m e n t, is a n in tr in s ic g ly c o p r o te in w h ic h s p a n s th e d isk
m e m b r a n e th ic k n e s s c ro ssin g th e m e m b r a n e i n te r f a c e sev eral tim e s. A m o d ifie d c iliu m c o n
n e c ts th e o u t e r s e g m e n t w ith th e i n n e r s e g m e n t, w h e re th e c ell sy n th e siz e s p ro te in s , p r o d u c e s
th e e n e rg y -ric h n u c le o s id e tr ip h o s p h a te s a n d a sse m b le s th e d isk m e m b ra n e s . B elow t h e n u c le
us, th e p h o t o r e c e p t o r a x o n p r o c e e d s to th e s y n a p tic te r m in a l, w h ic h fo rm s sy n a p se s w ith th e
n e u r o n s o f th e i n n e r r e tin a a n d c o n ta in s th e s p e c ia liz e d b io c h e m ic a l m a c h in e r y n e e d e d fo r
n e u r a l sig n a lin g .
 INHERITED RETINAI, DEGENERATIONS IN THE MOUSE 127

Choriocapillaris
RPE

Inner nuclear
layer

Inner plexiform
layer

G anglion cell
layer

F ig u re 3 L ig h l m ic r o g r a p h o f th e r e tin a fr o m a 30-day-old rd m o u s e (c o u rte s y o f Dr. J o h n

F la n n e r y ) , sh o w in g its to ta l la c k o f p h o t o r e c e p t o r cells. As a c o n s e q u e n c e th e i n n e r n u c le a r
lay e r is a p p o s e d to th e r e tin a l p ig m e n t e p ith e liu m (R P E ).

sected samples consisting o f the p h o to rec ep to r layer and the in n er layers o f the
rd retina showed th at cyclic GMP accum ulates exclusively in the p h o to rec ep to r
cells (Farber and Lolley, 1974). This elevation in cyclic GMP levels is the result o f
a deficiency in the activity o f the ro d p h o to recep to r cyclic GM P-phosphodi-
esterase, the enzyme th at hydrolyzes cyclic GMP to 5'-GMP (Farber and Lolley,
1976). Cyclic GMP is synthesized norm ally by the rd visual cells via the guanylate
cyclase reaction from the onset of differentiation (Farber and Lolley, 1976).
Cyclic G M P-phosphodiesterase activity could never be d etected when it was as
sayed in hom ogenates o f freshly dissected retinas obtained from rd m ice of any
age. But if kinetic studies were carried out using freeze-dried retinas o f 12-day-old
rd mice, the enzyme was found to have a M ichaelis constant (Km) sim ilar to that
of p h o to recep to r cells o f control retina, although its m axim um velocity (Vmax)
was well below norm al (Farber and Lolley, 1977). T he abnorm ality in cyclic nucle
otide hydrolysis seems to be restricted to the retinal photoreceptors. Phosphodi
esterases in o th er tissues of the rd m ouse such as m uscle, skin, brain and several
blood com ponents - plasma, serum , lymphocytes and erythrocytes - have activities
com parable to those present in the corresponding tissues o f control mice (Lolley
and Farber, 1980).
128 DEBORA B. FARBER AND MICHAEL DANCIGER

Rod-specific cyclic G M P-phosphodiesterase is com posed of four subunits. The

two larger subunits (a: 88 kDa and (3: 84 kDa) have catalytic activity, an d the two
identical, sm aller y subunits (11 kDa) have inhibitory activity (Baehr, Devlin and
A pplebury, 1979; Fung, Young, Yamane a n d Griswold-Prenner, 1990). This en
zyme plays a key role in the p h ototransduction process o f rod visual cells, a cascade
of events which occurs in response to light. T he cascade begins with the isom eriza
tion o f the visual pigm ent rhodopsin (from 11-ris retinal to all-/w.s retinal), and
ends with the degradation o f cyclic GMP and the hyperpolarization of the rod p h o
to receptor m em brane, along with the inhibition o f n eu ro tran sm itter release at the
synaptic term inal of the ro d cell. T he activation of cyclic GM P-phosphodiesterase
by light is m ediated by rhodopsin, GTP and the h eterotrim eric protein transducin
and its deactivation requires rhodopsin kinase, ATP a n d arrestin (also re ferred to
as S-antigen o r the 48-kDa pro tein ).
Rod cyclic GM P-phosphodiesterase, which has been localized within the ph o to
recep to r o u ter segm ents (Robb, 1974), can also be activated in the rd m utant by
the phototransduction proteins. However, this activation is m inim al and it occurs
only during the postnatal period when the p h o to recep to r cells have m orphologi
cally intact o u ter segm ents. This is indicated by a small reduction in cyclic GMP
co n ten t during light adaptation (Farber an d Lolley, 1977).
Based on all of the above inform ation, it was inferred eith er that 1) the rd retina
h ad to have an abnorm ality in the cyclic G M P-phosphodiesterase m olecule itself
or 2) any o f the proteins involved in its activation or deactivation could be dysfunc
tional (Farber, 1989). O th er alternatives to consider were: 3) the rd enzyme could
have an excess of y subunit, in which case its activity could be greatly inhibited; 4)
the biosynthesis o f the rd enzyme could start later in rd than in norm al m ouse ret
ina o r it could be halted d u rin g the rd m ouse early postnatal life; 5) there could
be a greater degradation o f cyclic G M P-phosphodiesterase in rd than in norm al
retin a p rio r to the degeneration of the rd visual cells. Many different types o f in
vestigations were p erfo rm ed to clarify these possibilities.
To d eterm in e w hether the y subunit o f cyclic GM P-phosphodiesterase had the
co rrect am ino acid sequence an d was p resen t in the right concentration in the rd
retina, the cDNA encoding this subunit was first isolated and characterized from
n o rm al m ouse retina (Tuteja and Farber, 1988). This cDNA was then used as a
probe for the detection o f the corresponding mRNAs from norm al and rd retinas.
T h e mRNAs in both types of retinas were found to have the same size (approxi
m ately 900 bp) and concentration (Tuteja, Tuteja and Farber, 1989). F urther
m ore, cloning and sequencing o f the cDNA for the y subunit o f the rd enzyme
confirm ed th at the deduced am ino acid sequences o f the rd and norm al m ouse
retin a y subunits are identical (Tuteja, T uteja an d Farber, 1989). Thus, the y sub
u n it of cyclic GM P-phosphodiesterase is n o t the problem in the rd m ouse disease.
Studies on the synthesis o f cyclic G M P-phosphodiesterase indicated th at it starts
at the same tim e (postnatal day 6) both in n orm al and rd tissues an d th at the same
am o u n t o f newly synthesized polypeptides - recognized by affinity purified anti
bodies to the com bined a and [3 subunits o f the enzyme - is m ade in bo th cases
 INHERITED RETINAL DEGENERATIONS IN THE MOUSE 129

during all the developm ental period in which the p h o to rec ep to r cells are viable
(Farber et al., 1988). However, m easurem ents of cyclic GM P-phosphodiesterase
levels by radioim m unoassay showed th a t the concentration o f the enzyme in the
rd retina is already lower than norm al at 6 postnatal days, w hen no signs of degen
eration are discernible, an d th at it rem ains at about half the norm al level until the
p hotoreceptors begin to die (Farber et al., 1988). These results suggested that rd
cyclic G M P-phosphodiesterase is abnorm ally labile and that it gets degraded very
soon after it is m ade. In agreem ent with this idea is the finding th at peptides from
im m ature rd retinas that are recognized by antibodies against cyclic GMP-phos
phodiesterase sedim ent on sucrose gradients m ore slowly than the corresponding
proteins from norm al m ouse retina. T hese proteins m igrate within the gradient
with an a p p a ren t m olecular mass o f 105 kDa, w hereas the proteins from the n o r
mal retina form a com plex with an a p p a ren t m olecular mass o f 170 kDa. T he 170
kDa com plex is never d etected in extracts from rd retina (Lee et al., 1988). Thus,
the subunits of cyclic G M P-phosphodiesterase do n o t assemble into a functional
com plex in rd photoreceptors. This w ould m ake them susceptible to proteolysis
once they are synthesized.
D uring the course o f the studies focused on d eterm in in g w hether the proteins
involved in the activation/deactivation of cyclic G M P-phosphodiesterase in the rd
retin a were functional, a defect in the process o f dephosphorylation of rd rh o d o p
sin was identified. A lthough the co n cen tratio n o f the visual pigm ent is com parable
in norm al an d rd ph o to recep to rs until 10-11 postnatal days (Caravaggio an d Bon-
ting, 1963; Shuster an d Farber, 1986), rd rhodopsin cannot be phosphorylated in
vitro at any age (Shuster an d Farber, 1986). This lesion is n o t caused by an abnor
mality in rhodopsin kinase (the enzyme which catalyzes the transfer o f phosphate
from ATP to rh o d o p sin ), since the activity of the rhodopsin kinases from norm al
and rd retinas is com parable during all the developm ental period in which the rd
photoreceptors are viable (Palczewski, Farber an d H argrave, 1991). N or does the
lesion result from a defective rhodopsin m olecule, since at the mRNA level and in
im m unocytochem ical reactions, the rhodopsins from norm al an d rd retinas are
identical (Bowes a n d Farber, 1988; Bowes, van Veen an d Farber, 1989). Lack of
phosphorylation o f rd rho dopsin stem s from abnorm al activity of protein phos
phatase 2A, the enzyme that dephosphorylates rhodopsin (Palczewski, F arber and
Hargrave, 1991). P rotein phosphatase 2A activity is twice as high in rd than in nor
mal retina betw een 5 an d 10 postnatal days, although the rrf enzyme seems to be
structurally norm al. Apparently, some regulatory co m p o n en t p resen t in the rd ret
inal extract may be responsible for its increased activity. In previous studies carried
ou t in o u r laboratory, rhodopsin phosphorylation in o u ter segm ents o f norm al
rod photoreceptors was shown to be inhibited by high concentrations o f cyclic
GMP (Shuster and Farber, 1984). A possible explanation for this p h en o m en o n is
that raised cyclic GMP could en h a n ce the activity of protein phosphatase 2A,
which would remove the phosphate from rhodopsin as soon as the visual pigm ent
is phosphorylated. O u r findings with the rd retin a are consistent with this hypoth
esis. T he abnorm ality in the rd p ro tein phosphatase 2A activity w ould then be sec
130 DEBORA B. FARBER AND MICHAEL DANCIGER

ondary to the cyclic G M P-phosphodiesterase deficient activity which generates the

high levels o f cyclic GMP in the rd retina.
F u rth er investigations on the com ponents of the p h o to recep to r cell involved in
the activation/deactivation o f cyclic G M P-phosphodiesterase ruled ou t rh o d o p
sin, the a , p an d y subunits o f transducin, the a and y subunits o f cyclic GMP-phos
phodiesterase, the 33-kDa p rotein (also re ferred to as phosducin) and arrestin as
the cause o f the rd disease. In the first place, the mRNAs encoding m ost o f these
p roteins are qualitatively norm al in the rd retin a and are p roduced in the same
am ount and with the same onset o f expression as in control retina (Bowes an d Far
ber, 1987; Bowes, van Veen and Farber, 1988; Tuteja, Tuteja an d Farber, 1989). In
addition, each of the proteins translated from these mRNAs has the sam e time of
appearance during developm ent both in norm al and rd retinas (Bowes, van Veen
an d Farber, 1988). In the second place, the genes encoding the cyclic GMP phos
phodiesterase-related proteins are located on m ouse chrom osom es d ifferent from
th at to which the rd gene h ad been m apped, nam ely chrom osom e 5 (Sidm an and
G reen, 1965). Screening o f the candidate genes was carried o u t using somatic cell
hybrid analysis an d by linkage analysis o f backcross progeny. T he results are sum
m arized in Table 1. These studies indicated that no n e o f the proteins investigated
is the prim ary lesion in the rd disorder.

Isolation o f a C andidate cDNA fo r the rd Gene

C oncom itant with the work described above, the search for the rd gene began by
following a totally different strategic plan. This plan was based on the assum ption
th at the mRNA encoded by the rd gene would be eith er absent o r expressed in
h ig h er o r lower levels in the rd ph o to recep to rs com pared to norm al visual cells.
T he approach was to obtain the cDNAs present exclusively in the norm al p h o to
receptors an d then to com pare these cDNAs with the same pool o f cDNAs
p resen t in rd visual cells in retinas of 9- to 11-day-old mice. N ine to eleven day-old
rd/+ an d rd/rd litterm ates were used for the experim ents, since the m axim um
am o u n t o f age-m atched retinal tissue can be obtained at this age, before the rd/rd
cells suffer advanced degeneration.
T he adult m utant retin a appeared to be the perfect starting m aterial, since it is
devoid of all the ph o to recep to rs and is constituted solely by the in n er retinal lay
ers. A dult rd/rd retin a cDNAs were subtracted from those o f 9- to 11-day old nor
mal, rd/+ m ouse retina, and the photoreceptor-enriched, subtracted cDNAs were
used to probe a library o f the 9- to 11-day-old rd/+ retina. From approxim ately
100,000 clones tested, a pool o f 588 p h otoreceptor-enriched cDNAs was obtained.
These clones were screened by differential hybridization with single-stranded
cDNAs from 9- to 11-day-old rd/+ and rd/rd retinas and from adult rd/rd retina.
C om parison o f the positive clones hybridizing to the young an d the adult retinal
cDNAs confirm ed the p h o to rec ep to r specificity o f 400 clones; the rest could be
ru led out, since any clone able to hybridize to adult rd/rd m ouse cDNAs had to be
long to the in n er retinal layers. O f these 400 clones, 3 clones hybridized differently
with the 9- to 11-day-old rd/+ and rd/rd cDNAs. However, only one o f these candi-
 INHERITED RETINAL DEGENERATIONS IN TH E MOUSE 131

T a b le 1 C h r o m o s o m a l lo c i f o r th e g e n e s o f p r o t e i n s in v o lv e d in th e v isu al cycle o f r o d p h o
to r e c e p to r s .

Gene Mouse
Protein symbol Chromosome Reference

R hodopsin Rho 6 E lliott et al., 1990

T ransducin a -su b u n it (also called
G uanine n ucleotide binding
p ro tein a-subunit, transducin-1) Gnat-1 9 D anciger et al. 1989a
T randucin (3-subunit (also called
G uanine n u cleotide binding
pro tein (3-subunit-l [G p l]) Gnb-1 4 D anciger et al. 1990a
G uanine n ucleotide b in d in g
p ro tein p-subunit-2 (Gp2) Gnb-2 5 D anciger et al. 1990a

G uanine n ucleotide b inding

pro tein (3-subunit-l related
sequence (G(3lrs) Gnb-1 rsl 8 D anciger et al. 1990a
G uanine n ucleotide b inding
pro tein j3-subunit-3(G(33) Gnb-3 6 D anciger et al. 1992a
cGMP- phosphodiesterase a -
subunit Pdea 18 D anciger et al. 1990c
cG M P-phosphodiesterase y-
subunit Pdeg 11 D anciger et al. 1989c
In te rp h o to re c e p to r retinoid
binding p ro tein (IRBP) Rlyp-3 14 D anciger e t al. 1990b
33-kDa p ro tein (also called
p h o sp h o re ce p to r p ro tein -1 o r Rpr-1 or
phosducin) Pdc 1 D anciger et al. 1991
48-kDa p ro tein (also called
arrestin o r S-antigen) Sag 1 D anciger et al. 1989b
C alciu m /calm o d u lin -d ep en d en t
p ro tein kinase II a-su b u n it Camk2a 18 D anciger et al., 1992b

date clones for the rd gene, zr.408, gave a different hybridization signal on N orth
ern blots containing the mRNA o f young rd/rd retinas com pared with the mRNA
of age-m atched, rd/+ control retinas (Bowes et al., 1989).

Characterization o f the Candidate cDNA fo r the rd Gene

T he zr.408 clone hybridized to a set o f apparently slightly larger mRNA tran

scripts from rd/rd retin a than from n orm al retin a (3.6 vs 3.3 kb). At that time, it
was th o u g h t that this difference in mRNA size could result from an altered poly-
adenylation site, alternative splicing o r structural differences in the rd gene. As
132 DEBORA B. FARBER AND MICHAEL DANCIGER

discussed below, the second a n d third possibilities have been shown to occur. In
addition, differences betw een the rd/rd and + /+ m ouse DNAs revealed by
genom ic S outhern blot analysis, confirm ed th at structural differences in d eed
exist between the m u tan t an d norm al genes hybridized by zr.408. Tissue specific
ity o f the candidate cDNA was d eterm in e d by hybridization on slot blots to RNA
from several m ouse tissues. Only retinal RNA gave a strong signal, w hereas brain,
kidney, liver, lung and spleen RNAs were negative (Bowes et al., 1989).
T he chrom osom al location o f the gene corresponding to the zr.408 cDNA was
established by hybridization o f the candidate cDNA with the DNA o f ham ster-
m ouse somatic cell hybrids. T he zr.408 clone m apped, w ithout any discordancy, to
m ouse chrom osom e 5, the same chrom osom e to which the rd locus h ad b een as
signed. Expression of the mRNA encoded by the putative rd cDNA was detectable
on slot blots on postnatal day 1 in both norm al and rd/rd retinas, an d it continued
to increase steadily during developm ent. However, the concentration o f zr.408
mRNA was always lower in the diseased than in the + / + retina. By postnatal day 14,
following the degeneration p attern o f the rd retina, the rd zr.408 mRNA level d e
creased sharply and, as expected, it was n o longer detectable at 31 days o f age.
T herefore, the expression of the candidate rd cDNA is abnorm al in the rd/rd reti
n a from postnatal day 1, several days before cyclic GMP levels becom e elevated in
the p h o to rec ep to r cells. This is the earliest detected m olecular lesion in the rd dis
ease (Bowes et al., 1989).

M olecular G enetic Studies

In o rd e r to fu rth e r confirm that zr.408 was a p art o f the rd gene, two d ifferent
investigations were carried out. T he aim o f the first was to establish w hether the
gene corresponding to the zr.408 cDNA a n d the rd gene m ap p ed to the same
location on chrom osom e 5. T he second study was to verify if there was cosegrega
tion o f the candidate rd gene with the expression o f the rd disease, p h o to rec ep to r
degeneration, in m ouse crosses. Interspecies backcrosses were used for both
projects, since the mice resulting from these crosses have been shown to give
restriction fragm ent length polym orphism s (RFLPs) m ore frequently than those
resulting from in b red crosses.
To establish the location o f the gene corresp o n d in g to the zr.408 cDNA within
chrom osom e 5, the RFLPs d etected by hybridization to the zr.408 cDNA were first
analysed in the DNAs from N F S /N an d Mus musculus m ice. These RFLPs were
th en identified in DNAs from 62 m ice which were the backcrossed progeny o f the
cross (N FS/N x Mus musculus)F 1 xM us musculus. This allowed us to d eterm in e how
m any o f the 62 progeny were hom ozygous or heterozygous for zr.408. T he same
type o f analysis was p erfo rm ed for RFLPs th at were hybridized by Afp and Gits, two
o th e r genes that lie on opposite sides o f the rd gene on chrom osom e 5. From a
com parison o f the in h eritan ce p attern o f the three genes, we established the nu m
b er of recom binants present, an d from the rate o f recom bination, the distance in
centiM organs o f each gene relative to the others (D anciger et al., 1990). In this
 INHERITED RETINAL DEGENERATIONS IN THE MOUSE 133

way, zr.408 was placed betw een Afp an d Gus at distances sim ilar to those established
for the rd gene (C hapm an et al., 1975).
To d eterm in e w hether the zr.408 cDNA cosegregated with the retinal degener
ation trait, we classified 72 mice, the backcrossed progeny o f the cross (C57BL/6J
rd/rd x Mus spretus)FI x C57BL/6J rd/rd, eith er as hom ozygous (rd/rd, affected with
retinal degeneration) o r heterozygous (rd/+, m orphologically norm al) by two dif
feren t m ethods. In the first m ethod, backcross progeny were scored by RFLP
S outhern blot analysis to find ou t if they were hom ozygous or heterozygous for
zr.408 hybridizing fragm ents co rresponding to the C57BL/6J strain. In the second
m ethod, we p erfo rm ed a histological exam ination o f each o f the retinas o f the 72
progeny mice. W hen the results o f both approaches were com pared, zr.408 was
found to cosegregate with the rd disease with no recom binants; that is, each of the
assignm ents to rd/rd or rd/+ d eterm in e d by S outhern blot hybridization with
zr.408 corresp o n d ed exactly to the histologic identification (D anciger et al.,
1990).
T he m olecular genetic results from these two d ifferent studies su pported very
strongly the n otion th at zr.408 was a p a rt o f the rd gene. As described below, this
has since been confirm ed in studies o f transgenic mice.

Identification o f the P roduct o f the Candidate rd Gene

T he initial search for hom ologies betw een zr.408 an d cDNAs encoding the a and
P subunits o f ro d cyclic G M P-phosphodiesterase an d the cone a 1 subunits o f the
enzyme showed strong cross-hybridization with the ro d (3 subunit cDNA. Since
zr.408 had been isolated from a cDNA library p rep ared with RNA from rd/+ reti
nas, this clone was used to screen n orm al m ouse (+/+) retinal cDNA libraries.
Sequencing o f several o f the isolated clones an d o f PCR-derived fragm ents, estab
lished th at zr.408 is a tru n ca ted clone which corresponds to 2 /3 o f the total
sequence o f the p subunit o f cyclic G M P-phosphodiesterase (from base 1,114 to
base 2,706). T here is only one nucleotide changed in the zr.408 sequence, which
may be due to a polym orphic difference in the m ouse strains th at were used for
the generation o f the libraries (Bowes et al., 1990).
In o rd e r to fu rth e r confirm that zr.408 is a p art o f the P subunit o f cyclic GMP-
phosphodiesterase, a com parison o f the patterns o f hybridization o f poly (A)+
RNA an d genom ic DNA from rd/rd, rd/+ an d + /+ m ouse tissues with zr.408 cDNA
an d with several m urine an d bovine cDNA probes o f the P subunit o f cyclic GMP-
phosphodiesterase was carried out. T he same transcripts as well as RFLPs were ob
served on the N o rth ern and S outhern blots with all d ifferent cDNA probes tested.
These observations, along with the m apping of zr.408 to the rd region on m ouse
chrom osom e 5, su p p o rted the conclusion th at the norm al co u n terp art of the rd
gene encodes the P subunit o f cyclic G M P-phosphodiesterase (Bowes et al., 1990).
134 DEBORA B. FARBER AND MICHAEL DANCIGER

Conclusive D em onstration that the (3 Subunit o f Cyclic GM P-Phosphodiesterase is

the Site o f the rd M utation

In o rd e r to obtain definitive p ro o f that the gene encoding the (3 subunit o f cyclic

G M P-phosphodiesterase is responsible for the retinal d egeneration o f the rd
m ouse, its norm al cDNA was expressed in transgenic rd mice. T he construct
injected into one-cell stage m ouse em bryos consisted o f the com plete cDNA of
the gene ligated on its 5' e n d to the 5' flanking region o f the m ouse rod opsin
gene (to target expression o f the transgene specifically to the p h o to recep to r
cells) an d on its 3 end to the simian virus 40 polyadenylation signal. Two lines of
hom ozygous rd anim als on different genetic backgrounds expressed the trans
gene. O ne o f these lines showed com plete rescue o f the p h otoreceptors whereas
the o th er only exhibited partial rescue o f the visual cells, with only those cells in
th e superior tem poral region o f the re tin a appearing norm al. T he p h o to recep
tors of the inferior nasal region had com pletely d egenerated an d those from the
central retin a showed an interm ediate level of degeneration, with their in n er seg
m ents intact but their o u ter segm ents eith er disorganized o r absent. Both lines of
transgenic m ice h ad integrated a single copy o f the transgene (Lem et al., 1992).
T he cyclic G M P-phosphodiesterase activity in the retinas of the two different
lines of transgenic mice an d in those o f th eir non-transgenic, age-m atched rd/rd
siblings was com pared, in o rd e r to co rrelate enzyme expression with p h o to recep
tor rescue. T he retinas of mice that had com plete rescue of the p hotoreceptors
h ad enzyme activity similar to those o f norm al (+/+) anim als of the same age,
whereas the retinas o f the non-transgenic rd/rd siblings had m inim al o r no activity.
T he retinas o f the anim als th at showed a g rad ien t in expression of the transgene
h ad h ig h er cyclic G M P-phosphodiesterase activity in the superior than in the infe
rior hem isphere. These results indicated th at the enzyme activity correlates with
the n u m b er o f rescued p hotoreceptors present in the retin a and, fu rth erm o re,
th at the transgene was capable o f restoring the activity o f cyclic GM P-phosphodi-
esterase in r/l retina (Lem et al., 1992). T he study o f the transgenic m ice allowed
us unequivocally to conclude that the retinal degeneration phenotype in the ho
mozygous rd m ouse arises from a defect in the cyclic G M P-phosphodiesterase p
subunit gene.
T he rd m utation is an old one that is p resen t in a wide variety o f m ouse inbred
strains. T he cyclic G M P-phosphodiesterase P-subunit gene is quite com plex. It has
22 exons varying in length from 48 bp to several h u n d re d base pairs. A nonsense
m utation in the seventh exon (codon 347), which is predicted to give rise to a
tru n ca ted gene product, has been fo u n d (P ittler and Baehr, 1991). This m utation
potentially elim inates over one-half of the peptide, including the putative catalytic
dom ain. We have recently re p o rted the presence in the rd retin a o f cyclic GMP-
phosphodiesterase p-subunit transcripts th at are incorrectly spliced (Bowes et al.,
1992). A b erran t processing an d expression o f these transcripts could result from
the integration o f an 8.5 kb m urine leukem ia provirus (Xmv-28) into the first in
tron (Bowes et al., 1992; 1993). A 100% correlation was found betw een the pres
 INHERITED RETINAL DEGENERATIONS IN THE MOUSE 135

ence o f Xmv-28 at this site and the rd m utation in 16 m ouse strains, while n o n e of
36 non-rd strains contained Xmv-28 (Bowes et al., 1993). T he Xmv-28/rd allele has
also been found in strains derived from wild mice caught recently in Asia, USA and
Britain, which raises questions ab o u t the evolutionary significance o f such a wide
spread and apparently ancient m utation. As m en tio n ed before, the levels o f [3-sub-
u n it mRNA are greatly red u ced in rd mice as well as abnorm al in size. It is
therefore unclear w hether the ab e rra n t transcription or the stop codon or both
give rise to the rd phenotype. We are currently investigating the m echanism s that
lead from cyclic G M P-phosphodiesterase deficiency to death o f rd photoreceptors.
A variety o f o th er anim al m odels o f retinal degeneration have been identified,
one o f which also appears to have a m utation in the P subunit of ro d cyclic GMP
phosphodiesterase (Farber et al., 1992). T he Irish setter dog suffers from an early
onset rod-cone p h o to rec ep to r deg en eratio n d u rin g the period o f o u ter segm ent
elongation. T he retinas o f these dogs are deficient in cyclic GM P-phosphodi
esterase activity and have greatly elevated levels o f cyclic GMP that precede m or
phological signs o f degeneration, sim ilar to rd/rd mice (Aguirre et al., 1978).
While the p h o to recep to r cells o f the diseased retinas are viable, the mRNAs for op
sin, transducin oc^ an d pj, arrestin, phosducin an d the a an d y subunits o f cGMP-
phosphodiesterase have norm al transcript sizes an d levels. In contrast, the cyclic
G M P-phosphodiesterase p-subunit mRNA is p resen t in levels lower than norm al
from early life (Farber et al., 1992). Since this abnorm ality is observed prior to any
signs o f arrested p h o to rec ep to r developm ent an d p rio r to the described biochem
ical defect (Aguirre et al., 1982), o u r results indicate a specific involvem ent o f cy
clic G M P-phosphodiesterase P-subunit in the Irish setter disease. Recently, Suber
et al.(1993) showed that this is associated with a nonsense m utation in the canine
hom ologue of the rd gene which is pred icted to elim inate 49 am ino acid residues
from the C term inal e n d o f the P subunit o f cyclic GM P-phosphodiesterase, includ
ing an isoprenylation addition site. This isoprenylation site o f the norm al p-sub-
unit may facilitate its interaction e ith e r with the disk m em branes or with o th er
protein subunits of the enzyme.
Finally, several m utations have recently been re p o rted in the hum an hom ologue
of the rd gene in patients with autosom al recessive retinitis pigm entosa (ARRP).
M cLaughlin et al. (1993) studied 7 o f the 22 exons of the hum an cyclic GMP-phos-
phodiesterase p-subunit gene in 99 un related patients. They fo u n d 4 m utations
that were n o t observed in 100 control individuals w ithout ARRP an d that co-seg-
regated with the disease in families. Two o f these were nonsense m utations (Gin
298X an d Arg 513X), one was a missense m utation (His 557 Tyr) an d one was a
one bp deletion (Pro 496). A lthough fu rth e r work is req u ired to establish the fre
quency o f these m utations in ARRP, these exciting findings provide fu rth e r valida
tion o f the rd m ouse as a m odel for hu m an retinal degeneration.
136 DEBORA B. FARBER AND MICHAEL DANCIGER

THE rds MOUSE

In the late 1970s, a new retinal d egeneration was described in the 020/A inbred
m ouse strain which was found to be due to a single defective gene in h erited as an
autosom al recessive (van Nie et al., 1978). T he gene was localized distal to and
n ea r the H-2 locus in the m iddle region o f m ouse chrom osom e 17 (D em ant
et al., 1979). Due to the relatively slow rate o f degeneration o f the retinal ph o to
receptors in the 020/A mice when com pared with the rate o f d egeneration of
these cells in hom ozygous rd mice, the m utation was nam ed retinal degeneration
slow or rds (van Nie an d D em ant, 1978).
D egeneration in hom ozygous rds mice is characterized by absence of the o uter
segm ents o f ro d p h otoreceptors and the gradual and com plete loss o f the rest of
these visual cells over time. T he rod o u ter segm ents, which norm ally begin to d e
velop during the first week of life, simply fail to form in the rds m ouse even though
the o th er retinal layers appear to develop norm ally. In place o f the rod o u ter seg
m ent, a bulb o f plasm a m em brane w ithout any disk structures accum ulates on top
o f the cilium. T he o u ter segm entless p h o to rec ep to r cells then gradually d egen
erate over a p erio d of 7 m onths to a year u n til they are no longer found in the ret
ina (for reviews of the rds phenotype, see Sanyal et al., 1985; Hawkins et al., 1985;
C ohen, 1983). A lthough the rds m utation was first decribed as a recessive, rds/+
heterozygotes show rod o u ter segm ents th at are reduced in length an d contain ir
regular, swollen and vacuolated disks. T he ph o to recep to rs also u n d erg o very slow
degeneration, so th at the rds m utation is sem i-dom inant.

Identification o f the Defective rds Gene

Over a period of several years, m any im m unocytochem ical studies were per
form ed which com pared the am o u n t an d localization o f several p h o to rec ep to r
proteins in rds and norm al retina. T he proteins investigated included the inter-
p h o to rec ep to r retinoid binding protein (IRBP), opsin, arrestin, transducin,
actin, an d sulfated proteoglycans (van Veen et al., 1988; Carter-Dawson and Bur
roughs, 1989; Schalken et al., 1990; Navon e t al., 1987; Jansen et al., 1990; C haitin
et al., 1988; Tawara an d Hollyfield, 1990; C antera et al., 1990). A lthough each of
these studies co ntributed to o u r un d erstan d in g o f the m echanism o f p h o to recep
tor degeneration, n o n e was able to specifically p o in t to the defective gene in the
rds retina. In fact, the rds gene was identified by a com pletely different approach,
again utilizing the techniques of m olecular biology (Travis et al., 1989).
Retinal cDNA was m ade from adult rd/rd mice which, as described earlier, do
n o t have any photoreceptors. This pool o f photoreceptorless cDNAs was used to
subtract non -p h o to recep to r messages from cDNAs isolated from norm al retina.
T he rem aining cDNAs from norm al retina, highly en rich ed for p h o to recep to r
cDNAs, were cloned an d screened with single stranded cDNAs from developing
norm al ds/rds retinas. Twelve clones were selected which seem ed to give d ifferent
hybridization signals with m u tan t an d norm al probes. N o rth e rn blot analysis and
 INHERITED RETINAL DEGENERATIONS IN TH E MOUSE 137

chrom osom al localization by m eans o f som atic cell hybrids revealed a single cDNA
clone that hybridized m ore weakly with rds/rds retinal mRNA (com pared to n o r
mal retinal mRNA) th at is increased in size an d m apped to m ouse chrom osom e
17. F u rth er study o f the rds m utation dem onstrated that the gene was defective
due to disruption of the coding region by insertion o f a 2.7 kb m ouse repetitive
elem ent at nucleotide position 899 (the entire coding sequence is 1038 bp, coding
for 346 am ino acids; Travis et al., 1989).
T he norm al pro d u ct o f the rds gene is a p h o to rec ep to r disk protein called rd s /
p erip h erin to distinguish it from an u n re la ted neurofilam ent protein called pe-
rip h erin (C onnell et al., 1991; Travis et al., 1992a; Arikawa et al., 1992). T he
bovine hom ologue o f rds/p e rip h e rin h ad been identified independently of the rds
study (C onnell and Molday, 1990). R d s/p e rip h e rin is a 39 kDa glycoprotein o f u n
known function, localized by im m unocytochem istry to the rims o f p h o to rec ep to r
disks (Molday et al., 1987) which is pred icted to have four m em brane-spanning
a-helices an d is expressed exclusively in the ro d an d cone p h o to recep to r disks.
T he rds m utation would be expected to tru n cate the protein p ro d u c t by 116 am ino
acids with loss o f the glycosylated region o f the intradiscal D2 loop, th o u g h t to be
im p o rtan t in interaction with o th er proteins, an d o f the fourth m em brane-span
ning segm ent and the cytoplasmic C3 segm ent (Travis et al., 1989).
C om plete rescue o f the m u tan t rds phenotype has recently b een achieved in
transgenic m ice th at h ad in co rp o rated norm al copies o f the rd s /p e rip h e rin cDNA
(Travis et al., 1992b). T he transgene contained upstream regulatory sequences
from the m ouse opsin gene fused to a norm al rd s /p e rip h e rin m ini-gene with the
SV40 early splice an d polyadenylation signals. T he construct was m icroinjected
into fertilized m ouse eggs from rds/+ m utants. T hree transgenic lines were ob
tained an d crossed with hom ozygous rds m utants to observe the effects o f the
transgene in a hom ozygous rds/rds background. Two high rd s/p e rip h e rin express
ing lines showed retinas th at were indistinguishable from the wild-type, with nor
mal o u ter segm ent m orphology an d no p h o to rec ep to r degeneration. An
interm ediate phenotype was seen in a low expressing line, with about 10% o f wild-
type rd s/p e rip h e rin levels, which showed whorls o f disorganized disks in the sub-
retinal space, representing dysplastic o u ter segm ents. N on-transgenic litterm ates
showed the typical rds/rds m u tan t phenotype. These results indicate th at the semi
d om inant rds phenotype probably results from haplo-insufficiency - lack o f suffi
cient norm al gene p ro d u ct even in the heterozygous state - ra th e r than a delete
rious effect o f the m u tan t p ro d u ct per se (dom inant negative m utation). This
would be consistent with the rds m utation being a null allele, with little or no ex
pressed product, as predicted from the m utation analysis.
T he introduction of upstream p ro m o ter sequences from the m ouse opsin gene
into transgenic mice coupled to a lacZ re p o rte r gene had previously been shown
to drive rod-specific transgene expression with onset shortly after the appearance
of o u ter segm ent structures (around the first postnatal week) (Lem et al., 1991;
Zack et al., 1991). T he transgenic rescue o f rds m utants would be expected to re
verse rod bu t n o t cone p h o to rec ep to r degeneration, since rds/p e rip h e rin is ex
138 DEBORA B. FARBER AND MICHAEL DANCIGER

pressed in both cell types and the opsin p ro m o ter is rod-specific. However, only
ab out 3% o f photoreceptors in mice are cones (Carter-Dawson et al., 1978) an d it
was not possible to confirm this prediction (Travis et al., 1992b).

T he H um an C ounterpart o f the Mouse rd,s G ene Causes H um an Retinitis

Pigm entosa and M acular Degenerations

T he nam e retinitis pigm entosa (RP) represents a heterogeneous group o f dis

eases that have in com m on a progressive retinal degeneration, the presence of
pigm ent deposits in the retina and night blindness. T he incidence in the US is
ab o u t 1 in 3,500 people and the in h eritan ce p attern is quite heterogeneous.
Autosom al recessive, autosom al dom inant, X-linked and sim plex (no family his
tory o f disease) are all com m on patterns o f inheritance o f the various form s of
RP. F urtherm ore, there are many clinical variations within each inheritance pat
tern.
Recently, the cause of a n u m b er of cases o f autosom al d om inant RP (ADRP) has
been assigned to defects in the rhodopsin gene. T he focus on rhodopsin started
with its localization to the long arm of hum an chrom osom e 3 (3q) (Sparkes et al.,
1986; N athans et al., 1986). This was followed a few years later by the discovery of
linkage of ADRP to the same region o f chrom osom e 3q in a large Irish family
(McWilliams et al., 1989) and then linkage to rhodopsin itself in the same pedi
gree (F arrar et al., 1990). From there, a n u m b er o f point m utations were found in
the rhodopsin gene th at cosegregated perfectly with the ADRP cases in particular
families (Dryja et al., 1990a,b; Keen et al., 1991; Gal e ta l., 1991; Inglehearn et al.,
1991; F arrar et al., 1991a). However, in o th e r families, genetic analysis revealed
th at the disease gene was specifically n o t linked to chrom osom e 3q (F arrar et al.,
1990; In g leh earn et al., 1990; B lanton et al., 1990). In fact, it has been estim ated
th at only about 20-30% o f the cases of ADRP are due to defects in the rhodopsin
gene (Kajiwara et al., 1991).
T he rds gene story follows a sim ilar line o f discovery as that o f the rhodopsin
gene. After the cloning o f the m ouse rd s/p e rip h e rin gene, its hum an hom ologue
(RDS) was m apped to chrom osom al region 6 p l2 (Travis et al., 1991). T h en , link
age o f ADRP to chrom osom e 6p and specifically to the R D S /p erip h erin locus was
dem onstrated in a large family (F arrar et al., 1991b; Jo rd a n et al., 1992). Finally,
as in the case o f rhodopsin, m utations in the p erip h erin gene that segregated with
RP were found in particular families (Kajiwara et al., 1991; F arrar et al., 1991c).
T h e m utations were eith er single base changes o r 3 base deletions an d were dis
covered by single strand conform ational polym orphism analysis. F u rth er analysis
of the R D S /p erip h erin gene in hum an retinal disease states has revealed fu rth e r
m utations associated with a surprising degree o f clinical heterogeneity. These m u
tations have been found in autosom al d o m in an t families with typical retinitis pig
m entosa, with various types of m acular dystrophy, w'ith eith er retinitis pigm entosa
or m acular dystrophy in different m em bers o f the same kindred, an d in a family
with retinitis punctata albescens.
 INHERITED RETINAL DEGENERATIONS IN TH E MOUSE 139

T he m ain difference betw een the course o f scientific discovery o f the rhodopsin
m utations and o f the R D S /p erip h erin m utations in m an was the direct involve
m en t o f the rds anim al m odel o f retinal degeneration in the latter case. H um an
m apping an d pedigree linkage analysis p u t rhodopsin and ADRP together. Since
rhodopsin is the m ost ab u n d a n t p rotein in ro d photoreceptors, it was a logical
choice for the site o f a gene defect. Interest in the hum an p erip h erin gene as a
candidate for RP m utations occu rred after it was dem onstrated that its m urine h o
m ologue was responsible for the rds disease, one o f several m ouse m odels o f RP.
This is a straightforw ard exam ple o f a case w here the hum an hom ologue o f a gene
responsible for an in h erited disease in an anim al is responsible for a sim ilar h u
m an disease. As discussed above, the hum an co u n terp art of the rd g ene also now
appears to cause retinal d egeneration in m an. It may well be that, w hen the m o
lecular basis o f the o th er rem aining m ouse retinal degeneration m utants becom es
known, fu rth e r hum an disease genes will be identified and explained.

MAPPING OF OTHER GENES EXPRESSED IN RETINAL TISSUES

T he gene for rhodopsin (Rho) was localized to distal m ouse chrom osom e 6 in the
vicinity of the m icroophthalm ia (mi) locus (Elliott et al., 1990), an in h erited
m urine defect which results in a sm aller eye. A nother gene expressed in the re t
ina has recently b een m apped to the same chrom osom al region, a third G p ro
tein p-subunit, Gnb-3 (D anciger et al., 1992a). F u rth er studies will n eed to be
carried ou t with both Rho and Gnb-3 now that they are potential candidates for mi
based on their chrom osom al location.
T he gene for the a-subunit of calciu m /calm o d u lin -d ep en d en t p rotein kinase II
(Camk2a ) has also recently been m ap p ed to m ouse chrom osom e 18 in the vicinity
of the bouncy (be) neurological m u tan t (D anciger et al., 1992b). T he Camk2a gene
p ro d u ct is p resen t in brain, re tin a a n d o th er tissues. Since the only published in
form ation about the be m u tan t is related to its neurological symptoms (bouncy gait
and trem or) or its breed in g capabilities (Lane et al., 1981; G reen, 1989), it is pos
sible that pathology involving o th er tissues (such as retina) may occur. T herefore,
by virtue of its chrom osom al location and expression in brain a n d o th er tissues,
the Camk2a gene is a potential candidate for the be m utation.
In the course o f m apping the transducin p-subunit gene (Gnb-1) to m ouse chro
m osom e 4, we localized two o th er hom ologous sequences to m ouse chrom osom es
5 and 8 (D anciger et al,, 1990a). C hrom osom al m apping was done by S outhern
blot analysis o f C hinese ham ster-m ouse som atic cell hybrid DNAs th at were
probed with the X TB112 cDNA for the Gnb-1 gene (provided by Dr. Melvin I. Si
m on [Sugim oto et al., 1985; Fong et al., 1986]; Figures 4 an d 5 an d Table 2 ). Link
age analysis with the m arker genes Afp an d Gus (Table 2) was used to localize the
chrom osom e 5 sequence (Gnb-2) to a region that was hom ologous to the region of
hum an chrom osom e 7 w here a second G p rotein p-subunit gene (GNB2) had
been m apped (Blatt et al., 1988; Hillyard et al., 1992; Lyon and Kirby, 1992; Searle
140 DEBORA B. FARBER AND MICHAEL DANCIGER

et a l , 1987). Linkage data was collected prim arily by m eans o f allelic RFLP analysis
o f S outhern blots (Figure 6 and Table 3). T he G n b -2 gene was in the vicinity of the
rd locus, but m ore than 10 cM distal to it (Hillyard e t al., 1992; Lyon an d Kirby,
1992). T herefore, fine m apping dem onstrated th at G n b -2 is n o t the ratgene - which
was later confirm ed by the identification o f the rd site as the cyclic GM P-phospho-
diesterase p-subunit gene - avoiding the n eed for fu rth e r effort on the G n b -2 gene.

1 2 3 4 5 6 7 8 9

- 23.1 kb

-- 9.4 kb

- 6.6 kb
8

5 -> -- 4.4 kb
4

S
2.3 kb
~ 2.0 kb

F ig u re 4 A u to r a d io g r a m o f a S o u th e r n b lo t o f h a m s te r-m o u s e so m a tic cell h y b rid D N A s d i

g e s te d w ith H in tM l a n d h y b rid iz e d w ith th e X TB112 cD N A p r o b e ; e a c h la n e h a s 6 |I g o f DN A.
L a n e 1, C h in e s e h a m s te r c e ll DN A; la n e 2, N F S /N m o u s e liv er D N A ; la n e s 3 -9 , re p re s e n ta tiv e
s o m a tic cell h y b rid D N As. T h e five a rro w s p o i n t to th e > 23 .1 -,1 5 .0-, 5.9-, 4.5- a n d 3.9-kb m o u s e
b a n d s u s e d to s c o re f o r th e p r e s e n c e o f h o m o lo g o u s s e q u e n c e s in th e h y b rid s. T h e n u m b e r s
o n th e r i g h t (in k b ) m a r k th e p o s itio n s o f X D N A f r a g m e n ts c r e a te d by d ig e s tio n w ith H in tM l.
T h e n u m b e r s o n th e le ft in d ic a te c h r o m o s o m e a ssig n m e n ts. D N A e le c tr o p h o r e s is w as c ar
r ie d o u t in 1 .2 % a g a ro s e g els a n d th e s e p a r a te d f ra g m e n ts w e re tr a n s f e r r e d to n y lo n
m e m b r a n e s by th e t e c h n iq u e o f S o u th e r n (1 9 7 5 ). H y b rid iz a tio n w ith la b e le d p r o b e w as c a r
r ie d o u t as d e s c rib e d p re v io u sly ( D a n c ig e r e t al., 1 9 9 0 a ).
F igu re 5 A u to r a d io g r a m o f a S o u th e r n b lo t o f D N A s f ro m th e m o u s e d ig e s te d w ith H b id lll,
h y b rid iz e d w ith th e A T B 1 12 cD N A p r o b e , a n d w a sh e d a t in c re a s in g s trin g e n c y f ro m le ft to
r ig h t; e a c h la n e w as o n a s e p a r a te s trip o f n y lo n m e m b r a n e a n d h a d 6 (J,g o f D N A . T h e n u m
b e rs o n th e le ft in d ic a te c h r o m o s o m e a s s ig n m e n ts . T h e n u m b e r s o n th e r ig h t (in k b ) m a rk
th e p o s itio n s o f X D N A f r a g m e n ts c r e a te d by d ig e s tio n w ith f t i n M l . In t h e h ig h e r strin g e n c y
w a sh e s o n ly th e f ra g m e n ts th a t m a p to m o u s e c h r o m o s o m e 4 re s is te d b e in g s e p a ra te d fro m
th e p r o b e . T h is in d ic a te s th a t th e c h r o m o s o m e 4 site is th e m o s t h o m o lo g o u s t o th e A, T B 1 12
p r o b e a n d su g g e sts th e r e f o r e , th e site o f t h e Gnb-1 g e n e . T h is a s s ig n m e n t is f u r t h e r c o r r o b o
r a te d by th e fa c t th a t Gnb-1 is lo c a liz e d to a re g io n o f m o u s e c h r o m o s o m e 4 t h a t is h o m o lo
g o u s to h u m a n c h r o m o s o m e 1, th e c h r o m o s o m e to w h ic h th e h u m a n h o m o lo g u e o f Gnb-1
(G N B 1) m a p s (B la tt e t al., 1988; D a n c ig e r e t al., 1990a; H illy a rd e t al., 1992; L yon a n d Kirby,
1992).
142 DEBORA B. FARBER AND MICHAEL DANCIGER

T a b le 2 C o r r e la tio n b e tw e e n sp e c ific m o u s e c h r o m o s o m e s a n d r e s tr ic tio n f r a g m e n ts hy

b r id iz in g to th e A.TB112 cD N A (Gnb-1, Gnb-2, G n b lrs - i, in C h in e s e h a m s te r- m o u s e s o m a tic
c e ll h y b rid s a) .

Gnb-1 Gnb-2 Gnblrs-1

Alouse Chromosome % Discordant1 %Discordanl) %Discordantb

1 33 50 38

2 46 58 39

3 38 38 33
4 0 36 24
5 33 0 29
6 42 62 35
7 50 77 65
8 20 40 4C

9 23 35 23
10 27 15 19
11 33 11 22
12 42 74 47
13 38 58 46
14 32 32 24
15 59 88 71
16 30 26 39
17 46 58 39
18 29 38 29
19 32 52 24
X 36 44 36

a T h e chrom osom e c o n te n t o f the som atic cell hybrids was d e te rm in e d by trypsin-Giem sa b an d in g fol
lowed by staining with H oechst 33258, o r by typing for specific m ark e r loci.
b T h e n u m b e r o f som atic cell hybrids th a t had the p a rticu la r m ouse chrom osom e b u t did n o t show the
m ouse hybridizing sequence o f th e gene, plus th e n u m b e r o f hybrids th a t did not have th e c h ro m o
som e b u t d id show the hybridizing sequence o f th e g en e, divided by the total n u m b e r o f hybrids eval
u a te d for th a t p a rticu la r chrom osom e.
c T h e 4% discordancy was d u e to o n e hybrid w hich c o n ta in ed Chr.8 , but did not show th e 5.9-kb scor
ing m ouse fragm ent. T h e discrepancy can be exp lain ed by the fact th at this hybrid line had a low
percentage o f cells c o ntaining C hr.8 (low copy n u m b er). F urther, the C hr.8 assignm ent was co rro b
o rated by m ultilocus linkage analysis o f backcross progeny (D anciger e t al., 1990a).
 INHERITED RETINAL DEGENERATIONS IN THE MOUSE 143

1 2 3 4 5

#
mm

F igu re 6 A u to ra d io g r a m o f a S o u th e r n b lo t o f te s t c ro ss p ro g e n y D N A s fro m th e c ro ss (N F S /
N x Al. m. m usculus)F I x M . in. musculus. D N A s w e re d ig e s te d w ith H in d lll a n d h y b rid iz e d w ith
X T B 1 12 cD N A ; e a c h la n e h a s 10 |ig o f D N A . L a n e 1, p a r e n ta l N F S /N DN A; la n e s 2-5, r e p r e
s e n ta tiv e b a c k c ro ss p ro g e n y . T h e a rro w s p o in t to a 14 k b A l m. m usculus f r a g m e n t ( A l m. m.)
p r e s e n t in all te s t c ro ss p r o g e n y a n d a 4.5 k b f r a g m e n t r e p r e s e n tin g th e u n i q u e N F S /N a lle le
(fo r Gnb-2 ) p r e s e n t o n ly in h e te ro z y g o te s . D N A s w e re e x tr a c te d fro m m o u s e livers, c le a v e d
w ith H in d lll, r u n o n 0.4% a g a ro s e gels, t r a n s f e r r e d to n y lo n m e m b r a n e s a n d h y b rid iz e d to
la b e le d p r o b e as d e s c rib e d by I l o g g a n e t al. (1 9 8 8 ).
144 DEBORA B, FARBER AND MICHAEL DANCIGER

T a b le 3 S e g r e g a tio n o f a 4 .5 -k b H in d lll h y b r id iz in g f r a g m e n t (Gnb-2) w ith

a lle le s o f A fp a n d G ut'' in th e c ro ss ( N F S /N x M .rn. m usculus) F I - M . m.
m usculus.

LOCI No. o f progeny

Afp Gnb-2 Gus

parentals
+b + + 21
- - - 23

single recombinants
+ -
- +
+ +
- -
double recombinants
- +
- 9
+ 6
- 3
+ 0
- 1

% R ecom bination
(Afp, Gnb-2) = 16/6 3 = 25 5.5C
(Gnb-2, Gus) = 4 /6 3 = 6 3 .i

a Afp a n d Gus alleles were scored by S outhern blot hybridization with ap p ro p ria te cDNA probes. DNA
was digested with EcoRI for Afp, and llirullil for Gus. A unique 6.0-kb N F S/N restriction frag m en t hy
bridized with a 960-bp cDNA p ro b e released from the pBR322-AFPl plasm id (provided by Dr. Shirley
T ilghm an, Princeton University [D Eustachio et al., 1981]). A u n iq u e 3.7-kb N F S/N restriction frag
m en t hybridized with a 1.45-kb cDNA probe c o rresp o n d in g to the Gus gene, released from the pGA l
plasm id (provided by Dr. G ordon W atson, UC Berkeley [Watson e t al., 1985]).
b T he symbol refers to progeny th at are heterozygous.
'P e rc e n t recom bination and standard e rro rs were calculated according to G reen (1981); all recom bi
n a n t fractions are significant to the 0.05 level.

M olecular Genetic Studies in the M ouse Could Increase our U nderstanding o f

H um an Diseases

T he hum an genom e can be viewed as a rearran g em en t o f chrom osom al pieces of

the m ouse genom e, o r vice versa. For exam ple, hum an chrom osom e 1 has large
regions o f hom ology with m ouse chrom osom es 1, 3 and 4, an d hum an chrom o
some 5 has large regions o f hom ology with m ouse chrom osom es 11, 13 an d 18
(Searle et al., 1987; Lyon and Kirby, 1992; Hillyard et al., 1992). T he rearran g e
m ents probably re p resen t a history of translocations an d o th er alterations in the
two genom es as they diverged from a com m on ancestor millions o f years ago. In
fact, all o f the genes that have been m apped by o u r laboratory eith er fell within,
o r extended in a distal or proxim al direction, these m o u se/h u m an hom ologous
chrom osom al regions. Accordingly, nearly any localization o f a m urine gene can
suggest the location o f its hum an hom ologue based on hom ologies betw een m an
and mouse.
 INHERITED RETINAL DEGENERATIONS IN THE MOUSE 145

O nce a gene has been localized in the m ouse genom e, it is an easy m atter to
check the chrom osom al m aps to see if the hom ologous region in the hum an con
tains any m utations. For exam ple, N orth C arolina m acular dystrophy has recently
been localized to hum an chrom osom al region 6q by linkage analysis of a single
large family (Small et al., 1992). H um an 6q has hom ology with small regions of
m ouse chrom osom es 4, 9 and 17 an d with a larger region o f m ouse chrom osom e
10. Thus, any m urine retinal gene m apping to these regions becom es a potential
candidate for this disease. T here are o th er in h erited retinal diseases that have
been linked to regions o f the hu m an genom e an d to which the same logic can be
applied: U sher syndrom e type II (Lewis et al., 1990; K im berling et al., 1990); sev
eral form s o f X-linked retinitis pigm entosa (M usarella et al., 1991; 1989; O tt et al.,
1990; Levy et al., 1989) an d the RP1 form o f autosom al d o m in an t retinitis pigm en
tosa (B lanton et al., 1991).
In summary, the advances in the fields of m olecular biology a n d m olecular ge
netics that have o ccu rred in the last few years, together with the availability o f an
imal m odels of retinal deg en eratio n - eith er in h erited or created by the
integration of m u tan t genes into the m ouse genom e - have o p en ed up trem en
dous possibilities for the u n d erstan d in g o f genetic diseases affecting the retina.
We are encouraged by the re cen t findings an d hope that in the n ear future no t
only the causes will be unravelled bu t also ways to prevent or cure the devastating
disorders that lead to blindness.

ACKNOWLEDGEMENTS

We wish to thank Dr. Cathy Bowes for h er co ntinued contribution to the study o f
the rd m ouse an d Dr. J o h n F lannery for providing us with the figures of the n o r
mal an d rd m ouse retinas. This work was supported by the N ational Institutes of
H ealth grants EY02651 an d EY08285 and Core g ran t EY0331; a C enter g rant from
the N ational Retinitis Pigm entosa F oundation; and a g ran t from the G eorge
G und F oundation. D.B.F. is the recipient o f a Research to Prevent Blindness
Senior Scientific Investigator Award.

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6. AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA

PETER HUMPHRIES, PAUL KENNA and G. JANE FARRAR

Depart ment of Genetics, Trinity College, Dublin 2, Ireland

SUMMARY

Retinitis pigm entosa (RP) is the collective term for a group o f in h erited diseases
of the eye involving a degeneration o f the retina, the light sensitive m em brane
carrying the ro d an d cone photoreceptors. As these cells die off, symptoms of
night blindness develop, usually followed by a gradual constriction o f the
patient's field o f vision. In addition, pigm entary deposits may build up in the
re tin a as a result o f d egeneration o f the neural epithelial cells, which has given
rise to the nam e retinitis pigm entosa. RP is a prevalent genetic disorder, affecting
approxim ately one in every four thousand persons (probably ab o u t one an d a
half m illion people th ro u g h o u t the w o rld ). It can be in h erited in an X-linked, an
autosom al d o m in an t or a recessive m ode. Approxim ately fifty p ercen t of cases
have no family history and are presum ed to be the result o f autosom al recessive
disease, reduced penetrance, new X-linked o r autosom al d o m in an t m utations or
phenocopies. It is in the autosom al d o m in an t form of the disease th at m uch
recen t progress has been m ade in o u r u n d erstanding o f the m olecular pathology
of RP. H ere, the genetic aetiology has been shown to have its origins in defects
either o f the rhodopsin o r rd s/p e rip h e rin proteins, both o f which are com po
nents of the rod p h o to rec ep to r o u ter segm ent disc m em branes. Recently rh o d o p
sin m utations have also been identified in patients with an autosom al recessive
form RP. G enetic linkage studies have identified the location of fu rth e r loci
responsible for adRP on chrom osom es 8, 7p, 7q an d 19q indicating th at fu rth e r
genes rem ain to be identified. An intriguing picture o f the m olecular basis for
the degenerative processes involved in RP is now em erging.

INTRODUCTION

The group o f degenerative conditions o f the eye term ed retinitis pigm entosa
(RP) represents the m ost prevalent retinopathy (disease o f the retina) showing
clear-cut M endelian inheritance. T he disease is at p resen t incurable, appears to
be p resen t in all of the w orlds populations an d currently afflicts up to 1.5 million
people. Pathological changes associated with the disease include the progressive
death of ro d p h o to recep to r cells. O f the two types o f p h o to rec ep to r within the
retina, rods an d cones, the rods are by far the m ost light-sensitive, containing the
visual pigm ent rhodopsin. R hodopsin is too sensitive to function in norm al day
light, b u t enables the eye to perceive light in dimly lit conditions (at night, for
 154 PETER HUM PHRIES ET AL.

exam ple). As the rod cells die off, night blindness, or nyctalopia, develops. In
some cases, the progression o f the disease is rapid, n ight vision problem s devel
oping within the first decade o f life. In o th e r instances, such symptoms may not
m anifest until the fifth decade o r beyond. Variability o f clinical expression is a
classical feature in some genetic types of RP, particularly the d o m in an t form . T he
d eath of the rod p h otoreceptors is followed by m ore extensive pathological
changes in the retina. C one cells, responsible for the perception o f colour, begin
to die off an d there is a gradual constriction o f the patient's visual fields (Figure
I)-

A
90

F igu re 1 D ia g ra m s o f th e v isu a l fie ld s o f th e le ft eye fr o m n o r m a l a n d RP p a tie n ts .

(A) show s th e visual fie ld o f a n o r m a l in d iv id u a l, u s in g ta rg e ts o f d e c r e a s in g size. T h e b la c k
a r e a is th e e x te n t o f th e p h y sio lo g ic a l b lin d sp o t, c a u s e d by th e o p tic n e rv e h e a d .
(B) show s th e visual fie ld o f a R P p a ti e n t w ith m o d e ra te ly a d v a n c e d r e tin o p a th y . T h e h a tc h e d
a r e a is th e e x te n t o f a n o n - s e e in g a r e a (s c o to m a ) in th e r e tin a l m id - p e r ip h e r y to th e la rg e st
siz e d ta rg e t. N o te also th e loss o f p e r ip h e r a l fie ld in th e lo w e r r ig h t q u a d r a n t.
(C ) show s th e gro ssly c o n s tric te d v isu a l field , to t h e la rg e s t siz ed ta r g e t o f a n R P p a ti e n t w ith
a d v a n c e d re tin o p a th y . T h is is th e sta g e o f tu n n e l v is io n .

T he retin a visibly thins an d the blood vessels supplying the retinal tissue begin
to attenuate. Gradually, black pigm entary deposits build up in the neural retina,
the result of dam age to the p h o to rec ep to r and pigm ent epithelial cells, which
 AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 155

leads to m igration o f pigm ent laden cells into the o u ter and in n er retinal layers.
Many patients eventually lose all o f th eir sight. Pathological changes in the retinal
fundus typical of RP are illustrated Figure 2.

V i f '
* I -V
"V j %
, 4 ; ' '

Figure 2 (A ): sh o w a n o r m a l o p tic d isc a n d re tin a l vessels.

While the symptoms o f RP clearly originate in the death o f rod cells, nothing
was known, until very recently, o f the m olecular events associated with this d egen
erative process. Rod cells carry a sophisticated battery of proteins form ing com po
nents o f the visual transduction cycle, the process through which light is absorbed
and converted into neu ro n al signals (C habre and D eterre, 1989; C habre and
Vuong, 1992). T he disease could, in some way, be associated with defects in this
cycle. Alternatively, defects could lie in structural com ponents o f the retinal cells
or even in cells of the underlying pigm ent epithelium . Possibly, the problem could
be one o f vascular supply. A gene, o r genes associated with the m aintenance o f the
fine netw ork of capillaries suffusing the re tin a could be defective, resulting in
gradual cell death through ineffectual blood supply. With no clues, the search for
the defective protein, or proteins, m ight seem virtually impossible. Fortunately, a
powerful technique is available to enable the localization o f defective genes, p ro
vided th at sufficiently large families can be located in which the disease is
156 PETER HUM PHRIES ET AL.

Figure 2 (B ): show s th e o p tic d isc p a llo r a n d a tt e n u a t io n o f re tin a l vessels in th e f u n d u s o f a n

RP p a tie n t. T y p ic a l in tr a -re tin a l b o n e sp ic u le p ig m e n ta r y d e p o s its a r e v isib le a r o u n d th e e d g
es o f th e p h o to g r a p h .
(C ): show s la rg e b o n e s p ic u le p ig m e n t d e p o s it in th e m id -p e rip h e ra l r e ti n a o f a n R P p a tie n t.
S e e C o lo u r P la te 1 a t th e back.
 AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 157

segregating. T he technique is called linkage analysis (C hapter 1). G enetic m arkers

representing alleles (alternative form s) o f a series o f given chrom osom al loci are
tested, essentially random ly, in o rd e r to d eterm in e w hether co-segregation can be
observed within families betw een m arker and disease locus. T he establishm ent of
linkage to a given m arker is the first step in the isolation o f the causative gene.
O nce the chrom osom al location o f the gene is established, the process o f posi
tional cloning (for review see Collins, 1992) can be deployed in o rd e r to isolate
the gene from cloned DNA covering the region in question.
Two prerequisites for linkage analysis are the availability both o f families with
the disease and of suitable genetic m arkers. T he first linkage o f a genetic m arker
to a disease locus was established as early as the m id 1950s by Jan M ohr an d col
leagues (Mohr, 1951). These workers fo u n d th at the d o m in an t m arker Se, now
known to reside on chrom osom e 19 an d governing the ability to secrete the A and
B blood g roup substances in bodily fluids and saliva, showed genetic linkage to the
m yotonic dystrophy locus in families segregating this disease. U nfortunately how
ever, the technique o f genetic linkage analysis was available before the em ergence
of suitable m arker technology. At the time o f the Ja n M ohr linkage, a m ere han d
ful o f genetic m arkers, com prising polym orphic serum proteins and blood groups,
had been characterized. W hile there were m any h ereditary conditions with large
families segregating the disease, an insufficient n u m b er of m arkers were available
to m ake the routine deploym ent o f linkage analysis feasible. T he technique had to
await the developm ent o f a new gen eratio n o f m arkers, which cam e with the estab
lishm ent o f RFLP (restriction fragm ent length polym orphism ) technology in the
late 1970s. H ere, polym orphism at the level of the DNA sequence, in the form of
variation in the size o f restriction enzyme cleavage fragm ents at given loci, may be
detected using the technique o f S outhern blotting (C hapter 1).
A rm ed with a growing n u m b er o f m arkers of the RFLP type, m em bers of o u r re
search team began, in the m id 1980s, to analyse DNA from a large family in Ireland
who suffered from an autosom al d o m in an t form o f RP. Many m arkers were analy
sed (Bradley et al., 1989; D aiger et al., 1989; F arrar et al., 1989), an d approxim ate
ly 60% o f the genom e was covered in the search for a disease locus. Eventually, the
m arker C l 7 (D3S42) was shown to co-segregate with the disease locus with zero
percen t recom bination (McWilliam e t al., 1989). A portion o f the family, illustrat
ing the segregation of the C l 7 RFLP with the disease phenotype, is shown in Fig
u re 3A. In all cases, allele A co-segregated with the disease, resulting in a lod score
o f 14.9. A lod score o f 3 is taken to indicate an approxim ately 95% probability of
linkage (C hapter 1). T he observed lod score o f 14.9 thus pro d u ced a very strong
indication of linkage, and enabled the m apping o f the disease locus to an interval
of a few m illions o f base pairs on e ith e r side of the m arker (F arrar et al., 1990).
C l 7 is located on the long arm o f chrom osom e 3 and a n u m b er o f additional
m arkers were very rapidly ru n over the region. O ne o f these m arkers h ap p e n ed to
be derived from the recently m ap p ed rhodopsin gene. In a m ultilocus analysis
(F arrar et al., 1990), in which data from a nu m b er o f m apped m arkers from the
158 PETER HUMPHRIES ET AL.

A B
tO

2
3

Figure 3 (A) show s a p o r tio n o f a n a d R P fam ily in w h ic h th e d ise a se p h e n o ty p e se g re g a te s

w ith a lle le A o f th e C l 7 RFLP.
(B) show s a p o r t io n o f a n o th e r a d R P fam ily in w h ic h th e d ise a se p h e n o ty p e s e g re g a te s w ith
a lle le 3 o f th e rd s p o ly T / A p o ly m o rp h is m .

long arm o f chrom osom e 3 were sim ultaneously analysed, the rhodopsin m arker
was shown to m ap to the disease locus with zero recom bination and a lod score of
approxim ately 20. These results very strongly suggested th at a m utation o f the
rh odopsin gene m ight be the underlying defect which caused RP in the Irish farn-
ily.

MUTATIONS OF THE RHODOPSIN GENE IN ADRP

An intensive search for m utations w ithin the rhodopsin gene in patients suffering
from autosom al d om inant RP (adRP) was m o u n ted as a result of the above obser
vations. A m utation was rapidly found in an Am erican p atien t (Dryja et al., 1990).
This was a single am ino acid substitution (a C ^ A transversion) at codon 23 of
the p rotein, resulting in the removal o f proline and its substitution by histidine.
T his m utation has now been found in up to 15% o f all d o m in an t cases o f RP in
the U nited States. Interestingly, the m utation is eith er totally absent within the
p o pulation o f Europe, or present at a very low frequency (F arrar et al., 1990).
Using a variety of new techniques, the rapid screening of DNA for disease-relat-
ed m utations is now possible. A com bination of such m ethodologies, including
single strand conform ational polym orphism (SSCP), d irect sequencing, h etero d u
plex analysis an d allele-specific oligonucleotide analysis have been used in screen
ing the rhodopsin gene from patients with RP. Using the first technique, SSCP
analysis (O rita et al., 1989), small sectors o f the gene (up to a few h u n d red s o f base
pairs) are am plified from genom ic DNA using the polym erase chain reaction. The
 AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 159

am plified DNA is d en a tu red an d subjected to electrophoresis in a neutral, n o n

denaturing acrylam ide gel. Because the two single strands of DNA may assume dif
ferent three dim ensional conform ations as a result of intra-strand pairing, they
often separate in the gel, som etim es by considerable distances. Even the presence
of a single base change in one o f the two hom ologous DNA m olecules is often suf
ficient to produce a radical mobility shift, sufficient to produce four bands (two
originating from each double strand) instead o f two. Evidence for the presence of
a m utation w ithin the rhodopsin gene, using this technique, is illustrated in Figure
4A. T he DNA may then be subjected to direct sequence analysis in o rd e r to con
firm the presence o f a m utation. Again, sequencing is p erfo rm ed on am plified ge
nom ic DNA. H ence DNA from both hom ologous loci are am plified and
sequenced at once (Figure 4B).

A B
1 2

Figure 4 (A) show s re su lts fro m a n S S C PE analysis. B a n d s 1 a n d 2 r e p r e s e n t th e two d e n a

tu r e d D N A s tra n d s fr o m e x o n 3 o f th e r h o d o p s in g e n e . A th ir d b a n d is p r e s e n t in tw o o f th e
la n e s, in d ic a tin g th e p r e s e n c e o f a s e q u e n c e d if f e r e n c e b e tw e e n th e a lle le s o f th e D N A a m p li
fie d fr o m th e s e in d iv id u a ls ( h ig h lig h te d by a n a r r o w ) .
(B) show s d ir e c t s e q u e n c e fr o m P C R a m p lif ie d D N A p r o d u c ts f ro m e x o n 1 o f th e rd s g e n e .
S a m p le 1 show s s e q u e n c e f r o m a n in d iv id u a l h e te ro z y g o u s f o r a 3 b p d e le tio n , sa m p le 2 show s
th e n o r m a l s e q u e n c e f r o m th e sa m e r e g io n . T h e site o f th e d e le tio n a t c o d o n s 1 1 8 /1 1 9 is in
d ic a te d by a n arrow .

P oint m utations are identified as double bands at the same position in the se
quencing gel. T he presence of a m utation may then be confirm ed by the use of
allele-specific oligonucleotides (ASOs). S hort prim ers specific eith er for the n o r
m al or m u tan t DNA are synthesised a n d allowed to hybridise to DNA from m em
bers o f the pedigree. DNA is usually im m obilised on filters in the form of dot
160 PETER HUM PHRIES ET AL.

blots. N orm al DNA hybridises only to the norm al ASO, while m utant DNA (be
cause it carries both norm al an d m u tan t alleles) will hybridise to both norm al and
m u tan t ASOs. An exam ple o f the detection o f a p erip h erin /R D S in m utation seg
regating in DNA from an RP pedigree using the ASO technique is illustrated in
Figure 5.

H i M

Figure 5 S e g re g a tio n o f th e d ise a se p h e n o ty p e a n d a m u ta tio n a t c o d o n 212 o f th e rd s g e n e

in a fam ily w ith a d R P is sh o w n . D N A was a m p lifie d a r o u n d th e site o f th e m u ta tio n fr o m all
m e m b e rs o f th e fa m ily a n d was th e n h y b rid is e d to b o th th e n o r m a l a n d m u ta n t a lle le sp e c ific
o lig o n u c le o tid e s (A S O ). O n ly a ffe c te d in d iv id u a ls h y b rid is e d to th e m u ta n t A S O (A ), b o th
n o r m a l a n d a ffe c te d in d iv id u a ls h y b rid is e d to th e n o r m a l A S O (151

Using a com bination o f these, and sim ilar techniques, at least 60 m utations with
in the rhodopsin gene have been identified in patients with d om inant RP over the
last three years (reviewed by H um phries et al., 1992). T he positions of these m u
tations within the p rotein are illustrated in gure 6. In o rd e r to gain some insight
into how such m utations m ight cause RP, an u n d erstanding o f the function of
rhodopsin within the visual transduction cycle is req u ired (C habre and D eterre,
1989; C habre and Vuong, 1992)
 AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 161

Figure 6 A d ia g ra m a tic r e p r e s e n ta tio n o l th e r h o d o p s in m o le c u le in d ic a tin g th e p o s itio n s a t

w h ic h m u ta tio n s in a d R P a n d a rR P p a tie n ts h a v e b e e n o b s e rv e d . T h e s e re s id u e s a re in d ic a te d
by th ic k e r fille d circles.

Rod photoreceptors consist o f an o u ter an d an in n er segm ent (Figure 7).

R hodopsin synthesis takes place in the in n er segm ent, and the protein is then
transported to the o u ter segm ent discs. T he discs are flattened structures stacked
on top o f each o th er ra th e r like thin wafers. Each rod cell contains many such
discs. T he rhodopsin protein becom es incorporated into the m em branes o f the
discs as illustrated in Figure 7, an d it is here th at light is absorbed. T he am ino ter
m inus o f the protein is situated in the lum en of the disc, while the carboxy term i
nus lies within the cell cytoplasm. T he protein has seven transm em brane dom ains.
W ithin the 7th transm em brane dom ain, at the lysine residue of position 296, a
m olecule o f the ch ro m o p h o re 11-cis retinal is bound. A bsorption o f a single p h o
ton o f light is sufficient to result in the isom erization o f the ch ro m o p h o re to the
all-trans form . This activates rho d o p sin as a result o f a change in its configuration,
and in this form it may react with the second com ponent o f the cycle, a G-protein
called transducin (Figure 8). T ransducin consists o f three com ponents, alpha,
beta an d gam m a subunits. As a result o f its interaction with activated rhodopsin,
the beta and gam m a subunits dissociate and GDP, on the alpha subunit o f the en
zyme, isreplaced by GTP. This activated alpha subunit o f transducin th en activates,
lb:! PF.TF.R HUMPHRIKS FT AL.

BOD PH O TO RECEPTO R PH O TO RECEPTO R O l TER

SEG M EN T DISC

P e rip herin/R D S

P e ripherin/R D S

F ig u re 7 In th is d ia g ra m , b o th th e r h o d o p s in a n d rd s p r o te in s a re se e n e m b e d d e d in th e p h o
to r e c e p t o r o u t e r s e g m e n t disc m e m b r a n e . B o th p r o te in s twist in a n d o u t o f th e lip id bilayCi
h a v in g 7 a n d 4 tr a n s m e m b ra n e s e g m e n ts re sp ectiv ely .

F ig u re 8 T h e r o d c ell o u t e r s e g m e n t d isc h a s r h o d o p s in m o le c u le s e m b e d d e d in th e m e m
b r a n e . P h o to a c tiv a te d rh o d o p s in m o le c u le s stim u la te tr a n s d u c in , w h ic h a c tiv a te s cG M P p h o s
p h o d ie s te r a s e a n d le a d s to lo w e re d levels o f cG M P in th e c y to p la sm h e n c e r e s u ltin g in c lo s u re
o f c G M P -g a te d io n c h a n n e ls . T h e re s u ltin g lo w e re d c a lc iu m levels le a d s to th e s tim u la tio n o f
re c o v e rin a n d g u a n y la te cyclase a ctiv ities a n d re su lts in cG M P p r o d u c tio n a n d r e o p e n i n g o f
th e c h a n n e ls .
 AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 163

in tu rn , the enzyme cyclic GMP phosphodiesterase (PDE). T he latter protein con

sists o f four subunits, alpha, beta and two gammas. Activation is achieved as a result
o f the removal from the p rotein o f the two inhibitory gam m a subunits. Activated
PDE is able to rapidly hydrolyse cyclic GMP, which can bind to a transm em brane
protein of the rod cell called the cyclic GMP-gated channel protein. W hile cyclic
GMP is b o u n d to this protein, the ch an n el in the m em brane o f the ro d cell is
open, across which m onovalent an d divalent cations may diffuse. Cleavage o f cy
clic GMP lowers the intracellular concentration available for binding to the chan
nel protein, which results in closure o f the channel. This results in a rapid
hyperpolarization o f the cell, red u cin g n eu ro tran sm itter release an d initiating a
signal which is transm itted to the brain. In o rd e r to com plete the cycle, cyclic GMP
m ust be reg en erated and rhodopsin deactivated. T he fo rm er occurs as a result of
co n tin u ed removal o f calcium ions from the rod cell via a sodium /calcium ex
changer in the cell m em brane. T he lowering o f calcium ion concentration results
in the activation o f the enzyme recoverin, which in tu rn stim ulates guanylate cy
clase. T he latter enzyme regenerates cyclic GMP. Cyclic GMP then rebinds to the
channel protein resulting in opening o f the ion gate. M eantim e rhodopsin is de
activated by the com bined activities o f two proteins, namely arrestin (S-antigen)
and rhodopsin kinase. T he cycle is re p resen te d diagram m atically in Figure 8.
M utations within the rhodopsin g en e in some way initiate a pathological process
which ultim ately results in the d eath o f the rod cell. Most o f the m utations so far
detected in cases o f adRP are single base pair substitutions (for a detailed list see
H um phries et al., 1992). These have been found within the carboxy and am ino
term inus dom ains and also in each o f the seven transm em brane dom ains of the
m olecule (Figure 6 ), Such m utations may have a n u m b er o f effects. Firstly, they
may interfere with the targeting a n d /o r stability o f the p rotein (e.g. thr-17-met, or
pro-43-his/leu). Secondly, they may in terfe re with the folding or integration o f the
protein into the bilayer o f the disc m em brane (e.g. pro-53-arg o r leu-125-arg).
Thirdly, they may prevent binding o f the ch rom ophore 11-cis retinal (e.g. lys-296-
glu). Fourthly, they may prevent rhodopsin from interacting with, or activating,
transducin (e.g. arg-135-leu/trp). Finally, some m utations toward the extrem e car-
boxy term inus of the m olecule (e.g. thr-340-frame shift) may prevent phosphory
lation of the m olecule by rhodopsin kinase o r its interaction with arrestin (S-
antigen). Sung et al. (1991) succeeded in expressing 13 d ifferent m utant rhodop-
sins responsible for adRP by transfection into cultured hum an em bryonic kidney
cells. T hree m utants resem bled wild-type rhodopsin in their yield o f pigm ent and
their ability to bin d 11-cis retinal an d release all-trans retinal in response to light
(Phe-45-Leu, Gln-344-ter, Pro-347-Leu). Two m utants pro d u ced photolabile pig
m ents at greatly reduced yield com pared with wild-type (Val-87-Asp, Gly-89-Asp).
T he rem aining eight m utants failed to pro d u ce any detectable photopigm ents on
incubation with 11-cis retinal (Thr-17-Met, Pro-23-His, Thr-58-Arg, Gly-106-Trp,
Arg-135-Trp, Tyr-178-Cys, Asp-190-Gly). T he last two groups were found to be inef
ficiently transported to the plasm a m em brane, rem aining prim arily in the en d o
plasmic reticulum (S u n g e ta l., 1991). This suggests the possibility o f a m echanism
by which the endoplasm ic reticulum retains an d degrades incorrectly assembled
164 PETER HUM PHRIES ET AL.

or unstable proteins, which may be harm ful to the photoreceptor. In addition to

its role in absorbing light, rhodopsin is also a co m p o n en t o f the o u ter disc m em
branes. Instability o f the protein o r in co rrect folding into the m em brane could in
terfere with the delicate m olecular architecture of the outer segm ent discs,
eventually resulting in rod cell destruction. Inhibition o f the transduction cycle, a
probable m anifestation o f some of the m utations described, m ight have sim ilar ef
fects.

TRANSGENIC MICE WITH THE ADRP PRO-23-HIS MUTATION

Olsson et al. (1992) have constructed a transgenic m ouse containing the Pro-23-
His m utation that is com m on in N orth America. T he en tire rhodopsin gene,
including 4-5 kb o f upstream and 6-8 kb o f dow nstream regulatory sequences, was
isolated from a p atien t with adRP who was heterozygous for this m utation. T he
no rm al and m u tan t genes were m icroinjected into the m ale pronuclei o f single
cell m ouse em bryos an d five lines o f transgenic m ice were studied from the
resultant progeny (two carrying the wild-type and four the m u tan t allele). All
th ree m u tan t lines developed p h o to rec ep to r degeneration, the degree o f d egen
eration correlating with the extent o f m u tan t allele expression. T he retinas o f
affected mice showed atten u ated vessels a n d geographic patches o f retinal pig
m ent epithelial hyper- or hypopigm entation, contrasting with the dendritic
clum ps o f pigm ent found in the hum an disorder. A m onoclonal antibody specific
to the hu m an ro d opsin detected expression of the m utant opsin in o u ter seg
m ents an d failed to find accum ulations in the endoplasm ic reticulum . This con
trasts with the findings of Sung et al. (1991) who showed a failure o f the Pro-23-
His opsin to be in corporated into plasm a m em branes in vitro and re te n tio n in
th e endoplasm ic reticulum . Olsson et al. (1992) unexpectedly fo u n d abnorm ally
located m u tan t opsin at the synaptic en d o f rods (outer plexiform layer), raising
the possibility o f disturbed intracellular transport.
Surprisingly, one of the two transgenics containing the wild-type allele also d e
veloped a very sim ilar retinal degeneration. T he ratio of hum an to m ouse ro d op
sin mRNA expressed in this line was 5:1 com pared to the unaffected wild-type
transgenic which expressed equal am ounts o f hum an an d m ouse transcript. T he
d egeneration was th o u g h t therefore to be related to overexpression o f the (hu
m an) opsin gene.
These experim ents show firstly that this m utation confers a degenerative p h en o
type, even in the presence o f two norm al m ouse genes, which excludes the possi
bility that it is a null allele which gives rise to adRP as a result o f a deficiency of
norm al gene product. Secondly, some proposed explanations for the d eg en era
tion, such as failure of RPE cells to phagocytose rod o u ter segm ents or inability of
the m utant protein to be in corporated into o u ter segm ent discs, ap p eared less
likely in the light o f these findings. Finally, the finding that overexpression even of
the norm al hum an gene p ro d u ct causes a severe retinal d egeneration perhaps
 AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 165

em phasises how finely tu n ed the retin a is for m aintaining norm al structure and
function.

A DIVERSE MOLECULAR PATHOLOGY IN ADRP

R hodopsin m utations account for only about 25% of all d o m in an t cases of RP.
Following the establishm ent o f the initial rhodopsin linkage, we u n d erto o k addi
tional linkage work in a second adRP pedigree o f Irish origin which showed
exclusion o f a locus from the long arm o f chrom osom e 3 (F arrar et al., 1990). In
this family the disease was o f m uch later onset than that in the first pedigree. RP
is clinically divisible into two m ajor form s, so called types 1 and 2, the form er
being the m ore severe (C hapter 3). T h e disease is also classifiable as eith er a dif
fuse o r a regional retinopathy, d e p e n d in g on the n ature and ex ten t of pathologi
cal changes in the retina. Diffuse a n d regional form s o f the disease usually
correlate with type 1 and 2 form s respectively. M em bers of the second Irish family
had a regional (type 2) form of RP with night blindness and field loss n o t usually
m anifesting until the fo u rth decade. By the tim e o f this second linkage study, a
new generation o f genetic m arkers had becom e available. Such m arkers are
called m icrosatellites (W eber an d May, 1989; Litt and Lutty 1989). H um an DNA
contains many (one h u n d re d thousand o r m ore) short blocks o f dim er repetitive
sequence o f the type, d(C A )n, interspersed th ro u g h o u t the genom e. T he num
bers o f CA repeats at any given locus usually varies on each hom ologous chrom o
some. O ligonucleotides representing the sequence o f DNA im m ediately flanking
such repeats can be synthesized a n d used as prim ers in the polym erase chain
reaction to amplify the segm ent o f the genom e spanning the block o f repetitive
DNA. Am plified DNA may th en be analysed by electrophoresis in acrylam ide
gels, sim ilar to the type used for DNA sequencing. Variation in the length o f the
repetitive block, even if this is as small as a single CA unit, is readily detectable.
M icrosatellites have two m ajor advantages over conventional RFLPs. Firstly, they
can be used w ithout the n eed for enzymatic digestion o r S outhern blotting, in
which lengthy gel separations, DNA transfer to m em branes, hybridisations and
autoradiography are required. Secondly, m icrosatellites are generally o f m uch
higher inform ation c o n te n t than conventional m arkers. This m eans that a pro
portionately greater n u m b er o f genetic crosses are inform ative for linkage in any
particular pedigree.
C ontinued linkage studies, based largely on the use o f such m arkers, resulted in
the localisation of a causative gene (F arrar et al., 1991; Jo rd a n e ta l., 1991; K um ar-
Singh et al., 1991). T he gene resided close to the HLA locus on th e short arm of
chrom osom e 6. Interestingly, the gene for an additional p rotein co m p o n en t o f the
rod o u ter segm ent discs, p erip h erin /R D S , h ad recently been m apped to the same
region (Travis et al., 1991). Co-segregation of the disease locus with the m icrosat
166 PETER HUMPHRIES ET AL.

ellite m arker at the 3 en d of the p erip h erin /R D S gene is illustrated in Figure 3B.
This was o f special significance, since the rd s /p e rip h e rin protein had previously
been im plicated in the cause of the naturally occurring retinopathy o f mice called
retinal degeneration slow (rds) (C hapter 5). T h e protein takes its nam e from this
ro d en t disease which, in many respects, is sim ilar to RP in man. T he rd s/p e rip h -
erin p rotein thus becam e a prim e candidate for hum an adRP and a m utation with
in the gene was soon fo u n d in a patient from an Irish adRP pedigree (Farrar
et al., 1991). T he m utation was a trinucleotide deletion o f codons 118/119, rem ov
ing o n e o f a pair o f conserved cysteine residues.
T he hum an p erip h erin /R D S gene is highly conserved in mice, cattle, rats and
hu m ans an d contains three exons, coding for a predicted 193, 83 an d 70 am ino
acid residues respectively. T he p rotein p ro d u c t is a 39 kDa glycoprotein b u t m uch
less is known o f its structure and function com pared with rhodopsin. It is n o t be
lieved to be a com ponent o f the visual transduction cycle but, like rhodopsin, it
resides within the m em branes o f o u ter segm ent discs (Figure 7). U nlike rh o d o p
sin, it is present in rod and cone p h o to rec ep to r o u ter segm ents (Arikawa et al.,
1992). To date, ab o u t th irteen m utations within the p erip h erin /R D S gene have
b een re p o rted in cases of adRP (F arrar et al., 1992; Kajiwara et al., 1992; S. Bhat
tacharya, A. Gal, personal com m unications). T he original Irish pedigree in which
linkage was established contains an am ino acid substitution at codon 212 (F arrar
et al., 1992). In addition, a n u m b er o f silent polym orphic am ino acid substitu
tions have b een detected which are n o t associated with a disease phenotype
(J o rd a n et al., 1992). T hree of these polym orphism s cluster toward the carboxyl
term inus o f the m olecule. (Interestingly, these silent polym orphic variations oc
cur at residues h ith erto considered to be conserved am ong m am m alian rd s /p e
rip h erin p ro te in s). C u rre n t m odels suggest th at the rd s /p e rip h e rin p rotein has
fo ur m em bane-spanning a-helical dom ains with both the carboxyl an d am ino acid
term inals facing the cytoplasmic side of the disc m em brance (Figure 9). T he pro
tein is believed to have two intradiscal loops, one m uch larger than the other. It is
possible that the protein may dim erise via at least o n e disulphide bridge, and it
may have a role in m aintaining disc structure.
It is too early to say what p ro p o rtio n o f adRP is caused by m utations within the
RDS gene. T he figure may well be sim ilar to the frequency o f rhodopsin-associated
RP. However, a n u m b er o f m utations in the p erip h erin /R D S gene have recently
been identified in families segregating dom inantly in h erited disorders o th e r than
RP. Kajiwara et al.(1993) screened over 350 patients with a variety o f hereditary
retinal disorders for p erip h erin /R D S gene m utations using SSCP analysis. Exons
1 an d 2 together with som e flanking DNA (intron or 5' u ntranslated) were
screened, om itting exon 3 which contains freq u en t polym orphism s in norm al in
dividuals. Only one patient, with autosom al d om inant retinitis punctata albescens,
was found to have a m utation. T he clinical phenotype consisted o f m acular hyper
pigm entation and atrophy associated with w idespread yellow-white flecks u n d er
the retina.
 AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 167

F igure 9 A d ia g ra m a tic r e p r e s e n t a t i o n o f th e r d s p r o t e i n in d ic a tin g th e a m in o a c id s in w h ic h

m u ta tio n s h a v e b e e n id e n tif ie d in a d R P p a tie n ts . T h e s e re s id u e s a re in d ic a te d by th ic k e r fille d
circles. N u c le o tid e c h a n g e s w h ic h a re s ile n t o r w h ic h a lte r a m in o a cid s b u t a re n o t a sso c ia te d
w ith th e a d R P p h e n o ty p e a re i n d ic a te d by c irc le s fille d w ith strip e s.

Affected individuals h ad progressive loss of central vision, abnorm al dark adapta

tion an d abnorm al rod an d cone ERG responses, together indicating a g enera
lised retinal degeneration. A 2-base pair deletion was found in codon 25 o f the
p erip h erin /R D S gene, representing a null allele since this would lead to a
severely tru n cated protein product, sim ilar to the m urine rds m utation. Wells
et al.(1993) re p o rted three families with autosom al d om inant m acular d egenera
tions associated with missense m utations at codon 172. They also re p o rted find
ing a nonsense m utation at codon 258, producing a prem ature stop codon, in a
168 PETER HUMPHRIES ET AL.

family with autosom al d om inant adult vitelliform m acular dystrophy. This may
also be a null allele, similar to th at in the p atien t with retinitis punctata albescens,
although the phenotypes are ra th e r different. Finally, Nichols et al. (1993)
described a family with autosom al d om inant butterfly-shaped pigm ent dystrophy
of the fovea associated with a missense m utation in p erip h erin /R D S codon 167,
which would substitute an aspartate for a highly conserved glycine residue. T he
m utation segregated with the disease (lod score = 4 at a recom bination fraction
o f zero) and was no t present in 100 control chrom osom es. T he disorder is char
acterized by accum ulations o f yellowish or pigm ented m aterial at the level o f the
retinal pigm ent epithelium (RPE), central visual loss apparently related to the
ex tent of accom panying RPE atrophy, n orm al dark adaptation thresholds and
electroretinogram s. H ence, like rhodopsin, m u tan t rd s /p e rip h e rin proteins show
a wide range o f phenotypic expression. Indeed, m ost recently families with
digenic adRP have been identified, in which only patients who in h erit m utations
in both the rd s/p e rip h e rin gene an d the gene encoding the rod o u ter m em
b ra n e (ROM) protein are affected (T. Dryja, T h e Association for Research in
Vision and O phthalm ology M eeting, 1994). Such families segregating with
digenic adRP show autosom al d o m in an t inheritance with reduced p e n e tra n c e .
A dditional RP families have been identified in which n eith er rhodopsin n o r
rd s /p e rip h e rin is involved. O ne such family form s the large adRP pedigree
UCLA1 from the U nited States. H ere, the causative gene has been localised by ge
netic linkage analysis to the pericentric region o f chrom osom e 8 (B lanton et al.,
1991). However, no potential candidate gene has, as yet, been identified. Most re
cently, two fu rth e r genes have been identified in large adRP families o n opposite
arm s o f chrom osom e 7. Inglehearn et al. (1993) found linkage to a gene on the
short arm of chrom osom e 7 (7p) in a large English adRP kindred, close to the
m arker D7S460 (Z = 5.60 at 0 = 0). A n o th er large kindred was fo u n d b y jo rd a n
et al. (1993) to have a disease gene linked to m arkers on the long arm o f ch ro m o
some 7, close to the m arker D7S480 (Z = 7.20 at 0 =0). Most recently an adRP
gene has been localised to the long arm o f chrom osom e 19 close to the m arkers
D19S180 an d D19S214 (Al-Maghteh et al., 1994). In addition, at least o n e o th er
adRP family currently u n d e r study in o u r own laboratory shows no evidence for
linkage on chrom osom es 3, 6, 7 o r 8, indicating the presence o f at least one m ore,
as yet unlocalised, adR P gene (Kumar-Singh et al., 1992, 1993;Jordan et al., 1992).
T hus in the d o m in an t form o f RP, a m inim um of six genes are responsible for
th e cause o f the disease in different families. Possibly about one third o f all dom
in ant cases can be accounted for by known m utations in the rhodopsin an d pe
rip h erin /R D S genes. It is o f interest that both of these genes code fo r proteins
which are located within the m em branes o f the o uter segm ent discs. W hether all
adRP proteins are located within these structures rem ains to be determ ined.
 AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA 169

CONCLUSIONS

In this review we have lim ited our coverage to the d o m in an t form o f RP, in which
two o f the first proteins responsible for RP were identified. Perhaps the most sur
prising finding in adRP has been the very extensive genetic heterogeneity. T here
now ap p ear to be a m inim um o f six loci responsible for this disorder, on chrom o
somes 3, 6, 7p, 7q, 8 an d at least o n e o th er as yet unidentified site. D ifferent
m utations in the rhodopsin gene on chrom osom e 3 give rise to very different
phenotypes, from a very early onset and severe degeneration in som e types of
adRP to the clinically norm al carriers o f the tru n cated m olecule associated
with autosom al recessive disease. In the case o f the p erip h erin /R D S gene on
chrom osom e 6, the phenotypic variation is proving even m ore diverse. D ifferent
m utations are associated eith er with predom inantly m acular o r p eripheral re tin o
pathy. Families with m acular dystrophy, adult vitelliform m acular dystrophy,
butterfly-shaped pigm ent dystrophy, retinitis pigm entosa and retinitis punctata
albescens have all been associated with R D S /p erip h erin m utations. In some
cases, m ore than one phenotype has been fo u n d within the same family. T he
clinical heterogeneity is as striking as the genetic heterogeneity.
D evelopm ents are rapidly taking place in o th er form s of retinitis pigm entosa
and in related conditions. T he locations of two X-linked RP genes have been
known for some tim e (O tt et al., 1990). In autosom al recessive form s o f the dis
ease, genes for U sher syndrom es type 1 an d 2 (RP associated with deafness) have
also been localised by genetic linkage analysis (Kim berling et al., 1990) (C hapter
15). M oreover, a m utation w ithin the rhodopsin gene an d several m utations within
the (3 subunit o f cyclic GMP phosphodiesterase have recently b een identified in
families displaying autosom al recessive RP (Rosenfeld et al., 1992; M cLaughlin
et al., 1993). O u r own studies o f a large family o f D utch origin indicate that there
is an additional autosom al recessive locus on lq close to the m arker F13B an d the
gene encoding the retinal protein phosducin (Bleeker-W agemakers et al., 1992;
van Soest et al., 1994). In addition, genes responsible for two o th er dom inantly
heritable form s of m acular deg en eratio n - N orth C arolina m acular dystrophy and
Bests disease - have been localised on chrom osom es 6 and 11 respectively (Small
et al., 1992; Stone et al., 1992) (C hapter 15). T he availability o f additional pedi
grees suitable for genetic linkage analysis, coupled with the now well developed
techniques for positional cloning an d rapid m utational screening, will ensure that
the many o f these genes will be characterised within the n ext few years.

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iesterase in p a tie n ts w ith a u to so m a l recessive re tin itis p ig m e n to sa . Investigative Ophthalmology
and Visual Science 34, 1148.
M cW illiam , P., F a rrar, G. J., K e n n a, P., Bradley, D. G., H u m p h rie s , M. M. , S h a rp , E. M., M cC on
nell, D .J., Law ler, M., Shiels, D., R yan, C., Stevens, K , D aiger, S. P. a n d H u m p h rie s , P. (1989),
A u to so m a l d o m in a n t re tin itis p ig m e n to sa (A D R P ): L o ca lisatio n o f a n A D RP g e n e to th e lo n g
a rm o f c h ro m o s o m e 3. Genomics 5, 619622.
M o h r,J. (1951) A cta P a th o l M icro b io l S c a n d 28: 207; ibid. 29, 339 TITLES.
O rita , M ., Suzuki, Y., Sekiy, A. T. a n d H yashi, K. (1989). R ap id a n d sensitive d e te c tio n o f p o in t
m u ta tio n s a n d DN A p o ly m o rp h ism s u sin g th e p o ly m e rase c h a in re a c tio n . Genomics 5, 8 7 4 -
879.
O tt,J ., B h a tta ch a ry a, S., C h e n ,J . D., D e n to n , M .J., D o n a ld , J., D ubay, C., F a rrar, G .J , F ish m an ,
G. A., Frey, D., Gal, A., H u m p h rie s , P., Ja y B .,Jay, M., L itt, M., M achler, M., M usarella, M.,
N e u g e b a u e r, M., N u ssb au m , R. L., T erw illiger, J. D., W eleber, R. G., W irth , B., W ong, F., W or
to n , R. G., a n d W right, A. F. (1 990). L o c a lisa tio n o f m u ltip le re tin itis p ig m e n to sa loci o n th e
X c h ro m o s o m e . Proc. Natl. Acad. Sci. 87, 7 0 1 -7 0 4 .
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R o se n fe ld , P. J., Cowley, G. S., M cG ee, T. L., S a n d b e rg , M. A., B erson, E. L. a n d D ryja, T. P.

(1 992). A nu ll m u ta tio n w ithin th e rh o d o p sin g e n e causes ro d p h o to r e c e p to r d y sfu n c tio n
a n d a u to so m a l recessive re tin itis p ig m e n to sa . N ature Genetics 1, 2 0 9-213.
Sm all, K. W., W eber, J. L, Roses, A., L e n n o n , F., V ance, J. M., a n d Pericak-V ance, M. A. (1992).
N o rth C a ro lin a M a c u la r D y strophy is assig n e d to c h ro m o s o m e 6 . Genomics 13, 681685.
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age o f vitellifo rm m a c u la r d e g e n e ra tio n (B est's disease) to c h ro m o s o m e 1 l q l 3 . N ature Genet-
?7s 1 ,2 4 8 -2 5 0 .
Su n g , (..-II., S c h n e id e r, B. G., A garw al, N., P a p e rm a ste r, D. S. a n d N a th a n s , ]. (1 991). F u n c tio n a l
h e te ro g e n e ity o f m u ta n t rh o d o p sin s re sp o n sib le fo r a u to so m a l d o m in a n t re tin itis p ig m e n
tosa. Proc. Natl. Acad. Sci. USA 8 8 , 8840-8844.
Travis, G. II, C h riste rs o n , L., D a n ielso n , P. E., Klisak, 1, S parkes, R. S., H a h n , L., D ryja, T. P. a n d
S u tc liffe ,J. G. (1991). T h e h u m a n re tin a l d e g e n e r a tio n slow (RDS) g e n e : C h ro m o so m e as
s ig n m e n t a n d s tru c tu re o f th e m RNA. Genomics 10, 7 3 3-739.
Travis, G. H ., S u tc liffe ,J. C. a n d a n d Bok, D. (1 991). T h e re tin a l d e g e n e ra tio n slow (rds) g e n e
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van Soest, S., van d e n B o rn , L. I., G al, A., F arrar, G. J ., B leeker-W agem akers L., W esterveld, A.,
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recessive re tin itis p ig m e n to sa (RP12) to c h ro m o s o m e Iq 3 1 -q 3 2 .1 in an in b re d a n d g e n e ti
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W eber, J. L. a n d May, P. E. (1989). A b u n d a n t class o f h u m a n DNA p o ly m o rp h ism s w hich can b e
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 7. THE MOLECULAR GENETIC APPROACH TO
MACULAR DEGENERATION

EDWIN M. STONE1, and VAL C. SHEFFIELD2

Departments of Ophthalmology1 and Pediatrics . the University of Iowa College of Medicine,
Iowa City, Iowa 52242, USA

INTRODUCTION

A nu m b er o f epidem iologic studies have shown that diseases affecting the retinal
m acula cause a significant p ro p o rtio n of severe visual loss in o ld er individuals
(Sorsby, 1966; Kahn an d M oorhead, 1973; Kahn et al., 1977; Leibowitz et al.,
1980; Ganley an d R oberts, 1983; Klein and Klein, 1982; M artinez et al., 1982;
G hafour et al., 1983; Som m er et al., 1991). For practical reasons, such studies
usually consider m acular disease in patients over about 50 years of age to be a sin
gle clinical entity now known as age-related m acular degeneration (AMD).
U nfortunately, the creation of a single epidem iologic pigeon h o le for older
patients with m acular disease, and the assignm ent of a single term to refer to it
have tended to obscure two im p o rtan t facts:
1) AMD is n o t a single disease at the clinical, cellular, o r biochem ical level; and
2) the predisposition to AMD is in h erited as an autosom al d o m in an t trait in a sig
nificant percentage o f affected patients.
In this chapter, we will review the published data that support these two asser
tions an d pu t fo rth o u r view o f th eir im portance to the design o f studies aim ed at
elucidating the pathogenesis o f - and developing a b etter treatm en t for - the dis
eases that m ake up AMD. We will then discuss the ways in which m olecular genet
ics can be used to exploit the heritable n atu re o f these disorders to identify the
precise m olecular m echanism s involved in their pathogenesis an d provide some
exam ples of recent progress that has been m ade with this approach.

DEFINITION OF AMD

Before one can address the heritability o r pathogenetic heterogeneity o f AMD,

one m ust decide what AMD is. U nfortunately, a universal definition o f age-related
m acular degeneration is elusive, an d the w orking definitions o f clinicians,
pathologists, epidem iologists an d m olecular biologists differ so widely that it can
be difficult for each group to in terp re t an d apply the findings o f the others. Most
clinicians use the term age-related m acular degeneration to refer to patients over
the age o f 50 with some degree o f visual loss associated with an ophthalm oscopic
174 EDWIN M. STONE AND VAL C. SHEFFIELD

abnorm ality o f the m acular retinal pigm ent epithelium (Kahn et al., 1977; Klein
an d Klein, 1982; Hym an et al., 1983). In m ost such patients, drusen are present,
and in 5-10% choroidal neovascularization eventually develops (Leibowitz et al.,
1980, Hym an et al., 1983). Even w hen signs o f a specific m acular disease are
present (old central serous retinopathy, the presum ed ocular histoplasm osis syn
drom e, angioid streaks, etc.) the diagnosis o f age-related m acular d egeneration
is often applied to patients over 50, w hereas the m ore specific diagnoses are m ore
likely to be used in patients u n d er 50.

HETEROGENEITY OF AMD

However pragm atic the foregoing definition is for the clinic, it poses some diffi
culties for investigators trying to work o u t the pathophysiology o f the diseases
that m ake up AMD. First, it is ap p a ren t from the designs of the two population-
based epidem iologic studies (Leibowitz et al., 1980, Ganley an d Roberts, 1983)
th at a variety o f d ifferent pathophysiologic entities were g ro u p ed u n d er this sin
gle heading. T he fraction o f AMD caused by each distinct m acular disorder can
n o t be deduced from such studies. This is im p o rtan t because if one assum es that
AMD is a single pathophysiological entity, an d designs experim ents to try to id en
tify a single disease m echanism , one is likely to fail if the study population actu
ally consists o f patients with clinically similar bu t biochem ically different
disorders.
For exam ple, suppose that 5% o f AMD is caused by a genetically d eterm in e d ab
norm ality o f a p h o to rec ep to r p rotein that increases the eyes vulnerability to ultra
violet light. If two groups o f AMD patients with different light exposure histories
are studied, the ap p a ren t effect of LTV light on the entire high ex posure group
m ight be insignificant even though a very significant effect is p resen t in the 5% of
the population with high UV vulnerability. An exam ple from the literature in
which the study p articipants specific diagnosis dram atically affected the apparent
effectiveness o f a treatm ent fo r m acular disease is the juxtafoveal arm o f the m ac
ular photocoagulation study (Figure 1). In this study, patients who had laser treat
m en t for juxtafoveal choroidal neovascular m em branes secondary to the
presum ed ocular histoplasmosis syndrom e (POHS) h ad a 31% rate o f m em brane
persistence o r recu rren ce over five years (M acular P hotocoagulation Study G roup,
1989) while patients with juxtafoveal m em branes secondary to non-PO H S m acu
lar d egeneration had a five-year rate o f persistence or recu rren ce o f 79% (M acular
P hotocoagulation Study G roup, 1990).
Over the years, a num ber o f d ifferent structural an d functional abnorm alities,
physical exposures and dietary deficiencies have been proposed to play a role in
the pathophysiology of m acular degeneration, including:
1) abnorm ality of the neurosensory retin a (G artner an d H enkind, 1981; Bird
and Marshall, 1982; Katz et al., 1986);
 THE M OLECUIAR GENETIC APPROACH TO MACUIAR DEGENERATION 175

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F ig u re 1 C o m p a r is o n of m e m b r a n e r e c u n v t u e fo llo w in g la s e r tr e a tm e n t o f ju x ta fo v e a l c h o
ro id a l n e o v a s c u la r m e m b r a n e s s e c o n d a r y to th e p r e s u m e d o c u la r h isto p la sm o sis sy n d ro m e
(P O H S ) a n d a g e r e la te d m a c u la r d e g e n e r a tio n . T h e fra c tio n o f tr e a te d p a tie n ts s u ffe rin g a
p e r s is te n c e (less th a n 3 m o n th s a fte r tr e a tm e n t) o r r e c u r r e n c e o f a c h o r o id a l n e o v a s c u la r
m e m b r a n e fo llo w in g la s e r tr e a tm e n t is g iv e n o n th e Y axis w h ile m o n th s fo llo w in g t r e a tm e n t
is g iv e n o n th e X axis. W ith in five y e a rs o f t r e a tm e n t, th e to ta l fra c tio n o f k ry p to n -tre a te d
P O H S p a tie n ts w h o h a d a p e r s is te n t o r r e c u r r e n t m e m b r a n e was 31% w h ile 7 9 % o f A M D p a
tie n ts sh o w e d p e rs is te n c e o r r e c u r r e n c e o f t h e i r n e o v a s c u la riz a tio n d u r i n g th is s a m e in te rv a l.
T h is f ig u re w as d ra w n u s in g d a ta f ro m tw o i n d e p e n d e n t r e p o r ts o f th e M a c u la r P h o to c o a g u
la tio n S tu d y G r o u p (se e t e x t ) .

2) abnorm ality o f the retinal pigm ent epithelium associated with a co-factor de
ficiency (Newsome et al., 1988), o r an inborn e rro r o f m etabolism (Deut-
m an an d Jansen, 1970);
3) abnorm ality of B ru ch s m em brane associated with a faulty prim ary structure
(e.g. pseudoxanthom a elasticum a n d A lport syndrom e) or by deposition o f an
abnorm al substance from an RPE o r vascular source (Bird, 1986; Newsome et
al., 1987);
4) abnorm ality of the choriocapillaris (Friedm an et al., 1963; Tso, 1985);
5) im m une system activation including developm ent of anti-retinal antibodies
(G urne et al., 1991) an d attraction o f m acrophages (Penfold et al., 1986);
6) abnorm al scleral rigidity (Friedm an et al., 1989);
176 EDWIN M. STONE AND VAL C. SHEFFIELD

7) m itochondrial dysfunction (Kuriyama et al., 1984; Modi et al., 1991);

8) light toxicity (Young, 1988; Taylor et al., 1992; Gottsch et al.. 1993); and
9) various nutritional deficiencies (Eye Disease Case-Control Study Group,
1993).
Each o f these hypotheses has some theoretical m erit an d some experim ental
support; and, in o u r opinion, there is currently little reason to com pletely exclude
any o f them from a significant role in a subset o f AMD patients. O ne o r m ore of
these m echanism s could have a n atu ra l age-related co m ponent and m ore than
o ne process could be active in any given patient. Thus, the dem onstration o f an
age-related increase in disease prevalence o r severity does n o t in and o f itself sup
p o rt or preclude the existence of any o f the previously listed m echanism s, n o r
does it preclude the existence of various types of genetic predisposition to the dis
ease.
O th er authors have previously expressed the opinion th at AMD is n o t a single
disease but is a com m on clinical m anifestation of a variety of biochem ical disor
ders (Delaney an d Oates, 1982; Elm an a n d Fine, 1989; Bird, 1991). If m ultiple
m echanism s are involved in significant subsets of AMD, one would expect to find
some clinical an d histopathologic evidence for heterogeneity in populations of
AMD patients. Such evidence exists bu t is often obscured by the varying defini
tions of d ifferent authors. For exam ple, Bressler et al. (1988) broadly define AMD
as features o f d rusen an d geographic atrophy of the retinal pigm ent epithelium
as well as retinal changes associated with choroidal neovascularization in individ
uals over 50 years o f age. In his atlas, Gass (1987) lists over 20 entities known to
be associated with choroidal neovascularization ranging from inflam m atory dis
eases, to drusen. If one were to take the latter half of the definition o f Bressler and
co-workers at face value, patients over the age o f 50 with any o f the d ifferent dis
o rders listed by Gass as com plicated by choroidal neovascularization could be con
sidered to have AMD.
Even within the relatively narrow subcategory of d ru se n there is quite a bit of
heterogeneity. Typical drusen can range from tiny hyaline bodies only a few RPE
cells in diam eter to relatively large detachm ents o f the retinal pigm ent epitheli
um. In addition, there is an ophthahnoscopically an d angiographically distinct
form known as cuticular o r basal lam in ar d rusen (Gass et al., 1985). In contrast
to the wide variation in the clinical appearance o f d rusen in the AMD population
as a whole, it has been o u r experience that within families with AMD, the affected
relatives are often distinctly sim ilar to one an o th er ophthahnoscopically (Figure
2 ).
T here is also som e histopathologic evidence that the susceptibility to drusen de
v elopm ent may n o t be uniform within the population. Feeney-Burns an d Eller-
sieck (1985) studied the ultrastructure o f B ruch's m em brane in 68 eyes spanning
10 decades o f life. T he authors assigned each specim en to one of six grades of ul-
trastructural abnorm ality an d showed that younger patients on average had lower
grades of abnorm ality than older patients (Figure 3 ). A lthough the authors did
 THE M O L E C l'IA R GENETIC APPROACH TO MACL'IAR DEGENERATION 177

M i I 1 1 I

Figure 2 O p h th a lm o s c o p ic sim ila rity o f m a c u la r d ise a se in re la tiv e s a ffe c te d w ith fa m ilia l m a c

u la r d e g e n e r a tio n . T h e p e d ig r e e is sh o w n a t th e to p . C lo se d sy m b o ls in d ic a te in d iv id u a ls w ith
m a c u la r d e g e n e r a tio n . F u n d u s p h o t o g r a p h s o f th e m o th e r (A) a n d five a ffe c te d c h ild r e n (B-
F) a re sh o w n in d e c re a s in g o r d e r o f a g e . T h e d r u s e n a re soft, c o n f lu e n t a n d lim ite d to th e c e n
tra l m a c u la . T h e p e r ip h e r a l f u n d u s is n o r m a l. T h r e e p a tie n ts (A, B a n d D) h a v e s o m e g e o
g r a p h ic a tr o p h y in th e c e n t e r o f th e a ffe c te d a re a s. T h e view o f th e f u n d u s in p a ti e n t A is
lim ite d by c a ta ra c t. S ee C o lo u r P la te If a t th e b a ck .
178 EDWIN M. STONE AND VAL C. SHEFFIELD

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Figure 3 P re v a le n c e o f u ltras!) tu t.urai a b n o rm a litic .s in B ru c h 's m e m b r a n e in p a tie n ts o f d if
f e r e n t ages. T h e f ig u re is d ra w n u s in g d a ta r e p o r t e d bv F e e n e y -B u rn s a n d E lle rsie c k (1 9 8 5 ).
T h e Y ax is gives th e f ra c tio n o f p a tie n ts In e a c h a g e g r o u p a ssig n e d to e a c h o f six g ra d e s o f
u ltr a s tr u c tu r a l sev erity th a t a rc d is tr ib u te d a lo n g th e X axis. W h ite b a rs in d ic a te p a tie n ts 1 -2 0
y e a rs o f ag e; g re y b a rs, 2 1 -6 0 y e a rs o f ag e; a n d , b la c k b a rs 61-100 y e a rs o f ag e. N o te t h a t 10%
o f t h e y o u n g e s t p a tie n ts h av e th e s a m e d e g r e e o f a b n o r m a lity (g ra d e 3) as 5 4 % o f th e o ld e s t
p a tie n ts .

n o t discuss the possibility o f varying susceptibility to drusen form ation within the
population, it is interesting that o ne patient (10%) from their youngest group had
the sam e ultrastructural severity as 20 patients (54%) from their oldest group. Pos
sible explanations for this observation are that Feeney-Burns and Ellersiecks
age-related sequence o f changes is 1) a com m on m anifestation o f a variety of dif
ferent biochem ical processes with d ifferent ages of clinical onset; o r 2) tru e age-
related p h en o m en a are superim posed on a background o f variable genetically-de
term in ed susceptibility.
A dditional insight into this possibility was provided by Coffey and Brown stein
(1986) who co unted drusen in serial sections of 23 pairs of postm ortem eyes. T he
unusual and im p o rtan t feature of this study is that th eir data were not averaged -
 THE M O LECUIA R GENETIC APPROACH TO MACUIAR DEGENERATION 179

they were re p o rted individually. Two of the 23 patients had an extraordinarily

large n u m b er of drusen which the authors speculated m ight be due to a genetic
predisposition.

HERITABILITY OF AMD

An im portant req u irem en t for a m olecular approach to any hum an disease is the
existence of families affected with a heritable form . T he way in which such fami
lies are used for the study o f disease will be discussed m ore fully below, b u t at this
ju n c tu re it m ight be reasonable to ask w hether there is any evidence that a subset
o f AMD is heritable.
T h ere are a n u m b er of dystrophies th at affect the m acula whose heritability has
been un d isp u ted for decades. Some o f these are clinically similar enough to AMD
that distinction from the latter is often difficult in affected patients over the age
of fifty, especially if the family history is n o t known. These include Bests disease
(Best, 1905), the p attern dystrophies (M arm or and Byers, 1977; Hseih et al. 1977;
Watzke et al., 1982; D ejong and D ellem an, 1982), Sorsbys m acular dystrophy
(Sorsby et al., 1949), and do m in an t d ru sen (Pearce, 1968; D eutm an an d Jansen,
1970). T he familial n atu re o f the latter entity was first described nearly 100 years
ago (Doyne, 1899), and at least o n e au th o r feels that it is clinically indistinguish
able from typical AMD (Gass, 1973). Bests, Sorsbys and the p attern dystrophies
also have some AMD-like features including an accum ulation o f abnorm al m ateri
al at the level of B ru ch s m em brane a n d the RPE as well as the eventual develop
m ent o f choroidal neovascularization o r geographic atrophy in a subset of
patients. N orth C arolina M acular Dystrophy can also mimic AMD ophthalm oscop-
ically b u t differs from the o th er dystrophies in that it is usually n o t progressive
(Small et al., 1991). T he specific genetic defects th at cause each o f these diseases
are o f great interest because the affected genes may be involved in a sizeable pro
portion of typical AMD. At the very least, identification o f a gene th at causes any
m acular dystrophy has the potential to greatly im prove o u r u n d erstanding o f the
cascade o f m olecular events th at occurs in AMD.
If m utations in m acular dystrophy genes play an im p o rtan t role in AMD, one
would expect to find evidence o f heritability in a significant p ro p o rtio n o f typical
AMD patients. However, it is im p o rtan t to realize that even if all AMD has a pri
marily genetic basis, this would n o t necessarily be clinically obvious. Since the dis
ease does n o t usually becom e sym ptom atic until the seventh o r eighth decade, the
parents o f affected patients are no t usually living and their siblings may also be de
ceased or otherw ise unavailable for exam ination. Also, since only a m inority o f af
fected patients experience severe visual loss, it is im possible by history alone to
determ ine w hether a siblings 2 0 /5 0 vision is due to AMD or some o th er cause;
and conversely, w hether a p aren t with excellent vision actually had num erous as
ym ptom atic drusen. C ertainly if one assumes that AMD is a natural aging p h en o m
enon an d does no t even question patients about their family, one is unlikely to
180 EDWIN M. STONE AND VAX. C. SHEFFIELD

discover evidence to support a genetic basis for the disease. To restate this im por
tant point: failing to look for evidence of heritability is not equivalent to carefully
looking for it w ithout success.
For exam ple, a recently published study exam ined AMD risk factors in over 400
patients and 600 controls (Eye Disease Case C ontrol Study G roup, 1992). T he sub
jects were questioned about cigarette use, alcohol an d coffee consum ption, sun
light exposure, m arital status, physical activity, the presence o f diabetes or
cardiovascular disease, p rio r hysterectomy, an d the use o f estrogen or birth con
trol pills. No questions regarding AMD in family m em bers were asked and it is thus
n o t surprising that these authors did not even m ention the possibility o f genetic
risk factors, m uch less find any evidence to su p p o rt them .
In contrast, w hen evidence for genetic factors has been looked for, it has been
found. In 1973, Gass re p o rted the results o f his study o f 200 patients with m acular
d egeneration who had been followed for an average o f fo u r years. He stated: al
though it was my initial im pression that few patients with d rusen an d disciform d e
tachm ent had a positive family history, m ore careful questioning o f the patients
and investigation o f the few relatives available during this study has revealed a sig
nificant incidence of familial involvem ent. Specifically, he obtained a positive
family history of central visual loss in 38 of the 200 patients (19%) an d was able to
confirm the diagnosis o f AMD in the relatives o f 10 o f these patients by personal
exam ination. In a case-control study by Hym an et al. (1983) the presence o f AMD
in parents and siblings of the study subjects was investigated via questionnaires dis
tributed to the subjects, th eir siblings and th eir siblings eye exam iners. Inclusion
o f the latter group was revealing because the eye exam iners o f the case siblings re
p o rted the presence of AMD alm ost twice as often as the case siblings themselves,
suggesting that patients are often not told that they have m acular degeneration
unless they have experienced visual loss. A ccording to the eye exam iners respons
es, 19.9% o f the siblings o f AMD cases had AMD themselves in com parison to 7.9%
of the siblings o f controls. T he age and sex distribution o f the case siblings were
the sam e as those o f the controls. W hen the parental and sibling data were com
b ined by defining a positive family history as eith er a p a re n t or a sibling with a his
tory o f the disease, the data were similar with 21.6% of the cases having a positive
family history as opposed to 8.6% of the controls.
T here are a few reports of identical twins with AMD com plicated by choroidal
neovascularization (Melrose et al., 1985; Meyers an d Zachary, 1988; Dosso and
Bovet, 1992). In all th ree of these reports, the second twin developed symptoms of
choroidal neovascularization within 16 m onths o f the first. T he twins re p o rte d by
Meyers and Zachary belonged to a sibship o f 13, and four of the o th e r 11 siblings
were also affected.
At least three studies have suggested that AMD is significantly m ore com m on in
white patients than in blacks (Chumbley, 1977; G regor an d Joffe, 1978; Som m er
et al., 1991). A lthough it is possible that increased pigm entation is directly respon
sible for the protective effect, it is also possible that AM D-predisposing genes are
less com m on in the black population.
 THE MOLECULAR GENETIC APPROACH TO MACUIAR DEGENERATION 181

U nfortunately, n eith er the F ram ingham study (Kahn et al., 1977; Leibowitz et
al., 1980) n o r the National H ealth a n d N utrition Exam ination Survey (Ganley and
R oberts, 1983) collected data regarding a family history of AMD. However, these
studies were already underw ay when Gasss 1973 paper was published an d were
com plete before the re p o rt o f Hyman et al. in 1983. It is less understandable why
m ore recent studies (W eiter et al., 1985; Bressler et al., 1989; Klein et al., 1991; Eye
Disease Case C ontrol Study G roup, 1992) have chosen to ignore the possibility of
genetic predisposition.
In any case, th ere seems to be sufficient evidence in the literature to support the
assum ption th at a significant fraction o f AMD is heritable and therefore am enable
to m olecular genetic attack. Moreover, the evidence that AMD is pathophysiolog-
ically h eterogeneous is also com pelling and we believe that som e m ethod of reli
ably dividing patients according to disease m echanism is essential for new
therapies to be rationally designed, tested and adm inistered.

M OLECUIAR APPROACHES TO DISEASE GENE IDENTIFICATION

Identification of genes that cause in h erited diseases has been accom plished with
a variety of approaches. T he first genetic disease genes were identified based on a
knowledge of the specific cellular proteins involved in the pathogenesis of the
diseases. Exam ples o f this are the globin genes involved in the thalassem ias and
sickle cell anem ia (Ingram , 1956; W eatherall a n d Clegg, 1981; B unn and Forget,
1986). For many genetic diseases, including hereditary eye diseases, inform ation
concerning proteins involved in the pathogenesis o f the disease is lacking or has
failed to aid in the identification o f the disease-causing locus. For such disorders,
the use o f genetic linkage analysis with polym orphic DNA probes to identify the
genetic location of the gene has proven valuable.
Figure 4 provides an overview of a stepwise genetic approach to m acular disease.
T he ideal starting m aterial is a large family affected with an autosom al dom inant
disease which can be used for linkage analysis. Linkage analysis does no t require
any specific hypothesis ab o u t the pathogenesis of the disease. It requires only the
availability o f a sufficient n u m b er o f correctly diagnosed individuals to allow a sta
tistically significant relationship between m arker alleles and the disease p h en o
type to be dem onstrated. Linkage data are expressed as lod scores which are
derived and in terp re ted as discussed in C hapter 1.
T he first m acular disease to be successfully linked to a genetic m arker was an
atypical vitelliform dystrophy linked to the m arker GPT-1 (Ferrell et al., 1983)
which is now known to lie on the long arm of chrom osom e 8 (Rocha et al., 1988).
More recently, Small and co-workers (1992) m apped N orth C arolina M acular Dys
trophy (Figure 5) to chrom osom e 6. Two m icrosatellite m arkers located in chro
m osom al region 6ql3-q21 (MFD171, MFD97) were found to be linked to the
disease locus with lod scores of 8.4 a n d 13.1 at recom bination fractions o f 0.004
and 0.017 respectively. A patient with a cone dystrophy and m ental retard atio n as-
182 EDWIN M. STONE AND VAL C. SHEFFIELD

r' i

CLIN IC AL DIAGNOSIS LINKAGE ANALYSIS PRIMARY LINKAGE

FUNCTIONAL POSITIONAL TISSUE-SPECIFIC

n___n

\ i / DGGE
TEGEN
CANDIDATE GENES
\ G A TC GATC

>
SINGLE AFFECTED PATIENTS
TRANSGENIC ANIMAL

DNA
(with or without family history)
Sequencing

/ \
SSCP

MUTATION SCREENING

Figure 4 D ia g ra m m a tic r e p r e s e n ta tio n o f a s te p w ise m o le c u la r g e n e tic a p p r o a c h to in h e r ite d

d ise ases. T h e u p p e r p a n e l d e p ic ts a fa m ily -o rie n te d a p p r o a c h . S ta rtin g w ith a la rg e fa m ily af
fe c te d w ith a n i n h e r i t e d d ise a se , lin k a g e an aly sis is u s e d to d e te r m i n e th e c h r o m o s o m a l lo c a
tio n o f th e d ise a s e -c a u s in g g e n e . T h e ab ility to re fin e th e lo c u s w ith fin e m a p p in g is
d e p e n d e n t u p o n fam ily size as w ell as th e d e n sity o f m a rk e rs in th e a re a o f p r im a r y lin k a g e .
T h e lo w e r p a n e l d e p ic ts a c a n d id a te g e n e a p p r o a c h to i n h e r i t e d d iseases. C a n d id a te g e n e s
a re o f th r e e types: 1 ) g e n e s e n c o d in g p r o te in s w h o s e fu n c tio n c an b e th e o re tic a lly a sso c ia te d
w ith th e d ise a s e p h e n o ty p e (e .g . r h o d o p s in a n d r e tin itis p ig m e n to s a ) ; 2 ) g e n e s k n o w n to m a p
a t a lo c u s lin k e d to th e d ise a s e p h e n o ty p e ; a n d , 3) g e n e s w h o se p r o te in s a re p rim a rily e x
p re s s e d o n ly in th e tissu e a ffe c te d by th e d ise ase. D N A sa m p le s fro m m e m b e rs o f fa m ilie s af
fe c te d w ith a n i n h e r i t e d d ise a se (as in th e to p p a n e l) o r sin g le a ffe c te d p a tie n ts w ith o u t a
fam ily h is to r y a r e u s e d f o r s c re e n in g th e s e c a n d id a te g e n e s fo r d ise a s e -c a u s in g m u ta tio n s via
d e n a t u r in g g r a d ie n t g e l e le c tro p h o r e s is (D G G E ) o r sin g le s tr a n d c o n f o rm a tio n a l p o ly m o r
p h is m (SSC P) analysis. D N A s e q u e n c in g is u s e d to d e te r m i n e th e e x a c t n u c le o tid e c h a n g e s
in e a c h m u ta tio n d e te c te d . T ra n s g e n ic a n im a ls c o n ta in in g th e m u ta te d g e n e c a n b e c r e a te d
a n d u s e d to stu d y th e p a th o p h y s io lo g y ' o f th e d ise a s e a n d c a n p o te n tia lly b e u s e d to te st e x
p e r im e n ta l th e r a p ie s as well.
 THE MOLECULAR GENETIC APPROACH TO MACUIAR DEGENERATION 183

F igure 5 N o r t h C a ro lin a m a c u la r d y stro p h y . T y p ic al f e a tu re s o f th e d ise a s e a re th e c e n tr a l

c r a te r w ith a sh in y b a s e s u r r o u n d e d by a g lio tic rim . S e e C o lo u r P la te III a t th e b ack .

sociated with a translocation breakpoint in the same region has been re p o rted by
T ranebjaerg et al. (1986). These rare chrom osom al rearrangem ents are potential
ly im p o rtan t for the task o f identifying the gene directly. Also in 1992, we (Stone
et al., 1992) an d an o th er g roup (Forsm an et al., 1992) independently m apped
Best's Vitelliform Dystrophy (Figure 6) to the long arm of chrom osom e 11.
This disorder has been studied histopathologically and found to be associated
with lipofuscin-like deposits within and beneath the retinal pigm ent epithelium .
O u r group m apped a large five-generation family with this early-onset autosom al
do m in an t form of vitelliform m acular dystrophy to the 11 q 13 region, between
m arkers INT2 and D11S871 (Zmax = 9.3 at 3.9 cM from m arker D11S871) (Stone
et al., 1992). T he o th er group m ap p ed the Best gene in a large Swedish family to
the same chrom osom al region (11 q 13) with a lod score of 15.12 at a recom bina
tion fraction o f 0.01 for the closest marker, I S'12 (Forsm an et al., 1992). A possible
candidate gene is the ROM-1 gene (Bascom et al., 1992) which m aps to same chro
m osom al region and shows similarity to the p erp h erin /R D S gene (see below).
T he gene product, rom-1, is a m em brane-associated protein o f rod o u ter segm ents
th at is o f sim ilar size and am ino acid sequence as rd s/p e rip h e rin .
O th er m acular diseases th at should be am enable to linkage analysis include
dom inant drusen (both typical and cuticular), Sorsbys fundus dystrophy, various
p attern dystrophies, an d S targardts disease.
IIS4 EDWIN M. STONE AND VAL C. SHEFFIELD

F ig u re 6 B e st's v ite llifo rm d y stro p h y . A classic e g g y o lk d e p o s it o f lip o l'u sc in -lik e m a te ria l is

p r e s e n t b e n e a th th e r e tin a l p ig m e n t e p ith e liu m o f th e c e n tra l m a c u la . S e e C o lo u r P la te IV at
th e b ack .

O nce a disease is m apped to a region, one may find that a candidate gene has
been previously m apped to the same location. This was the case for autosom al
d o m inant retinitis pigm entosa in which the rhodopsin gene had been localized to
the long arm o f chrom osom e 3 (Sparkes et al., 1986) before linkage analysis sug
gested that a disease-causing m utation m ight exist at the same locus (McWilliam
e ta l., 1989 and C hapter 6).
An alternative m ethod used to identify disease-causing genes is the candidate
g e n e approach. Genes that are likely candidates for playing a role in a given ge
netic disease are selected and tested for th eir involvem ent in the disease eith er by
m utation analysis o r by linkage studies that use polym orphism s previously identi
fied w ithin the gene sequence. A candidate gene can be selected based on its func
tion, its chrom osom al location, or the tisstie in which it is prim arily expressed.
Successful identification o f a gene involved in hum an retinal disease was recently
accom plished using the candidate gene approach. Specifically, the m urine RDS
gene was shown to be m utated in anim als with retinal degeneration (Travis et al.,
1989) and this finding m ade the hum an hom ologue (Travis et al., 1991) a good
candidate gene for retinitis pigm entosa. This was confirm ed when m utations in
 THE MOLECULAR GENETIC APPROACH TO M ACITAR DEGENERATION 185

the hum an RDS gene were identified in patients with autosom al do m in an t retini
tis pigm entosa (F arrar et al., 1991; Kajiwara et al., 1991; Wells et al., 1993). Using
the same reasoning, we recently identified two different m utations in the RDS
g en e (Nichols et al., 1993a and 1993b) in families affected with an autosom al dom
in an t p attern dystrophy (Figures 7 and 8) resem bling D eu tm an s butterfly dystro
phy of the fovea (D eutm an et al., 1970). T he first was a G to A substitution at
codon 167 th at substitutes an aspartate for a highly conserved glycine residue. T he
m utation segregated with the disease in eleven affected and fo u r unaffected (>55
yrs) family m em bers (Zmax = 4.0 at 6 - 0). T he glycine1*^ residue is also conserved
in the peripherin-related p rotein rom-1, which may associate non-covalently with
p erip h erin in rod p h o to recep to rs (Bascom et al., 1992). T he phenotype in this dis
o rd e r involves accum ulation o f yellow m aterial within and beneath the retinal pig
m en t epithelium in the perifoveal region. T he parallels with the rds/+ m ouse are
interesting, since the latter shows dysplastic o u ter segm ents which appear to be u n
stable and to shed at an increased rate. T he underlying pigm ent epithelium con
tains large and abnorm al phagosom es which may result from im paired digestion
o f the abnorm al o u ter segm ents (Sanyal and Hawkins, 1988). It is possible that
accum ulation of such phagosom es in the m acular pigm ent epithelium gives rise

F ig u re 7 B u tte rfly d y stro p h y . T h e r e is a yello w ish su b fo v e a l le s io n w ith five r a d ia tin g a rm s . In

a d d itio n , r o u n d p ig m e n t d e p o s its th a t a re la r g e r th a n typ ical d r u s e n a re p r e s e n t j u s t p o s te ri
o r to th e m a jo r v a sc u la r a rc a d e s . F ro m N ic h o ls e t al., N ature Genetics (1 9 9 3 ); u s e d w ith p e r
m issio n . S ee C o lo u r P la te V a t th e b ack .
186 EDWIN M. STONE AXD VAL C. SHEFFIELD

I H------ 1 i---------- Ml-----------1

II 12 14 1 3 7 11 15 18
i O O t t t t t t t

F ig u re 8 D e n a tu r in g g r a d ie n t g el e le c tr o p h o r e s is o f PC R p r o d u c ts c o n ta in in g R D S e x o n 1 se
q u e n c e s . C lin ically a ffe c te d in d iv id u a ls a r e in d ic a te d by c lo s e d sy m b o ls w h ile c lin ically u n a f
fe c te d in d iv id u a ls a re in d ic a te d by o p e n sym bols. S p o u ses a re in d ic a te d by sy m b o ls w ith o u t
n u m b e r s w h ic h a re c o n n e c te d to a n a ffe c te d p a tie n t's sym bol by a h o riz o n ta l lin e . E ac h gel
la n e c o n ta in s a s a m p le fro m th e in d iv id u a l w h o se p e d ig r e e sy m b o l is d ire c tly ab o v e. A ffe cte d
in d iv id u a ls h a v e m u ltip le b a n d s o n th e g el (b o th h o m o d u p le x e s a n d h e te r o d u p le x e s ) w h ile
n o r m a l in d iv id u a ls h a v e o n ly o n e b a n d c o r r e s p o n d in g to n o r m a l h o m o d u p le x m o le c u le s.
F ro m N ic h o ls, e t al., N ature Genetics (1 9 9 3 ); u s e d w ith p e rm iss io n .

to the deposits of yellowish m aterial seen in the butterfly lesions (Nichols et al.,
1993a). The second m utation seen in a family with butterfly-shaped pigm ent dys
trophy of the fovea was caused by a 2 base pair deletion in the RDS gene overlap
ping codons 299 and 300 that results in a translational frameshift. This m utation
 THE M OLECUIAR GENETIC APPROACH TO MACULAR DEGENERATION 187

causes 56 incorrect am ino acids to be translated before a stop codon is en co u n

tered 131 nucleotides past the en d o f n orm al translation.
Kajiwara et al. (1993) screened over 350 patients with a wide variety of re tin o p
athies for m utations in the p erip h erin /R D S gene using single strand conform a
tion polym orphism (SSCP) analysis an d found one m utation in a family with
retinitis punctata albescens. This condition is a progressive disorder o f the central
and peripheral retin a associated with subnorm al ro d and cone ERGs and subreti-
nal white or yellow punctate deposits. These accum ulations may rep resen t the
same m aterial as found in the family o f Nichols et al. (1993a). T he m utation gives
rise to a prem ature stop codon an d is therefore probably a null allele which tru n
cates the rds p erip h erin p rotein to 42 am ino acids instead of the norm al 346. Sim
ilarly, Wells et al. (1993) screened R D S /p erip h erin exons for m utations in 100
patients with RP and 13 with various m acular dystrophies. They fo u n d a 3 base pair
deletion associated with loss o f a cysteine at codon 118 o r 119 that segregated with
typical autosom al d o m in an t RP in o n e family. In three families with autosom al
d o m in an t m acular dystrophy, they fo u n d missense m utations at codon 172 o f the
RDS gene, associated with a p red icted substitution o f glutam ine for arginine in
one family and a tryptophan substitution in the o th er two. Finally, a family with
adult vitelliform m acular dystrophy was found to have a nonsense m utation at
codon 258 (Tyr 258 stop) which w ould lead to a tru n cated p rotein product, similar
to that in the rds/+ heterozygous m ouse (C hapters 5, 6). In this case, the m acula
shows a fried egg appearance again with yellow deposits at the level o f the retinal
pigm ent epithelium ra th e r than in the p h o to rec ep to r layer. O ne m em ber o f the
family with butterfly-shaped pigm ent dystrophy o f the fovea described by Nichols
et al. (1993a) also showed bilateral vitelliform lesions rem iniscent o f Best's disease
while others showed eith er punctate o r p attern ed lesions. T he single affected pa
tient with the Tyr 258 stop m utation was only aware o f a relatively m ild central vi
sion loss. H er father h ad a sim ilar disorder but no o th er affected relatives were
available to confirm segregation o f the m utation with the illness. However, the se
vere n ature o f the m utation seems to leave little d o u b t th at it is causally related to
the disorder.
This surprising finding th at m utations in the same gene can cause phenotypes
as widely differing as butterfly dystrophy an d retinitis pigm entosa, serves to illus
trate two im p o rtan t points. First, c u rre n t clinical and electrophysiological tech
niques are no t capable o f separating diseases into m olecularly sim ilar groups. T hat
is, m utations in the rhodopsin gene on chrom osom e 3 cause a phenotype that is
clinically and electrophysiologically m uch m ore sim ilar to RDS-associated retinitis
pigm entosa than the pattern dystrophies caused by m utations elsewhere in the
RDS gene. Second, clinical, psychophysical, an d electrophysiologic m ethods are
n o t capable of predicting the prim ary site of expression o f the disease causing
gene. T hat is, p rio r to the discovery o f the vitelliform an d p attern dystrophy asso
ciated RDS m utations, m ost retinologists would have predicted the m olecular de
fect in these dystrophies to exist at the level of the retinal pigm ent epithelium or
perhaps B ruch's m em brane. In this case, m olecular biology was capable of dem
188 EDWIN M. STONE AND VAL C. SHEFFIELD

onstrating th a t the prim ary expression o f the genetic defect is actually in the neu-
rosensory retina.
Some caution should be introduced at this point: no t all sequence changes in
candidate genes are disease-causing m utations. W hen h u n d red s o f patients are
screened for changes in alm ost any gene sequence, some variation is likely to be
fo und. A change is likely to be a disease causing m utation if it has the following
characteristics.
1) It results in an altered am ino acid (this is especially convincing if the m utation
results in a stop codon, a fram eshift, o r a dram atic change in charge, size or
hydrophobicity o f a single am ino a c id ).
2) It is not found in norm al individuals to any m easurable extent. For exam ple,
if screening fifty n orm al controls (100 chrom osom es) does n o t reveal a similar
sequence change, it is less likely to be a clinically silent polym orphism .
3) T he m utation segregates with the disease phenotype in a statistically signifi
cant fashion (Figure 8 ) .
T he latter criterion is im p o rtan t because if a heterozygous polym orphism is de
tected in an affected p atien t the probability th at a second affected sibling o r par
en t will h arb o r the same polym orphism ju s t by chance is 50%; the probability that
a third would have the polym orphism is 25%, and so on. Thus, pedigrees contain
ing only one or a few affected patients can n o t by themselves prove the participa
tion o f a given candidate gene in the disease. It is rem otely possible for a DNA
sequence change to satisfy all three of the above criteria w ithout actually causing
any disease. For exam ple, suppose the RDS sequence change illustrated in Figure
8 was actually a silent polym orphism , an d that the true disease-causing m utation
was in a n o th er gene a centiM organ o r two from RDS. If the polym orphism s were
in phase with the disease-causing m utation, o n e would get exactly the sam e result
as shown in Figure 8, even although the RDS gene was n o t really involved in the
disease. O f course, the likelihood of this hypothetical situation occurring (espe
cially in two d ifferent families) is so low th at statistically significant segregation o f
an am ino-acid-changing m utation is usually accepted as tentative p ro o f o f the m u
tation's involvem ent in the disease. In fact, once a given phenotype has b een sig
nificantly associated with a m utation in a given gene, it is probably sufficient to
satisfy only the first two criteria. Each new family with a given phenotype that har
bors a d ifferent m utation in the same candidate gene lends additional support to
the hypothesis that the m utations are the prim ary cause o f the disease.
T he ultim ate p ro o f th at a given m utation actually causes the disease is the cre
ation o f the disease in a transgenic anim al by the introduction of a specific m uta
tion initially identified in an affected hum an. T he existence o f such anim als also
provides a platform for studying a disease in ways that are im possible in hum ans.
For exam ple, no hum an eyes with butterfly dystrophy have ever been studied his-
topathologically, and if such specim ens do eventually becom e available they will
likely be from an elderly p atien t with advanced atrophic disease. T he existence of
a transgenic anim al m odel would allow the m orphological features o f the disease
 THE MOI .EClI.AR GENETIC APPROACH TO MACULAR DEGENERATION 189

to be worked out in great detail, including the tem poral changes from birth to ad
vanced disease. U ltrastructural, biochem ical, and cell biological studies using
transgenic anim als would have a m uch greater chance o f elucidating the m olecu
lar sequence of events that cause the clinically evident disease than would similar
studies in the hum an population. In addition, once the disease m echanism was
well worked out, physical, dietary o r pharm acologic interventions could be de
vised and tested m uch m ore readily in a transgenic anim al m odel than in the h u
m an population.
Figure 4 dem onstrates two o th er im p o rtan t features o f the m olecular genetic
approach to hum an disease; the d ep e n d en ce at nearly every level on the availabil
ity o f actual hum an patients affected with various disorders an d the n eed for
skilled clinicians to correctly diagnose these patients and carefully define their
phenotypes. It is the clinician who initially discovers a family that is suitable for
linkage analysis. M ore im portantly, if such an analysis is to be successful, each pa
tient m ust be diagnosed with a high degree o f accuracy. For exam ple, it could be
very dam aging to a linkage study to incorrectly diagnose a p atien t with the pre
sum ed ocular histoplasmosis syndrom e as affected in a family with p attern dys
trophy. In a fully inform ative sibship o f 11 sibs and 2 living parents, such a
m isdiagnosis could decrease the lod score from 3 to 1.3. If the nearest genetic
m arker was 10 centiM organs from the disease gene, a single m isdiagnosis could
result in a m axim al lod score o f only 0.5 which m ight not even result in additional
intensive searching in th at area.
At the candidate gene level, clinicians can participate by co ntributing patients
with various phenotypes fo r m utation searches. W hen such m utations are found,
clinical ophthalm ologists can attem pt to correlate specific features o f the disease
with individual m utations. Such correlations have the potential to m ake m olecular
diagnosis a very useful clinical tool. For exam ple, if certain m utations are found
to be associated with a high risk o f exudative m acular degeneration while others
ten d to be associated with slowly progressive geographic atrophy, such inform a
tion could be used to counsel patients as well as to help plan follow-up visits so that
the greatest am o u n t o f scrutiny could be focused upon the patients at greatest risk.
Two recent advances have greatly facilitated both linkage analysis an d the can
didate gene approach. T he developm ent o f highly polym orphic DNA m arkers
known as m icrosatellites (W eber and May, 1989; Litt an d Luty, 1989; Weber, 1990)
has greatly sim plified genetic linkage studies. M icrosatellite m arkers are superior
to restriction fragm ent length polym orphism s (RFLPs) because they are m ore in
form ative and they can be assayed using the polym erase chain reaction (C hapter
1). T he second advance has been the developm ent o f m ethods for screening sin
gle-base sequence substitutions. These m ethods include d en atu rin g gradient gel
electrophoresis (DGGE) (Fischer an d Lerm an, 1980; Meyers e ta l., 1987; Sheffield
et al., 1989), single-strand conform ational gel analysis (SSCP) (O rita et al., 1989),
chem ical cleavage of m ism atch (C otton et al., 1988), and hetero d u p lex analysis
(Keen et al., 1991). As a group, they allow rapid screening o f a candidate gene for
m utations in m ultiple individuals. These techniques decrease the am o u n t o f labor
190 EDWIN M. STONE AND VAL C. SHEFFIELD

intensive DNA sequencing th at needs to be perfo rm ed and thereby increase the

n u m b er o f affected individuals an d controls that can be screened.

THERAPY FOR AMD

Everyone involved in the study of age related m acular degeneration is hoping

th at m ore effective therapy can be developed. As outlined at the beginning o f
this chapter, a variety of pathogenic m echanism s are likely to be involved in the
large clinical entity known as AMD an d it is unrealistic to expect that any given
treatm en t will be effective for all of them . It is essential to develop m ethods to
reliably subdivide patients into pathophysiologically sim ilar groups. W ithin such
g roups (e.g. within a family whose affected m em bers all carry the same d o m in an t
m utation) it is com m on to find that som e patients do relatively well clinically
while others do poorly. This suggests that dietary, physical or additional genetic
factors are capable o f m odulating the effect o f the disease gene. Such m odulators
have the potential to be the basis of effective therapy. T hat is, the pathological
process does no t have to be totally overcom e in a given type o f m acular d egenera
tion to m ake a trem endous difference in these patients. If the rate o f progression
of the disease can be altered by only 10 or 15%, it would make a significant nu m
b er o f affected patients asym ptom atic for th eir entire lives.
M olecular genetics can facilitate the developm ent o f effective therapy in the fol
lowing ways.
1) W hen patients can be divided into pathophysiological groups based on an ob
jective m olecular diagnosis, risk fa cto r studies will be m ore likely to be able
to identify agents that accelerate o r retard the disease process.
2) T he identification o f disease causing genes will suggest specific agents that
m ight be investigated. For exam ple, if a disease is fo u n d to result from a m u
tation in a zinc-requiring enzyme of the RPE, zinc augm entation m ight be a
reasonable approach to therapy in these patients.
3) Transgenic anim als will allow experim ental therapies to be investigated rapid
ly an d in a controlled fashion.
4) T he ability to identify disease-causing m utations in presym ptom adc individu
als will allow specific therapy to be adm inistered over a longer period of time.
For exam ple, if light exposure plays a role in a subset of AMD, sunglasses
would be m ore likely to m ake an im pact if they were prescribed for children
with the genetic predisposition than if their use was delayed until the clinical
signs o f m acular degeneration were evident.

CONCLUSION

T he hum an genetic m ap is being refined to the centiM organ level with highly
polym orphic genetic m arkers and many genes are being characterized and
 THE MOLECULAR GENETIC APPROACH T O MACULAR DEGENERATION 191

placed on this map. T he increasing availability o f good m arkers an d the increas

ing resolution of the m ap greatly facilitates the m apping of disease-causing m uta
tions as well as the isolation of the affected genes themselves. In the past two
years alone, the genes for two types o f m acular disease have been m ap p ed and
specific m utations involved in a third have been identified.
A decade ago, the prospect of u n d erstan d in g any form o f m acular d egeneration
at the m olecular level seem ed rem ote. However, recent progress on the clinical,
genetic and m olecular fronts m akes it likely that such u n d erstanding will be fo rth
com ing in the n ear future an d m akes us hopeful that m ore effective therapy will
follow closely on its heels.

ACKNOWLEGEMENTS

P reparation o f this ch ap ter was su p p o rted in p a rt by the N ational Retinitis Pig

m entosa F oundation Fighting Blindness; the G eorge G und Foundation; the C. S.
O Brien C enter for M acular Diseases; the Forrest L attner Foundation; Public
H ealth Service Research grants EY08426 (EMS), P50HG00835 (VCS) and
HG00457 (VCS), an d an u nrestricted grant from Research to Prevent Blindness,
New York, N.Y.

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 8. MOLECULAR GENETICS OF RETINOBLASTOMA

JO H N K. COWELL

ICRF Oncology Group Institute of Child Health

30 Guilford Street, London WC1N1EH, UK

INTRODUCTION

It appears that many hum an solid tum ours result from the inactivation o f critical
genes which are responsible for the norm al developm ent of the particular tissue
in question. Because these genes, th ro u g h their norm al function, ensure histio-
genesis an d prevent tum origenesis, they have been called tu m o u r suppressor
genes or recessive oncogenes. T he first such gene to be isolated was that
responsible for the developm ent of the ch ild re n s eye cancer, retinoblastom a.
T he study of this gene and the application o f this knowledge to the clinical m an
agem ent o f the disease has led, and continues to lead, the way in o u r u n d erstan d
ing o f m olecular events which result in tum our developm ent. T he details about
the isolation an d characterisation o f this gene is the subject o f this chapter.

RETINOBIASTOMA GENETICS

As the nam e implies, Rb is a tu m o u r of retinal cells and, with only rare excep
tions, affects children u n d e r the age o f 5 years. Individuals can present with Rb at
birth, dem onstrating that the tum ours have been growing since early fetal life.
This view' is sup p o rted by the histopathology o f the tum our which dem onstrates a
relatively undifferentiated, em bryonic-like organisation, im plying an arrest in
developm ent o f a retinal precursor cell. Thus, pools o f cells are frozen in a state
in which fu rth e r genetic changes can occur, giving rise to the full tum our p h en o
type. T he exact identity o f these p recursor cells, however, rem ains unknown.
Approxim ately 10% of patients will have a p rio r family history, the rest being ap
parently sporadic. Since the new m utation rate is relatively high (Vogel, 1979)
many o f these apparently sporadic cases will carry new germ line m utations. In
the familial form , the tu m o u r phenotype segregates as an autosom al dom inant
trait (Figure 1). This m eans th at inheritance o f a single m utant gene is apparently
sufficient to result in tum origenesis. In fact, pedigree analysis (Figure 2) shows
that, in 10% o f cases, individuals who inherit the m utant gene do no t develop a
tum our - so called incom plete p e n e tra n c e - so it is clearly only a predisposition
to tum origenesis that is in h erited an d o th er genetic events m ust happen. Thus, at
the cellular level, Rb gene m utations act in a recessive m anner. In fact, this must
be the case since no t all retinal cells in predisposed individuals develop into tu
m ours.
198 JO H N K. COWELL

* j O

C h ri l- jo

O r^BnO i in
i n f l

F ig u re 1 F am ily p e d ig r e e s h o w in g th e s e g r e g a tio n o f th e R b tu m o u r p h e n o ty p e . All in d iv id

u a ls a re a ffe c te d b ila te ra lly as in d ic a te d by th e b la c k sym bols;
= m a le = fe m a le

In a consideration of how many additional events are required, K nudson analy

sed the incidence an d age distribution o f tum our developm ent to produce the
now classic m athem atical theory the two-hit hypothesis (Knudson, 1971). In h e
reditary cases, the first m utation is present in all cells o f the body and only one ad
ditional m utation is req u ired for tum our developm ent. Note th at this event only
affects the developing retina suggesting a highly specific role for this gene in
retinal developm ent (but see later). Since only one additional m utation is re
q u ired in these cells - the chances of which are high hereditary Rb is character
ised by the presence o f m ultiple tum ours in both eyes. For this reason all
bilaterally affected individuals m ust be considered carriers o f Rb gene m utations
which accounts for 40-50% o f all patients. In truly sporadic cases, both m utations
m ust occur in the same gene, in the same cell, d u rin g an early stage o f develop
m en t and the possibility o f this occurring by chance is relatively small. Sporadic
cases, therefore, are generally characterised by the presence o f unilateral, unifocal
tum ours. However, we know empirically th at approxim ately 10-15% o f families
have unilaterally affected individuals and, therefore, som e unilaterally affected,
sporadic cases will carry a predisposing m utation although it is difficult to identify
which ones. It m ight be expected, for exam ple, that early onset unilateral cases
would potentially be m u tan t gene carriers. In o u r experience, this g roup probably
represents less than 5%. In some families, apparently unaffected individuals have
been seen to have retinal scars which resem ble successfully treated tum ours. These
have been described as benign tum ours - retinom as (Gallie et al., 1982) - o r as
regressed tum ours. Occasionally, several affected children can be b o rn to unaf
fected parents with no prior family history. O ne possibility in these cases is that an
unusual insertional translocation is segregating in the family (see later) and they
should certainly be referred for cytogenetic analysis. It is also possible, however,
 MOLECULAR GENETICS OF RETINOBLASTOMA 199

Figure 2 F a m ily p e d ig r e e s h o w in g in c o m p le te p e n e t r a n c e o f th e R b p re d is p o s itio n g e n e .

T h e m a le , in d ic a te d by th e a rro w , h a s i n h e r i t e d th e p r e d is p o s itio n g e n e fr o m h is a ffe c te d fa
th e r a n d p a s s e d it o n to h is a ffe c te d so n , a lth o u g h h e is n o t a ffe c te d h im se lf. H is s e c o n d so n
is also a n u n a f fe c te d g e n e c a r r ie r sin c e h is d a u g h te r is a lso a f fe c te d . N o te th a t tw o in d iv id u a ls
in th is fam ily a r e o n ly u n ila te ra lly a f fe c te d (h a lf-fille d sy m b o ls). T h e a ffe c te d tw ins h a v e b i
la te ra l d ise a s e (b la c k sy m b o ls). F o r f u r t h e r d e ta ils see tex t.

th at one o f the parents is a tissue m osaic (see Ribeiro et al., 1988 for review) car
rying the m utation in the germ line b u t n o t in th eir own retinal cells.
If tum ours are d etected early, they are usually m ore easily treated than those p re
senting later, although exactly w here the tu m o u r arises in the eye is im p o rtan t in
this regard. T reatm ent o f small tum ours usually involves cryosurgery, photocoag
ulation o r radiation therapy, whereas larger tum ours usually require enucleation.
Tum ours left to develop in the eye will eventually m etastasise, often down the optic
nerve, and prognosis in these cases is very poor indeed. Since early diagnosis of
fers a b etter prognosis, all at risk patients are screened regularly during the first
years o f life. In practice, this involves all relatives o f Rb patients since, because of
the possibility o f incom plete penetrance, tum our form ation is, for m ost patients,
the only unequivocal m eans o f identifying m u tan t gene carriers. Clearly a system
to identify those patients with germ line m utations would m ake the clinical m an
agem ent o f this disease m ore efficient (see below).

FINDING THE RB GENE

O ur u nderstanding o f the finer details of the genetics of Rb came with the clon
ing o f the predisposition gene, RBI. T h e circum stances leading to the actual clon
ing o f RBI resulted from painstaking analysis o f many patients over many years.
Thirty years ago Stallard an d colleagues (Stallard, 1962) showed that the associ
ation betw een Rb an d phenotypic abnorm alities such as m ental retardation, dis
tinctive dysm orphic features and, som etimes, abnorm al gonadal developm ent
200 JOHN K. COWELL

were due to the presence o f constitutional chrom osom e deletions in these individ
uals. T he deletions involved one o f the D-group o f chrom osom es (Lele et al.,
1963), later shown to be chrom osom e 13 (Yunis and Ramsay, 1978). In all cases,
region 13ql4 was involved (Figure 3), indicating the site o f the RBI gene. Since
the esterase-D gene (ESD) was also shown to be located in 13ql4 (Sparkes et al.,
1980), deletion carriers could be detected by a quantitative assay for this enzyme,
which form ed the basis for population studies (Cowell et al., 1986). D eletion car
riers, who have 50% o f norm al enzyme levels, constitute approxim ately 3% o f all
Rb patients (Cowell et al., 1989). Close genetic linkage between Rb and ESD was
re p o rte d by Sparkes et al. (1983), dem onstrating the unequivocal location o f the
h ereditary form o f the disease and, to date, there have been no re p o rted recom
bination events between the two loci (Cowell et al., 1987b). T he exact orientation
o f the two genes was established by the analysis of a p atien t with a constitutional
13ql4-q31chrom osom e deletion and norm al ESD levels (Cowell et al., 1987a). Us
ing somatic cell hybrids the ESD gene was shown to rem ain on the deletion chro
m osom e b u t the RBI gene did not. This placed the RBI gene distal to ESD
(M itchell an d Cowell, 1988), probably in 13ql4.3.
Sporadic tum ours from individuals heterozygous for polym orphic ESD alleles
(allowing each o f the parental chrom osom es to be distinguished) often lost one
allele (G odbout et al., 1983). This loss o f heterozygosity was also dem onstrated
using polym orphic DNA probes (Cavenee et al., 1983, Dryja e ta l., 1984). C hrom o
some analysis of these tum ours showed that two copies of 13 were still present. T he
in terp retatio n o f this observation was that an acquired m utation in retinal p recu r
sor cells was duplicated at some stage an d the norm al hom ologue was then lost. In
this way, the cell becom es hom ozygous for the initial loss o f function m utation in
RBI an d the cells do n o t have a functional protein. T he m echanism s by which this
loss o f heterozygosity (LOH) occurs were m ost frequently due to chrom osom e
non-disjunction and m itotic recom bination (Cavenee et al., 1983). Cavenee et al.,
(1985) showed that, in a tu m o u r from a p atien t with heredutary Rb, the chrom o
some retained in the tu m o u r was th at transm itted by the affected parent. It
appears that up to 70% of tum ours experience this LOH (Cavenee et al., 1983).
These kind o f analyses allowed the origin o f the parental m utation to be deter
m ined (Dryja et al., 1989). In sporadic cases there was no differential susceptibil
ity to som atic m utation between the hom ologous copies of the gene. However, for
new germ line m utations, the heritable m utation arose on the paternally derived
chrom osom e. These findings and those o f Zhu et al. (1989) argue against
genom ic im printing being im portant in Rb tum origenesis but point to new m uta
tional events arising predom inantly d u rin g sperm atogenesis. It has not, however,
been possible to attribute these to a paternal age effect (M atsunaga et al,, 1990).

CYTOGENETIC ANALYSIS OF RB TUMOURS

T he m echanism s leading to LOH can clearly occur w ithout structural chrom o

some rearran g em en t. It was hardly surprising, therefore, th at chrom osom e 13
 MOLECULAR GENETICS OF RETINOBLASTOMA 201

9 *

GOS 36 GOS 115

8 % g 9
GOS 46 GOS 122

I a 5
GOS 49 GOS 140

S fc |
v
GOS 50
GOS 173

F ig u re 3 C h ro m o s o m e 13 p a irs f ro m p a tie n ts w ith c o n s titu tio n a l d e le tio n s . T h e n o r m a l c h r o

m o s o m e is sh o w n o n th e le ft in all cases. D e le tio n s f r o m th e f o u r d if f e r e n t p a tie n ts G O S 36,
46, 49 a n d 50 c a r r y la rg e d e le tio n s , w h e re a s th e o t h e r f o u r p a tie n ts o n ly sh o w sm a ll d e le tio n s
in v o lv in g re g io n 1 3 q l4 .

abnorm alities were found infrequently in tum our cells, although several interest
ing observations have com e ou t of these cytogenetic analyses (see Cowell and
Hogg, 1992, for review). T he m ost consistent finding was the presence o f (usually
two copies of) an isochrom osom e 6p (iso-6p) and trisomy for all or part of the
long arm of chrom osom e 1 (lq + ), in 45% an d 44% o f tum ours respectively.
C hrom osom e abnorm alities involving chrom osom e 13, usually resulting in
m onosom y 13, was only found in 20% o f cases. A bnorm alities involving lq are the
202 JO H N K. COWELL

m ost com m only seen in all tu m our cells. By contrast iso-6p is less frequently
observed, being restricted, largely, to Rb an d m alignant m elanom as (B echer et
al., 1983). It is possible, therefore, that duplication o f certain genes on the short
arm o f chrom osom e 6 are im p o rtan t in tu m o u r progression. However, since this
analysis is usually p erfo rm ed on advanced stage tum ours, it is difficult to deter
m ine w hether these changes are causal in tum origenesis or consequences of it.
C hrom osom e analysis o f in d ep en d e n t tu m o u r foci from a bilaterally affected
p atient showed that each had distinct abnorm alities, suggesting an in d ep en d e n t
origin for them (Squire et al., 1985). Tien et al. (1989) analysed a large, appar
ently unilateral, tum our and found cytogenetically distinct clones. This was inter
p reted to m ean that the tum ou r probably arose as the result of fusion of several
foci. This has im p o rtan t im plications for counselling since unilateral tum ours are
th o u g h t to be associated, predom inantly, with sporadic events. Multifocal
tum ours, however, even in only one eye, probably identifies th at patient as a
h ereditary case, especially if these tum ours have a relatively early age of onset.
W hether this analysis is justified for the relatively small re tu rn in term s of
im proved m anagem ent is questionable.

ISOLATION OF THE RBI GENE

Following the random isolation of only 12 DNA probes from a flow-sorted chro
m osom e 13-specific DNA library (Lalande et al., 1984) one, H3-8, was shown to
be within the smallest of constitutional deletions in Rb patients. A djacent
sequences (Dryja et al., 1986) were shown to be within a gene which was soon iso
lated (Friend et al., 1986) and which detected structurally abnorm al mRNA in Rb
tum ours with varying frequencies (Friend et al., 1986; G oddard e ta l., 1988). Sim
ply isolating a gene from a particular p art o f the chrom osom e, however, is not, in
itself, conclusive evidence for the authenticity o f the gene. A djacent genes would
also be candidates although the identification of structural rearrangem ents o f
this gene in Rb tum ours strongly su pported its candidature. T he tissue distribu
tion o f expression o f RBI, however, was slightly surprising, being present in rela
tively high levels in all tissues exam ined (Friend et al., 1986). This was
u n expected since the hypothesis was that this gene controls im p o rtan t aspects of
the developing fetal retina. T he dem onstration of predisposing m utations involv
ing RBI provided m ore convincing evidence for its authenticity. In patients show
ing constitutional, predisposing reciprocal translocations the breakpoints on
chrom osom e 13 always in te rru p te d the RBI gene (Higgins et al., 1989; M itchell
an d Cowell, 1989). Thus, adjacent genes could n o t be involved.
A few cases have been re p o rted where the translocation p artn e r chrom osom e is
the X (Ejima et al., 1982; H ida et al., 1980; Nichols et al., 1980). Because random
X-inactivation occurs in females, w hen the derivative chrom osom e is inactivated in
retinal precursor cells the position o f the b reakpoint on 13 is no t im portant, since
the whole chrom osom e experiences genetic silencing, thereby constituting the
 MOLECULAR GENETICS OF RETINOBLASTOMA 203

first hit. In two families in o u r series, with t( 1:13) (q22:ql4) and t (13:20) (q l4 :p l2 )
translocations, the p atien t with Rb in h erited the rearran g em en t from a p aren t
who was n o t affected (B. Gibbons, pers. com m ). A sim ilar situation was discussed
by Dryja et al. (1984) for 13ql4 deletions, in that deletion carriers often only have
unilateral, unifocal tum ours. In o u r survey (Cowell et al., 1989) half o f the 16 cases
re p o rted had unilateral tum ours. T h ere are also reports of deletion patients who
have never developed tum ours (Cowell et al., 1988; Fukushim a et al., 1987). T he
age o f onset of tum ours from patients with chrom osom e 13 abnorm alities also ap
pears to be later than those with germ inal m utations (Ejima et al., 1988). T he ex
planation for the low p en e tran ce in deletion patients is n o t clear although one
proposal is th at these deletions expose lethal m utations which are deleterious to
the rapidly growing tum our cells (Dryja et al., 1984). This does n o t explain the low
p en etran ce of the reciprocal translocation carriers, unless large deletions are as
sociated with the rearrangem ent. In o n e case o f a t (1:13) rearra n g em en t (Mitchell
and Cowell, 1989), although a deletion was associated with the translocation, it was
maximally 8 kb long and confined to the RBI gene. Keith et al. (1985) described
a p atien t with a 13ql4 deletion with a single tu m o u r in one eye and a retinom a in
the other, both o f which are considered to be 'm ild' form s o f the disease.

MUTATIONS IN RB TUMOURS

Only 20% o f tum ours showed structural abnorm alities of RBI an d very few p re
disposing m utations involve chrom osom e translocations. Clearly, the majority of
m utations were m ore subtle. T he n a tu re o f these m utations in tum ours has been
dem onstrated in a variety o f ways. D unn and colleagues (D unn et al., 1989) anal
ysed RNA from tu m o u r cells, although this is n o t always possible for reasons dis
cussed by Cowell an d Hogg (1992). N onetheless a variety o f d ifferent m utations
in tum ours and cell lines were rep o rted . T he RBI cDNA is 4.7 kb long (Friend
et al., 1986) and consists o f 27 exons clustered into three groups each separated
by two very large introns (Figure 4 ). T he 27 exons rep resen t the fragm ented cod
ing region of the gene as they ap p ear on the chrom osom e which are subse
quently jo in e d together in the final mRNA. In the absence o f mRNA, each
individual exon can be sequenced from DNA by first amplifying the exon and
flanking intron regions using the polym erase chain reaction (PCR). D irect
sequencing o f these PCR products would identify m utations by com parison with
the norm al sequence. This approach has been successful (Yandell et al., 1989)
and was im proved using the single strand conform ation polym orphism (SSCP)
technique to prescreen the PCR am plified exons before sequencing, which iden
tified those DNA m olecules m ost likely to carry m utations (Hogg et al., 1992).
In sum m mary, the available data shows th at there is no ap p a ren t hot-spot for
m utations within RBI. T he m ajority o f m utations are insertions, deletions or sin
gle base pair substitutions which result in the p roduction o f prem ature stop
codons. These m utations would be predicted to result in structurally grossly
204 JOHN K. COWELL

200 kb ------------------------------------------

36 kb 70 Kb

V V
H I I 114 II II I II I I M I I III 111
12 3 4 56 7 89 1011 12 13-17 18 19 20 21-23 24-26 27

F ig u re 4 D ia g ra m m a tic r e p r e s e n ta tio n o f th e RBI g e n e . T h e 27 in d iv id u a l e x o n s, r e p r e s e n t e d

by th e v e rtic a l b a rs, a re c lu s te re d a lo n g a 2 0 0 k b re g io n o f t h e c h r o m o s o m e a n d s e p a ra te d by
tw o re la tiv e ly la rg e (36 kb a n d 70 k b lo n g ) in tr o n s .

abnorm al proteins, which is consistent with antibody analysis where Rb tum ours
mostly had n o detectable Rb protein (Horowitz et al., 1990). In approxim ately
10% of tum ours, the m utations affect the correct processing of the mRNA (splic
ing) by dam aging sequences essential for this process. Missense m utations, simply
substituting one am ino acid for another, appear to be less com m on. In o u r own
survey of hereditary Rb patients, one exam ple was found in exon 20, which was
associated with a low p e n e tra n c e phenotype (O nadim et al., 1992). It is tem pting
to speculate that the substitution of a single am ino acid only com prom ises the
function o f the protein and, unless the second m utation in the tu m o u r precursor
cell causes loss of RBI function, duplication of the weak m utation allows a suffi
ciently functional Rb protein to be produced, so preventing tum origenesis. This is
consistent with o u r observation in this particular family since many o f the m utant
gene carriers were eith er unaffected or had regressed tum ours (O nadim et al.,
1991). Sakai et al. (1991) also investigated low pen etran ce families and found m u
tations in recognition sequences for different transcription factors - the pro m o ter
-w h ic h lie at the beginning o f the gene and which controls the production of RBI
mRNA. Again the suggestion is that, as a result, a quantitative decrease in tran
scription occurs ra th e r than com plete inactivity. Sufficient pRB is produced, how
ever, and any phenotypic consequences are mild. W hether single am ino acid
changes will generally be found in patients with m ild phenotypes is still n o t clear.

THE FUNCTION OF THE RBI GENE

T he fact that Rb tum ours are relatively u ndifferentiated, histopathologically, sug

gests an arrest in developm ent of a retinal precursor cell at an early stage. By-
inference it m ight appear, therefore, th at RBI controls the transition from this
 MOLECULAR GENETICS OF RETINOBIASTOMA 205

im m ature precursor cell to a p h o to rec ep to r o r o th er neuroepithelial cell in the

retina. E ither directly, or indirectly, RBI m ust therefore control this differentia
tion process although analysis o f the structure of the gene does n o t reveal any
tell-tale motives im plicating it as a reg u lato r of transcription. T he first clues to
the function o f pRB cam e from the dem onstration that it could bind to proteins
from certain DNA tum our viruses which dom inantly transform norm al cells into
cancer cells. After invading cells, DNA tum our viruses produce an early set of
proteins which trigger the norm ally quiescent cell into division. This is essential
for successful virus propagation. Thus, the E l A protein from adenovirus (Whyte
e t al., 1988), Large-T-antigen (LT) from SV40 (D eCaprio et al., 1988) an d E7 pro
tein from hum an papillom a virus (Dyson et al., 1989), all form com plexes with
pRB. All o f the viral transform ing proteins share conserved regions which are
necessary for their transform ing function. M utations in these conserved regions,
which prevent cellular transform ation, also prevent binding to pRB (Whyte et al.,
1989). pRB can be phosphorylated at many positions. In resting cells pRB is
u n phosphorylated but, as the cell moves into S-phase of the cell cycle, pRB
becom es phosphorylated until the e n d o f mitosis where it is dephosphorylated
again (M ihara et al., 1989). This led to the suggestion th at the unphosphorylated
form o f pRB prom otes cell quiescence, i.e. prevents cell growth.
By selectively deleting parts o f the RBI gene, two non-contiguous regions were
identified which were necessary for E la and LT binding (IIu et al., 1990; H uang
et al., 1990; Kaelin et al., 1990). These protein binding dom ains com prised am ino
acids 393-572 and 646-772 respectively, which have collectively b een called the RB
pocket. It appears, therefore, that by sequestering pRB from the cell during G l,
the viral transform ing proteins allow the cell to en ter S-phase. T he association of
the viral early genes with pRB is alm ost certainly an in vitro p h en o m en o n , since it
is unlikely that fetal retinal cells have b een infected with these viruses. Rather, this
m odel system points to associations o f pRB with o th er naturally occurring pro
teins. In fact, it has em erged that pRB participates in the establishm ent o f protein
com plexes which associate and dissociate during the cell cycle (Figure 5 ). Many of
these processes are u n d e r tight control an d the whole system appears to be regu
lated by the biochem ical m odification o f the participants in these com plexes and
their availability to jo in the com plex. T he actual m echanism s, real and im plied,
have been discussed in many re c e n t reviews an d the m inutiae o f these m odels will
n o t be discussed h ere (Horowitz, 1993). In essence, pRB can bind to many cell
cycle regulating proteins an d regulate th eir function. T he n et result is th at if pRB
is missing, several genes are left active which should not be so and the cell contin
ues to divide instead of going into quiescence.
pRB has also been shown to com plex with a developm entally regulated cellular
transcription factor, DRTF-1 (Bagchi et al., 1991; B andara et al., 1991; Partridge
and L aT h an g u e, 1991). T he suggestion is that, by binding with a sequence-specif-
ic transcription factor, pRB can control transcription o f target genes. Since DRTF
is developm entally regulated, and its expression is tissue d ep en d en t, this associa
tion suggests a role for pRB in differentiation (B andara an d La T hangue, 1991;
P artridge and L aT h a n g u e, 1991).
206 JO H N K. COWELL

T ranscription

Go G1 S G2 M

F ig u re 5 S c h e m a tic r e p r e s e n ta tio n o f th e f u n c tio n o f th e R b p r o te in . T h e u n p h o s p h o r y la te d

p r o te in p r e s e n t in GO b in d s a t r a n s c r ip tio n fa c to r, E2F, a t th e tr a n s itio n to G l , in a c tiv a tin g it.
T h e R b p r o te in is th e n p h o s p h o r y la te d , possib ly m e d ia te d by a n a s so c ia tio n w ith c d c 2 k in a s e
a n d cyclin A a n d E 2F is re le a s e d , fo r a b r i e f p e r io d , to a c tiv a te o t h e r g e n e s w h ic h a re p r e s u m
ably r e la te d to c ell d iv isio n . A fte r th is b r i e f p e r io d o f f r e e d o m , E 2F is a g a in c a p t u r e d by th e
R b -re la te d p r o te in , p l 0 7 , w h ic h t h e n fo rm s a p r o t e i n c o m p le x re p re s s in g E 2F activity. E x actly
w h a t h a p p e n s b e tw e e n G 2 a n d GO is u n c le a r.

RBI MUTATIONS IN OTHER TUMOURS

It has been known for some time that m u tan t RBI gene carriers also develop sec
o n d non-ocular tum ours; the conclusion always being that RBI also controls the
n o rm al developm ent of these tissues. Patients carrying constitutional RB gene
m utations are at significantly higher risk for the developm ent o f second, non-ocu
lar tum ours later in life (D raper et al., 1986) .These are usually osteosarcom as and
soft tissue sarcomas. Both o f these tum ours were shown to lose heterozygosity for
m arkers on chrom osom e 13 (Dryja et al., 1986; Friend et al,, 1987). T he same
classes of tum ours also show freq u en t structural an d transcriptional abnorm ali
ties o f RBI suggesting it plays a role in establishing the m alignant phenotype in
these cells. T he risk o f second tum ours is en h a n ced within the irrad iated tissues
following radiation treatm ent. This risk is greater for patients with germ line
m utations in RBI. This raises im p o rtan t issues ab o u t w'hether to treat these
patients with radiation although the risks m ust be offset with the benefits.
 MOLECULAR GENETICS OF RETINOBLASTOMA 2 07

In addition to those tum ours known to be associated with RBI gene m utation
carriers, structural abnorm alities were also found in breast cancer (Lee et al.,
1988; T'Ang et al., 1988) and small cell lung carcinom a (H arbour et al., 1988) cells
at high frequency. A series o f o th er tum ours showing less freq u en t involvem ent
was presented by Horowitz et al. (1990). It is likely, however, that somatic RBI m u
tations in these o th er tissues only contribute to tu m o u r progression since, in many
cases, the frequency of tum ours with m utations is still relatively low.
O ne interesting tum our associated with hei'editary Rb is pineoblastom a. Be
cause the pineal is th o u g h t to be a vestigial p h o to rec ep to r organ, patients with ret
inal an d pineal tum ours have been described as having trilateral retinoblastom a
(Bader et al., 1982; Holladay et al., 1991). T he chances o f two such rare tum ours
occurring coincidentally is negligible an d suggests that the RBI gene also contrib
utes to the developm ent of pineoblastom a.

CLINICAL APPLICATIONS OF RBI ISOLATION

T he standard of m etaphase chrom osom e analysis is sufficiently good these days

in m ost ro u tin e labs that patients carrying the 13ql4 deletions will be readily
identifiable. Because o f the cost and effort in setting up ESD quantitation studies
for those rare cases with small deletions, we consider it no longer necessary to
p erfo rm ESD quantitation on all Rb patients. In the majority o f cases, because
m ost o f these have associated congenital abnorm alities typical o f 13q patients,
cytogeneticists will be alerted to the possibility of a deletion. In situ hybridisation,
using cosmids for RBI, to m etaphase chrom osom e from Rb patients could easily
identify small deletions where conventional G banding is equivocal. In this case
hybridisation would not occur to one (the deletion) chrom osom e. For the clini
cal m anagem ent o f Rb, it is essential to be able to predict accurately who is a
m utant gene carrier and who is not. Linkage analysis using the ESD protein poly
m orphism (Cowell et al., 1987b) has also b een superseded by DNA probe analy
sis. Using a series o f five unique DNA sequences, derived by Wiggs et al., (1988)
from within the RBI gene, the m ajority o f individuals with a family history of Rb
can be successfully analysed. These DNA probes all recognise restriction frag
m en t length polym orphism s (RFLPs) with varying frequencies and, to date, no
recom bination has been re p o rted between any of these probes an d the Rb p h e
notype (O nadim et al., 1990; Scheffer et al., 1989; Wiggs et al., 1988). This analy
sis has since (Figure 6) been ex ten d ed to prenatal an d perinatal screening
(M itchell et al., 1988; O nadim et al., 1992). In our own experience, approxi
mately 85% o f families would qualify fo r prenatal screening using these 5 probes
alone. T he developm ent o f o th er highly inform ative polym orphism s, based on a
single base pair polym orphism (Yandell and Dryja, 1989) an d a small variable
n u m b er tandem re p eat (Figure 7) in the intron adjacent to exon 20 (McGee et
al., 1989), now m eans th at virtually all gene carriers in families with a history of
Rb can be identified (O nadim et al., 1992b). Family pedigree analysis, however,
208 JO H N K. COWELL

1-8Kb -1 -75 Kb
1-7Kb-

F ig u re 6 P r e n a ta l s c re e n in g u s in g th e p o ly m o r p h ic D N A p r o b e RS2. T h e p e d ig r e e o f a fam ily

r e q u e s tin g p r e n a ta l s c r e e n in g is sh o w n a b o v e . T h e tr a n s m ittin g p a r e n t is h e te ro z y g o u s f o r th e
1 .7 5 /1 .7 k b a lle le s. T h e u n a f fe c te d f a th e r is h e te ro z y g o u s 1 .7 /1 .8 kb. S in c e th e a ffe c te d first
b o r n c h ild is h o m o z y g o u s fo r th e 1.7 k b a lle le , th e c h r o m o s o m e fro m th e m o th e r c a r ry in g th e
1.7 k b a lle le a lso c a rrie s th e m u ta n t R b g e n e . T h e c h o r io n ic villus s a m p le fr o m t h e s e c o n d
p re g n a n c y was sh o w n to in h e r it t h e 1.75 k b a lle le f ro m th e m o th e r a n d h e n c e th e fe tu s h a s
n o t i n h e r i t e d th e m u ta n t g e n e .

has shown considerable heterogeneity in the Rb phenotype which has significant

im plications for clinical m anagem ent. Using intragenic polym orphic DNA
probes, we have shown that it is possible to dem onstrate that individuals with
regressed tum ours in Rb families carry the m u tan t allele (Figure 8) and have
identified unaffected gene carriers whose children are at high risk o f tu m o u r
developm ent (O nadim et al., 1991). T he prenatal screening option for these fam
ilies is to have chorionic villus sam pling after 10 weeks o f pregnancy. In m ost
cases the results are available after 2-5 days. We have recently published our
experience with prenatal screening using DNA probes (O nadim et al., 1992b)
where all patients have been followed for at least 18 m onths and so far all p redic
tions have been correct.

RISK ASSESSMENT

T here have been many estim ates of the relative risks for the developm ent o f Rb in
the children and siblings o f affected individuals. Many o f these calculations are
 MOLECULAR GENETICS OF RETINOBIASTOMA 209

reviewed by Vogel (1979) bu t the figures do vary from study to study and alm ost
certainly reflect referral bias. In a review of the UK population o f Rb patients
involving over 900 cases over a 25 year period, D raper an d colleagues (D raper et
al., 1992) have re-evaluated the figures. Clearly, w here there is a family history the
chances o f inheriting the m u tan t gene is 50% but, because only 90% of gene car
riers actually develop a tum our, the actual risk is n ea rer 45%. It was n oted some
time ago (Cowell et al., 1986) that bilaterally affected m u tan t gene carriers gener
ally have bilaterally affected children and th at unilaterally affected gene carriers
have a greater chance o f having unilaterally affected children, o r som etim es unaf
fected gene carriers (O nadim et al., 1991). T he risk o f tum origenesis for the chil
d ren of unilaterally affected m u tan t gene carriers is therefore lower, estim ated by
D raper and colleagues at aro u n d 30%. In the UK series, where there is no family
history o f Rb the probability of a sibling being affected is 1.1% if the pro b an d is
bilaterally affected an d 0.6% if the p ro b a n d is unilaterally affected. T he differ
ence, however, is no t statistically significant. Finally, using the m ethod o f maxi
m um likelihood estim ation the probability of a unilateral case with no family
history being a gene carrier was calculated to be 1.7% giving th eir children a risk
o f approxim ately 0.8%.

11 12 111 1111 II2

F ig u re 7 P e d ig re e analysis u s in g th e R b l .20 p o ly m o rp h is m . T h e a ffe c te d g r a n d p a r e n t 1.2 is

h e te ro z y g o u s ( 3 / 5 ) a n d passe s th e 3-allele to h e r s o n w h o is a ffe c te d . H e in t u r n p a sse s th is
s a m e a lle le to h is d a u g h te r w h o is a lso a ffe c te d .
210 JO H N K. COWELL

to

' X *
I I
F ig u re 8 E x a m p le o f a fam ily p e d ig r e e sh o w in g m ild e x p re s s io n o f th e RB p h e n o ty p e a n d a
m is se n se m u ta tio n in e x o n 20. D N A s e q u e n c e analysis show s th e m u ta n t g e n e is p r e s e n t in
th e g r a n d p a r e n ts ( h a tc h e d sym bols) w h o b o th h a d re g re s s e d tu m o u r s as w ell as in th e u n a f
fe c te d in d iv id u a ls in d ic a te d by th e a rro w s. A ffe c te d in d iv id u a ls a re sh o w n w ith so lid sym bols.
In d iv id u a ls w ith u n ila te r a l R b a re r e p r e s e n t e d by h a lf-fille d sym bols, th o s e w ith b ila te ra l R b
by so lid sym bols.

IDENTIFICATION OF CAUSATIVE MUTATIONS

T he big challenge still, however, is to d eterm in e which o f those apparently spo

radic cases represent germ line m utations, w'hose children are at risk o f tu m o u r
developm ent. To achieve this with conventional linkage analysis, the at risk par
en t m ust have an affected child or o th er relative, in o rd e r to establish the linkage
phase (C hapter 1). If the causative m utation can be characterised, however,
m u tan t gene carriers can be identified unequivocally w ithout extensive pedigree
analysis. Since only a m inority o f tum ours are removed, these m utations m ust be
identified in constitutional cells. T he PCR-sequencing procedure described ear
lier has m ade this possible and m utations have been described in individuals p re
disposed to Rb (Hogg et al., 1992; O nadim et al., 1992a; Yandell and Dryja,
1989). A lthough all co n cerned paediatricians and ophthalm ologists would con
sider that th eir patients should benefit from this screening procedure, it is a very
tim e consum ing process and m ust be carried o u t in specialist laboratories. It is
unlikely th at sufficient funds will be available in the short term to screen all
patients in a cost effective way and some logical rationalisation, therefore, should
occur. For exam ple, perhaps only those patients with a family history for whom
linkage analysis is no t available should be subm itted for m utation analysis
although there is a reasonable case for investigating those families with unusual
family pedigrees to confirm the gene carrier status o f unaffected family m em
bers.
All apparently sporadic, bilaterally affected cases m ust be considered carriers o f
germ line m utations and should probably have m utational analysis perform ed. In
 MOLECULAR GENETICS OF RETINOBLASTOMA 211

o u r series, priorities are given to those individuals approaching childbearing age

although, ideally, all individuals should be analysed. U nilaterally affected individ
uals, for whom the risk o f being carriers of gene m utations is low should possibly
be assigned a lower priority given lim ited resources although, if their tum ours had
an early age of onset, there is perhaps a m ore u rg e n t n eed to carry ou t the study.
Since the clinical screening program m e for Rb patients with an affected relative is
well established, it is usually the unilateral cases which present late and experience
enucleation. T he im portance o f exam ining the tum our cells from these patients
cannot be overem phasised. Since approxim ately 70% o f these tum ours will be h o
mozygous for the causative m utation it is im p o rtan t to study the RBI gene. If a m u
tation is identified that is n o t present in constitutional cells from the same
individual, then the patient can be classified as truly sporadic. It rem ains to be
seen w hether the n atu re o f the m utations being identified in gene carriers will al
low us to predict outcom e, prognosis and susceptibility to second tum ours. A re
view of the types o f m utation which cause Rb shows a variety o f d ifferent types. In
some cases, the m utations coincidentally create o r destroy a restriction enzyme
site. In these cases, subsequent analysis o f family m em bers m erely requires diges
tion o f the PCR p ro d u ct with the appropriate enzyme, and m u tan t gene carriers
will have the abnorm al restriction p attern (O nadim et al., 1992a). In constitution
al cells the m utation will always be heterozygous which makes the analysis o f se
quencing gels m ore difficult. T he fact that several exons can be analysed
sim ultaneously makes the p ro ced u re less labour intensive. PCR analysis can also
be carried ou t on form alin-fixedparaffin-em bedded tissue sections (O nadim and
Cowell, 1991), which m eans archival m aterial can also be used to identify causative
m utations. In practice, it is n o t u rg e n t to identify the m utation in sporadic cases,
since those individuals will no t be considering children for 16-20 years although
they may be at increased risk of developing second tum ours.
T he availability o f prenatal screening clearly raises ethical issues. At present,
screening is only offered to parents who are considering term inating the pregnan
cy if the fetus carries the m utation. O therw ise the test can be p erfo rm ed perina-
tally. T he justification for considering term ination is that, although 90% curable,
Rb is a lethal tum our if it escapes the confines o f the eye. Even if the patient sur
vives the tu m o u r th ere may be side effects o f the treatm ent and no guarantee that
the sight will be preserved. In addition, survivors o f Rb are at risk o f developing
second tum ours later in life for which there is no effective screening program m e.
A lthough first trim ester screening is preferable to am niocentesis, there may be
psychological effects on parents who may change their m inds ab o u t term ination.
Given these considerations, there is a possibility o f perform ing preim plantation
screening in som e cases, especially w here the n atu re o f the m utation is known. It
should soon be possible to study m utations using PCR in single cells which could
be derived from in vitro fertilised em bryos. Only m utation-free em bryos w ould be
reim planted although the success o f this procedure rem ains to be evaluated.
212 JO H N K. COWELL

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 9. COLOR VISION DEFECTS

JAY NEITZ an d MAUREEN NEITZ

Medical College of Wisconsin, Department of Cellular Biology and Anatomy, and Department
of Ophthalmology, 8701 Watertown Plank Road, Milwaukee, W I53226, USA

SUMMARY

In this chapter, the light sensitive visual pigm ents that underlie norm al and
defective color vision, an d the genes th at encode them are reviewed. New u n d er
standing o f these has developed quickly in the last few years since the techniques
o f m olecular biology have been b ro u g h t to bear on long p o n d ered problem s.
Decades before m olecular m ethods w ere available, a theory to explain the biolog
ical basis o f norm al and defective color vision was widely accepted. A favored ver
sion o f that theory held th at everyone with norm al color vision has, in com m on,
three types of cone photoreceptor, each containing a d ifferent type o f visual pig
m ent. T he com m on color defects were proposed to occur w hen one type o f cone
pigm ent was lost, or w hen the p h o to p ig m en t in one o f the cone types was
replaced by an abnorm al (or anom alous) pigm ent, d ifferent in absorption spec
trum from any o f the three stereotyped norm al pigm ents. However, over the
years, results have accum ulated th at are difficult to fit within the fram ework of
the classic view. Thus, an alternative theory is outlined. Its essentials are as fol
lows.
(1) H um ans can have many genes on the X-chrom osom e th at encode cone p h o
topigm ents (as many as ten are n o t uncom m on).
(2) Spectrally distinct subtypes o f pigm ents occur within each of the two classes of
X -encoded cone pigm ents. Both n orm al an d color defective observers draw
from the same pool o f pigm ents.
(3) People with norm al color vision all have at least one pigm ent from each of the
two X -encoded pigm ent classes, b u t they can have m ore than o n e subtype
from eith er class and, thus, can have m ore than three spectrally distinct cone
types.
(4) All of the com m on color vision deficiencies can be explained by a m utational
pathway in which genes that produce pigm ents req u ired for norm al color
vision are deleted by u n equal crossing over betw een norm al pigm ent gene ar
rays.
This is n o t offered as the only possible m odel, b u t h ere it serves as a fram ework
aro u n d which the facts o f color vision phenotypes an d genotypes can be organized
and re-evaluated.
218 JAY NEITZ AND MAUREEN NEITZ

INTRODUCTION

C olor vision capacities are characterized by the abilities to distinguish lights that
differ in th eir wavelength com positions. A lthough hum an color capacities are
extensive, there are many differences in wavelength com position to which we are
all quite blind. Deficiencies in o u r ability to tell spectrally different lights apart
are exploited, for exam ple, in color television th at employs m ixtures o f ju st three
color prim aries, red, green, and blue to synthesize a wide range o f colors. T he
wavelength com position o f the light from a banana seen on television is quite dif
feren t from a real banana, bu t we are nearly blind to the physical difference.
Inabilities to see the physical differences betw een lights th at differ in wavelength
co n ten t have proved convenient for classifying d ifferent form s o f color vision.
For exam ple, given a test light o f any wavelength com position, hum ans with nor
mal color vision can make an identical color m atch using ju s t three appropriately
chosen color prim aries, e.g., red, green, a n d blue - eith er by m ixing the right
am ounts o f the three prim aries, o r by m ixing two prim aries and adding the third
to the test light. This three-dim ensionality or trichrom acy is the hallm ark o f nor
mal hum an color vision. O th er form s of color vision (or color vision deficiency)
can be similarly classified by the nu m b er o f prim aries req u ired in color m atches.
An individual who is com pletely color-blind, classified in this way, is a m onochro-
mat. A m o n ochrom at can exactly m atch the appearance of any light, regardless
of its wavelength content, with a single fixed prim ary light by simply adjusting the
intensity o f the com parison prim ary. T he interm ediate category, dichom ats, need
only two prim aries. This form of color vision seems to be a standard am ong o th er
species (Jacobs, 1981). For exam ple, am ong m am m als, trichrom acy has been
fo und to occur only am ong prim ates. Many o th er fam iliar m am m als, e.g., dogs,
cats, an d squirrels, are dichrom ats. D ichrom acy is also fairly com m on am ong
hum ans. In Caucasian populations ab o u t 1 person ou t of every 100 is a dichro-
mat.
T he term s m onochrom acy, dichrom acy and trichrom acy strictly refer to the
n u m b er of prim ary colors req u ired in color m atches, however, these term s are of
ten (mis-) used to refer to the nu m b er of spectrally d ifferent cone pigm ents an in
dividual has, or is believed to have. W hile it is true that three pigm ents are the
m inim um required for trichrom atic color vision an d at least two are n eeded for
dichrom acy, there may be hazards in equating visual capacities with the nu m b er
of cone pigm ent types. It is possible, for exam ple, to im agine a system with dozens
of spectrally different receptors that still has m onochrom atic (color blind) vision,
since color vision requires th at the receptors be w ired in a way th at yields infor
m ation ab o u t spectral differences. Or, m ore to the p o in t in this review, the sim
plest (and favorite) theory of hum an color vision holds th at we have th ree types of
receptors each tu n ed to a d ifferent region o f the visible spectrum . Trichrom acy is
a fact o f norm al hum an color vision. No doubt, we have at least three types o f re
ceptors. But, we could have m ore than th ree and still be trichrom ats if the signals
from m ultiple receptors were channeled into three outputs at any stage in the
 COLOR VISION DEFECTS 219

visual system. As will be seen, re cen t evidence suggests that many people may, in
deed, have m ore than three spectrally d ifferent cone pigm ents.

Classes o f Cone Pigments

H um an color vision is based on light sensitive pigm ents th at absorb maximally in

different regions o f the visible spectrum (D artnall, Bowmaker, & M ollon, 1983;
Schnapf, Kraft, & Baylor, 1987; O prian, Asenjo, Lee, & Pelletier, 1991; Merbs &
N athans, 1992a). Each pigm ent m olecule is com posed o f an apoprotein com po
nent, opsin, linked to the light absorbing ch rom ophore 11-cis retinal. S tructural
differences between the opsins govern the absorption properties o f the pigm ents.
Pigm ents can be classed according to the spectral region o f maxim al absorption.
N ot everyone agrees on the ap p ro p riate term s for these different pigm ent
classes. T he term s red, green an d blue are som etim es used, however, there has
been a move to avoid them since they can be m isleading if they are taken to m ean
that the different pigm ent classes are m ost sensitive to the regions of the spec
trum seen as red, green, an d blue, which is n o t true. For exam ple, the re d pig
m ents have peak absorption n ear 560 nm , a wavelength that appears greenish-
yellow, n o t red, an d the blue pigm ent peaks som ewhere below 430 nm, a spectral
region th at appears violet. T he sensation o f redness com es ab o u t because o f the
way o u tp u t from the receptors is processed by the nervous system, n o t because
hum ans have a recep to r th at is m ost sensitive to the spectral region that appears
red. A nother com m on m isunderstanding is th at people who are missing a re d
pigm ent can see green and blue b u t not red, while those missing a g re e n pig
m en t see red an d blue b u t n o t green. This is n o t an adequate conception. In
people with norm al color vision the ability to distinguish lights with wavelengths
longer than ab o u t 550 nm , e.g., red, orange, yellow, and yellowish-green comes
from com parison, by the nervous system, of outputs from the re d an d g re e n
cones. T he absence of eith er cone type results in dichrom atic color vision in
which all m iddle-to-long wavelengths are virtually indistinguishable. C onfusion
can be m inim ized if color term s are avoided when referring to pigm ents. H um an
pigm ents can be classed into three categories: long-wavelength sensitive (pig
m ents that peak near 560 nm ), m iddle-wavelength sensitive (pigm ents that peak
near 530 nm ), an d short-wavelength sensitive (pigm ents that peak below 430 nm )
and conveniently abbreviated (e.g., following M ollon, 1991) long-wave, m iddle-
wave, and short-wave. These term s will be used here.

Form s o f congenital color blindness

(1) Monochromacy

Am ong hum ans, com plete color blindness (m onochrom acy) is extrem ely rare,
affecting ab o u t 1 in 100,000 o f the general population. W ithin this group are
three types o f congenital m onochrom acy (reviewed by N athans et al., 1989;
220 JAY NEITZ AND MAUREEN NEITZ

McKusick, 1992). Individuals with congenital rod m onochrom acy (congenital

achrom atopsia) lack all cone-m ediated responses b u t have norm al rhodopsin and
rod function. C ones are present bu t functionally defective. These individuals
function poorly in bright light and see b etter in dim light. In some cases, the cen
tral cones m ediate a light response with the action spectrum of rhodopsin, sug
gesting that rhodopsin may be present in rods an d cones (typical rod
m onochrom acy). In others, typical cone pigm ents are present, suggesting that
the defect lies beyond the point o f light absorption (atypical rod m o n o ch ro
macy). Both conditions are non-progressive and are in h erited as autosom al reces
sive traits.
Blue-cone m onochrom ats only have functional rods an d short-wavelength
cones. This is an X-linked condition m ost com m only associated with one o f two
classes o f g ene rearran g em en t, eith er (1) the reduction, presum ably by unequal
hom ologous recom bination, to a single X-linked pigm ent gene (as in dichrom acy)
plus a deleterious m utation in the rem aining functional gene, o r (2) the deletion
o f the locus-control region (LCR). T he LCR is located about 4 kb upstream of the
transcription initiation site and is necessary for the correct transcription o f the X-
linked pigm ent genes (N athans et al., 1989; Wang et al., 1992). Both long- and
middle-wave genes are presum ed to becom e inactive as a result of LCR deletion.
Some patients who are reduced to a single X-linked pigm ent gene lose the func
tion o f the rem aining gene as a result of a p o in t m utation changing a cysteine to
an arginine at am ino acid position 203, a highly conserved site am ong G-protein
coupled transm em brane receptors (N athans et al., 1989). This residue is appar
ently involved in form ing a disulfide linkage that is essential for the assembly o f a
functional visual pigm ent (Karnik and Khorana, 1990).
In all three types o f m onochrom acy, affected individuals have p o o r visual acuity,
cannot distinguish colors and often show nystagm us and photophobia. Some blue
cone m onochrom ats develop a slowly progressive central retinal degeneration.

(2) Dichromacy

T he m ost severe form s o f congenital color blindness that are com m only en co u n
tered, are the dichrom acies. As th ere are three classes o f hum an cone pigm ents,
th ere are three different types o f dichromacy. P rotanopes have a single m iddle-
and a single short-wave pigm ent b u t no long-wave pigm ents. D euteranopes have a
single long- an d a short-wave pigm ent bu t lack middle-wave pigm ent. T ritanopes
are missing short-wave receptors but have the o th e r two classes. T ritanopia is a
rare condition that is in h erited as an autosom al d om inant disorder. It is m uch
less com m on than the X-linked form s of color blindness bu t in some populations
the incidence is as high as 1 in 500 (van H eel et al., 1980). Recently, three differ
en t m utations have been identified within the gene encoding the short-wave pig
m en t in individuals with tritanopia, which segregated with the disorder and were
absent from general population controls (Weitz et al., 1992a; Weitz, W ent &
Nathans, 1992b). In each case, a non-conservative am ino acid substitution was
 COLOR VISION DEFECTS 221

predicted (Gly-79-Arg, Ser-214-Pro, Pro-264-Ser). O ne o f these residues (Pro-264)

is highly conserved both in visual pigm ents and in G-protein coupled receptors.
P rotanopia an d d euteranopia each occur at a frequency o f about 1% in Cauca
sian males.

(3) Trichromacy

Less severe form s o f color vision deficiency are the anom alous trichrom acies.
A nom alous trichrom ats, like n orm al trichrom ats, require three prim aries in
color m atches. However, they generally have p o o re r color discrim ination ability
than norm al. T he term s for the anom alous trichrom acies: protanom aly, deutera-
nomaly, an d tritanom aly, parallel those for the dichrom atic types. D euteranom aly
is the m ost com m on form o f color vision deficiency affecting about 5% of males
in Caucasian populations. T he frequency o f protanom aly is about 1% o f males.
Tritanom aly is a rare condition.
T here is considerable variation in the ability to discrim inate colors within each
class of X-linked anom alous trichromacy. Some protanom als and deuteranom als
have re d /g re e n color vision that is nearly as p o o r as a dichrom at's. O thers have
m uch m ore m ild color deficiency; the color discrim ination ability of some ap
proaches norm al. P erceptual tests th at involve color m atching were useful for
broad classification o f color vision types. C olor m atching tests are also used in the
diagnosis o f color anom alies. A dram atic difference betw een the m ild anom alous
trichrom acies an d norm al can be seen in behavior in a color m atching task that is
referred to as the Rayleigh color m atch (after Lord Rayleigh, 1881, who intro
duced the use of this color m atch for diagnosing red-green color vision varia
tions). A person is given ju st two prim ary lights, a red and a green and is asked to
mix them in a p ro p o rtio n that will exactly m atch the appearance of a m onochro
matic yellow com parison light. Only two prim ary lights are req u ired in this color
m atch because the short-wave cones are very insensitive to the lights used in this
test. The observer needs only to satisfy the req u irem en t th at the m iddle- an d long
wave pigm en ts absorb photons at the same rate when stim ulated by the re d /g re e n
m ixture as they do when presented with the m onochrom atic yellow' light. Similar
to norm als, mildly anom alous trichrom ats can find a fairly narrow range o f re d /
green m ixtures th at identically m atch the yellow com parison. However, they
choose a ratio o f red to green light that is very different from norm al. Mildly pro-
tanom alous observers require considerably m ore red light and mildly deutera-
nom alous observers, considerably m ore green light in the m ixture than observers
with norm al color vision. For exam ple, mildly deuteranom alous observers typical
ly m atch the m onochrom atic yellow light with a re d /g re e n ratio that is 4 to 5 times
lower than norm al. People with m ore extrem e color anom alies, because o f their
p o o r color discrim ination ability, will accept a wide range o f re d /g re e n ratios as
indistinguishable from the yellow standard, often including the norm al re d /g re e n
ratio an d the ratio o f the corresponding m ild anomaly.
222 JAY NEITZ AND MAUREEN NEITZ

C olor m atches occur at the level of the pigm ents. In the color m atch described
above, the m iddle- and long-wave pigm ents each absorb some pro p o rtio n o f inci
d e n t photons from the m onochrom atic yellow light. T he m ixture ratio of red and
green light can be adjusted to produce the same p ro p o rtio n of quantal absorption
in these pigm ents as the yellow light does. T he im portant consequence o f the fact
th at the m atch occurs at the level o f pigm ent absorption is that once the lights are
set to m atch in their effects on the pigm ents, the two lights cannot be m ade distin
guishable by any trick of the nervous system, o r by changing eith er the ratio of
m iddle-to-long wave cones or the distribution o f pigm ents am ong the cone recep
tors. For exam ple, a pigm ent m atch m ade by a person who has long-wave pigm ent
in one set o f cones and middle-wave pigm ent in a separate set o f cones, would be
the same as a (hypothetical) person who has one cone type with long-wave pig
m ent and a second cone type containing a m ixture o f m iddle- and long-wave pig
m ents.
Individual differences in the density o f spectrally selective ocular filters, such as
the yellow pigm ents in the lens and m acula, do produce slight individual differ
ences in the ratio o f red-to-green light reaching the pigm ents, and these can influ
ence the color m atch. Also, differences in the effective optical density o f the visual
pigm ents within the cones can produce slight differences in the absorption spec
tra o f the receptors. O th er than the slight influences o f these factors, however, two
people can have different color m atches only if they have com plem ents o f pig
m ents th at differ in absorption spectra. T he fact th at anom alous trichrom ats re
quire extrem ely abnorm al ratios o f red-to-green light in color m atches indicates
that they have very different com plem ents o f pigm ent spectral sensitivities than
norm als.
T he visual pigm ents that u nderlie the two X-linked anom alous trichrom acies
have, at least to a first approxim ation, been characterized (e.g. DeM arco, Pokorny,
& Smith, 1992; Pokorny & Smith, 1977; P iantanida, 1976; Pokorny, Smith, & Katz,
1973). Protanom alous observers, like protanopes, have no long-wave pigm ents b u t
they m aintain two slightly d ifferent pigm ents th at peak in the m iddle wavelengths.
T hese two pigm ents are sufficiently different in their spectral properties to sup
p o rt some color discrim ination in the m iddle-to-long wavelengths. T he absence of
long-wave pigm ents in protanom alous observers confers a loss in sensitivity to very
long-wavelength (red) lights. In color m atching, protanom alous observers com
pensate for their insensitivity to red light by adding a m uch higher p ro p o rtio n of
the red prim ary to the m ixture. In contrast, deuteranom alous observers lack any
m easurable contribution to their vision from middle-wave pigm ents, b u t they
m aintain two types o f long-wave pigm ents. T he spectra o f the two long-wave pig
m ents expressed in deuteranom aly are similar bu t their difference supports some
degree of red-green color vision th at is variable across individuals.
Until recently it was believed that anom alous trichrom acy was caused by either
the replacem ent o f the gene th at encodes norm al long-wave pigm ent (in p rota
nomaly) o r the gene th at encodes norm al middle-wave pigm ent (in d eu teran o m
aly) with an abnorm al gene. T he abnorm al gene (previously believed to be allelic
 COLOR VISION DEFECTS 223

with the norm al gene) was th o u g h t to produce an anom alous pigm ent with a spec
trum shifted from th at of norm als. Thus, for exam ple, deuteranom alous observers
were conceived as having a m u tan t (anom alous) middle-wave pigm ent an d a nor
mal long-wave pigm ent. Similarly, protanom alous observers were conceived as
having a norm al middle-wave pigm ent and a m u tan t (anom alous) long-wave pig
m ent. This is now th o u g h t to be in co rrect (N athans et al., 1986a). C u rre n t u n d er
standing suggests, to us, that a m uch m ore useful conception is that anom alous
trichrom ats, like th eir dichrom atic counterparts (deuteranopes an d protanopes),
are each missing all m em bers o f one class o f cone pigm ent. D euteranom alous ob
servers have no middle-wave pigm ents, however, unlike deuteranopes, they m ain
tain two slightly different long-wave pigm ent types. P rotanom alous observers have
no long-wave pigm ents b u t they m aintain two slightly d ifferent middle-wave pig
m ent types.

Variation in norm al color vision and in norm al pigm ents

Dichromacy and anom alous trichrom acy are phenotypic color vision variants that
occur with relatively high frequency in the hum an population. People with these
color vision deficiencies have different com plem ents of cone pigm ents than nor
mal. In addition to the large color vision differences between norm al an d color
defective vision, m ost people with norm al color vision have experienced disagree
m ents with others ab o u t colors. Are som e o f these norm al individual differences
in color perception caused by individual differences in cone pigm ents? R ecent
evidence indicates that they are.
Individual differences in norm al color vision have been long recognized. How
ever, only recently has there been renew ed interest in these norm al variations and
their cause. Fifteen years ago, A lpern and colleagues m easured individual differ
ences in color defective observers th at they attrib u ted to variation in the cone pig
m ents (Alpern & Moeller, 1977; A lpern & Pugh, 1977; A lpern & Wake, 1977).
They concluded th at the long-wave pigm ents of different d eu teran o p es are vari
able in their spectral position as are the middle-wave pigm ents o f protanopes.
They reasoned th at if the dichrom acies are reduced form s of norm al vision, then
the m iddle- and long-wave pigm ents o f norm als m ust vary as well. They em pha
sized th at a finding o f individual differences in norm al pigm ents would dictate m a
jo r alterations in theories o f the basis o f color deficiency. However, the idea that
norm al pigm ents vary in spectral sensitivity has been controversial over the past
decade an d a h alf and the possibility o f variation in norm al pigm ents has contin
u ed to be ignored in the form ulation o f favored theories to explain color defects.
W hile all the controversies have yet to be settled, results have now accum ulated
to provide strong evidence th at the spectra o f norm al cone pigm ents do vary
across individuals. T he picture th at em erges is that am ong color norm als there
may be discrete form s o f both the long-wave pigm ents an d the middle-wave pig
ments. Most com m on are, perhaps, two m ajor alternative form s o f the long- and
two m ajor alternative form s o f the middle-wave pigm ent. T he idea o f discrete vari
224 JAY NEITZ AND MAUREEN NEITZ

ation in norm al hum an pigm ents was suggested earlier by W aaler (1967) from
analysis o f norm al color m atches, by Eisner & M acLeod (1981) who used psycho
physical m ethods to isolate responses from norm al m iddle- and long-wave pig
m ents and by Dartnall, et al. (1983), whose m icrospectrophotom etric study o f the
spectra from individual hum an ph o to recep to rs suggested two types o f m iddle- and
two types o f long-wave receptors am ong people with norm al color vision.
T he largest n u m b er o f recen t studies that provide evidence for variation in nor
mal pigm ents have exam ined norm al individual differences in color m atching.
T he Rayleigh color m atch (described earlier) where a m ixture o f red an d green
light is adjusted to m atch the appearance o f a m onochrom atic yellow light, has
long served to classify abnorm al vision. Since the re d /g re e n ratio set in the m atch
is directly related to the absorption spectra of the pigm ents, this test can be a very
sensitive indicator o f individual differences in norm al m iddle- and long-wave pig
m ents as well. If the cone pigm ents of norm al observers vary in spectral position,
the distribution of norm al color m atches will reflect that variability. R ecent studies
specifically designed to exam ine such norm al individual differences agree that
th ere is considerable variability in norm al color m atches, an d the range o f differ
ences is sim ilar across studies (Neitz & Jacobs, 1986; Eisner & Burns, 1987; Jo rd a n
& M ollon, 1988; W ebster & MacLeod, 1988; Lutze, Cox, Smith, & Pokorny, 1990;
Neitz & Jacobs, 1990; Lindsey, W inderickx, Sanocki, Teller, Deeb, & Motulsky,
1992; Shevell & He, 1992; W m derickx, Lindsey, Sanocki, Teller, Motulsky, & Deeb,
1992b; P iantanida & Gille, 1992). These differences in norm al color vision can be
striking. N orm al observers at one extrem e o f the distribution require a re d /g re e n
ratio that is m ore than twice as high as those at the o th er extrem e. This m eans that
a re d /g re e n m ixture that a norm al person from one e n d o f the distribution sees
as exactly m atching the m onochrom atic yellow is seen as a very conspicuous red-
o range m ism atch to a norm al person that falls at the o th er extrem e.
A greem ent about the issue o f discretely different pigm ents has been a d ifferent
m atter. If there are discrete form s of the n orm al m iddle- and long-wave pigm ents,
and if the steps in spectral peak are large enough, then different groups o f indi
viduals with distinct pigm ent com plem ents should be evident in the distribution
of norm al color m atches. Evidence for such m odality in the distribution o f color
m atches is claim ed by some studies (e.g. Neitz & Jacobs, 1986; Neitz & Jacobs,
1990; Eisner & Burns, 1987; Lindsey, et al., 1992; W inderickx, et al., 1992b;
P iantanida & Gille, 1992) bu t clear evidence o f m odality is lacking, o r at least, is
n o t as evident in o th er studies (e.g. Jo rd a n 8c M ollon, 1988; Lutze, et al., 1990;
Webster, 1992).
In the past year, the idea th at a small n u m b er o f discretely different types o f pig
m ents occur am ong observers with norm al color vision has gained considerable
im petus from studies that reveal a strong correlation between individual differ
ences in color vision and a polym orphism at a single am ino acid position in the
m iddle and long-wave pigm ents (Neitz, Neitz, & Jacobs, 1991c; Lindsey, et al.,
1992; W inderickx, et al., 1992b; Merbs & N athans, 1992a; Neitz, Neitz, &: Jacobs,
1993). These are discussed in detail in the section on spectral tuning, below.
 COLOR VISION DEFECTS 225

PIONEERING STUDIES OF THE MOLECULAR GENETICS OF COLOR VISION

N athans et al. (1986a; N athans, Thom as, & Hogness, 1986b) were the first to iso
late and characterize genes that encode cone visual pigm ents. They described the
goal o f their studies: to test the hypothesis th at hum an color vision is m ediated
by a family o f rhodopsin-like m olecules, the apoproteins of wLiich are encoded by
the corresponding m em bers o f a family o f g en e s... [and the] related hypothesis
that the com m on in h erited variations in hu m an color vision (color blindness)
are due to m utations in the m em bers of this gene family. A long history of
research on color vision and color blindness m ade clear predictions as to what
they would find. F undam ental to theories o f color vision is the idea that norm al
hum an vision is m ediated by three pigm ents, each presum ed to be en co d ed by a
single gene. C olor vision deficiencies that involve the short-wave pigm ent are
in h erited in an autosom al fashion whereas red-green color vision deficiencies are
X -chrom osom e linked. Thus, it was expected that two cone pigm ent genes, one
encoding a long-wave, and the o th er a middle-wave pigm ent w ould be found on
the X-chrom osom e, and a third, encoding the short-wave pigm ent, would be
found on an autosom e.
N athans et al. (1986b) isolated clones containing fragm ents o f at least th ree pig
m ent genes th at m ap to the X -chrom osom e from a genom ic DNA library of a m ale
( J. N athans) who has norm al color vision. They concluded th at the cloned frag
m ents, from this one person, co rresp o n d ed to one gene encoding the long-wave
pigm ent and two copies o f a gene encoding the middle-wave pigm ent. They also
isolated three X-linked pigm ent genes from a cDNA library m ade from several
dozen hum an eyes obtained at autopsy. They no ted th at these X-linked genes were
polym orphic. All the genes were slightly d ifferent in nucleotide sequence.
T he finding o f m ore than two X-linked cone pigm ent genes from one norm al
m ale was quite unexpected. Subsequently, S outhern hybridization analysis of the
X-linked pigm ent genes from a sam ple o f males with norm al color vision suggest
ed that the presence o f e x tra pigm ent genes was no t uncom m on, an d th at the
n u m b er o f pigm ent genes was variable across individuals. N athans an d colleagues
concluded th at everyone had a single copy of the long-wave gene, b u t individuals
could have eith er one, two, or three copies o f the middle-wave gene. Two copies
o f the middle-wave gene were proposed to be the m ost com m on arrangem ent.
In a second study (N athans, et al., 1986a), the genes of 25 subjects with congen
ital color defects were exam ined using S outhern hybridization analysis. It was long
believed that X-linked color vision deficiencies are caused by alterations in genes
that encode m iddle- and long-wave pigm ents and, indeed, in every case, the gene
arrangem ents o f the color blind subjects, as in ferred from S outhern analysis, were
different from any observed in the N athans et al. (1986b) sam ple o f norm als. T he
results an d conclusions can be sum m arized as follows.
1) D euteranopia. Six o f nine d eu teran o p es exam ined h ad a single long-wave
gene an d no middle-wave genes. T he three o th er d eu teran o p es h ad m ultiple
226 JAY NEITZ AND MAUREEN NEITZ

X-linked pigm ent genes b u t none of those genes corresponded, in structure,

to the prototypical middle-wave genes, i.e., no n e had a restriction fragm ent,
from near the 3 end, identified as th at derived from a norm al middle-wave
gene. It was concluded th at the color defect in this sam ple o f d euteranopes
was associated with the absence o f all genes th at encode middle-wave pig
m ents.
2) P rotanopia. T he genes from six p rotanopes were exam ined. Two had a single
X-linked pigm ent gene. T hree others h ad m ultiple genes but all th eir genes
had a restriction fragm ent, n ear the 3 end, corresponding to N athans ow'n
middle-wave gene. T he color defect in these five observers seem ed associated
with the absence of all genes that could encode long-w^ave pigm ent. Inexplica
bly, the sixth p ro tan o p e had all the fragm ents corresponding to an intact long
wave gene. H e also h ad m ore middle-wave genes (perhaps six) than h ad been
attrib u ted to any norm al observer.
3) Protanom aly. N either o f the two protanom alous observers had any genes with
3' fragm ents identified as being derived from long-wave genes. It appeared,
however, that com pared to N athans own norm al genes, each h ad one gene
whose 5' sequences were derived from a long-wave gene and 3 sequences de
rived from a middle-wave gene. It seem ed logical th at these 5 long- 3 m iddle-
wave fusion genes would encode a pigm ent with a spectrum interm ediate to
the two norm al pigm ents. It was concluded that while the protanom alous ob
servers were lacking any norm al long-wave genes, they had norm al m iddle-
wave genes and, in addition, fusion genes to encode the anom alous pigm ents
th o u g h t to characterize protanom aly.
4) D euteranom aly. T he results from deuteranom alous subjects were surprising.
They continue to be the greatest challenge to o u r u n d erstan d in g o f the genet
ic m echanism s that u nderlie color vision. At the same tim e, solving the co n u n
d rum s o f deuteranom aly may be a key to un d erstan d in g the relationship
betw een pigm ent genes an d color vision. T he reason for color deficiency can
be understood easily in the case o f only one deuteranom alous subject in
N athans' study. This one deuteranom alous observer has several genes bu t
no n e with a 3 fragm ent identified as being derived from a middle-wave gene.
He, thus, has no intact genes that could encode norm al middle-wave pigm ent.
T he o th er seven deuteranom alous observers all h ad fragm ents corresponding
to the identified com plete norm al long- an d com plete norm al middle-wave
genes. In addition, from the stoichiom etries of long- and middle-wave gene
fragm ents, it appeared th at all the deuteranom alous subjects had one or m ore
fusion genes with a 5 en d derived from a norm al m iddle- and 3 en d derived
from a n orm al long-wave gene. T he fusion genes could encode a pigm ent with
a slightly d ifferent spectrum from the pigm ent encoded by the norm al long
wave gene and, thus, explain the anom alous pigm ents that were believed to
characterize deuteranom aly. But, how could the presence o f norm al m iddle-
wave genes be reconciled with the deuteranom alous phenotype? N athans
et al. (1986a) offered the explanation th at the fusion genes would be ex
 COLOR VISION DEFECTS 22 1

pressed in the green cones to g eth er with the norm al green pigm ent gene or
genes. This arran g em en t th erefo re confers a G R+ [mild] or G R+ [extrem e
deuteranom alous] phenotype. This cannot, however, explain the deu tera
nom alous phenotypes. As described earlier, the X-linked pigm ents that u n d e r
lie deuteranom aly are sim ilar in spectrum . Close enough, in fact, th at they can
b oth be classed as long-wave pigm ents. Mixing both long- an d middle-wave
pigm ents in the same middle-wave cones would n o t cause subjects to set the
abnorm al Rayleigh m atches th at characterize deuteranom aly. As discussed
earlier, this is because color m atches occur at the level of the pigm ents and not
the cones. Individuals who expressed both norm al m iddle- and deu teran o m a
lous pigm ents in the same middle-wave cones would set norm al or n ear nor
mal pigm ent m atches. From a study th at investigated this issue in m ore detail
(Neitz, Neitz, & Jacobs, 1991b) it was concluded th at deuteranom alous sub
jects do n o t express any detectable am o u n t o f middle-wave pigm ent even
though they often appear, by S outhern analysis, to have m ultiple norm al mid-
dle-wave genes.
With the exception o f an adequate explanation for the behavior o f deu teran o m
alous observers, the in terp retatio n s o f N athans an d colleagues seem to offer sim
ple explanations o f the genetic basis for red-green color blindness. O ne greatly
simplifying aspect o f their theory is th at some o f the color-blind genotypes are ex
plained as arising from unequal b u t hom ologous recom bination of norm al gene
arrangem ents. This can be simply diagram m ed as in the exam ple o f Figure 1.
T he im portance o f the work of N athans an d colleagues c an n o t be underestim at
ed. They established the prim ary structures of the pigm ent genes an d their chro
m osom al locations. They suggested th at the X-linked pigm ent genes are in a
tandem array, and dem onstrated that m ost people have m ore pigm ent genes than
previously believed. They also d em onstrated differences betw een norm al gene ar
rangem ents an d those o f color blind observers and illustrated how some o f the
gene arran g em en t m ight com e about by hom ologous recom bination.
N athans et al. (1986a,b) came to som e new conclusions about the genetic m ech
anisms o f color vision: 1) m any X-chrom osom es o f norm al observers were pro
posed to have an extra copy (or two) o f the middle-wave gene, 2) the abnorm al
pigm ents o f anom alous trichrom ats were proposed to result from recom bination
between the long- and middle-wave genes, an d 3) many deuteranom alous observ
ers were proposed to have both n orm al m iddle- and long-wave genes in addition
to m iddle- /long-wave fusion genes. W ith these exceptions, th eir results were in
terp reted as generally confirm ing conventional theories. Botstein (1986), fo r ex
am ple, in terp re ted the m olecular genetic results as, at last, confirm ing the Young-
H elm holtz theory of color vision in which the existence o f three types of light ab
sorbing particles (one for each prim ary color) are proposed to explain why nor
mal hum an vision is trichrom atic (Young, 1802). F urtherm ore, inheritance of
m u tan t pigm ents with abnorm al spectra, different from any expressed in norm al
observers, was in terp re ted as the cause o f anom alous trichromacy.
228 JAY NEITZ AND MAUREEN NEITZ

Normals

Deuteranope

Normal with two

middle-wave genes

Key: Long-Wave gene

d> Middle-wave Gene

Figure 1 U n e q u a l c ro ssin g o v e r b e t w e e n X -lin k e d p ig m e n t g e n e a n avs c a n e x p la in p o ly m o r

p h is m in h u m a n p ig m e n t g e n e a r ra n g e m e n ts . In th is e x a m p le , a n in te r g e n ic r e c o m b in a tio n
b e tw e e n tw o g e n e a rra y s th a t c o n f e r n o r m a l c o lo r v ision p r o d u c e s o n e X - c h ro m o s o m e w ith
a sin g le lo n g - a n d n o m id d le-w av e g e n e s a n d a n o t h e r X -c h ro m o s o m e w ith o n e lo n g - a n d two
m id d le -w av e g e n e s . A m a le w h o in h e r its a n X -c h ro m o s o m e w ith o n ly a sin g le p ig m e n t g e n e
w ill h a v e d ic h r o m a tic c o lo r v isio n . O n e w h o in h e r its o n e lo n g - a n d tw o m id d le -w av e g e n e s
w ill h a v e n o r m a l tric h ro m a tic c o lo r vision.

Now, seven years later, the body of experim ental results th at relate to the m olec
ular biology o f color vision has grown. It may be time to reconsider the theories
p u t forth in 1986. A new theory may b etter explain the experim ental results - a
theory in wrhich all the ideas in the preceding paragraph w ould be held as substan
tially incorrect. As will be elucidated in subsequent sections, the evidence suggests
th at it is likely that people with norm al color vision have, on average, m ore X-
linked pigm ent genes than suggested by N athans and colleagues. F u rth erm o re,
m any observers wdth norm al color vision may have m ultiple long- as well as m ulti
ple middle-wave genes. T he facts are consistent with an alternative theory in which
the fusion genes n eith er produce abnorm al pigm ents, n o r do they cause color de
fects. T hat is, fusion genes an d the variant pigm ents they produce may be found
an d expressed in individuals with norm al color vision. Finally, results from m olec
ular biology may refute, ra th e r than confirm , the Young-Helmholtz theory as the
explanation for hum an trichromacy.
 COLOR VISION DEFECTS 229

MUTATIONAL PATHWAYS: FROM NORMAL TO DEFECTIVE GENOTYPES

T he discoveries that the m iddle- an d long-wave pigm ent genes are highly hom ol
ogous, lie closely spaced in a tandem array, and are variable in n u m b er suggested
a m utational pathway to gene arrangem ents that confer color defects that is sim
ple and com pelling - the arran g e m e n t o f the pigm ent genes is one that would
prom ote a high frequency o f u n equal but hom ologous recom bination, a m echa
nism that could increm ent and d ecrem en t gene nu m b er an d produce fusion
genes, all seemingly, ju st as req u ired to explain the gene arrangem ents that
underlie color defects. This idea is illustrated in Figure 1; an intergenic exchange
between two norm al arrays transfers a middle-wave gene from one chrom osom e
to the other, giving rise to one chrom osom e with only a single long- and no m id
dle-wave pigm ent genes. T he o th er chrom osom e that results from this crossover
has one long- an d two middle-wave genes. In a stroke, both the occurrence of
d euteranopes with only a long-wave gene an d norm al trichrom ats with extra mid-
dle-wave genes are explained.
Difficulties arise, however, when one tries to explain the gene arrangem ents of
o th er color defective observers by sim ilar one-step m utational pathways. For exam
ple, several o f the genotypes in ferred for deuteranom alous subjects cannot result
from ju st one crossover betw een norm al arrays (see Figure 2). Fusion genes can
result from crossovers between norm al gene arrays bu t no m ore than one fusion
gene can be produced in each crossover. Yet, frequently, deuteranom alous sub
jects seem to have m ultiple fusion genes. A crossover betw een two norm al arrays
is illustrated at the top o f Figure 2. T he pro d u ct of the crossover that is assem bled
with a 5 m iddle- 3 long-wave fusion gene also has the com plete long-wave gene
from one parental chrom osom e an d the com plete middle-wave gene from the
other. To get two fusion genes by crossing over, an arran g e m e n t th at already has
one fusion gene (Figure 2) m ust u n d erg o a second recom bination. T he same type
of recom bination m ust occur three separate times for three fusion genes. T he
probability o f a recom bination occurring twice is expected to be the square of the
probability o f one occurrence ancl the likelihood of accum ulating three fusion
genes by this m echanism is expected to be equal to the cube o f the probability of
a single occurrence. T he frequency of deuteranom alous phenotypes should equal
the sum of the frequencies o f each of the different genotypes. Satisfying these re
quirem ents yields that, in the population, 95.2% o f deuteranom alous subjects
should have a single fusion gene, 4.5% should have two fusion genes, an d only
0.2% should have three fusion genes.
Table 1 shows com putations o f the n u m b er o f fusion genes for each o f the eight
d euteranom alous subjects exam ined by N athans et al. (1986a). T he calculations
suggest th at two subjects may have two fusion genes an d two m ore may have three
fusion genes each. This is wildly d ifferent from what would be expected if the in
heritance o f a single fusion gene was sufficient to cause deuteranom aly. If 95% of
d euteranom alous individuals in the population have only a single fusion gene,
then the probability of drawing four subjects (out of eight) with m ultiple fusion
230 JAY NEITZ AND MAUREEN NEITZ

N orm als

D euteranom alous?

D euteranom alous?

D euteranom alous?

Key:
Long-Wave gene i N Middle-wave Gene Fusion Gene

Figure 2 M u ta tio n a l p ath w ay s to d e u te r a n o m a ly a re n o t k n o w n . T w o g e n e a r r a n g e m e n ts th a t

c o n f e r n o r m a l c o lo r v isio n (sh o w n a t th e to p ) c o u ld c ro sso v e r to p r o d u c e a n X -lin k e d p ig
m e n t g e n e a rra y th a t h a s a 5 m id d le - 3 long-w ave fu s io n g e n e in a d d itio n to n o r m a l m id d le -
a n d n o r m a l long-w ave g e n e s . T h is g e n e a r r a n g e m e n t h a s b e e n p r o p o s e d to c o n f e r a d e u te r
a n o m a lo u s p h e n o ty p e . I f all o b s e rv e rs w ith n o r m a l c o lo r v isio n h a v e o n ly a sin g le long-w ave
g e n e as h a s b e e n p r o p o s e d by N a th a n s e t a l. (1 9 8 6 b ) , t h e n to p r o d u c e a g e n e a rra y w ith two
fu s io n g e n e s r e q u ir e s a g e n e a r r a n g e m e n t w ith o n e fu sio n g e n e to u n d e r g o a s e c o n d c ro ss
o v e r (illu s tra te d as c ro sso v e r n u m b e r 2 ). F o r a n a rra y to h a v e th r e e fu s io n g e n e s r e q u ir e s
t h r e e i n d e p e n d e n t c ro sso v e r e v en ts. I f th e p r e s e n c e o f a fu sio n g e n e c o u ld c a u s e d e u te r a
n o m a ly th e n d e u te r a n o m a lo u s o b s e rv e rs w ith o n e fu s io n g e n e w o u ld b e e x p e c te d to s u b s ta n
tially o u t n u m b e r th o s e th a t h av e a c c u m u la te d m u ltip le fu s io n g e n e s . In s te a d ,
d e u te r a n o m a lo u s o b s e rv e rs w ith m u ltip le fu s io n g e n e s a p p e a r to b e c o m m o n , su g g e stin g
th a t th e i n h e r ita n c e o f a fu s io n g e n e d o e s n o t c a u s e c o lo r d e fec ts.

genes an d two of those with three fusion genes is less than one in a m illion. Earlier,
it was n o ted th at a m ysterious feature o f deuteranom aly is the presence o f seem
ingly norm al middle-wave genes. Now we have an added curiosity. D euteranom a
lous subjects seem to have m ore fusion genes than would be expected from a
m odel in which the inheritance of a fusion gene causes deuteranom aly.
 COLOR VISION DEFECTS 231

Table 1. The number of fusion genes in deuleranomalous observers are calculated from
the results of Nathans et al. (1986a). The total number of X-linked pigment genes in each
subject was determined from his Ag/Ar fragment ratio. The ratio of band intensities was
translated to a gene fragment ratio using the intensity ratios suggested to correspond to a
1:1 gene ratio (Ar:Ag = 0.97; Dr:Dg = 1.24). Gene number was taken to equal Ag/Ar + 1; these
values were rounded to the nearest whole number. The number of genes that contain frag
ment Dr was calculated using the equation: Genes with Dr = Total number of X-linked pig
ment genes / (Dg/D r +1 ). These were rounded to the nearest whole number. The number
of fusion genes equals 1 less than the number of genes with fragment 1). because one gene
with Dr is the normal long-wave gene. These calculations assume that all subjects have ont}
one copy of fragment A,.. If some subjects have more than one copy of A,, the number of
fusion genes would be higher.

Subject Number of Dg/D, Number of

X-linked long-wave
pigment genes fusion genes

34 5 0.27 3
35 3 0 2
36 6 0.57 3
37 6 1.61 1
38 5 0.84 2
39 3 0,32 1
40 3 0.45 1
41 4 1.32 1

N athans et al. (1986a) exam ined the genes of twenty-five color deficient males.
We have organized the gene arran g em en ts th at can be inferred from their results
into 10 d ifferent categories. These are illustrated in the left-hand colum n of Fig
ure 3. T he po p u lar idea is that color defective arrangem ents can be explained by
simple, one-step recom binations betw een norm al arrays that result in gene dele
tions or produce a fusion gene. A rran g em en t num ber 1 of Figure 3, can be pro
d uced by a crossover in which bo th products o f the recom bination fit perfectly
with this idea (as was shown in F u re 1); one pro d u ct confers d eu teran o p ia and
the o th er has two middle-wave genes ju st as fo u n d to occur am ong norm als. The
o th er nine color-blind gene arran g em en ts are m ore difficult to fit with this
scheme. T he p ro tan o p e arrangem ents, like num bers 2 and 3 an d the protanom a-
lous arrangem ents, like num ber 4 o f Figure 3 can arise by a sim ple crossover. How
ever, two distinct gene arrangem ents arise from every crossover. W hen a protan
arran g em en t is produced, an o th er array with an intact norm al long-wave gene
from one parent, all the norm al middle-wave genes from the o th er parent, and a
5 m iddle- 3long fusion gene is also produced. T he 5 m iddle- 3 long fusion gene
is expected to encode a pigm ent with a long-wave spectrum . P rotanope gene ar
rangem ents are missing sequences th at encode a long-wave pigm ent, b u t in their
production, an o th er chrom osom e effectively gains the long-wave gene (as a fusion
gene) th at the p ro tan o p e lost.
232 JAY NEITZ AND MAUREEN NEITZ

Color Defects Normals

10

F ig u re 3 A n e w th e o r y o f th e p h o to p ig m e n t g e n e a r r a n g e m e n ts th a t u n d e r lie n o r m a l c o lo r
v isio n allow s a v e ry s im p le e x p la n a tio n f o r h o w c o lo r d e fe c ts a rise. T h e in f e r r e d g e n e a r r a n g e
m e n ts o f th e 25 c o lo r d e fe c tiv e o b s e rv e rs s tu d ie d by N a th a n s e t a l. (1 9 8 6 a ) c a n b e c la sse d in to
10 c a te g o rie s sh o w n in th e le ft-h a n d c o lu m n (a b b re v ia tio n s : D, d e u te r a n o p e ; P p r o ta n o p e ;
PA, p ro ta n o m a lo u s ; D A d e u te r a n o m a lo u s ) . I n s o m e cases, th e re s u lts fro m S o u th e r n analysis
in d ic a te th e p r e s e n c e o f a n in ta c t g e n e w h o se e x p re s s io n is n o t e v id e n t in th e su b je c ts' b e h a v
ior. F o r e x a m p le , th e c o lo r v isio n o f d e u te r a n o m a lo u s o b s e rv e rs is th a t p r e d ic te d b y th e e x
p r e s s io n o f tw o slig h tly d if f e r e n t long-w ave p ig m e n ts b u t n o m id d le-w av e p ig m e n ts , h ow ever,
d e u te r a n o m a lo u s su b je c ts o fte n h a v e all th e g e n e fra g m e n ts c o r r e s p o n d in g to in ta c t m id d le -
w ave g e n e s . A lig h tn in g b o l t sym bol d ra w n t h r o u g h a g e n e in d ic a te s lac k o f e x p re s s io n o r
f u n c tio n . T h e g e n e a r r a n g e m e n ts o f c o lo r d e fe c tiv e su b je c ts a r e o fte n c o m p lic a te d , h a v in g
m a n y g e n e s , a p p a r e n tly u n e x p r e s s e d g e n e s , a n d m u ltip le fu s io n g e n e s . T h e s e s u g g e st a p re v i
o u sly u n r e a liz e d c o m p le x ity in th e n o r m a l g e n e a r ra n g e m e n ts fr o m w h ic h th e y a ro s e . T h e
r ig h t- h a n d c o lu m n show s h y p o th e tic a l g e n e a r r a n g e m e n ts th a t c o u ld c o n f e r n o r m a l c o lo r
visio n . E a c h p a ir o f n o r m a l g e n e a r ra n g e m e n ts c o u ld u n d e r g o a sin g le c ro sso v e r to y ie ld th e
c o lo r-d e fe c tiv e g e n e a r r a n g e m e n t sh o w n d ire c tly to its left. In th is n e w th e o ry , n o r m a l o b s e rv
e rs a re p r o p o s e d to c a rr y d e fe c tiv e g e n e s s o m e tim e s b u t in e a c h case th e y h a v e a t le a s t o n e
f u n c tio n a l m id d le - a n d o n e f u n c tio n a l long-w ave p ig m e n t g e n e . S o m e n o r m a l o b s e rv e rs a r e
sh o w n to h a v e f u s io n g e n e s . T h e e x p re s s io n o f th e s e g e n e s in n o r m a l o b s e rv e rs is p r o p o s e d
to c o n tr ib u te to th e o b s e rv e d v a ria tio n in n o r m a l c o lo r v isio n b u t d o e s n o t in te r f e r e w ith n o r
m a l c o lo r vision. P re v io u s th e o rie s r e q u ir e d p r o ta n o m a ly a n d d e u te r a n o m a ly to a ris e by d if
f e r e n t m u ta tio n a l p athw ays, o fte n w ith m a n y ste p s. In c o n tra s t, th e c ro sso v e rs sh o w n h e r e all
give rise to c o lo r d e fe c ts by th e sa m e o n e -s te p m e c h a n is m . T h e y p r o d u c e g e n e a r r a n g e m e n ts
t h a t lack e it h e r f u n c tio n a l m id d le -w av e g e n e s ( d e u ta n d e fe c ts ) o r long-w ave g e n e s ( p r o ta n d e
fe cts).
 COLOR VISION DEFECTS 233

It seems that either one o f two alternatives m ust be true with regard to the gene
arrangem ents that are pro d u ced as co u n terp arts to the protan gene arrays. O ne is
th at the inheritance o f a 5 m iddle- 3 long-wave fusion gene usually prevents the
expression o f the otherw ise norm al middle-wave genes and, thus, d eutan gene ar
rangem ents are pro d u ced as the com plem ent to protan ones. This would explain
why deuteranom alous subjects, like those illustrated by arran g em en t num ber 5 of
I- ' lire 3, can often have n orm al middle-wave genes. T he alternative is that the
gene arrays th at are pro d u ced in the same crossovers that give rise to protan d e
fects can confer norm al color vision. This would allow the possibility that norm al
observers can have, a n d may express fusion genes. T he question o f w hether or not
color norm al observers can have a n d express fusion genes is central to u n d er
standing the m olecular genetics o f color vision.
The rem aining five categories o f gene arrangem ents illustrated in Figure 3 are
all bew ildering because of their com plexity relative to the sim ple gene arran g e
m ents that have been proposed to u n d erlie norm al color vision. N one of them can
be explained by a sim ple single crossover between two of the proposed com m on
norm al gene arrays.
N um ber 6, a protanom alous gene arrangem ent, apparently has at least six
genes. Protanom alous arrangem ents are proposed to arise from deletion of the
one norm al long-wave gene. To result from a single crossover, an arran g em en t like
n u m b er 6 would have to arise from a norm al arran g em en t with at least seven
genes. T he suggested counts of the n u m b e r o f pigm ent genes in norm als would
indicate that the o ccurrence of norm als with seven genes is exceptionally rare.
Such a p rotanom alous arran g e m e n t ca n n o t have arisen by a single crossover from
any of the proposed com m on norm al gene arrays.
N um ber 7, a pro tan o p e, also has m any m ore genes (perhaps seven) than are
proposed for color norm al observers. In addition, he appears, by S outhern analy
sis, to have a norm al long-wave gene. A likely explanation o f his protanopia, is that
his long-wave gene has a m utation th at ren d ers the pigm ent p ro d u ced non-func
tional, perhaps similar to the m utations fo u n d to occur in blue-cone m o n o ch ro
m ats (N athans et al., 1989) (as are discussed earlier). In Figure 3, genes which do
n o t appear to contribute to the phenotype have a lighting b o lt symbol drawn
through them . A deleterious m utation may explain the loss o f long-wave pigm ent
function. But, the extrem ely high gene num bers fo u n d in p ro tan subjects relative
to those proposed for norm als are n o t w hat would be predicted from a single
crossover betw een com m on norm al arrays.
Some deu teran o p es (num ber 8) have only two genes, a norm al long-wave gene
and a 5 m iddle- 3long-wave fusion gene. Middle-wave genes cannot be deleted in
the same crossover th at p ro d u ced the fusion gene. N athans an d colleagues no ted
that eith er two in d ep en d e n t crossovers o r a gene conversion are req u ired to ex
plain these arrangem ents.
N um ber 9, a deuteranom alous arran g e m e n t with m ultiple fusion genes, and no
middle-wave genes can only be explained by some com bination of crossovers, or
234 JAY NEITZ AND MAUREEN NEITZ

crossovers an d gene, conversions. This is a n o th e r exam ple o f an arran g em en t that

m ust be proposed to arise by a m utational pathway with many steps.
As discussed at the begining o f this section, m any deuteranom alous observers
do n o t have one fusion gene, which is expected to be by far the m ost com m on ar
rangem ent, bu t m ultiple fusion genes (N um ber 10).
T he idea that color defects can be explained by a one-step recom bination is an
attractive one. If this explanation is correct, then nearly all the observed color
blind arrangem ents should be explainable by a one-step path. This is no t the case.
Instead, the arrangem ents associated with color deficiencies often have too many
n o rm al genes, too many fusion genes, or they lack genes th at would be expected
from one-step m utational pathways. O f course, m ore com plicated arrangem ents
should occasionally occur am ong color deficient observers, bu t they would be tre
m endously o u tn u m b ered by sim pler arrangem ents.
T h ere is a sim ple solution. If color-deficient phenotypes arise via one-step re
com bination of norm al gene arrangem ents, then the m odel for what constitutes
a n orm al array m ust be revised. T hat is, the com plexity o f the color-blind gene ar
rangem ents provides a unique window through which we can glim pse a previously
unrealized com plexity in the norm al arrangem ents from which they were derived.
In developing their m odel o f norm al gene arrangem ents, N athans an d colleagues
m ade two assum ptions: 1) everyone with norm al color vision has a single long
wave gene, an d 2) fusion genes cause color defects and they, thus, do n o t typically
o ccur in norm al arrays. However, the evidence may favor a radically different view
of the genes th at underlie norm al color vision; one that does no t rely on these as
sum ptions a n d that allows all color defecient phenotypes to be explained as aris
ing from one-step recom binations.

A new concept o f norm al genes makes abnorm al gene arrangem ents easily
explainable

T he right h an d colum n of Figure 3 illustrates a revised m odel of what constitutes

n o rm al arrays that enorm ously simplifies the explanation of how the color defi
cient genotypes are produced. In this new m odel, all the gene arrangem ents in
the rig h t h an d colum n of Figure 3 are proposed to confer norm al color vision
and overall these m ight typify the arrangem ents o f people with norm al color
vision. These were constructed to obey the following rules. 1) To confer norm al
color vision an individual m ust have at least one functional long- an d one func
tional middle-wave pigm ent gene. 2) C olor norm al individuals can have ten or
m ore X-linked pigm ent genes in total, b u t to fall within the ratios o f m iddle- to
long-wave genes suggested from S outhern analysis, the ratio of m iddle- to long
wave genes is typically no larger than four to one. 3) N orm al arrays can have m ul
tiple long-wave genes but the additional long-wave genes are always illustrated as
5 middle- 3 long-wave fusion genes to em phasize that they include, a n d /o r are
p receded by 3 sequences that have b een believed, in norm al subjects, to be
exclusive to middle-wave genes. 4) N orm al subjects can have genes with defects
 COLOR VISION DEFECTS 235

th at interfere with their expression o r function; such m utations would be

expected by genetic drift if people usually have additional genes to provide the
function.
In Figure 3 a single crossover is illustrated for each pair o f chrom osom es in the
right-hand colum n. Each crossover produces the color-blind genotype shown to its
left. All com m on form s of color-blindness can arise by the same m echanism - an
unequal hom ologous recom bination that deletes a gene (or genes) required for
norm al color vision. Q uite simply, if n orm al arrangem ents are as proposed in this
new m odel, illustrated on the right-hand side o f Figure 3, then the color-blind
gene arrangem ents could:
1) all arise from m utation o f norm al arrays,
2) all com e about by the same one-step pathway, and
3) all be red u ced form s o f norm al arrangem ents.
Notably absent from this schem e are the com plications th at deuteranom alous
and protanom alous arrang em ents com e ab o u t by d ifferent m echanism s, that col
or-blindness can be caused by the addition o f fusion genes to norm al arran g e
m ents, and th at m any color-blind arrangem ents n eed to be explained by
im probable m utational pathways from norm al arrangem ents.

Results from Southern analysis re-exam ined

G enetic m echanism s that give rise to color defects could be quite simple, if: 1)
people with norm al color vision can often have m ultiple long-wave genes, 2)
color norm al people often have fusion genes, an d 3) the average nu m b er o f pig
m en t genes p er X -chrom osom e o f color norm al observers is substantially larger
than previously im agined. T he c u rre n t view o f norm al gene arrangem ents comes
from the analysis o f pigm ent gene fragm ents by S outhern hybridization
(Nathans, et al., 1986b; Vollrath, N athans, & Davis, 1988; D rum m ond-B org,
Deeb, & Motulsky, 1989; Deeb et al., 1992). Art exam ination o f those results lends
insight into w hether o r n o t they may have been m isinterpreted. For exam ple, is it
likely that a norm al array with m ultiple long-wave genes an d with fusion genes
m ight have been in terp re ted as having only a single long-wave gene, and no
fusion genes?
Figure 4 shows the restriction m aps for m iddle- and long-wave genes. Four re
striction fragm ents, labeled A, B, C, an d D have been used in S outhern analysis to
distinguish m iddle- from long-wave genes. T he A fragm ent from the long-wave
gene (A, s is larger than the A fragm ent from the middle-wave gene (Ag) because
of an insert (of about 1.9 kb, in the first intron) about one th ird o f the way into
the gene. This same insert accounts for the size difference betw een the C g and C r
fragm ents. T he fragm ent, B r , is sm aller than B^ because it is produced by cleav
age at a Bam HI site that occurs in un iq u e sequences flanking the 5 end o f the
array. These unique sequences are n o t part of the tandem ly rep eated sequences
236 | \Y M l IV \M> MAI RI 1- \ Ni l : /

B B B Middle-wave

F igu re 4 (T op ) R e s tric tio n m a p s oi m id d le - a n d long-wavi? g e n e s sh o w in g f ra g m e n ts th a t have

b e e n u s e d to d is tin g u is h th e two g e n e s in S o u t h e r n analysis. A n in s e r t in th e long-w ave g e n e
is re s p o n s ib le f o r th e size d if f e r e n c e b e tw e e n f r a g m e n ts A,, a n d A g a n d b e tw e e n C,. a n d C
T h e f r a g m e n t B i s f o r e s h o r te n e d c o m p a r e d to th e c o r r e s p o n d in g f r a g m e n t f r o m th e m id d le -
w ave g e n e , B b e c a u s e o f a B am H I site (B) th a t o c c u rs u p s tr e a m o f th e first g e n e , o u ts id e o f
th e r e p e a te d p h o t o p ig m e n t g e n e array. T h e B, f r a g m e n t o c c u rs o n ly o n c e in e a c h g e n e c lu s te r
a n d c a n n o t b e d u p lic a te d o r d e le te d by h o m o lo g o u s r e c o m b in a tio n .

(B o tto m ) B e c a u se th e in s e r t th a t d is tin g u is h e s fr a g m e n ts A r a n d C r f ro m A g a n d C ,, o c c u rs
w ith in th e g e n e , a c ro sso v e r a n y w h e re in a b o u t th e first th ir d o f th e g e n e w ill d u p lic a te th e
long-w'ave g e n e in c lu d in g fra g m e n ts A ,., C r . a n d D r . T h e d u p lic a te d long-w ave g e n e w o u ld
h a v e f r a g m e n t B . It m ay also h a v e e x o n 1 d e r iv e d fro m a m id d le-w av e g e n e b u t th a t e x o n is
id e n tic a l b e tw e e n m id d le - a n d long-w ave g e n e s . T h u s , th e d u p lic a te d long-w ave g e n e a n d th e
long-w'ave g e n e fro m w h ic h it was d e riv e d will h a v e id e n tic a l c o d in g s e q u e n c e s .
 COLOR VISION DEFECTS 237

th at constitute the array. Thus, a corresponding Bam HI site is n o t found flanking

the genes o th er than the first in the array.
T he sequences flanking the first gene in the array are n o t hom ologous with any
that are within the array. Thus, the fragm ent Br can n eith er be deleted o r dupli
cated by hom ologous recom bination. O n the o th er hand, the insert that distin
guishes Ar from Ag and Cr from Cg will be duplicated by any crossover that occurs
in the first one-third o f the gene. N athans et al. (1986a,b) proposed th at observers
with norm al color vision have a single long-wave gene. They explain th at because
o f its position at the 5' edge of the array, the long-wave gene can never be com
pletely duplicated or deleted by hom ologous exchange. T he lower half o f Figure
4 shows that, while this is true, the sequences that distinguish Ap Cp and Dr will all
be duplicated by any crossover in the first third of the gene. T he fusion gene that
results may have exon 1 derived from a middle-wave gene, but N athans et al.
(1986b) found exon 1 to be identical betw een m iddle- an d long-wave genes. Thus,
long-wave genes can be duplicated. T he long-wave gene duplicated by any cross
over in the first third o f the gene will have a coding sequence th at is identical to
its p aren t long-wave gene. If the likelihood of a crossover in any hom ologous re
gion is related to the length o f th at region, then about one-third o f all crossovers
between m iddle- an d long-wave genes should duplicate the coding sequence of
the long-wave gene in its entirety. People with m ultiple long-wave genes are ex
pected from the same logic th at explains m ultiple middle-wave genes, i.e., the ho
mology and arran g e m e n t o f the pigm ent genes prom otes a high frequency of
hom ologous recom bination.
Figure 5 shows the effect m ultiple long-wave genes would be expected to have
on the results from S outhern analysis. T hree gene arrays are shown with one, two,
and three long-wave genes respectively. Even though they differ in gene num ber,
all would have Ag/A r fragm ent ratios of 2.0. They are similarly identical in their
C g/C r and Dg/ D r ratios of 2.0. T he only indicator o f the fact that they are different
is the Bg/ B r ratio, which increases with gene num ber. Previously, the Ag/A r ratio
was used to calibrate the absolute n u m b er o f pigm ent genes (N athans, et al.,
1986a; N athans, e ta l., 1986b; Vollrath, e ta l., 1988; D rum m ond-B org, e ta l., 1989).
Thus, all three arrangem ents o f Figure 5 would be in terp re ted as having one long
wave gene and two middle-wave genes. However, because Ar is expected to be du
plicated (perhaps frequently) by recom bination; Ag/Ar cannot be taken as a reli
able indicator o f gene num ber.
T he Bg/ ! r ratio, on the o th er hand, is predicted to be a reliable indicator o f dif
ferences in gene num ber. Suppose th at the observers with norm al color vision ex
am ined previously by S outhern analysis vary in their nu m b er of long-wave genes
and th at the total n u m b er o f genes people have is often m uch higher than is indi
cated by their A /A r ratio. Since the Bg/ B r ratio is an indicator o f gene num ber,
then there are two predictions about the B fragm ent ratio. First, Bg/ B r should be
greater in m agnitude than the A o r C ratios reflecting that the average gene num
ber is higher than Ag/A r would predict. Second, the B fragm ent ratio should be
m ore variable than the A or C fragm ent ratios reflecting variability in the n u m b er
238 JAY NEITZ AND MAUREEN NEITZ

A g / A r =2 B g / B r =8 C g / C r =2 Dg / D r =2

Figure 5 T h e p r e s e n c e o f m u ltip le long-w ave g e n e s in m a le s w ith n o r m a l c o lo r v isio n w o u ld

n o t b e e v id e n t fro m th e ra tio s o f A, C, a n d D g e n e f ra g m e n ts v isu a liz ed in S o u t h e r n analysis.
T h e g e n e a r r a n g e m e n ts sh o w n a re d ra m a tic a lly d if f e r e n t fr o m o n e a n o th e r , yet, th e y h ave
id e n tic a l ra tio s o f A g/ A r , C g ./ C r , a n d Dg / D r . I n S o u th e r n analysis, o n ly th e B g / B r ra tio
w o u ld re v e a l th e d iffe re n c e s a m o n g th e s e a rra y s. P re v io u s in te r p r e ta tio n s o f S o u t h e r n b lo t
d a ta h a v e e m p h a s iz e d th e A g / A r f r a g m e n t r a tio in th e c a te g o riz a tio n o f n o r m a l g e n e a rray s.
T h a t p r a c tic e w o u ld le a d to th e i n c o r r e c t c o n c lu s io n th a t th e t h r e e g e n e a rra y s sh o w n h e r e
a re id e n tic a l to o n e a n o th e r . E a c h w o u ld b e i n te r p r e t e d a s h a v in g o n e long-w ave g e n e , two
m id d le -w av e g e n e s a n d n o fu s io n g e n e s .

o f long-wave genes within groups that are classified by A g / A r ratio. This is exactly
what is found. For exam ple, N athans et al. (1986a) found that for people with a
1:1 A g / A r ratio ( m ean A g / A r = 0.97) the o th er fragm ents had the following-
m eans and standard deviations: Bg /B r , 2.00 0.49;C g /C r , 0.88 0.18; Dg / D p
1.24 0.30J u st as predicted if some people have m ultiple long-wave genes and,
thus, their higher Bg /B r ratios are included in the sam ple, the m ean Bg / B r ratio
is high, m ore than twice the A o r C ratios. As predicted by the idea that the sam ple
is inhom ogeneous in gene num ber, the B ratio is highly variable across subjects.
T he B g /B r ratio has a standard deviation 2.7 times greater than the C g / C r ratio.
If everyone had a single long-wave gene, B g/ B r would be expected to be always
equal to A g/ A r and C g / C r both in m agnitude an d in degree o f variability. Nei
th e r o f these are b o rn e out by experim ental results.
Two specific exam ples can serve to illustrate, in detail, how nicely a new inter
p retation o f norm al gene arrangem ents can explain color defective genotypes and
can explain results from S outhern analysis o f norm al subjects as well. C onsider two
cases from N athans et al. (1986a), subject #27, a protanope, and subject #40, a
d euteranom alous observer. Figure 6 shows gene arrangem ents that could yield the
fragm ent stoichiom etries observed for each subject. Both are problem s for earlier
theories because the arran g em en ts can n o t com e ab o u t by any simple pathway
 COLOR VISION DEFECTS 239

from norm al arrangem ents. Also shown for each subject are theoretical norm al ar
rangem ents that could crossover ju st once to yield the color defective genotype.
These differ from the norm al genotypes proposed previously in th at they have
m ore genes (as many as eight) an d they all have one o r m ore 5 m iddle- 3 long-
wave fusion genes. C ould such arran g em en ts really be typical o f color norm al ob
servers? D rum m ond-B org et al. (1989) exam ined the pigm ent genes of 134 Cau
casian males using S outhern analysis. Subjects were classified according to their
A g /A r ratios. T he results for the m ales who were considered to have norm al gene
arrangem ents with nom inal A g / A r ratios o f 3:1 (about 20% of th eir subjects) are
illustrated in the lower half o f Figure 6. T he observed m ean for each of the A, B,
an d D fragm ent ratios is shown together with bars that extend two standard devi
ations on each side o f the m ean. T he C ratio is om itted because the inform ation
it yields about the genes is re d u n d a n t with the A ratio. O verlaid are the fragm ent
ratios that co rresp o n d to the two hypothetical norm al arrangem ents illustrated
(in the u p p e r half of Figure 6) to give rise to the p ro tan o p e (squares) an d the two
proposed to give rise to the deuteranom al (circles) (m iddle panel, Figure 6). Even
though the arrangem ents are very different from those previously th o u g h t to u n
derlie norm al color vision, the stoichiom etries are consistent with those consid
ered norm al. This suggests th at the norm al gene arrays exam ined previously
probably included such arrangem ents - e.g., with large num bers of genes, m ulti
ple long-wave genes and fusion genes. It would seem that norm al arrangem ents
similar to those illustrated in Figure 6 were in terp reted as having only a single
long-wave gene an d no fusion genes because the interp retatio n em phasized the re
sults for the A fragm ents.
Each recom bination illustrated in Figure 6 simply deletes genes req u ired for
norm al color vision b u t the process alters the fragm ent stoichiom etries substan
tially, such th at those o f the color defective observers fall outside n orm al limits.
This is illustrated in the bottom panel o f Figure 6. T he p ro tan o p e inherits only
one long-wave gene (a non-functional one). Even though his X -chrom osom e has
the same n u m b er o f pigm ent genes (seven) as one o f the norm al arrangem ents
from which it is proposed to arise, the reduction to a single long-wave gene in
creases his A g /A r an d D g /D r ratios to the high value considered rare for norm als.
In the case of the prod u ctio n of the deuteranom alous arrangem ent, previous in
terpretations would be oblivious o f the fact th at the two norm al arrays shown as
giving rise to the deuteranom alous genotype have fusion genes, b u t the crossover
im parts a deuteranom alous gene arran g em en t in which the presence of fusion
genes becom es obvious because o f the extrem ely low Dg/ D r ratio (even though no
additional fusion genes were p ro d u c e d ).
Pigm ent gene fragm ent stoichiom etries o f color-deficient observers alm ost al
ways differ from those of typical norm al observers. However, there are exceptions;
Deeb et al., (1992) found four o f 129 norm al observers in which the presence o f
fusion genes was obvious from the fragm ent stoichiom etries. It was previously be
lieved th at the abnorm al stoichiom etries were the result of fusion genes present
in color defects but nearly always absent in norm al subjects (Nathans, et al.,
240 JAY NEITZ AND MAUREEN NEITZ

Ll_
OL
A g/A r B g /B r Dg/ D r

Figure 6 Two specific examples o f how a new theory o f norm al gene arrangem ents can ac
count for results from color defective and color n o rm al subjects. T he results from S outhern
analysis for two subjects exam ined by N ath a n se t al. (1986a) are as follows: subject #27 (a pro
tanope), A g /A r = 6.08, B g / B r = 5.40, C g / C r = 3.77, D g /D r = 5.87; su b ject#40 (deuteranom
alous), A g /A r = 2.31, B g /B r = 5.36, C g/ C r = 1.42, Dg/ D r = 0.56. Those fragm ent
stoichiom etries suggest the gene arrangem ents shown (m arked #27 and #40). T he o rd er of
the genes was arbitrarily chosen. Above each color-defective gene array, a pair of hypothetical
arrays proposed to confer norm al color vision is shown. T he pairs of norm al gene clusters
could each undergo a single crossover to yield the observed color defective genotypes. Each
norm al array illustrated has m ore pigm ent genes than previously proposed for norm als and
has m ultiple genes that encode long-wave pigm ents. T hree o f the norm al arrays have defective
genes in addition to both norm al m iddle- and long-wave genes. Plotted in the lower h alf o f the
figure are the ranges of fragm ent stoichiom etries (bars, drawn as the m ean2 SD) d eterm in ed
for genes, considered to confer norm al color vision, from a sample of males that had nom inal
A g /A r fragm ent ratios of 3:1 (from D rum m ond-B org, e t al., 1989). Superim posed on those
ranges are the fragm ent ratios for the four hypothetical norm al gene arrangem ents shown in
the u p p er half o f the figure (circles, parents to the deuteranom alous gene arrangem ent;
squares, parents to the protanope gene arran g em en t). Even though the hypothetical norm al
gene arrangem ents are m uch different than those previously proposed for norm al subjects
they yield fragm ent stoichiom etries that are consistent with those previously considered nor
mal. Fragm ent stoichiom etries for the two color defective gene arrangem ents are plotted in
the bottom -m ost panel (circles, deuteranom alous; squares, pro tan o p e). They fall outside the
norm al limits.
 CO LO R VISION DEFECTS 241

1986a). It can be seen th at the presence o f (even m ultiple) fusion genes in an ar

ray is not sufficient to im part stoichiom etries th at fall outside norm al limits. In
fact, it appears that the presence o f fusion genes in a large subset o f norm al sub
jects is req u ired to account for the observed range of norm al stoichiom etries.
Thus, for exam ple, in the deuteranom alous observer, it is the small n u m b er of
middle-wave genes relative to the n u m b er of long-wave and fusion genes that ac
counts for his abnorm al stoichiom etries. This occurs because o f the deletion of
norm al middle-wave genes n o t because o f the production o f fusion genes. This
would suggest that the fusion genes do n o t cause the color deficiency; the loss of
norm al middle-wave genes does.
T he theory th at is presented here allows all color defective arrangem ents to
arise from norm al arrays by one-step deletions o f genes req u ired for norm al color
vision. N othing could be simpler. M ore than that, however, the theory is attractive
because it can account fo r facts earlier theories would have difficulties explaining.
In previous sections we discussed th at earlier theories have difficulties explaining,
1) why a deuteranom alous observer could seemingly in h e rit all the norm al m id
dle-wave genes from a norm al array yet n o t express any o f them or 2) why so many
color-blind observers have m ore com plex gene arrangem ents than can be ex
plained by the sim ple addition o r deletion of a gene from norm al arrays. Now we
see th at earlier theories would have difficulty explaining a third observation, why
the B g/B r ratio is both so high an d so variable com pared to the o th er fragm ent
ratios. All these can be explained by a new interp retatio n of the norm al gene ar
rangem ents.
Earlier theories posed a deleterious role for the fusion genes, their presence was
believed to cause color anomaly. To the contrary, in this new schem e, the fusion
genes have a beneficial role. For exam ple, if fusion genes that encode slightly dif
feren t pigm ents were not p resen t in n orm al observers, th en the loss o f functional
middle-wave pigm ents would always cause deuteranopia. Instead, many people are
protected by the presence of fusion genes, and in h erit the often m uch m ilder
color deficiency deuteranom aly when they suffer the loss o f functional m iddle-
wave genes.

D efective genes

We suggest th at defective genes are com m on am ong color defective and color
norm al populations. By defective we m ean th at individual genes have altered cod
ing or regulatory sequences, as m ight com e about by point m utations o r dele
tions, th at prevent expression or function of expressed product. Such defects
m ight be expected to be especially com m on in the X-linked pigm ent genes
because often people have genes to spare (perhaps, som etim es as m any as seven
or eight m ore than are req u ired to sup p o rt trichrom atic color vision). Thus,
defects m ight be acquired with little consequence to vision. We propose that d eu
teranom alous individuals with middle-wave genes may be people who in h erit all
their middle-wave genes as defective genes. They could arise from norm al gene
2 42 JAY NEITZ AND MAUREEN NEITZ

arrangem ents with defective genes by u n fo rtu n ate recom binations (as illustrated
in Figure 6) th at leave no functional middle-wave genes. R ecent evidence sug
gests that defects in individual genes are a com m on cause of o th er visual defects.
It is, thus, plausible that defective genes underlie deuteranom aly as well. T hree
d ifferent p oint m utations within the gene encoding the short-wave pigm ent have
been fo u n d to be associated with tritanopia (Weitz et al., 1992a; Weitz, Went, &
N athans, 1992b). P oint m utations within X-linked pigm ent genes have also been
found associated with some cases o f blue cone m onochrom acy (N athans, et al.,
1989). An exam ple o f defective genes in red-green color blindness has also been
found. W inderickx et al. (1992c) exam ined the middle-wave pigm ent genes of a
d eu tan observer who had an apparently norm al gene array, i.e., by S outhern
analysis he had long- and middle-wave genes an d no fusion genes. From these
results he would be expected to have norm al color vision. Analysis of his m iddle-
wave genes revealed sequences encoding the same cysteine to arginine substitu
tion observed in blue cone m onochrom ats. These m utations probably explain
the lack of middle-wave pigm ent function in this observer. T he same alteration
was detected in one middle-wave gene o f a mildly deuteranom alous observer. It
was also detected in a middle-wave gene o f one out o f 52 norm al subjects dem o n
strating that defective genes can occur in observers with norm al color vision.
T h e possibility th at defective cone pigm ent genes may be fairly com m on in ob
servers with norm al color vision may have im p o rtan t clinical im plications in light
o f the discovery th at p h o topigm ent gene defects cause retinal degeneration. At
least 32 m utations in the rhodopsin gene have been found associated with autoso
mal d o m in an t retinitis pigm entosa (e.g., Dryja et al., 1990; Sung et al., 1991a;
Sung et al., 1991b). C one pigm ent gene defects may similarly cause cone-specific
degenerations. Some blue cone m onochrom ats show retinal degeneration
(N athans, et al., 1989). Cone degeneration has also been observed in one family
w here functional middle-wave genes were present but there was a deletion in the
long-wave gene (Reichel, Bruce, Sandberg, & Berson, 1989). A possibility that
seems w orth considering is that while in m ost cases the inheritance o f a defective
cone pigm ent gene in an array o f otherw ise norm al genes may have no serious
consequences for vision in young eyes, som e cone pigm ent gene defects may cause
late-onset retinal (m acular) degenerations.
A nother re cen t study relates to the issue o f defective pigm ent genes and their
expression. T he expression o f photopigm ent genes was investigated in an exam i
nation o f cone pigm ent mRNA transcripts from hum an retinas (W inderickx, Bat-
tisti, Motulsky, & Deeb, 1992a). A com m on silent polym orphism (A vs. C at the
th ird position o f codon 283) occurs in exon 5 o f the middle-wave gene. G enom ic
DNA derived from blood samples and cDNAs from retinas were exam ined from 13
males. Both the C and the A versions of the middle-wave genes were detected in
the genom ic DNA from 10 of the males. Middle-wave cDNAs were detected in sam
ples from all eyes, however, middle-wave cDNAs with C at the third position of
codon 283 were no t detected in the samples from any o f those 10 males. T he ex
pression o f the C allele was detected in only one subject, a male who had no A
 COLOR VISION DEFECTS 243

alleles o f the gene. O ne possible explanation for this result is th at the C serves to
m ark middle-wave genes th at have a n o th er m utation that interferes with their ex
pression. If this is true, middle-wave genes with such defects occur at a high fre
quency in the norm al population, as we propose. An alternative explanation
(p referred by W inderickx, et al., 1992a) is that only the first two genes are ex
pressed from the pigm ent g ene arrays of all observers. T here is good evidence for
a locus-control region (LCR) about 4 kb upstream of the transcription initiation
site (N athans, et al., 1989; W ang et al., 1992). An interaction betw een the LCR and
a pigm ent gene is proposed to be re q u ired to switch on transcription. W inderickx
et al. (1992a) suggest th at the LCR allows transcription o f only the two genes clos
est to the LCR; m ore dow nstream genes are supposed no t to interact with the LCR
an d are, thus, n o t transcribed. If so, it is curious that in individuals with m ultiple
middle-wave genes, the expression o f only the A allele was observed. C onsidering
the high frequency o f recom bination betw een opsin genes, one m ight expect that
eith er allele could occur in the second position, and across individuals both alleles
would be found to be expressed.
T here is p re ced e n t from o th er systems for the idea th at m ore proxim ally located
genes have a com petitive advantage to interact with the LCR. It is im aginable that
the probability o f a particular pigm ent gene being expressed in a cone is p ro p o r
tional to its proxim ity to the LCR, i.e., the m ost 5 gene would be expressed in the
highest n u m b er o f cones, the second gene expressed in fewer cones than the first
and so on, u ntil the m ost distal genes o f large arrays are expressed in very few
cones. Wre have suggested th at the n u m b er o f genes p er array m ight often be larg
er than has been suggested from previous interpretations o f results from S outhern
analysis. It could be th at the A alleles o f the middle-wave genes usually o u t n u m b er
the C alleles in individual arrays an d the C alleles may tend to occur at m ore distal
positions relative to the LCR, where they are expressed at low levels. This m ight
explain why W inderickx et al. (1992a) detected the expression o f only the A ver
sion in their subjects who carry both alleles. Such a proposal is attractive, and it
does n o t conflict with a theory in which m ore than two X-linked pigm ent genes
can be expressed in an individual eye. Even the expression o f a gene at very low
levels is expected to have im p o rtan t consequences for vision in some cases. T here
are about 5 m illion cones in each eye. If a gene were expressed at a level o f only
5%, it m ight n o t be detected in experim ents like those o f W inderickx et al.
(1992a) bu t a person would still have a quarter-m illion cones with th at expressed
pigm ent. It is virtually certain that a p erson would no t suffer extrem e deu teran o m
aly if he h ad that n u m b er o f norm al middle-wave cones. Extrem ely biased cone ra
tios still seem to support norm al color vision. For exam ple, Pokorny, Smith &
Baron (1988) re p o rte d the case of a fem ale heterozygous carrier o f a protan color
defect who, they concluded, has an extrem ely small n u m b er o f long-wave cones;
the subject has norm al color vision. T he possibility th at levels o f expression d e
pen d on gene position (W inderickx, et al., 1992a; Deeb, et al., 1992) is interesting
an d extrem ely im portant, b u t it needs to be tested fu rth e r before the consequenc
es for genotype-phenotype relationships in color vision are understood.
244 JAY NEITZ AND MAUREEN NEITZ

SPECTRAL TUNING AND NORMAL COLOR VISION VARIATIONS

Red-green color vision depends on having at least two pigm ents with different
spectral sensitivities in the m iddle-to-long wavelengths. U nderstanding the
genetic basis of norm al color vision and the cause o f color defects d epends criti
cally on knowing which am ino acid differences betw een the cone pigm ent opsins
are responsible for giving the m iddle-to-long wave pigm ents different spectra. A
com parative study o f the genes th at encode eight pigm ents with various spectra,
two from hum ans, an d three each from two species o f South A m erican monkey,
indicated that am ino acid substitutions at ju st three am ino acid positions could
account for the spectral difference betw een hum an long- and middle-wave pig
m ents (Neitz, et al., 1991c). T he positions o f these substitutions in the opsin are
illustrated in Figure 7. Two am ino acid differences betw een long- an d m iddle-
wave pigm ents, at positions 277 and 285 are encoded by nucleotides in exon 5 of
the pigm ent genes. A substitution o f Tyr for Phe at position 277 produces a spec
tral shift o f ab o u t 10 nm, and a substitution o f T h r for Ala at 285 produces a shift
o f about 15 nm. Together, these two substitutions shift pigm ents from ones that
can be categorized as middle-wave to those th at can be categorized as long-wave,
confirm ing an earlier conclusion that substitutions in exon 5 are principally
responsible for the difference between m iddle and long-wave pigm ent classes
(Neitz, Neitz, & Jacobs, 1989). Thus, if all com m on hum an X-linked pigm ents are
categorized into two classes, m iddle- o r long-wave sensitive, any gene th at has
exon 5 from a long-wave gene will encode a long-wave pigm ent. Any gene that has
exon 5 derived from a middle-wave gene will encode a middle-wave pigm ent. A
third am ino acid substitution, of Ser for Ala at position 180, encoded by a nucle
otide substitution in exon 3 o f the genes is responsible for a sm aller spectral shift
of ab o u t 4-6 nm. This is one o f the substitutions that appears to be responsible for
spectral differences within the classes o f m iddle- and long-wave pigm ents. Sub-
types o f long-wave pigm ents and sub-types o f middle-wave pigm ents th at have dif
ferences at position 180 could arise from point m utations o r from crossovers
betw een the m iddle- and long-wave genes occuring dow nstream o f exon 3 bu t
upstream o f exon 5.
T he finding that substitutions at the three positions, 180, 277, and 285 are re
sponsible fo r spectral differences am ong the pigm ents has been confirm ed (C han,
Lee, 8c Sakmar, 1992; Ibbotson et a l , 1992; Merbs & N athans, 1992a; Merbs &
N athans, 1992b; Williams et al., 1992). However, som e studies suggested th at these
th ree are n o t the only substitutions involved in tuning the spectra o f m iddle to
long-wave pigm ents. Specifically, several results (Ibbotson, et al., 1992; Williams,
et al., 1992; W inderickx, et al., 1992b) h in ted that o n e o r both of two closely sep
arated substitutions, at positions 230 an d 233 encoded in exon 4 m ight also be in
volved. Recently, Merbs and N athans (1992b) have m easured the absorption
spectra of hybrid pigm ents expressed in vitro. Pigm ents en co d ed by genes with
long-wave exon 4 were found to have spectra that differ by ab o u t 2 -4 nm from
those en co d ed by genes with middle-wave exon 4. Further, they found that substi-
 COLOR VISION DEFECTS 245

Figure 7 Amino-acid substitutions at three sites can account for most of the differences in
spectral sensitivity among middle-to-long wavelength sensitive visual pigments. The pigment
molecules consist of seven membrane-spanning helices. The filled square in the seventh he
lix indicates the lysine residue that binds the chromophore, 11-cis-retinal. Filled circles indi
cate the positions of three amino acid residues that participate in controlling the pigment
spectrum. The identities of the two residues in helix six determine whether a pigment will
fall into the middle- or the long-wave class. Both residues are encoded in exon 5 of the gene.
Substitutions at the site in helix four (position 180) produce a smaller spectral shift that ac
counts for spectral differences between pigment subtypes within each of the middle- and
long-wave classes. In addition to the three residues indicated, substitutions at other sites en
coded by exons 2 and 4 of the genes are associated with small spectral shifts that are likely to
contribute to spectral variation within the middle- and long-wave classes.

tutions in exon 2 were also associated with small shifts in the spectral peak o f the
long-wave pigm ents.
T he small spectral shifts p roduced by nucleotide substitutions in exons 2, 3 and
4 may seem hardly significant, bu t together they may occasionally be responsible
for im p o rtan t differences betw een subtypes within each of the classes o f m iddle-
and long-wave pigm ents. For exam ple, we would class as long-wave pigm ents, all
pigm ents pro d u ced by genes with a long-wave exon 5, however, a gene with m id
246 JAY NEITZ AND MAUREEN NEITZ

dle-wave exons 2, 3, and 4, bu t a long-wave exon 5 is predicted to have a spectrum

as m uch as 11-12 nm shorter than the longest long-wave pigm ent that occurs in
hum ans (Merbs & N athans, 1992b). It is an attractive idea th at spectral differences
within each o f the classes o f m iddle- an d long-wave pigm ents may be responsible
for the differences between m ild and extrem e form s o f color anomaly. For exam
ple, a deuteranom alous observer who in h erited two well separated long-wave pig
m ents m ight be expected to have b e tte r color vision, i.e., a m ilder form of
deuteranom aly, than one who draws a set o f long-wave pigm ents th at are very sim
ilar in spectra. However, there are several lines of evidence th at would seem to con
tradict this as the com plete explanation (e.g., H urvich, 1972; Nagy, 1982; Pokorny
& Smith, 1982). They suggest that factors o th e r than the spectral separation o f the
pigm ents contribute to differences in discrim ination ability am ong anom alous ob
servers.
As we pointed out earlier, and as shown in Figure 8, there is considerable vari
ability in the Rayleigh color m atches o f p eople classed as having norm al color vi
sion. It has now been dem onstrated that substitutions at a single am ino acid
position, residue 180, are largely responsible for those individual differences. Two
observations suggested to us (Neitz, et al., 1991c) that substitutions at position 180
p ro d u ce norm al color vision variations, 1) substitutions o f Ser for Ala at position
180 o f the X-linked pigm ents produce a shift in spectrum (Neitz, et al., 1991c;
Neitz, Neitz, & Jacobs, 199 Id ), and 2) the long-wave pigm ent cDNAs (presum ably
isolated from norm al observers) analyzed by N athans et al. (1986b) differed from
N athans' own genom ic long-wave gene at position 180, suggesting th at the am ino
acid at this position was polym orphic in the color norm al population. We tested
the hypothesis th at spectral variations in both the m iddle- and long-wave pigm ents
p roduced by S er/A la substitutions at position 180 can explain norm al color vision
variations by com paring individual differences in Rayleigh m atches with indivi
dual differences in the relative p ro p o rtio n o f X-linked pigm ent genes that encode
Ala 180 vs. Ser 180. T here was a high correspondence betw een the two m easures in
dicating th at individual differences in the p ro p o rtio n o f X-linked pigm ent genes
th at encode Alai 80 vs. S eri 80 are, alone, sufficient to predict 89% o f the variance
in the observed norm al color m atches (see Figure 9) (Neitz, et al., 1993). In a sim
ilar experim ent, W inderickx et al. (1992b) used two different prim ers in the poly
m erase chain reaction (PCR) to specifically amplify two subsets of DNA fragm ents
containing the codon for am ino acid 180 from the X-linked p igm ent genes in each
individual. They used single strand conform ation polym orphism (SSCP) analysis
to exam ine individual differences and found that the m igration p atterns o f the
DNAs th at they believed w ere from long-wave genes correlated with individual dif
ferences in Rayleigh m atches. Even though W inderickx et al. (1992b) em phasized
a conclusion that polym orphism at position 180 o f the long-wave genes influences
the Rayleigh m atches o f color norm al males, they also found th at the fragm ents
th at they believed were from middle-wave genes were polym orphic at position 180;
while those gene from 84% o f individuals were concluded to have only Ala, 16%
were concluded to have genes with Ser am ong at least some o f the putative m iddle-
 COLOR VISION DEFECTS 247

wave genes. Psychophysical results (e.g., W ebster & MacLeod, 1988; Neitz & Ja
cobs, 1990) suggest th at individual differences in both m iddle- an d long-wave
genes underlie norm al color vision variation. A lthough the exact frequencies still
n eed to be clarified, it appears that there are two subtypes o f m iddle- and two sub-
types of long-wave pigm ents with respect to the identity o f the am ino acid, Ser or
Ala, at position 180 in the population with norm al color vision.

25

20

JdL
I 15
LU
3
O
10

i i i i i i________i i i i j

0.42 0.46 0.5 0.54 0.58

R/R+G
Figure 8 Individual differences am ong males with norm al color vision. Com plem ents of mid-
dle-and long-wave pigm ents vary am ong people with norm al color vision. These are manifest
as individual differences in a color m atching task in which subjects are asked to adjust the pro
portion of red light in a m ixture of red and green so that it exactly m atches the appearance of
a m onochrom atic standard light. Illustrated is the distribution o f Rayleigh color m atches for
males with norm al color vision com piled from Neitz and Jacobs (1986; 1990). In these studies
most males fell into one o f two large groups. However, a few subjects fell into smaller groups
th at flank the m ajor modes. A bout 8% o f males fell into a group (circled) who require less red
light in a re d /g re e n m ixture th an the o th er norm al observers. Results from experim ents d e
signed to distinguish individual differences in color vision pro d u ced by shifts in the spectral
peaks of the m iddle- and long-wave pigm ents from those p roduced by o th er factors suggest
that individuals in the small group are different from the larger groups in their com plem ents
of pigm ents as are the two large groups different from one an o th er in their pigm ents (Neitz
& Jacobs, 1990).

As discussed earlier, one o f the m ost engaging questions raised by the finding
that m ost people have extra X-linked pigm ent genes is; w hat are the extra genes
doing? Are they expressed; and if so is each gene expressed in its own subset of
cones? T he investigations o f polym orphism in norm al color vision lend insight
into these questions. O ne idea is th at only the first two genes in the array are ex
pressed (Deeb, et al., 1992; W inderickx, et al., 1992a). If the favored view of nor
248 JAY NEITZ AND MAUREEN NEITZ

mal gene arrangem ents was true and alm ost everyone with norm al color vision has
only a single long-wave gene, the first in the array, then the second gene could al
ways be a middle-wave gene in norm als. If only the first two genes were expressed,
then everyone with a norm al gene arran g em en t would express one long- an d one
middle-wave gene. This would fit well with the traditional view of norm al color vi
sion in which everyone is supposed to express only three different cone pigm ents.
F u rtherm ore, this m echanism could account for the fact that deuteranom alous
observers do n o t express the norm al middle-wave genes they are often believed to
carry. T he idea is that, in deuteranom aly, the addition o f a 5m iddle- 3long-wave
fusion gene in the second position of the array displaces the norm al middle-wave
genes to m ore 3 positions w here they are no t expressed (Deeb, et al., 1992; W in
derickx, et al., 1992a). Conversely, however, we have proposed here that many col
or norm al subjects have m ultiple long-wave genes. In Figure 3, the extra long-wave
genes are illustrated as occuring in the second position of the array to explain how
crossovers between norm al gene arrangem ents can produce color defects. This
would seem to preclude the possibility that only the first two genes are expressed.
N orm al individual differences lend insight into this issue as follows. We find that
the norm al color m atches o f m ost males fall into one o f two m ain groups as shown
in Figure 8 (Neitz & Jacobs, 1986; Neitz & Jacobs, 1990). However, there is a small
g roup of males, circled in Figure 8, who require less red light in the Rayleigh mix
ture than those in eith er m ain group. T he differences in color vision betw een in
dividuals in the two m ain groups correlate w'ell with am ino acid differences at
position 180 (Neitz, et al., 1993; W inderickx, et al., 1992b). We find that the m ore
extrem e color m atches are also predicted by the p ro p o rtio n o f X-linked genes with
Ala vs. Ser at position 180 (Neitz, et al., 1993). It has b een suggested (M erbs &
N athans, 1992a; Merbs & N athans, 1992b; W inderickx, et al., 1992b) that the two
large m ain groups of observers differ because individuals in the large g ro u p who
req u ire less red light in the m ixture, express a long-wave gene with S e ri80 and
middle-wave genes with A la i80 while those in the o th er main group have Ala at
position 180 o f both m id d le-an d long-wave pigm ents. Since the m ost extrem e col
or m atches also correlate with the pro p o rtio n o f genes encoding A la i80 vs.
S e ri80, in o rd e r to be consistent with the idea that only two genes are expressed,
people who fall into the extrem e group (circled in Figure 8) m ust have Ser at po
sition 180 o f both their expressed m iddle- and long-wave genes. W inderickx et al.
(1992b) suggest that although about 60% o f the long-wave genes encode Ser at po
sition 180, only a small percentage of the middle-wave genes have S eri 80. They
used nucleotide differences in exon 2 to identify m iddle- from long-wave genes. It
is unlikely th at those changes would reliably distinguish m iddle- from long-wave
genes, but nonetheless, it does appear, from th eir experim ent, that Ala is m uch
m ore com m on than Ser at position 180 am ong the middle-wave pigm ent genes.
From this, u n d e r the hypothesis that everyone has a single long-wave gene and
only the first two genes are expressed, the com m on gene arrangem ents that
should give rise to the extrem e color m atches (circled in Figure 8) can be predict
ed. They are illustrated in ] gure 9, the first two genes m ust have Ser at 180 but
 COLOR VISION DEFECTS 249

the other, unexpressed genes are likely to have Ala. It can be seen that if people
are allowed to have unexpressed genes with A la i80, the pro p o rtio n of genes that
encode Ala vs. Ser at 180 should vary widely for this extrem e phenotype d ep e n d
ing on how many unexpressed middle-wave genes a person has. Indeed, there
should be very little co rresp o n d en ce betw een genotype and phenotype for this
group. To the contrary the correlation is very high. O u r experim ent suggests that
all these extrem e subjects have num erous genes (perhaps, betw een 4 an d 10) of
which the vast p ro p o rtio n have Ser at position 180. T he chance o f obtaining this
result if only the first two genes were expressed is vanishingly small. Conversely,
however, if all the functional genes are expressed, then the only way a person
could fall into this extrem e group is if the prep o n d eran ce o f his genes have
S erl80, ju st as is observed.
If m any o r all the functional pigm ent genes are expressed, then because o f the
polym orphism in the norm al pigm ents it seems likely th at many people would in
dividually have and express genes for both subtypes of m iddle- a n d /o r bo th sub-
types o f long-wave pigm ents. T hat is, contrary to the long favored view th at only
three pigm ent types u n d erlie norm al color vision, many people could have four
or five spectrally d ifferent pigm ents. This is consistent with the distribution of col
or m atches am ong color norm al males (Neitz, et al., 1993). A nother recen t result
suggests that the expression of additional pigm ents is also evident in the behavior
of individual males. A fundam ental property o f color vision based on three cone
types is that color m atches m ade with the un ad ap ted eye will persist after selective
color adaptation (Brindley, 1970; Jam eson & H urvich, 1972). To the contrary, in
an experim ent designed to test the idea that norm al males have m ore than three
spectrally d ifferent cone types, it was fo u n d that some norm al males change their
color m atches after adaptation to re d light, as predicted if they have m ore than two
different types o f long-wave cones (Neitz, Neitz, & Jacobs, 1991a).

No anom alous pigm ents in color anomaly

Protanom alous observers can be th o u g h t of as having two subtypes of m iddle-

wave pigm ents and deuteranom alous observers, two subtypes o f long-wave pig
m ents. Merbs an d N athans (1992b), recently poin ted out that a deuteranom alous
observer could inherit two norm al long-wave genes that differ at position 180
(one norm al and one hybrid gene with only exon 1 from the middle-wave gene).
In such a case, n eith er deuteranom alous pigm ent is an abnorm al one, instead he
has variants o f the long-wave pigm ents found in norm al trichrom ats, consistent
with the m odel for anom alous trichrom acy proposed years ago by A lpern an d col
leagues (Alpern & M oeller, 1977; A lpern & Pugh, 1977; A lpern 8c Wake, 1977).
Similarly, a protanom alous observer could in h erit two norm al middle-wave genes
that differ at position 180 and, thus, n o t have an anom alous pigm ent.
We would take the idea th at anom alous observers express norm al variants o f the
m iddle and long-wave pigm ents several steps further. T he results of Merbs and
N athans (1992b) suggest that substitutions in exons 2 an d 4 also influence the pig-
250 JAY XF.IT/. AND M Al'REEX NEITZ

1.0

CD 0.8

0
0.6

0.4

0.2

0.0

Color Match (R / R + G)

Proportion of
Genes with
Aia1SO

F ig u re 9 Individual differences at a single am ino acid position (180) of the m iddle- and long
wave pigm ents can account for individual differences in norm al red-green color vision. A G /
T polym orphism at nucleotide position 1032 o f the genes produces a S er/A la 180 polymor
phism am ong the X -encoded pigm ents. Filled circles plot the pro p o rtio n of red light in a mix
ture o f red and green light that each subject req u ired to m atch the appearance of a
m onochrom atic standard light versus the relative p ro p o rtio n of X -chrom osome pigm ent
genes that have G at position 1032. T he six subjects with the smallest (R/R+G) settings in the
color m atch would fall into the extrem e group th a t is circled in Figure 8. Each has a low pro
portion o f genes with G at position 1032 am ong his total X-chrom osome pigm ent genes, i.e.,
they all have a p reponderance o f genes encoding Ser 180. C om pare this result with the pre
diction from the hypothesis that males with norm al color vision each express only the two 5-
m ost genes in their array, one long- and one middle-wave gene. If only two o f the genes were
expressed, to explain their color matches, these extrem e subjects m ust have S erl8 0 for both
the expressed m iddle- and long-wave pigments, i.e., they m ust express the m ore long wave
length sensitive subtype of each pigm ent. Because o f a prepo n d eran ce o f genes th at encode
Alai 80 in the population, the unexpressed genes are expected to m ost often specify Alai 80.
The m ost com m on gene arrangem ents predicted for these extrem e subjects are illustrated at
the bottom o f the figure. This distribution p red icted by the hypothesis that only two genes are
expressed, is superim posed in histogram form , on the observed results in the u p p e r graph.
T he descrepency between the prediction an d the observed results suggests that most or all of
the functional X -chrom osome genes are expressed and contribute to the observed behavior.
 COLOR VISION DEFECTS 251

m en t spectra. A lthough they recognize position 180 as a polym orphic site in the
norm al pigm ents, long-wave genes th at include the active substitutions in exons 2
or 4 of the middle-wave genes would have to be fusion genes. If norm al observers
do n o t express fusion genes, then only a subset of the pigm ents that occur in
anom alous observers would co rresp o n d to n orm al pigm ent variants, others, p ro
duced by genes with fusions betw een exons 2 an d 5 would be exclusively expressed
in anom alous trichrom ats. To the contrary, here, it is suggested that fusion genes
are characteristic of norm al gene arrangem ents. We fu rth e r suggest that norm al
observers can express their fusion genes along with their o th er m iddle- and long
wave pigm ent genes. T he expression o f fusion genes in norm al subjects merely
contributes to the variability in n orm al color vision. Thus, norm al observers can
have and express any o f the pigm ents that occur in anom alous trichrom acy and
vice versa. In the traditional sense, anom alous pigm ents have been conceived as
pigm ents th at differ in spectrum from those that ever occur in norm al eyes. If the
picture o f the pigm ents and genes o f n orm al color vision presented here is the cor
rect one, then anom alous pigm ents do n o t exist. As originally envisioned by Alp
ern an d colleagues, anom alous trichrom ats draw from exactly the same set o f long-
and middle-wave pigm ents as do n orm al trichrom ats. Thus, there are no pigm ents
that are unique to color anom aly - i.e., no anom alous pigm ents. T he difference
between norm al an d anom alous trichrom acy is that for X-linked pigm ents, n o r
mal subjects draw a set th at includes o n e or m ore m em bers from each o f the m id
dle- and long-wave classes; deuteranom alous observers have functional pigm ents
from only the long-wave class; protanom alous observers have pigm ents from only
the middle-wave class.
In summary, the traditional theory held that the replacem ent o f a norm al pig
m en t with an anom alous one caused color anomaly. This thinking has carried over
in m ore recen t explanations o f anomaly, i.e., th at the fusion genes cause anom a
lous trichromacy. We hold that the fusion genes do not, in any way, cause color
anomaly. Q uite the opposite, the norm al variant pigm ents, including those p ro
duced by fusion genes, serve the advantageous role, of protecting som e observers
who loose the genes for (or function of) one class o f pigm ents, from becom ing
dichrom ats. They can in h erit anom alous trichrom acy instead. T he advantage of
anom alous trichrom acy com pared to dichrom acy may be quite significant for in
dividuals who in h erit the m ildest form s o f anomaly. This advantage may explain
why polym orphism s within the m iddle- and long-wave pigm ent classes have been
m aintained in the norm al hum an population.
O u r view o f the relationship betw een genes, cone pigm ents, an d color vision is
a simple one (illustrated in Figure 10). T he cone pigm ents are encoded by a gene
for the short-wave pigm ent on chrom osom e 7 and an array of genes on the X-chro
m osom e. T he n u m b er o f X -chrom osom e pigm ent genes is variable, with a range
across individuals, o f one to ten (or m o re ). Genes for middle-wave pigm ents are
m ore num erous than genes for long-wave pigm ents, b u t people with m ultiple
long-wave genes are n o t unusual. T he genes that encode middle-wave and long
wave pigm ents are highly polym orphic. An enorm ous variety o f pigm ent genes
252 JAY NEITZ AND MAUREEN NEITZ

SUM M ARY A T H E O R Y O F T H E C O N E S , C O N E P IG M E N T S A N D
C O N E P IG M E N T G E N ES O F H U M A N C O L O R V IS IO N

Normal
Genes

Pigments

650 400 650

Wavelength (nm)
Cones

liSi~
KPr
Color Defects

Protanope Deuteranope Protanom alous Deuteranom alous

r
Key: Middle-wave subtypes: Q Q Long-wave subtypes: tj:

Figure 10 Sum m ary of a new theory of the genes, pigm ents, an d cone types that underlie nor
mal and defective hum an color vision. Among observers with norm al color vision th ere is an
enorm ous variety of different pigm ent gene arrangem ents. Some are relatively simple as illus-
 COLOR VISION DEFECTS 258

with different structures exist because of p o in t m utations and because of freq u en t

recom binations th at have occurred between the long- and middle-wave genes over
the span o f evolutionary time. Some o f these differences am ong the genes are re
sponsible for spectrally d ifferent subtypes o f m iddle- an d long-wave pigm ents in
the population, and within individuals. Perhaps, two subtypes o f long-wave and
two subtypes o f middle-wave are m ost com m on. D eleterious m utations in the pig
m en t genes o r their regulatory sequences th at prevent expression, or interfere
with norm al pigm ent function are n o t uncom m on. These may have accum ulated
particularly am ong the X-linked genes because m ost people have many m ore
genes than are necessary to serve n orm al function. A lthough some of these may
ultim ately lead to retinal disorder, o n e or m ore gene defects can be tolerated
w ithout serious consequences to vision through the reproductive years o f life.
W ith the exception o f genes with defects th at specifically affect expression, m any
or all of the X-linked pigm ent genes are expressed (although the level of expres
sion may vary). Only one pigm ent gene is expressed in any one cone so that each
gene is expressed in its own subpopulation o f cones. Thus, contrary to the long
held idea th at norm al trichrom ats have only three cone types, individual males
and females with norm al color vision can, som etim es, have four or m ore spectrally
different cone types.
To have norm al color vision, a person m ust express a short-wave pigm ent an d at
least one pigm ent subtype from both the middle-wave class and the long-wave
class. All in h erited color vision deficiencies are caused by the loss o f pigm ents re
quired for norm al color vision. T he m ost com m on color deficiencies can be ex-

trated by the exam ple in the u p p er left. Many are m ore com plicated and the gene com ple
m ents can include fusion genes an d genes with defects, as illustrated by the m ore right-hand
u pper gene arrays. Most or all of the functional genes are expressed. T he pigm ents produced
by the X -chrom osome genes fall into two classes, middle- an d long-wave, b ut each of these
classes is com posed of subtypes. Perhaps, two major svibtypes of middle-wave and two m ajor
subtypes of long-wave pigm ent are m ost com m on. Exam ples o f absorption spectra of the ex
pressed pigm ents are shown directly below each exam ple gene array. Each includes a short
wave-pigment (encoded by a separate gene on chrom osom e 7) an d at least one pigm ent from
each of the middle- and long-wave classes. However, norm al individual males can express m ul
tiple m iddle a n d /o r m ultiple long-w'ave pigm ent subtypes. Below the spectral curves, small
patches o f the cone mosaic that would result from each gene arrangem ent, are diagram m ed.
Each cone is illustrated as a circle. D ifferences in fill a n d outline illustrate cones with spectrally
different pigm ents (short-wave cones, solid black; middle-wave cone types, dark outline; long
wave cone types; no outline; subtypes have different fills). Each gene is expressed in its own
subpopulation of cone, thus, individuals can have as many as four or five spectrally different
cone types. T he situation in the color defects is simple. Each o f the four illustrated is reduced
in functional genes, in pigments, and in cone types from the norm al arran g em en t shown
above it. All have a short-wave pigm ent. T he p ro tan o p e and deu teran o p e are each red u ced to
a single type of X -encoded pigm ent. T he protanom alous observer is red u ced to only norm al
middle-wave subtypes, the deuteranom alous observer to only norm al long-wave subtypes.
254 JAY NEITZ AND MAUREEN NEITZ

plained by the loss o f norm al pigm ent genes. All such losses can occur by one-step
recom binations in which norm al genes are deleted from gene com plem ents that
u n derlie norm al color vision. Anom alous trichrom ats with red-green deficiencies
m aintain two or m ore o f eith er the middle-wave subtypes or the long-wave pig
m ent subtypes that are expressed in norm al color vision. D ichrom ats with red-
g reen defects have X-linked genes that pro d u ce only a single type o f cone pig
m ent.
We offer this m odel as an alternative to conventional theories o f the basis of nor
m al an d defective color vision. It has been useful for us in providing a fram ework
for organizing and re-exam ining what is known about the biological m echanism s
that u nderlie norm al an d defective color vision. We believe it is the sim plest m odel
th at can explain the facts as we un d erstan d them . This m odel will, no doubt, be
controversial. If it stim ulates fu rth e r research directed toward un d erstan d in g the
intriguing mysteries of color vision, it will have served its purpose.

ACKNOWLEDGEMENTS

S u pported by NIH grants EY09303, EY09620 an d a RPB career developm ent

award to M. N.

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 10. X-TINKED RETINITIS PIGMENTOSA

M. A. ALDRED1, M. JAY2 and A. F. W RIGHT1

1MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU,
UK and
2 Department of Clinical Ophthalmology, Moorfields Eye Hospital, City Road, London EC1V
2PD, UK

SUMMARY

X-linked retinitis pigm entosa (xlRP) is one o f the m ost severe form s o f this group
of retinal dystrophies. It has been fo u n d to be genetically heterogeneous, with
two loci (RP2 an d RP3) on the short arm o f the X-chrom osom e. T he RP3 gene
has been localised by genetic linkage analysis an d m ore finely m apped by analysis
of deletion patients. T he RP2 locus is less well defined by linkage analysis alone
and the lack o f associated cytogenetic abnorm alities is likely to m ake the identifi
cation o f this gene m ore problem atic. H eterogeneity is a source of difficulty in
genetic counselling an d gene m apping studies, since many families cannot be
unam biguously assigned to one or the o th er locus.

INTRODUCTION

Genetic disorders are an im p o rtan t cause o f blindness in W estern countries. A

study in N ew foundland by G reen et al. (1986) showed th at 24% o f all blindness
h ad an established m onogenic cause, while ab o u t 40% o f blindness in children is
genetically d eterm in ed (Fraser and F riedm ann, 1967). T apeto-retinal dystrophies
form the m ost com m on genetic cause o f blindness (W arburg, 1990), with 31% of
all genetic blindness being due to retinitis pigm entosa o r rod-cone dystrophy and
22% due to cone, cone-rod or m acular dystrophy (G reen a n d Jo h n so n , 1983).
Retinitis pigm entosa (RP) is a g roup o f progressive retinal degenerations char
acterised by n ight blindness, pigm entary retinopathy and visual field loss. D eteri
oration o f central vision com m only occurs in the later stages o f the disease and RP
is one o f the m ajor causes o f blindness in m iddle age in the UK. T here is wide vari
ation in clinical expression b u t typically, an initial ring scotom a is found in the
m id-periphery an d this slowly enlarges both towards the m acula an d peripherally
(Pagon, 1988). T he scotopic electroretinogram (ERG) is abnorm al in the early
stages o f the disease and soon becom es unrecordable (Heckenlively, 1990). Dark
adaptation is also abnorm al, with raised thresholds (D eutm an, 1977) and night
blindness is one o f the earliest symptoms o f the disease. O phthalm oscopy reveals
depigm entation and atrophy o f the retinal pigm ent epithelium (RPE) and m igra
260 M. A. ALDRED ET AL.

tion o f pigm ent laden cells into the retina. P igm ent often adheres to the narrow ed
retinal vessels giving a characteristic bone-spicule appearance, but it may also be
diffusely scattered. T he retinal blood vessels, particularly the arterioles, gradually
constrict as the disease progresses (Heckenlively, 1988).
RP may be in h erited as an autosom al dom inant, autosom al recessive o r X-linked
recessive trait. In general, the d ifferent m odes of inheritance cannot be distin
guished clinically, although carriers o f the X-linked disorder may show a charac
teristic fundus appearance (see below). T he m ajority o f cases with bilateral,
symmetrical disease probably have a genetic basis (see Jay, 1982). However, 40-50%
o f patients have no family history o f RP (sim plex RP) (Fishman, 1978; Jay, 1982;
Bundey and Crews, 1984b; Kaplan et al., 1990).

X-LINKED RP

X-linked RP (xlRP) is one of the m ost severe form s o f RP. In the first decade of
life, affected males com m only have night blindness, an d early visual field loss may
be ap p a ren t in the child tripping over things or appearing clumsy. If th ere is a
family history of RP, then the significance o f these symptoms may be recognised
and a diagnosis m ade as early as age four years (Heckenlively, 1988). In the teens,
d eg eneration of the RPE becom es apparent, visual field loss is m ore m arked and
visual acuity may deteriorate. T he p atien t starts to experience problem s in bright
light as well as in the dark. In the third decade, the loss in visual acuity progresses
and visual fields may be red u ced to 10-15. Secondary cataracts may develop. By
the age of forty, visual acuity is often re d u ced to counting fingers o r the p ercep
tion o f light and searching nystagmus may develop.
RP patients tend to be myopic and those with high myopia have the ad d ed prob
lem o f myopic degeneration. This is particularly tru e o f X-linked RP. Sieving and
Fishm an (1978) found th at 75% o f all RP patients (268 eyes) and 90% o f xlRP pa
tients (41 eyes) were myopic, com pared to 12% o f the general population. xlRP
patients had, on average, h ig h er m yopia (m ean 5.51D, SD 4.04D) than those
w'ith non-X-linked RP (m ean T.20D, SD 2.79D). T here was a suggestion th at the
X-linked group m ight be divided into two subgroups with low o r high m yopia re
spectively, bu t the sample size was small. RP patients were also found to have a
m uch hig h er frequency o f astigmatism, with 74% having cylindrical errors o f
>0.5D com pared to 19% o f the general population. T here was no significant dif
ference betw een the X-linked an d non-xlRP groups with regard to astigm atic cor
rection.
In fem ales who are heterozygous for xlRP, random inactivation of one o r o th er
o f the two X chrom osom es in each cell d u rin g early em bryogenesis (Lyon, 1961)
causes the phenotype to range from asym ptom atic to severely affected, d ep en d in g
on the p ro p o rtio n o f retinal cells with an active disease-bearing X-chrom osom e
(Bird, 1975; Fishm an et al., 1986). Many carriers over the age o f 20-30 years have
one or m ore of the following features: p erip h eral retinal d egeneration with pig
 X-LINKED RETINITIS PIGMENTOSA 261

m ent deposition, ERG abnorm alities o r a tapetal reflex in the m acular region. T he
latter is a golden m etallic sheen which is seen in the fundus o f some, bu t n o t all,
carriers (Fishman et al., 1986) an d occasionally also in young hemizygous males
(Heckenlively, 1988; van Osch et al., 1990). However, its detection can be subjec
tive an d the reflex dim inishes with age, so it is not always a helpful indicator o f car
rier status (Bird, 1975). C arriers may have clinical signs at any age but tend not to
becom e sym ptomatic until m iddle to late life (Bird, 1975). Severely affected carri
ers may be n ight blind, have reduced visual field and som etim es loss of visual acu
ity. Like affected males, myopia and astigmatism are com m on in heterozygous
females an d carriers may show myopic degeneration of the fundus (Fishman et al.,
1986).
Fishm an et al. (1986) exam ined 33 obligate carriers (i.e. females with an affect
ed father, son, or both) from 22 families, ranging from 13 to 83 years o f age. T he
carrier state was detectable by the presence o f pigm ent a n d /o r an abnorm al ERG
in 28 patients (85% ), although 3 o f these (aged 28-45) showed only myopic
changes of the fundus and the abnorm al ERG may have been due to high myopia
rath er than RR A fourth patient (aged 31) who showed myopic degeneration as
the only fundus abnorm ality refused ERG. T he rem aining four patients showed a
b orderline norm al phenotype. T hree had norm al ERG and the only fundus abnor
mality was a tapetal reflex. O ne o f these patients was only 18 years old, bu t the o th
er two were bo th aged 49. T he rem aining patient h ad a com pletely norm al fundus
appearance and was only abnorm al in one o f five ERG tests, at age 16 years. Details
o f this study are sum m arised in Table 1.

Table 1 - Phenotypes of 33 obligate xlRP carriers (summarised from Fishman et al. (1986))

RP-type changes (27 patients) No RP-type changes (6 patients)

Fundi Peripheral Tapetal reflex Myopia only(4) Normal (2)

pigm ent (16) only ( 11)

ERG Abnormal (14) Abnormal (8) Abnormal (3) Abnormal (1)

(5 tests) Refused (2) 5 /5 N ormal (3) Refused (1) 4 /5 Normal (1)

T herefore, 10-20% o f obligate carriers show a norm al or near-norm al p h en o

type when assessed in specialist centres (Fishman et al., 1986; Friedrich et al.,
1985), an d the percentage may be considerably higher outside such centres. This
has im portant im plications for carrier detection in females who are at 50% risk of
being heterozygous for xlRR It ca n n o t be assum ed that those who have no detect
able carrier phenotype do n o t carry the gene, and while retinal changes may be
com e ap p aren t in later life, the question of genetic status is m ost im portant to
those o f child-bearing age.
262 M. A. ALDRED ET AL.

PREVALENCE

T he overall prevalence of RP is about 1 in 4000 of the general population

(B oughm an et al., 1980; Bundey and Crews, 1984a). Estimates of the proportions
o f the different genetic form s vary betw een countries and with the m eth o d of
ascertainm ent. T he high p ro p o rtio n o f simplex cases is an additional com plica
tion.
In the UK, X-linked inheritance accounts for about 15% o f all RP families (Jay,
1982; Bundey and Crews, 1984b). xlRP seems to be m ore com m on in the UK than
the USA, w here it accounts for only ab o u t 8% o f all families with RP (Fishman,
1978). A higher p ro p o rtio n o f xlRP is fo u n d in genetic clinics (>20% o f families),
due to ascertainm ent bias, an d com pares with 11% fo u n d in an electrodiagnostic
clinic (Bird, 1975;Jay, 1982).

GENETIC MAPPING OF XLRP GENES

xlRP was first localised by B hattacharya et al. (1984), who re p o rted linkage of
xlRP in five kindreds to DXS7, a m arker localised in X p ll.4 - p ll.3 (Figure 1).
However, ensuing reports were in conflict over the location of xlRP relative to
DXS7. Friedrich et al. (1985) and W right et al. (1987) found that xlRP was m ost
likely to be proxim al to DXS7, while others favoured a m ore distal location
betw een DXS7 and DXS84, tightly linked to OTC (Nussbaum et al., 1985; D enton
et al., 1988; M usarella et al., 1988, 1989b). This distal localisation was su pported
by two patients who had deletions of Xp21 associated with RP (Francke et al.,
1985; de Saint-Basile et al., 1988). W right et al. (1987) suggested that xlRP is
genetically heterogeneous and C hen et al. (1989) found evidence o f genetic het
erogeneity in nine xlRP families. M eitinger et al. (1989) re p o rted linkage o f xlRP
to DXS255, a probe 15-20cM proxim al to DXS7. F our o f their five families
showed no recom binants betw een DXS255 and xlRP in a total of 17 meioses, but
th e o th er family contained two such recom binants in 12 meioses. O ne o f these
also recom bined with DXS7, placing xlRP distal to DXS7 (Figure 1) in th at fam
ily. Several authors suggested th at xlRP with tapetal reflex in one o r m ore carriers
was a distinct genetic form m apping to the distal location (Nussbaum et al., 1985;
D enton et al., 1988; Curtis and Blank, 1989; M usarella et al., 1989b), although
one ou t o f 14 carrier females in a family with the proxim al locus has been
re p o rted to show a tapetal reflex (Friedrich et al., 1985).
M ultipoint linkage analysis o f 62 families with xlRP gave overw helm ing evidence
for the existence o f two xlRP loci, with odds o f 6.4 X 109:1 (O tt et al., 1990). O ne
locus, term ed RP2, was m apped to X pl 1.3-pl 1.2. T he m ost likely location o f RP2
was 2cM proxim al to DXS14, bu t the confidence limits were wide, extending to
3cM proxim al to DXS7. T he second locus, RP3, was m apped to X p2 1 .1 -p ll.4 at a
position IcM distal to OTC (Figure 1). T h ere was also evidence that the propor-
 X-LINKED RETINITIS PIGMENTOSA 263

DXS85
DXS41
DXS28
r DXS164
DMD
DXS206
0XS84
CYBB ____
RP3
L OTC
- DXS228
DXS7
MAOA
SYN/ARAF1
__TRP2
DXS426
l CSNB
TiMP
L 0ATL1
DXS225
DXS146
DXS14
DXS1

21.1

21.2
21.31
21.32
21.33

22.1
22.2
22.3

26.1
26.2
26.3

27.1
27.2
27.3

Figure 1 L o catio n o f g en es a n d m a rk e r loci in X p. T h e figure shows th e o rd e r a n d locatio n

o f m a rk e r loci u se d in linkage analysis o f X -linked RP a n d th e localisations o f th e disease g en es
relative to th ese m arkers.
264 M, A. ALDRED ET AL.

don o f RP2 and RP3 families varied between investigators, explaining the previ
ously discrepant results from d ifferent groups.
M usarella et al. (1988) had previously proposed a n o th er xlRP locus located be
tween DXS28 and the DMD locus. O tt et al. (1990) found some evidence for a
th ird locus, located in the same region, with odds o f 293:1, while M usarella et al.
(1990) found weaker evidence from an analysis o f 20 xlRP families. However, pa
tients deleted for this region have n o t b een re p o rted to have RP (Clarke et al.,
1986; Francke et al., 1987; reviewed by W right, 1990), which argues against an
xlRP gene in this location.
T he following sections sum m arise o u r c u rre n t knowledge with regard to the lo
calisations o f RP3 and RP2.

RP3

T he region containing RP3 has been defined both by linkage analysis and by
identification o f patients with deletions o f the X-chrom osom e. Linkage analysis
indicates that RP3 is proxim al to DXS84 an d distal to OTC (Nussbaum et al.,
1985; D enton et al., 1988; M usarella et al., 1988 and 1989b), a genetic distance of
ab o u t 6cM. This localisation is su pported by two patients that were identified with
RP an d an associated deletion. Patient BB (Francke et al., 1985), a m ale with a vis
ible deletion in Xp21, suffered from D uchenne m uscular dystrophy (DMD),
chronic granulom atous disease (CCD), M cLeod phenotype, RP and m ental retar
dation. A nother male, SB, was re p o rted with CGD, RP and M cLeod phenotype
(de Saint-Basile et al,, 1988). No visible cytogenetic abnorm ality was detectable in
SB, b u t both the CGD locus (CYBB) an d DXS140 were deleted from his DNA.
T h ere was evidence of X-linked transm ission, as his m o th er had dim inished neu
trophil cytochrom e b content, weak expression o f Kell antigens an d some bilat
eral retinal pigm entation, indicating that she is a carrier o f all three traits.
O th er patients with deletions overlapping those of BB and SB bu t who do n o t
have RP have been reported. P atient NF, a nine year old male, was re p o rted with
DMD and CGD (B aehner et al., 1986), while p atien t OM, a twelve year old male,
h ad CGD an d M cLeod phenotype b u t n o t RP (Bertelson et al., 1988). Both pa
tients had deletions th at included an d ex tended distally from CYBB, indicating
th at RP3 is proxim al to CYBB (Figure 2 ). T hus physical and genetic linkage data
indicate th at the region containing the RP3 gene is flanked distally by DXS84 and
CYBB, an d proxim ally by OTC an d the proxim al BB deletion breakpoint.
As a first step towards isolating the RP3 gene, the sequences at the proxim al BB
deletion b reakpoint have been cloned (B runs et al., 1988; M usarella et al., 1991)
an d used as a m arker for long-range physical m apping and chrom osom e walking.
A detailed physical m ap spanning the region assum ed to contain the M cLeod lo
cus (XK) an d RP3 has been constructed (M usarella et al., 1991; Ho et al., 1992)
and five CpG islands have been identified th at could m ark candidate genes for XK
an d RP3. O ne o f these islands is located 50 kb distal to the BB proxim al breakpoint
 X-LINKED RETINITIS PIGMENTOSA 265

and an adjacent DNA fragm ent showed cross-species hom ology (McDowell et al.,
1990). This conserved fragm ent was used to screen retinal and retinal pigm ent ep
ithelial cDNA libraries. A 2.1 kb cDNA clone was identified. It is expressed in re t
ina an d brain, and shows hom ology to the m ouse t com plex sterility locus
(McDowell et al., 1990). However, extensive exam ination of the gene in RP3 pa
tients has failed to identify any m utation that would indicate that this is the RP3
gene (R o u x e ta l., 1992).

LOCUS BB SB NF OM S/H
BB SB NF OM S/H

DXS164 - NT - NT +

DXS142 - NT - NT +

DXS84 - + - + +

DXS141 - NT - + -

CGD cDNA - - - - +

DXS140 - - - - +

OTC + + NT NT NT

DXS7 + + NT NT NT

Figure 2 D eletio n analysis a n d RP3. S ch em atic d iag ram show ing She e x te n t o l d e le tio n s in 5
patien ts, BB (F ran ck e e t a l., 1985; K unkel e t a l., 1985), SB (d e Saint-Basile e t a l,, 1988), NF
(R oyer-Pokora e t a l., 1986), O M a n d S /H (B ertelso n e t a l., 1988) ( S /H a re first cousins with
M cL eod p h e n o ty p e a n d have id en tical d e le tio n s). In terv als I to VIII c o n ta in th e follow ing lo
ci: D u c h e n n e m u scu lar d y stro p h y (DMD) (I), DXS84 (II), DXS141 (III), M cL eod p h e n o ty p e
(XK) (IV), c h ro n ic g ra n u lo m a to u s disease (CG D) (V), DXS140 (VI), RP3 (VII) a n d O T C a n d
DXS7 (V III). NF an d O M d o n o t have RP, in d ic atin g th a t th e RP3 g e n e is p ro x im al to CGD,
while d e le tio n s in O M a n d S /H d e fin e th e sm allest re g io n o f overlap th a t m u st co n ta in th e
M cL eod locus, distal to CGD, T h e tab le shows th e p re se n c e o r ab sen ce o f various m ark ers in
DNA fro m th ese p atie n ts, su m m arised fro m th e re fe re n c e s d e ta ile d above. N T = n o t
tested.

T he RP3 gene may be associated with one of the o th er CpG islands, b u t there
could be o th er candidate genes betw een CYBB an d the BB breakpoint th at do n o t
have CpG islands. It is also possible th at p art of the RP3 gene is situated proxim al
to the BB b reakpoint an d that BB was only deleted for the term inal p art of it. Al
ternatively, the deletion in th at p atien t may have caused a position effect, such that
a change in the chrom atin structure influenced the expression o f the RP3 gene,
even though it lay outside the deletion. However, the p atien t BB suffered from a
very severe form o f RP, suggesting th at a com plete loss of gene function was m ore
likely than red u ced expression d u e to a chrom osom al position effect.
266 M. A. ALDRED ET AL.

RP2

T he RP2 locus has been m apped by linkage analysis alone, since no associated
cytogenetic abnorm alities have been rep o rted . As m entioned above, O tt et al.
(1990) localised RP2 to a broad region extending from DXS7 to the centrom ere.
M ore re cen t data have refined the localisation o f RP2 to a position proxim al to
DXS7 an d distal to DXS255 (Friedrich e t al., 1992; A ldred et al., subm itted).
However, this still represents a region o f 13-18cM (M ahtani et al., 1991). Cole
m an et al. (1990) suggested that RP2 is localised distal to DXS426 on the basis of
a recom binant in one family. However, th at family has since been shown also to
be linked to m arkers in the RP3 region (L. Hardwick, personal com m unication)
and therefore is n o t unam biguously RP2-type.
O ne of the m ain problem s with the genetic m apping o f RP2 is the difficulty in
identifying a hom ogeneous subset o f families to study. Bayesian probabilities of
RP2 versus RP3 can be calculated for individual families (W right et al., 1991; van
D orp et al., 1992), b u t the p osterior probabilities are very sensitive to the assum ed
locations o f these loci, which in the case o f RP2 is n o t accurately known.
T he m ost robust m ethod o f classifying families is to inspect the haplotypes and
identify recom binant meioses. T hose families that have recom bination events lo
calising the gene relative to OTC a n d /o r DXS7 can be unam biguously classified,
as illustrated in Figure 3. However, only a very small n u m b er o f families can be
classified in this m anner. A recen t m ulti-point analysis o f seven such RP2 families
th at includes both new and previously published data indicates th at the m ost likely
localisation for RP2 is proxim al to DXS7 a n d distal to DXS255, with the m axim um
lod score at the TIMP locus (Aldred et al., su b m itted ). This com pares well with the
data from a heterogeneity analysis of 37 predom inantly British xlRP families, in
which the m ost likely location for RP2 was 3.5 cM distal to TIMP (Teague et al.,
1994).
T here is still some scope for refining the localisation o f RP2 fu rth e r by linkage
analysis, bu t given the classification difficulties outlined above, the resolution of
the genetic localisation is unlikely to be b etter than 5 cM. F u rth er m apping studies
will increasingly rely on physical m apping techniques, such as the developm ent of
a YAC contig spanning the region. C andidate genes could be identified by search
ing for CpG islands or for sequences with cross-species homology. However, de
tailed physical m apping of a 5 M egabase (Mb) region is a m ajor undertaking. An
alternative approach is to identify expressed sequences from the region of inter
est. H um an-specific cDNAs could be cloned from a YAC intro d u ced into a ro d e n t
cell line (Eliceiri et al., 1991), o r alternatively from a h um an-rodent som atic cell
hybrid by subtractive hybridisation or by cDNA synthesis from unprocessed h eter
ogeneous nuclear RNA (Sive an d St Jo h n , 1988; Liu et al., 1989; C orbo et al.,
1990).
 X-LINKED RETINITIS PIGMENTOSA 267

I ff I S13
! 2
i 1
I 1
1
! 2

I II $ i* $ j m
L j
i

2 ' 1 1
2! 1 2
1 i 1 2
2 1 | 2
3 i 1 3
2 ' 1 2
I I1! 1 1

IV
Markers:
DXS164

1
2 DXS84 1
2 DXS7 2
2 TIMP 1
3 DXS255 i 4
2 DXS14
DXYS1X ii
F igure 3 D istin g u ish in g b etw een RP2 a n d RP3 types o f xlRP by h a p lo ty p e analysis. Sections o f
two xlRP fam ilies illu strate th e use o f h a p lo ty p e analysis to d e te rm in e w hich locus is seg reg at
ing. In (a), u n a ffe c te d m ale filjj.h a s th e affe c te d h ap lo ty p e a t DXS7 a n d m o re distal m ark ers,
in d ic atin g th a t this is an RP2 family. T h is is su p p o rte d by a n o th e r re c o m b in a n t in individual
III 13, a fem ale with p h e n o ty p ic ev id en ce o f th e c a rrie r state. A g ra p h o f th e m u ltip o in t linkage
d ata fro m this fam ily co n firm s a b ro a d p e a k in th e RP2 re g io n (W right et a l, 1991).

T he search would be greatly facilitated by the identification o f a cytogenetic

abnorm ality associated with RP2, which m ight reduce the region o f search to less
than 1 Mb. However, no n e have b een identified. A ldred et al. (1994) have
described a family in which X-linked RP co-segregates with m ental retardation
and in which linkage analysis indicates th at it may be RP2-type. This raises the
possibility th at there is a sub-m icroscopic deletion involving the RP2 gene in this
family. If this proves to be the case, th e deletion could significantly reduce the
region o f search for the RP2 gene, as with RP3.
268 M. A. ALDRED ET AL.

1
II
Markers:
0
DXS84 2
CYBB 2
OTC 1
DXS7 2
OATH 2
DXS255 3

III
(O (?)

2 6 |7
IV 1I
2 2 2
2 - 2
1 - 1
2 1 2
2 - 1
3 3 2

F igure 3 (b) shows th e reverse situ atio n , in w hich affected m ale IVg is re c o m b in a n t w ith DXS7
a n d DXS255, in d ica tin g th a t th e disease locus is distal to DXS7, co n sisten t with a n RP3 local
isation. It sh o u ld be n o te d th a t th e m ajority o f fam ilies (8 6 % in this la b o rato ry ) d o n o t show
re c o m b in a n ts th a t allow such u n a m b ig u o u s classification. W hile p ro b ab ilities can be calcu lat
ed, assig n m en t difficulties re m a in o n e o f th e m ajo r p ro b le m s in resea rc h a n d co u n se llin g in
xlRP.

PROBLEMS CAUSED BY HETEROGENEITY

A n u m b er o f problem s arise from genetic heterogeneity in RP and in the X-

linked form in particular. T he different M endelian form s can n o t be distin
guished on clinical grounds alone an d the pedigree structure often fails to pro
vide unequivocal evidence o f the m ode of inheritance. Linkage analysis can
som etim es resolve this and establish w hether the disease is X-linked o r autosom al
d om inant, but genetic heterogeneity within the X-linked group is a fu rth e r com-
 X-LINKED RETINITIS PIGMENTOSA 269

plication. Many X-linked families can n o t be unam biguously assigned to one or

the o th er locus as they lack the recom binants necessary to make a clear distinc
tion. T he difficulty th at this raises in m apping studies has been discussed above,
b u t it is also a m ajor problem in genetic counselling. In some cases, this can be
circum vented by exam ining polym orphic m arkers spanning the entire R P3/RP2
interval (Bergen et al., 1991). Provided that there is no recom bination in this
interval, which would be tru e in ab o u t 70% o f cases (Aldred et al., subm itted),
prenatal diagnosis, presym ptom atic tests and carrier identification may be possi
ble w ithout d eterm in in g which locus is involved.

GENOTYPE-PHENOTYPE CORRELATIONS IN XLRP

T here are no unequivocal phenotypic differences between RP2 an d RP3 that

allow them to be distinguished on clinical grounds alone. However, clinical stud
ies in families that can be distinguished genetically have led to some tentative cor
relations betw een genotype and phenotype. A tapetal reflex, when reliably
observed in xlRP carriers, seems to correlate with an RP3 localisation (Nussbaum
et al., 1985; D enton et al., 1988; Curtis an d Blank, 1989; M usarella et al., 1989b),
but the absence o f a tapetal reflex does no t exclude this locus. W right et al.
(1991) re p o rted th at the presence o f m oderate to high m yopia is a characteristic
feature o f the RP2-type disease in the affected males o f one large kindred, bu t it
was n o t clear w hether this was related to the prim ary disease process or was sec
ondary to severity o r early visual dysfunction. Kaplan et al. (1992) have found
that if xlRP families were classified according to the onset symptom in affected
males, families showing night blindness as the initial symptom at about age 10
years were linked to the RP3 region, while families with m yopia presenting at the
age of about 3 years showed linkage to RP2. F u rth er clinical studies are required
to confirm this proposed distinction, but the confounding effect o f allelic differ
ences within each genetic type may m ake it im possible to clarify the issue until
the genes are isolated.

WHAT ARE THE RELATIVE PROPORTIONS OF THE RP3 AND

RP2-TYPE OF XLRP?

Since it is difficult to distinguish the two xlRP loci, both clinically an d genetically,
the answer to this interesting and im p o rtan t question rem ains som ewhat elusive.
In the m ulticentre analysis of O tt et al. (1990), 75% o f families were RP3-type
with confidence limits extending from 45% to 90%. However, the relative p ro p o r
tions varied between different investigators. T he highest p roportion o f RP2 was
found in families contributed from this laboratory, whereas M usarella et al. (1988
and 1990) found no evidence o f the RP2 locus am ongst 20 families. This raised
the possibility that RP2 m ight be m ore com m on in Britain than elsewhere.
270 M. A. ALDRED ET AL.

To investigate this further, published families that included linkage data for two
or m ore m arkers between DXS84 an d DXS255 inclusive were reviewed. W here
possible, they were tentatively classified as RP2 o r RP3-type on the basis o f haplo-
types and lod scores. As detailed above, families showing recom bination with OTC
a n d /o r DXS7 can be unam biguously classified. In addition, for this study, families
th at showed one o r m ore recom binants with DXS84 but were linked to the RP2
region (TIMP-DXS255) were classified as possible RP2 families an d vice versa.
T hose families showing no recom bination, or recom bination in both regions, and
those with recom binants that only involved m ore proxim al or distal m arkers were
n o t classified. Families which were n eith er RP2 n o r RP3 (four from this laboratory
an d one re p o rted by M usarella et al. (1988 and 1990)) were excluded.
T he results (Table 2) show that overall, 50 o f the 83 families (60%) can be clas
sified. C om paring Britain with the com bined data from the rest o f E urope and
Scandinavia, the proportions o f classified families are very similar, as are the rela
tive proportions of RP2 and RP3 am ongst the classified families. Thus there is no
evidence th at RP2 is m ore com m on in Britain than the rest o f Europe on the basis
o f this approxim ate classification.
T he com bined British an d E uropean data suggest that 38% of the 34 classified
families are RP2-type. This is alm ost twice the pro p o rtio n o f RP2 found in Austra
lia (22%) and nearly three times that in A m erica (14% ). RP2 may therefore be
m ore com m on in Europe than elsewhere, bu t the num ber of classified families
from Am erica and Australia is small (n = 16), so it is n o t clear w hether this re p re
sents a true difference.
D eterm ining the relative proportions o f the two genes and w hether there is vari
ation betw een countries is an im portant issue with im plications for genetic coun
selling. Overall, 32% of families in this study were RP2-type, agreeing with Teague
et al. (1994) who found 30% RP2 in a heterogeneity analysis o f 37 predom inantly
British families. However, given the difficulties in categorising families and the
small sam ple sizes, the question o f ethnic differences may n o t be resolved until
one of the genes is cloned.

RP2, XL-CSNB AND MYOPIA

It is interesting that X4inked congenital stationary n ight blindness (XL-CSNB)

m aps to the same region as RP2 (Gal et al., 1989; B ech-Hansen et al., 1990, 1992;
M usarella et al., 1989a, 1992; A ldred et al., 1992). Both XL-CSNB and RP2 are
associated with m oderate to high myopia an d night blindness, raising the possi
bility th at the two conditions could be allelic (Wright, 1990). Perhaps som e m uta
tions could cause rod cell death and progressive retinal degeneration, as seen in
RP, while o th er m utations in the same g en e m ight produce a stationary disorder
with non-functional ro d cells bu t no p h o to rec ep to r death.
 X-LINKED RETINITIS PIGMENTOSA 271

Table 2 Proportions of RP2 and RP3 in published xlRP data

Nationality Total no. Total no. No. RP2 No. RP3 Reference
classified unclassified families families

Danish 1 0 1 0 Friedrich et al. (1985)

Danish 1 0 1 0 Friedrich et al. (1992)
Swedish 3 0 2 1 Dahl et al. (1991)
German 1 0 0 1 Meitinger et al. (1989)
French 5 4 2 3 Kaplan eta l. (1992)
Dutch 1 0 0 1 Wirth eta l. (1988)
Dutch 6 8 2 4 Bergen et al. (in press)
Dutch 1 0 0 1 van Dorp et al. (1992)
Italian 1 0 0 1 footnote 3

Total Europe 20 12 (38% )* 8 (40%)2 12 (60%)

excl. Britain

British 13 8 5 8 Wright et al. (1987,1991);

Aldred eta l. (subm itted);& 3
British 1 0 0 1 Curtis and Blank (1989)

Total Britain 14 8 (36%) 5 (36%) 9 (64%)

Mixed origin 5 13 0 5 Musarella et al. (1988, 1989b,

1990)
Latin-American 1 0 0 1 Nussbaum etal. (1985);
Musarella et al. (1989b)
American 1 0 1 0 Litt et al. (1987)

Total America 7 13 (65%) 1 (14%) 6 (86%)

Australian4 9 0 (0%) 2 (22%) 7 (78%) Denton eta l. (1988);

Wirth eta l. (1988);
Chen etal. (1989)

Combined 50 33 (40%) 16 (32%) 34 (68%)

Totals

'Expressed as a percentage of total families.

Expressed as a percentage o f all classified families.

3And additional unpublished data from this laboratory.
4All British origin.

It has been suggested th at th ere are closely linked genes for CSNB and myopia
on the X-chrom osom e an d that hyperopic XL-CSNB patients result from crossing-
over between these genes (K houri et al., 1988). However, although X-linked myo
pia has been docu m en ted (Bartsocas and Kastrantas, 1981), a linkage study of a
family with two cousins affected by XL-CSNB, one myopic an d one hyperopic,
showed no evidence o f exchange of flanking m arkers (Dry et al., 1993). In both
CSNB and RP, m yopia may be a pleiotropic effect o f the m utation, perhaps influ
272 M. A. ALDRED ET AL.

encing the growth o f the eye pre- a n d /o r post-natally. Since ocular refraction is a
m ultifactorial trait, the variation in refraction observed in RP and CSNB could be
explained by m odulation o f the disease phenotype by the genetic background. Al
ternatively, m yopia m ight be secondary to the defective vision. It has been shown
th at neonatal lid fusion in m onkeys causes the eye to enlarge and becom e myopic
(Wiesel and Raviola, 1977). It is therefore possible that the early visual defect in
CSNB o r RP causes myopia. This m ight explain why Miyake et al. (1986) found
high m yopia to be associated with a total lack o f rod function in CSNB, while those
with some residual function ten d ed to have sm aller refractive errors, and why pa
tients with non-X-linked RP have lower m yopia that those with the m ore severe X-
linked disease (Sieving and Fishm an, 1978). However, the question o f allelic rela
tionships between these disorders will n o t be answered definitively until the genes
are isolated.

CONCLUSION

Following the identification o f genetic heterogeneity in X-linked retinitis pig

m entosa, genetic m apping studies have also revealed extensive genetic hetero g e
neity in autosom al form s o f the disease. C onsiderable progress has b een m ade
towards the identification o f the RP3 gene, located in the Xp21.1 region, due
largely to the availability of patients with deletions in this region. T he RP3 type
appears to be the m ost com m on form o f xlRP, affecting approxim ately two-thirds
o f families that can be classified, although there may be ethnic differences. The
RP2 gene was localised by linkage analysis to X plLA -pI 1.2 and has been shown to
lie in the DXS7-DXS255 interval. It has b een m ore difficult to refine this localisa
tion because o f genetic heterogeneity a n d the absence o f chrom osom al rear
rangem ents affecting this locus.

ACKNOWLEDGEMENTS

We are grateful to the British Retinitis Pigm entosa Society, the N ational Retinitis
Pigm entosa Foundation an d G eorge G und Foundation for financial support.

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keys. Nature 266, 6668.
W irth B., D enton M .J., C h e n J. D., N eu g eb au er M., Halliday F. B., Van Schooneveld M., D onald
J., Bleeker-wagemakers E., Pearson P. 1 .. Gal A. (1988), Two differen t genes fo r X-linked re
tinitis pigm entosa. Genomics 2, 263-266.
W right A. F. (1990). Towards the identification o f genes in X-linked retinitis pigm entosa. Prog.
Retina Res. 9, 1-31.
W right A. F., B hattacharya S. S., Clayton J. F., D em pster M., T ip p ett P., Mckeown C. M. e., Jay M.,
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W right A. F., B hattacharya S. S., A ldred M. A., Jay M., C arothers A. D., T hom as N. S. T., Bird A.
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11. CONGENITAL STATIONARY NIGHT BLINDNESS:
A CRITICAL REVIEW FOR MOLECULAR APPROACHES

ELISE HEON and MARIA A. MUSARELLA

The Hospital for Sick Children, Research Institute, Departments of Ophthalmology

and Genetics, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada

SUMMARY

In this chapter, the g roup o f disorders re ferred to as congenital stationary night

blindness (CSNB) is reviewed. T he term CSNB will be used to describe a sub
group o f in h erited disorders characterized by non-progressive night blindness,
norm al retinal appearance and M endelian inheritance. T he un d erstan d in g o f
CSNB requires knowledge o f tests th at m easure the function o f the retina in vari
ous conditions of light- and dark-adapted states. T he principles o f electrophysio-
logical testing are reviewed. T he re a d e r is also in troduced to the classifications o f
CSNB based on these tests. T he ch a p te r discusses two anim al m odels for CSNB,
linkage studies done on CSNB families, and cu rren t developm ents in the rela
tionship of CSNB to Aland Island eye disease an d D uchenne m uscular dystrophy.

INTRODUCTION

N ight blindness, o r nyctalopia, is a sym ptom o f a wide group o f retinal disorders.

It can be acquired o r inherited, a n d if inherited, can be eith er progressive or
non-progressive (stationary). U nfortunately, no uniform classification has been
developed for the g roup o f disorders known as stationary night blindness, and
the nom enclature is often confusing. For instance, the French literature uses
h em eralo p ie (Franceschetti, Babel & Francois, 1963) to describe night blind
ness (Larousse & Auge, 1974), w hereas in English, hem eralopia refers to day
blindness. This ch ap ter focuses on a group of non-progressive in h erited retinal
disorders, present at birth, in which the retin a has a norm al appearance. This
group is re ferred to as congenital stationary night blindness (CSNB).
Classifications for CSNB are provided that are useful in un d erstan d in g the vari
ous aspects o f these diseases. We also review the historical background, including
the exceptionally w ell-docum ented pedigrees that have given CSNB a special place
in hum an genetics. T he fundam entals o f electrodiagnostic tests are discussed,
since they are crucial no t only to the diagnosis bu t also to an un d erstan d in g o f the
pathogenesis of CSNB. Finally, we provide a sum m ary of the present state of
knowledge o f m olecular studies carried ou t to m ap the genes for CSNB. A lthough
278 ELISE HEON AND MARIA A. MUSARELLA

some work has been done on the autosom al d o m in an t CSNB (Yijian et al., 1991),
most o f the linkage studies have been co n ducted on the X-linked form (Volker-
D ieben & W ent, 1975; M usarella et al., 1989; B ech-Hansen et al., 1990; Schwartz &
Rosenberg, 1991; A ldred et al., 1992; Bech-H ansen, M oore & Pearce, 1992;
M usarella et al., 1992). T he results suggest a single gene may be responsible for
several clinical entities on the X-chrom osom e.

CLASSIFICATION OF INHERITED STATIONARY NYCTALOPIA BASED

ON FUNDUS APPEARANCE

We define any non-progressive n ight blindness th at is in h erited an d p resen t at

b irth as in h erited stationary nyctalopia. This category is fu rth e r divided into two
groups d ep en d in g on the appearance o f the retinal eyegrounds (also known as
the fu n d u s). T he first g roup includes patients with a norm al fundus, who are clas
sified as having congenital stationary n ig h t blindness (CSNB), a term initially
used by N ettleship (1912) and later by C arr (C arr & Siegel, 1964; Carr, 1974) to
designate a congenital non-progressive nyctalopia with norm al or abnorm al fun
dus. To avoid confusion, in this ch ap ter CSNB will only be used to define a group
o f in h erited retinal disorders characterized by non-progressive night blindness
p resen t at birth, norm al daylight vision an d visual fields, norm al appearance of
the fundus, and specific results on electroretinographic (ERG) an d psychophysi
cal testings. CSNB may be in h erited through one o f the three m odes o f M ende-
lian inheritance (Table 1).

T able 1 I n h e r ite d sta tio n a ry n ig h t b lin d n e ss. C lassificatio n by fu n d u s a p p e a ra n c e

Normal fundus Abnormal fundus

Congenital stationary night blindness Oguchi's disease (autosomal dominant)

Autosomal dom inant Fundus albipunctatus (autosomal recessive,
Autosomal recessive autosomal dom inant uncommon)
X-linked recessive Flecked retina of Kandori (autosomal recessive?)

T he second group, in h erited stationary nyctalopia associated with abnorm al

fundus appearance (Carr, 1974; Vaghefi et al., 1978; Jim enez-Sierra, O gden & Van
Boem el, 1989), includes three specific disorders that will be briefly discussed for
the sake o f com pleteness: O guchis disease, fundus albipunctatus an d flecked re t
ina o f Kandori. In O guchis disease (O guchi, 1907), the fundus is characterized by
a peculiar greenish-golden discoloration. After prolonged dark adaptation (2-3
hrs), this m etallic sheen disappears, leaving a perfectly norm al-looking retina.
This change in the retina is called the Mizuo p h e n o m e n o n (Mizuo, 1913). If the
fundus shows flecks, a diagnosis of eith er fundus albipunctatus (small flecks)
(M ooren, 1882;Lauber, 1910) o r flecked retin a of K andori (large irregular flecks)
 A CRITICAL REVIEW FOR MOLECULAR APPROACHES 279

(Kandori, 1959; Kandori, Setogawa & Tam ai, 1966; K andori et al., 1972) should be
considered.

INHERITANCE OF CSNB: HISTORICAL BACKGROUND

T hree M endelian form s o f in h eritan ce o f CSNB have been described: autosom al

dom inant (Cuniers, 1838), autosom al recessive (Gassier, 1925) an d X-linked
recessive (D onders, 1855). T he pedigrees for CSNB include some o f the m ost
extensive an d w ell-docum ented in hum an genetics. CSNB has supplied us with
one o f the m ost com plete genealogical records of an autosom al d o m in an t disor
der. R. C uniers, a Belgian ophthalm ologist (C uniers, 1838), was the first to rep o rt
this fam ous pedigree o f the oldest family known to be affected with CSNB. It has
been traced back to Je a n N ougaret, a butcher, who lived in the latter p a rt o f the
17th century in the small F rench village o f Vendem ian. T he pedigree was discov
ered only in the early 1830s, when a fifth-generation descendant o f N ougaret
claim ed exem ption from the F rench arm y because o f p o o r n ight vision. T he
exam ining surgeon fo u n d that the young m an could read by candlelight, thought
he was m alingering, fo u n d him fit for service and enrolled him in the arm y for 7
years. It was n o t until years later, w hen the m an was re-exam ined, th at he con
veyed the familial n atu re o f his night blindness an d was finally exem pted from
m ilitary duty (Snyder, 1963; Krill, 1977).
In 1907, N ettleship published an u p d ated pedigree o f the N ougaret family and
confirm ed the stationary n ature o f th eir n ig h t blindness (N ettleship, 1907). This
study encom passed nine generations and included 2121 persons, o f whom 135
were affected (Carr, 1974). An attem p t was m ade in 1949 to up d ate this pedigree
fu rth e r (D ejean & Gassenc, 1949), b u t the two world wars an d changing lifestyles
had led to the dissolution o f the original genetic isolate. Today this form o f CSNB
(dom inantly inherited) is referred to as the N ougaret variety (Carr, 1974). Many
o th er autosom al d o m in an t CSNB pedigrees have been described (Stievenart,
1847; Sedan, 1885; H udson, 1903; B essonnet, 1904; Sinclair, 1905; Snell, 1907;
Bordley, 1908; C arroll & Haig, 1952; Latte, M anunta & Pisano, 1954; Ram busch,
1954; Noble, C arr & Siegel, 1990; Yijian et ah, 1991).
T he first X-linked form o f CSNB was re p o rted by D onders (1855); the m ost fa
m ous pedigree of this form is that o f Pfluger-Am man-Kleiner (1898). T he first au
tosom al recessive form o f CSNB was re p o rte d by Gassier in 1925.

CLINICAL MANIFESTATIONS

Because o f the paucity o f symptoms a n d ocular findings, CSNB is probably one of

the m ost frequently m isdiagnosed g ro u p o f ocular disorders. T he m ost com m on
com plaint is decreased n ig h t vision, b u t because n ight blindness is p resen t at
birth, patients are often unaw are o f th eir handicap until later in life. Since the
280 ELISE HEON AND MARIA A. MUSARELLA

m ost freq u en t sign is nystagmus (jerky eye m ovem ents), patients are often ini
tially diagnosed as having ocular albinism o r congenital nystagmus. It may be dif
ficult to differentiate these conditions from CSNB w ithout p ro p e r electrodiag
nostic evaluation.
T he phenotypic distinction o f each p attern o f in h eritan ce is not strict and over
laps do occur. However, both visual acuity and refraction b ear some relationship
to inheritance. Visual function varies from norm al acuity (20/20) to legal blind
ness (20/200). In autosom al d om inant CSNB, im paired night vision is often the
m ain defect (C uniers, 1838). Visual acuity is usually within the norm al range (2 0 /
2 0 -2 0 /4 0 ) with a m ild refractive e rro r ( 0.50 D - +0.50 D) if any. Myopia is rarely
seen. In the autosom al recessive phenotype, vision is usually m oderately im paired
(about 2 0 /8 0 o r b etter), an d the myopia tends to be m ild to m oderate.
In males affected with X-linked CSNB, visual loss can show intra- and inter-famil-
ial variability (Pearce, Reedyk & C oupland, 1990). In these cases vision is usually
abnorm al, ranging from 2 0 /3 0 to 20/200. Visual acuity o f 2 0 /2 0 appears to be
rare in this subgroup. A lthough refractive e rro r is usually o f high myopia, it may
range from m ild hyperopia (+0.50) to severe myopia (up to 20 D) (D onders,
1855; C arroll & Haig, 1952; Francois, Verriest & De Rouck, 1965; Carr, 1974; Krill,
1977; Haim , 1985; K houri et al., 1988; Fishm an, 1990; Noble, C arr & Siegel, 1990;
Pearce, Reedyk & C oupland, 1990).
Nystagmus is a com m on finding in patients born with subnorm al vision (less
than 20/60) (Krill, 1977). In m ost cases o f CSNB the nystagm us is horizontal and
may be fine o r coarse. Fine rotary nystagmus has also been described (M erin et al.,
1970). Strabism us is often present, bu t th ere is no predom inance o f esotropia or
ex otropia (N ettleship, 1912; M erin et al., 1970; Carr, 1974; Krill, 1977; M erin,
1991). A nisom etropia (a difference in refractive e rro r greater than 1.5 D between
eyes) an d astigm atism are usually m ild (1.0-2.0 D). Amblyopia, which is n o t spe
cific, may also be seen (M erin et al., 1970; Krill, 1977; K houri et al., 1988; Pearce,
Reedyk & C oupland, 1990).
Findings o f the fundus exam ination are generally norm al. However, w hen myo
pia is m ore than 4.0 D, the retin a may show characteristic changes o f myopia, such
as a tigroid fundus and thinning o f the retina, but blood vessel caliber is always
n o rm al and no pigm ent clum ping is seen. Cases have been re p o rted o f the optic
disc being tilted, pale or dysplastic (Mintz H ittner, Borda & Justice, 1981; H ecken
lively, M artin & R osenbaum , 1983). C arr (1966b) also describes an occasional in
crease in granularity of the macula, which can alter the norm al foveal reflex.
Barricks et al. (1977) described paradoxical pupillary responses, in which the
pupil constricts in the dark, in three boys with CSNB of unspecified inheritance
(Barricks, Flynn & Kushner, 1977). Since this p h en o m en o n is no t seen in older
affected males (> 55 y.o.), it is considered a useful diagnostic finding in young chil
d ren w hen present (Khouri et al., 1988).
Vitreous fluorophotom etry, a useful tool in the evaluation o f the blood-retinal
barrier, was p erfo rm ed on three types o f n ight blindness: O guchis disease, X-
 A CRITICAL REVIEW FOR MOLECULAR APPROACHES 281

linked CSNB an d fundus albipunctatus. All eyes showed norm al values, strongly
suggesting th at the blood-retinal b a rrie r is intact (Miyake et al., 1983).
C olour vision is usually unaffected in CSNB, bu t occasionally a m ild tritan defect
can be seen (C arroll & Haig, 1952; A rm ington & Schwab, 1954; Franceschetti, Ba
bel & Francois, 1963; Miyake e ta l., 1986). R ed-green deficiency has been re p o rt
ed, regardless o f the p attern o f in h eritan ce (M erin et al., 1970; Ponte, Lodato &
Lauricella, 1974; Miyake et al., 1986), an d is n o t felt to be related specifically to
CSNB bu t ra th e r to an u n related red-green colour gene defect located at Xq 28
(McKusick, 1990a).
Visual fields are usually norm al w hen tested u n d e r lighted condition (photo-
pic). However, if patients are tested in dim illum ination, visual field constriction
may be no ted (Carr, 1974; Krill, 1977; Miyake et al., 1986).
Physical anom alies are usually n o t re p o rted in patients affected with CSNB;
however, M aeder (1946) re p o rted a family of autosom al d om inant CSNB in four
generations w here p lan tar syndactyly (fusion o f two toes) was a regular finding.
U nfortunately, no long-term follow-up of this family is available.

ELECTROPHYSIOLOGIC EVALUATION OF CSNB

T he diagnosis o f retinal diseases can be substantially aided by the use o f electro-

physiologic tests. E lectrodiagnostic procedures used to study the retina will be
briefly described as a p reparation for detailed discussion of th eir im portance in
the diagnosis o f CSNB.

Basics o f Electroretinography

T he electroretinogram (ERG) is a recording o f a flash-evoked electrical potential

that arises in the retina. T he m ost effective stim ulus is delivered by a full-field
hom ogeneous illum ination known as a Ganzfeld system. T he electrical response
is recorded from the co rn ea with a double-electrode contact lens, in tro d u ced by
Riggs in 1941, and represents an objective m easurem ent o f total mass response of
the retina. Localized diseases th erefo re may not show any changes on ERG test
ing. T he o u tp u t o f this response is am plified an d displayed on an oscilloscope.
T he norm al ERG is m ade up o f several com ponents th at can be separated,
d ep en d in g on the stim ulus presented, by varying the intensity a n d /o r wavelength
as well as the frequency o f the stim ulus and the state o f the retinal adaptation
betw een light and dark (Fishm an, 1985). These differences form the basis o f tests
designed to activate rods only (scotopic), bo th rods an d cones (m esopic), or
cones only (photopic) (Fishman, 1985; O gden, 1989) (Figure 1). T he ability to
separate rods and cones perm its m any retinal disorders to be differentiated. Rod-
specific responses can be elicited after a retin a has been dark-adapted for 30 to 40
m inutes with a single flash o f blue light (usually less than 470 nm ) o r a very low
intensity white light. A bright white flash produces responses from both cones
282 ELISE HEON AND MARIA A. MUSARELLA

and rods, as does a single red flash on a dark-adapted retina. Pure cone responses
are o btained with either a red or white light flickering at a frequency o f 30 cycles
p er second (H z), o r from a light-adapted retin a following a single flash o f yellow-
re d o r blue-green stimulus (Fishman, 1985).
T he three main com ponents of the ERG (Einthoven & Jolly, 1908; Jim enez-
Sierra, O gden 8c Van Boemel, 1989) are the a-wave, b-wave an d c-wave (Figure 1).
T h e a-wave, the initial cornea-negative com ponent, is g enerated by the p h o to re
cep to r layer. It is followed by a cornea-positive b-wave that arises from the in n er
nuclear layer (bipolar, M uller and am acrine cells). T he c-wave, p roduced by the
retinal pigm ent epithelial (RPE) cells, is a cornea-positive response th at is n o t rou
tinely used clinically (Fishman, 1985; Jim enez-Sierra, O gden & Van Boemel,
1989). T he ganglion cell layer does no t contribute to the ERG.
T he m ost freq u en t m easurem ents taken with the ERG are the a- and b-wave am
plitudes and the b-wave im plicit time (tim e from flash to peak o f b-wave). In
CSNB, the b-wave is m ost severely affected, an d in some form s of the condition, its
am plitude can be red u ced so drastically that it becom es negative. Several o th er
co m ponents o f the ERG are clinically useful. For exam ple, the am plitudes o f the
oscillatory potentials (OPs) (O gden, 1973), wavelets on the ascending limb o f the
b-wave, can be affected by diseases that are due to neurotransm ission defects in
the vicinity o f the in n er plexiform layer (Lachapelle, Little & Polom eno, 1983;
Miyake, Yagasaki & H origuchi, 1987c).
 A CRITICAL REVIEW FOR MOLECULAR APPROACHES 283

Electroretinography and CSNB

T he ERG plays a key diagnostic role in CSNB. Characteristically, in any o f the

in h erited stationary night blindness disorders, the scotopic or ro d ERG is pre-
dom inandy affected (Ponte, L odato & Lauricella, 1974). However, in CSNB the
dysfunction is n o t lim ited to the scotopic system (G oodm an & B ornschein, 1957).
T he cone photoreceptors also show a variable degree of involvem ent (Auerbach,
Godel & Rowe, 1969; Kawasaki, T usuchida & Jacobson, 1971; Krill & M artin,
1971; Lachapelle, Little & P olom eno, 1983). Two patterns o f ERG have been
described for CSNB: the Schubert-B ornschein an d the Riggs types (Table 2 and
Figure 2).

Table 2 Congenital stationary night blindness. Classification by electroretinography (ERG)

Schubert-Bornschein ERG Riggs ERG

Scotopic a-wave: norm al to subnormal Scotopic a-wave: norm al to subnormal

Scotopic b-wave: reduced to absent,
smaller than scotopic a-wave
b /a < 1
i. complete (no rod adaptation)
ii. incomplete (rod adaptation present)
Scotopic b-wave: near norm al to reduced
in amplitude, larger than scotopic a-wave
b /a > 1

T he first authentic case o f CSNB studied electrophysiologically was that o f Schu

b e rt an d B ornschein (S chubert & B ornschein, 1952; B ornschein & Vukovich,
1953) (Figure 3). T he S chubert-B ornschein ERG is a negative type in which the
am plitude o f the a-wave (norm al o r slightly subnorm al) is larger than that o f a re
duced b-wave (Vukovich, 1952; B ornschein & Vukovich, 1953). T h e Riggs-type
ERG is characterized by a reduced scotopic b-wave th at rem ains larger in am pli
tude than the a-wave (which is eith er norm al or subnorm al) (Riggs, 1954;
Fishm an, 1990). A lthough m ost cases o f the Riggs type have frequently been asso
ciated with autosom al d om inant CSNB and those of the S chubert-B ornschein
type (negative b-wave ERG) described for all form s of CSNB inheritance, Auer
bach et al. (1969) illustrated th at the division into groups derived from the ERG
p atterns had no relation to heredity (A uerbach, Godel & Rowe, 1969). Miyake et
al. (1986) subdivided the patients with S chubert-B ornschein ERG into two cate
gories, com plete an d incom plete, based on the presence or absence of rod func
tion on ERG and psychophysical d ark adaptation (Miyake et al., 1986) (Table 3).
T he com plete form o f CSNB lacks ro d function entirely, has no dem onstrable
scotopic rod-m ediated ERG b-wave, a n d produces night blindness. Visual acuity is
m oderately p o o r a n d psychophysical d ark adaptation o f rods is absent. Patients af
fected by this condition are known to have m oderate to severe m yopia (m ean SD;
7.9 8.1 D), an d oscillatory potentials are m arkedly reduced (Hill, Arbel & Ber-
son, 1974; Heckenlively, M artin & R osenbaum , 1983; Lachapelle, Little & Polom-
284 KI.ISK HKON AND MARIA A, MI sARKI I A

Figure 2 S cluibei l-B o rn si hem S i t . versus Ui^gs-lypc* ERG.

eno, 1983). A lthough this phenotype is m ost frequently seen with X-linked
in h eritance, it has also been re p o rted in autosom al d om inant an d autosom al re
cessive pedigrees (Noble, C arr & Siegel, 1990; Yijian et al., 1991).
Patients with incom plete CSNB exhibit m easurable, albeit reduced, scotopic b-
wave am plitudes, decreased night vision sensitivity an d less severe m yopia than
those with the com plete form (m ean 2SD; 0.8 10.8 D) (Heckenlively, M artin
& R osenbaum , 1983; Miyake et al., 1987a; Miyake, Yagasaki & H orogushi, 1987b).
Visual acuity may be norm al o r n ear norm al. Oscillatory potentials are m ore easily
reco rded than in com plete CSNB (Lachapelle, Little & Polom eno, 1983; Miyake,
Yagasaki & H origuchi, 1987c; Miyake & Kawase, 1984). This phenotype has also
been described in the three M endelian patterns of inheritance. Initially, the in
com plete CSNB was th ought to be a variant o f the com plete form , distinguished
mainly by the severity of functional disturbance. However, Miyake et al. (1986) fur-
 A CRITICAL REVIEW FOR MOLECULAR APPROACHES 285

T ab le 3 S ub classificatio n o f S c h u b e rt-B o rn s c h e in ERG

Complete Incomplete

Dark Adaptation absent present

ERG
Bright flash negative negative
* Oscillatory potentials absent present
30 Hz flicker norm al poor

th er characterized both subgroups an d provided evidence that the com plete and
the incom plete types o f CSNB are different clinical entities. O thers have debated
w hether these types are phenotypically an d genotypically distinct (Khouri et al.,
1988; Pearce, Reedyk & C oupland, 1990). In considering these questions, we be
lieve it is im portant to recognize th at the ERG test stim ulus used by Miyake et al.
(1986) is unique. T h eir use of a rectangular ra th e r than a flash-evoked stimulus
resulted in d ifferent ERG patterns, m aking com parison with o th er groups diffi
cult. This may contribute to th eir distinction betw een com plete and incom plete
CSNB. As we note later, linkage studies o f Miyake's incom plete families do no t
support separation into two disorders.

Figure 3 E le c tro re tin o g ra p b ic c o m p ariso n o f th e S c h u b e rt-B o rn ste in c o m p le te a n d in co m

p lete CSNB.
286 ELISE HEON AND MARIA A. MUSARELLA

T he photopic (cone) system is also affected in CSNB. Patients with negative

ERGs have been shown to have red u ced am plitudes o f their p hotopic (cone) ERGs
and an altered flicker response (Krill & M artin, 1971; Hill, Arbel & Berson, 1974;
L achapelle, Little & Polom eno, 1983; Miyake et al.. 1986, 1987a). These changes
are m o re severe in incom plete CSNB, in which cone am plitudes are m arkedly re
du ced along with the 30 Hz flicker cone responses (Miyake et al., 1986, 1987a). In
com plete CSNB, the photopic system is essentially norm al (Figure 3).
W hen Miyake (1987) fu rth e r studied patients with incom plete CSNB, he was
able to d em onstrate a peculiar p h en o m en o n o f the ERG waves after the eye was
dark-adapted an d stim ulated continuously for 12 to 15 m in with a 30 Hz flicker.
T he ERG showed an exaggerated increase in am plitude and a characteristic
change in ERG wave shape, a separation p h e n o m e n o n n o t seen in norm al sub
jects, com plete CSNB, o r any o th er kind of retinal dystrophy exam ined. This
change appears unique to incom plete CSNB (Miyake et al., 1987a) and may result
from a type o f rod-cone interaction specific to this type (Granit, 1938; H ood,
1972a,b). A lthough there has been controversy (Krill & M artin, 1971; Hill, Arbel
& Berson, 1974) about the norm ality of the im plicit tim e o f the scotopic ERG in
CSNB, it has been re p o rted to be norm al in m ost cases.
In com plete CSNB, the associated decreased am plitude b-wave has been inter
p reted as reflecting a focal destruction o f retinal elem ents (Berson, Gouras &
Hoff, 1969; Berson & Howard, 1971; Krill & M artin, 1971). This has led some in
vestigators to postulate a decrease in the cone population or at least an im pair
m en t in the photopic (cone) system (Hill, Arbel & Berson, 1974). However,
affected patients do not re p o rt visual anom alies that could be explained on the ba
sis o f a decrease in the cone population. For exam ple, photopic visual fields and
colour vision are usually within the norm al range (A rm ington & Schwab, 1954;
Krill, 1977). F urtherm ore, decreased vision is n o t a m ajor com plaint in patients
with CSNB (Krill, 1977).
By studying on- and off-responses u n d e r photopic conditions, Miyake, Yagasaki
& H origuchi (1987c) were able to d eterm in e the site o f im pairm ent o f the p h o to
pic visual pathway. T he rapid off-response represents the prim ary co m p o n en t of
the b-wave, and the late off-response reflects the decay o f the late recep to r p o ten
tial o f the cones (Kawasaki, Tusuchida & Jacobson, 1971). T he positive on-re-
sponse (b-wave) was significantly sm aller than norm al in both com plete and
incom plete CSNB. T he rapid off-response was norm al in the com plete type but
significantly sm aller than norm al in the incom plete type. These results indicate
that, in the com plete type, the photopic visual disturbance is in the m iddle retinal
layer, and the cone function is essentially intact. In the incom plete type, however,
the visual disturbance is in the cone itself.
A lthough the negative ERG is characteristic of CSNB, it can be seen in o th e r dis
orders. T he m ost im portant differential diagnoses o f the negative ERG to be con
sidered are listed in Table 4 (Miyake, Yagasaki & H orogushi, 1987b; W eleber et al.,
 A CRITICAL REVIEW FOR MOLECULAR APPROACHES 287

1989). O f these, special atten tio n will be given to Aland Island eye disease (AIED)
an d D uchenne m uscular dystrophy (DMD) in a later section.

T ab le 4 D iffe re n tia l d ia g n o sis o f N egative ERG s

1) Stationary
1.1 complete congenital stationary night blindness
1.2 incomplete congenital stationary night blindness
l.SO guchi's disease
2) Retinal dystrophies
2.1 Early retinitis pigmentosa
2.2 Infantile Refsums disease
2.3 Goldman-Favre vitreoretinopathy
2.4 Juvenile retinoschisis
2.5 Progressive myopia
3) Vascular disorders
3.1 Ischemic central vein occlusion
3.2 Ischemic central artery occlusion
4) Retinal toxicity
4.1 Quinine
4.2 Vincristine
5) Paraneoplastic melanoma
6) Familial optic nerve atrophy
7) Duchenne muscular dystrophy
8) Aland Island eve disease

Modified from Weleber, RG; Pillers, D -A; Powell, B R, H anna, CE, Magenis, R E, Buist, N R M (Wele
ber, 1989)

Electrophysiology o f the X-Linked C arrier

Most carriers o f X-linked CSNB are asym ptom atic. However, because o f random
inactivation o f the X chrom osom e (lyonisation), they may show signs and symp
toms o f the disease. Some may show evidence o f nystagmus a n d /o r ERG changes
(Pearce, Reedyk & C oupland, 1990), b u t the majority of these fem ale carriers are
n o t identified by routine ocular exam ination (Miyake, Yagasaki & H orogushi,
1987b; Bech-Hansen e ta l., 1990). Subtle changes have been d o cum ented by ERG
testing. Miyake an d Kawase (1984) re co rd ed statistically sm aller oscillatory p o ten
tials (OPs) and am plitudes in carriers o f X-linked CSNB than in non-carrier
women. T he im plicit tim e o f the OPs, a n d the am plitudes o f the a- and b-waves,
were within norm al limits. Young et al. (1989) established that the OPs are opti
mally reco rd ed in the X-linked carriers when the dark-adapted eye is stim ulated
by a blue flash.
288 ELISE HEON AND MARIA A. MUSARELLA

Recently, R uttum , Lewandowski and B atem an (1992) re p o rted the clinical find
ings an d the results of ERG testing and dark adaptom etry in four sym ptomatic car
rier fem ales from a five-generation family with X-linked CSNB. The scotopic ERGs
showed a negative b-wave consistent with the S chubert-B ornschein type of CSNB.
T he p o o r rod-m ediated dark adaptations found in some sym ptomatic carriers are
th o u g h t to be a consequence o f lyonization.

SPECIAL CONSIDERATIONS WITH AIED AND DMD

A lthough many o f the disorders causing a negative ERG can be recognized by

an adequate evaluation, a gray z o n e does exist; this m ust be kept in m ind when
evaluating these cases. We will focus o u r atten tio n initially on AIED, a disorder
m arked by controversy. It has been th o u g h t to be related to incom plete CSNB
an d later to DMD because o f a unique case with a deletion o f Xp21, com plex glyc
erol kinase deficiency, DMD, and a negative ERG.
T he family in whom AIED was first described (Forsius & Eriksson, 1964) origi
n ated in the Aland Islands in the Gulf o f B othnia, and was affected with an X-
linked ocular disorder th o u g h t to be a form o f ocular albinism, OA2 (Forsius &
Eriksson, 1964; McKusick, 1990b). OA2 is characterized by hypopigm entation of
the fundus, hypoplasia o f the fovea, im paired vision, nystagmus, progressive myo
pia, astigm atism an d dyschrom atopsia. In 1967, W aardenburg, Eriksson an d For
sius (1969) reviewed the features present in 10 affected males and 2 carriers from
the original pedigree of Forsius an d Eriksson. Visual acuity was variable in affected
males, ranging from 20/45 to 20/400. Myopia ranged from -1.50 D to 19 D, with
regular astigmatism between 1.25 D an d 7.0 D. Except for a m ild increase o f axial
m yopia in o lder patients, OA2 was known to be a non-progressive disorder
(W aardenburg, Eriksson & Forsius, 1969; McKusick, 1990b). T he irides transillu
m inated to a variable degree in half o f the patients, but fu rth e r studies showed
that, unlike what is seen in Nettleship-Falls ocular albinism, hypopigm entation
varied in d ifferent areas o f the fundus and was mostly lim ited to the posterior pole
(W itkop, Q uevedo & Fitzpatrick, 1983). Fem ale carriers dem onstrated m ild co
lo u r vision defects an d fine nystagmus, bu t did no t d em onstrate the characteristic
m osaic fundus p attern o f Nettleship-Falls ocular albinism (Forsius & Eriksson,
1964). T he work o f O D onnell et al. (1980) showed that, unlike Nettleship-Falls
albinism, carriers and affected OA2 patients did not show m acrom elanosom es on
skin biopsy (Writkop, Q uevedo & Fitzpatrick, 1983). In addition, van D orp et al.
(1985) dem onstrated th at optic nerve fibre m isrouting does no t occur in OA2, as
it does in all types o f albinism (van D orp et al., 1985). This finding strongly sug
gested th at OA2 was n o t an albinotic disorder. Consequently, the disease symbol
was changed from OA2 to AIED at the T enth In tern atio n al H um an G ene M apping
W orkshop in 1989 (Rosenberg, Schwartz & Sim onsen, 1990).
C onfusion arose w hen W eleber et al. (1989) re p o rted a child, O regon J R, with
a d eletion at Xp21 an d glycerol kinase deficiency, congenital adrenal hypoplasia,
 A CRITICAL REVIEW FOR MOLECULAR APPROACHES 289

an d AIED (W eleber et al., 1989). T he ERG responses o f this child were sim ilar to
those for AlED an d identical to those re p o rte d for incom plete CSNB, as described
earlier. It was suggested that AlED an d incom plete CSNB m ight be the same dis
ease, thus initially localizing this form o f CSNB to Xp21 (W aardenburg, 1970;
W eleber et al., 1989). F u rth er m olecular studies o f this patient localized the dele
tion to the subbands Xp21.3-p21.2, betw een DXS67 an d DMD (Miyake, Yagasaki
& H orogushi, 1987b; Pillers et al., 1989a, 1989b, 1990a, 1990b; W eleber et al.,
1989) (Figure 4). In 1991, linkage studies were re p o rted on two families with AlED
(Alitalo et al., 1991; Schwartz & Rosenberg, 1991). Both studies positioned AIED
on the proxim al p a rt o f the short arm o f the X chrom osom e. A lthough these re
sults did n o t support the work o f Pillers et al., th eir data suggested th at AIED and
com plete CSNB may be allelic and th at the ocular findings in the p atien t de
scribed by W eleber were probably p a rt o f the syndrom e resulting from the dele
tion at Xp21, ra th e r than AIED. To clarify the m atter, the ocular disorder of this
p atien t was nam ed O regon eye disease, and the origin o f the negative ERG of
O regonJR w as suggested to lie in the Xp21.3-p21.2 region, the location of the glyc
erol kinase deficiency and DMD genes.

Figure 4 L o calization o f X -linkcd CSNB.

Since then, the gene for DMD has been isolated and its gene product, dystro
phin, has been identified. A ntibodies to dystrophin were used to d eterm in e
w hether it was expressed in the retina, and dystrophin was identified in the o uter
plexiform layer, the putative site for CSNB (Bulm an et al., 1992). In o u r experi
290 ELISE HEON AND MARIA A. MUSARELLA

ence, m ost young DMD patients tested by ERG were found to have a significantly
red u ced b-wave am plitude, suggesting th at dystrophin may play a role in norm al
retinal neurotransm ission (M usarella et al., unpublished d a ta ). F u rth er studies to
d eterm in e the role o f dystrophin in the retina may contribute to a b etter u n d er
standing o f the X-linked form s o f CSNB.

PSYCHOPHYSICAL EVALUATION OF CSNB

Basics o f Dark Adaptation

Because dark adaptom etry m easures the ability o f the p hotoreceptors to adapt
over time in the dark, it is mainly indicated for patients suffering from nyctalopia.
In this test the patient is initially dark-adapted for 2 m inutes, then light-adapted
for 10 m inutes to bleach the photoreceptors. T he background light is then extin
guished an d the p atien t is asked to identify the test target, which has adjustable
intensities ( Jim enez-Sierra, O gden & Van Boemel, 1989; Fishm an, 1990). The
ability to dark-adapt is then m easured over a 30-m inute period with the dark
adaptom eter. A norm al curve has a b ipartite shape; the first 10-minute segm ent
rep resents the cones adaptation, an d the rest o f the curves rep resen t the rods'
adaptation (Figure 5).

Legend:------- normal curve

o I------------ 1------------ 1----------- 1________ I________l________ L

5 10 15 20 25 30

Time of Dark Adaptation (min)

F igure 5 N o rm al d a rk a d a p ta tio n curve.

 A CRITICAL REVIEW FOR MOLECULAR APPROACHES 291

Dark Adaptation and CSNB

T he initial psychophysical studies o f CSNB p erfo rm ed by D ieter (1929) and later

by G arabedian an d M eunier (1942) identified two types o f dark adaptation
curves in CSNB patients. F u rth er studies by A uerbach, Godel an d Rowe (1969)
and by Miyake et al. (1986,1987b) substantiated the existence o f these two curves,
which today are correlated with the presence or absence of rod function (Miyake,
Yagasaki & H orogushi, 1987b). T he curve may have a bipartite shape if some rod
adaptation is p resen t or a m onophasic shape with n o co n e -ro d break if it is
absent.
T he dark-adaptation curves of the Riggs and incom plete S chubert-B ornschein
types of ERG have a sim ilar shape (Miyake et al., 1986). Since som e rod adaptation
is still present, a bipartite co m p o n en t is preserved, although the overall adapta
tion threshold is elevated (Miyake, Yagasaki & H orogushi, 1987b). In the com plete
S chubert-B ornschein ERG, w here th ere is no rod adaptation, the dark-adaptation
curve is m onophasic and the residual cones threshold is elevated (Miyake, Yagasa
ki & H orogushi, 1987b) (Figure 6).

Legend:------- normal curve

o "complete" CSNB

. J _____________ i____________ I __ I | i

5 10 15 20 25 30

Time of Dark Adaptation (min)

F ig u re 6 D ark a d a p ta tio n curves in CSNB.

CLINICAL EVOLUTION OF CSNB

By definition CSNB is a stationary disorder, an d long-term follow-up o f the well-

d o cum ented pedigrees have shown lack o f progression (Franceschetti, Babel &
292 ELISE HE ON AND MARIA A. MUSARELLA

Francois, 1963; Carr, 1974; Krill, 1977). However, there appears to be a subgroup
of retinitis pigm entosa (RP), a form of n ig h t blindness, m arked by extrem ely slow
progression. This forme fruste of retinitis pigm entosa should n o t be confused with
CSNB (M aeder, 1946; Babel, 1963; Franceschetti, Babel & Francois, 1963; Auer
bach, Godel 8c Rowe, 1969). Because m any tapetoretinal degenerations may show
n o rm al retinal findings, usually in the early stages, it is essential to rep eat retinal
exam inations periodically. If progression is docum ented, the diagnosis o f CSNB
m ust be re-evaluated an d a tapetoretinal degeneration considered (France
schetti, Babel & Francois, 1963). T he ERG is useful in distinguishing these disor
ders. However, cases o f patients with initial findings consistent with CSNB (Zorn,
1920; A m m ann, 1946; Frangois, 1961; A uerbach, Godel & Rowe, 1969), including
the ERG characteristics, have been rep o rted , bu t in these cases the condition
developed very slowly, like a progressive retinitis pigm entosa. Sieving et al. (1992)
re p o rted a family that fell betw een the definitions for CSNB an d RP, since they
d em onstrated ERG criteria for CSNB and a progressive course consistent with RP.
In m ost family m em bers, this autosom al d o m in an t pedigree has no detectable
rod vision, norm al cone function and n orm al G oldm ann visual fields. O lder
m em bers have bone-spicules, a sign usually seen in retinitis pigm entosa. Since all
family m em bers tested had norm al (or n ear norm al) cone function, they are
believed to have a congenital night blindness, probably progressive (a type o f rod
dystrophy) ra th e r than retinitis pigm entosa (which would imply a progressive
rod-cone dysfunction). M olecular studies o f this family will be discussed later.

HISTOPATHOLOGY

In 1957, G oodm an and B orschein suggested that the n ea r absence o f scotopic

function was evidence o f a pathological abnorm ality in the distal segm ents o f the
ro d photoreceptors. This theory, however, has never been su pported by histo-
pathological findings; reports by Babel (1963) and Vaghefi et al. (1978) dem o n
strated relatively well-preserved in n er segm ents o f the p h o to rec ep to r cells. Babel
also n oted that num erous cone nuclei were displaced an terio r to the external
lim iting m em brane. No m ention was m ade o f the o u ter segm ent. T he internal
retina, retinal pigm ent epithelium , and the choroid were norm al.

PATHOGENESIS

Fundus reflectom etry is a laboratory test for evaluation o f rhodopsin concentra

tion and kinetics. It was introduced by Ripps and Weale (1969) to study patients
with night blindness (C arr & Ripps, 1967; Ripps et al., 1984). T he test depends on
the ability o f the retina to absorb light. W hen light traverses the retina, it is par
tially absorbed by the pigm ent epithelium and the visual pigm ent. With reflecto
metry, light o f d ifferent spectral wave lengths is transm itted into the eye and
 A CRITICAL REVIEW FOR MOLECULAR APPROACHES 293

m easured as it exits. C om parison o f m easurem ents after bleaching to those after

dark-adaptation indicates the am o u n t o f rhodopsin present (Ripps & Weale,
1969).
T he inability of nyctalopic patients to see at low levels o f illum ination is often
attrib u ted to a defect involving the biosynthesis of the visual pigm ent, rhodopsin.
However, a photochem ical defect was ruled out when the concentration, spectral
position and kinetics o f reg en eratio n were found to be norm al regardless o f the
p attern o f CSNB inheritance (Ripps & Weale, 1969). In view o f norm al ph o to
chemistry, analysis o f the ERG suggests that both the a-wave o f the ERG an d the
light rise in the standing potential originate proxim al to the o u ter segm ent o f the
receptors. This suggests that the origin o f the defect in CSNB is one of neural
transm ission.

ANIMAL MODELS

Two anim al m odels have been used in the study o f CSNB. In the first, Witzel et al.
(1978) studied electroretinographically nyctalopic A ppaloosa horses. U nder pho
topic and scotopic conditions, ERG changes were sim ilar to those in hum ans with
the S chubert-B ornschein type o f CSNB. T he photopic abnorm alities consisted of
red u ced b-wave and slower-than-norm al im plicit time. T he dark-adapted ERGs
showed a sim ple negative potential, the b-wave being non-recordable. F urther
m ore, the presence o f a norm al a-wave in the dark-adapted eye suggested that the
rod photoreceptors were responding norm ally an d that the rod-RPE relationship
was n o t disturbed. Absence of the scotopic b-wave localizes the functional defect
to the o u ter plexiform layer a n d /o r in n e r nuclear layers. Histological and ultra-
structural studies showed no abnorm ality.
T he second anim al m odel studied was the pearl m u tan t m ouse (Balkema,
M angini Sc Pinto, 1983; Pinto et al., 1985). T he m ouse is known to have no gross
m alform ations, decreased sensitivity in dark-adapted conditions, an abnorm al
ERG a-wave, absence of p h o to rec ep to r degeneration, an d norm al rhodopsin con
centration. T he pearl is an autosom al recessive m utation in the m ouse that has
been m apped to m ouse chrom osom e 13. T he m ouse is characterized by hypopig
m entation o f the RPE an d reduced ipsilateral retinofugal projections, as observed
in albinos. This phenotypic characterization o f the pearl m ouse raises questions
ab o u t w hether it should be considered an anim al m odel o f CSNB.

MAPPING OF THE GENES FOR CSNB

X-linked CSNB is distinguished from o th e r h ereditary types by its association with

high m yopia (Gal et al., 1989). It has been suggested that this association is due
to close linkage o f these two traits on the X-chrom osom e, b u t no data are avail
able to support this observation. M oreover, several families with X-linked CSNB
294 ELISE HEON AND MARIA A. MUSARELLA

do n o t show an association with m yopia (Cutler, 1895; Newman, 1913; Khouri et

al., 1988) a finding that could be explained by a crossing-over event betw een the
two genes.
Initial linkage studies p erfo rm ed by W hite et al. in 1940 excluded linkage to the
loci of colour blindness (X qter). Subsequently, Volker-Dieben and Went (1975)
failed to show linkage to the XG locus (X pter). More recently, linkage studies in
volving the locus for X-linked CSNB have been conducted by three in d e p e n d e n t
groups. Gal et al. (1989) studied three large E uropean families affected with X-
linked CSNB and m apped this gene to locus DXS7 at X p l 1.3. A lthough no recom
binations (0) were detected betw een the disease locus an d DXS7, the LOD score
was 2.02. M usarella and coworkers (1989) assigned the gene for com plete CSNB
(CSNB1) to the X p ll.3 region, very close to the DXS7 locus (LOD 7.35 at 0 =
0.00), by p erfo rm in g linkage analysis on eight families with X-linked CSNB (seven
with the com plete type and one with the incom plete). R ecom binations were not
d etected between the disease locus and DXS7 but were present distal an d proxi
mal to this locus. These eight families were subjected to heterogeneity testing but
heterogeneity was not found to be significant. Recently, the linkage analysis of
these families was ex tended to include synapsin, m onoam ine oxidase (MAO) and
the sy n ap to p h o rin /A raf 1 gene loci. R ecom binations o f CSNB1 with all o f these
gene loci excluded them as the candidate gene for X-linked CSNB (M usarella,
p ersonal co m m u n icatio n ). Analysis of inform ative crossovers places the gene for
CSNB proxim al to MAO an d distal to TIM R This localization was confirm ed by
linkage studies done by B ech-Hansen and colleagues (1990), an d m ore recently
by A ldred et al. (1992). Both groups fo u n d no distinction between the com plete
an d the incom plete form s o f CSNB in th eir families.
T he question of w hether incom plete CSNB (CSNB2) is localized to Xp21 o r to
X p l 1.3 was recently addressed (M usarella et al., 1992). In two o f the families p re
viously studied by Miyake, tight linkage was found between CSNB2 an d m arkers
located at X p ll.3 - p ll.2 . These data su pported the localization o f CSNB2 to the
same region as CSNB1 an d AIED. Interestingly, this locus has two o th er X-linked
eye disorders closely linked to it, X-linked retinitis pigm entosa (RP2) (B hatta
charya e ta l., 1984) and N o rries disease (Bleeker-W agemakers et al., 1985). T he
reason for this clustering rem ains to be elucidated. F u rth er testing with m ore poly
m orphic m arkers in families with CSNB1, AIED an d CSNB2 will be n eed ed to d e
term ine if these are allelic o r are tightly clustered on the short arm o f the X-
chrom osom e.
Few studies have been done on the autosom al d o m in an t form s of CSNB. In a
large C hinese family with typical d o m in an t CSNB, close linkage betw een autoso
m al d o m in an t CSNB and m arkers o f PGM1 (chrom osom e 1), ABO (chrom osom e
9) and MN (chrom osom e 4) was excluded (Yijian et al., 1991). Sieving et al.
(1992) carried out m utational analysis on rhodopsin in an autosom al dom inant
congenital n ig h t blindness pedigree discussed earlier and found a single base sub
stitution in codon 90 o f rhodopsin, resulting in a change in am ino acid from gly
cine to asparagine.
 A CRITICAL REVIEW FOR MOLECUIAR APPROACHES 295

CONCLUSION

A lthough th ere are distinct clinical entities re ferred to as CSNB, litde is known
about the m olecular defects o r even the defective neurotransm itters that are
responsible for any o f their forms. T he localization of putative genes for the auto
somal form s o f CSNB rem ains unknow n. T he X-linked type o f CSNB is located at
X p l 1.3 in a region n ea r m onoam ine oxidase A an d B, synapsin an d synapto-
phorin. However, linkage studies d o n e with these m arkers have excluded these
neurotransm itters as putative sites for this form o f CSNB. At this point, little else
rem ains at this location as possible candidate genes for X-linked CSNB. T he iden
tification o f dystrophin in the o u ter nuclear layer o f norm al retinas may indi
rectly provide clues to this enigm atic g ro u p of disorders.

ACKNOWLEDGEMENTS

This work was sup p o rted by the M acLaughlin F oundation (E. H.) an d the R P
F oundation o f C anada (MAM). This ch a p te r was p rep ared with the assistance of
Medical Publications, T he H ospital for Sick C hildren, Toronto.

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 12. CHOROIDEREMIA

FRANS P. M. CREMERS and HANS-HILGER ROPERS

Department of Human Genetics, University Hospital Nijmegen
P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

SUMMARY

C horoiderem ia (CHM) is an X-linked progressive degen erad o n o f the retinal pig

m ent epithelium (RPE), choroid a n d retina. M icroscopically visible deletions
associated with contiguous gene syndrom es have assigned the CHM gene to
Xq21. D eletions in patients with classical choroiderem ia were instrum ental in
refining this assignm ent and in identifying DNA m arkers in the vicinity o f the
CHM gene locus. Using evolutionary conserved sequences from the critical
region, CHM cDNAs were isolated which encode an ubiquitously expressed
mRNA o f approxim ately 6 kb. Thus far, 4.7 kb o f cDNA containing a 1.3 kb open
reading fram e (ORF) as well as 9 corresponding exons of the structural gene
have been cloned an d characterized.
In the W estern E uropean p o pulation 14% o f patients with CHM have deletions
encom passing p art o f the CHM gene an d 21% have subtle m utations. Invariably,
these m utations give rise to tru n cated proteins. Missense m utations have n o t been
observed. A closely related gene, CHML, was isolated and m apped to lq42-qter in
the vicinity o f the gene for U sher syndrom e type 2 (USH2). W hether CHML has a
role in USH2 is n o t yet known.
T here is re cen t evidence that the CHM gene codes for a com p o n en t o f a gera-
nylgeranyl transferase (G G T ). Six tryptic peptides o f co m p o n en t A o f ra t Rab GGT
show alm ost com plete identity with the m ouse CHM hom ologue. Rat Rab GGT is
a m ultisubunit enzyme th at prenylates Rab3A, RablA and possibly o th er small
G TP-binding proteins. In addition, the CHM an d CHML proteins show weak ho
mology to bovine Rab3A GDI, a p ro tein th at inhibits the exchange of b o u n d GDP
by GTP on Rab3A. Since Rab3A an d Rab GGT are expressed in a variety o f tissues,
their specific role in the aetiology o f CHM rem ains to be elucidated.

CLINICAL ASPECTS

C horoiderem ia was first described by M authner in 1872 who coined the nam e of
this disease because o f presu m ed congenital absence o f the choroid. Progressive
degeneration o f the choroid, retinal pigm ent epithelium (RPE) and retin a was
established only m uch later (Bedell, 1937; Schutzbach, 1938; Friedm an, 1940;
304 FRANS P. M. CREMERS AND HANS-HILGER ROPERS

M cCulloch and McCulloch, 1948). C horoiderem ia is an X-linked disorder with

full m anifestation in males and m inor clinical signs in fem ale carriers (W aarden
burg, 1942; G oedbloed, 1942). T he incidence o f CHM is estim ated as 1 in
100,000 (Heckenlively et al., 1988).
T he clinical picture and rate of progression can vary between affected individu
als from d ifferent families, bu t also within the same family. In affected males, fine
pigm ent m ottling in the m idperiphery is the first sign o f the disorder. Gradually,
th ere is atrophy o f the RPE which is accom panied o r directly followed by atrophy
o f the capillaries o f the choroid. These initial fundus changes cause a diffuse dys
function o f the photoreceptors, and as a result, m ost patients experience night
blindness since early childhood (Bedell, 1937; Francois, 1968). At a later stage, the
o u ter retinal layers and large choroidal vessels becom e atrophic, first in the m id
periphery, an d then centrally towards the macula. Consequently, vision deterio
rates and often results in so-called tunnel vision. O th er patients develop ring
scotom as (Karna, 1986).
Fem ale carriers are mostly asym ptom atic, bu t th eir fundi show characteristic
p igm ent changes in the m idperiphery closely resem bling the fine m ottling ob
served in the initial stage o f the disease in males. Occasionally, female carriers
have serious visual im pairm ent (Fraser a n d Friedm an, 1968; H arris an d Miller,
1968) which can be ascribed to skewed X-inactivation. Severe m anifestation o f the
disease in fem ales can also result from d isru p tio n of the CHM gene by X-autosome
translocations (Kaplan et al., 1989; Siu et al., 1990). In these females, X -chrom o
some inactivation is non-random : the n orm al X is preferentially inactivated while
b o th translocation fragm ents rem ain active.

PHYSICAL MAPPING OF THE CHM LOCUS

Contiguous G ene Deletion Syndromes

Several males have been described who show cytogenetically detectable deletions
o f (parts of) the Xq21 ban d which are associated with com plex phenotypes
(Tabor et al., 1983; R osenberg et al., 1986,1987; Schwartz et al., 1986; H odgson
et al., 1987; Nussbaum et al., 1987; Wells et al., 1991; R eardon et al., 1992). These
phenotypes alm ost invariably include CHM, congenital m ixed deafness, m ental
retard atio n (MR), and in a few cases cleft lip and palate (CLP), obesity, u rin ary
tract abnorm alities, hypertelorism , m acrocephaly, an d myopia. In o rd e r to posi
tion the CHM locus m ore accurately, these deletions were characterized in great
detail em ploying S outhern analysis (Schwartz et al., 1986, 1988; H odgson et al.,
1987; C rem ers et al., 1988, 1989a; M erry et al., 1990; Yang et al., 1990; Bach et al.,
1992). Based on these results, the Xq21 region was subdivided into a n u m b er of
intervals. As depicted in Figure 1, the sm allest region of deletion overlap th o u g h t
to contain the CHM gene encom passes interval 3 which is defined by the loci
DXS233 (pJL68), ZNF6 (CMPX1), DXS165 (p lb D 5 ), and DXS95 (pXG7c). The
 CHOROIDEREMIA 305

LG L 7 .6 3 3 .1 D20 DM XL62 NP X q
2906
lo cus p ro be 25,6 3.5 MBU SD XL45 RvD

DXS 7 2 p X 65H 7
DXS169 pX104f
DXS 2 6 PHU16
DXS 2 3 2 pJL.68
DXS12 1 p784
DXS2 33 p JL 8
ZN F6 CMPX1
DXS1 65 p 1bD5

DXS 9 5 pXG7c
DXS1 10 p 72 2
pXG8b
DXYS1 PDP34
DXYS5 p 4 7 b /p 3 1
DXS2 14 pPA20
DXYS12 pSt2 5.2
DXS 1 1 2 p753
DXS 3 p 19.2
DXS 7 3 P X20R 42
DXS 9 6 pXG3b
DXS 1 18 p776
pF8
pF 1

Figure I D eletio n m ap o f th e Xq21 re g io n . T h e DNA p ro b e s u sed in S o u th e rn analysis are

in d ic ated at th e left. D e le te d ch ro m o so m a l se g m e n ts a re in d ic a te d by vertical bars. T h e sh ad
e d h o riz o n ta l b a r re p re se n ts th e critical re g io n fo r th e C H M locus. T h e p ro b e o rd e r fo r in
tervals 6 a n d 7 is arb itra ry ; in terv als a n d b a n d in g p a tte rn a re n o t draw n to scale. CLP = cleft
lip a n d palate.

onlY discrepancy in this assignm ent is the absence of clinical signs o f CHM in
patient RvD, who, at the age of 15, does n o t show the characteristic features of
CHM despite the fact th at interval 3 is com pletely absent (J. J. P. van de Kamp
and E. M. Bleeker-W agemakers, personal com m unication). All o th er patients
show CHM or fundus abnorm alities reflecting an earlier stage o f CHM because of
their young age.

Chrom osom al A bnorm alities Associated with Classical CHM

In an attem p t to identify deletions in patients with classical CHM, the DNA o f 80

m ale CHM patients was investigated fo r the absence o f three loci from interval 3,
i.e. DXS233, DXS165, and DXS95. Five males were identified with deletions span
ning one o r m ore o f these DNA m arkers (Figure 1; C rem ers et al., 1987, 1990b).
In four of them , i.e. patients LGL2905, 25.6, 7.6, an d 3.5, the DNA m arker plbD5
(DXS165) was deleted, suggesting a location n ea r or in the CFIM gene. Addi
tional DNA probes were isolated in the vicinity o f the DXS165 locus em ploying
the following m ethods.
306 FRANS P. M. CREMERS AND HANS-HILGER ROPERS

1. Chromosomal walking and jumping

Em ploying the DNA m arker plbD5 as a probe on a hum an genom ic cosmid

library a 36 kb genom ic DNA fragm ent was isolated. Invariably, single copy
probes located proxim al to DXS165 (probe p lbD 5) were found to be p resen t in
the DNA o f one p atien t (7.6 in Figure 2), whereas DXS165 itself was deleted.
T hus, the critical region harboring the CHM gene could be positioned distal to
DXS165. Em ploying this m arker as a probe, two clones, i.e. p j l l and pJ15 (Figure
2), were isolated (C rem ers et al., 1989b) from a hum an chrom osom al ju m p in g
library (Collins et al., 1987). To gen erate starting probes for fu rth e r jum ps
towards the telom ere, probe pJ15 was subsequently used to isolate two overlap
ping clones spanning a total o f 23 kb. R escreening the ju m p in g library with two
single copy clones yielded clones pJ59 a n d pJ60, the form er of which could be
positioned proxim al to p j l l (C rem ers et al., 1989b) while the latter was m apped
distal to a n o th er deletion (patient 3.5 in Figure 2). Thus, this ju m p provided the
distal dem arcation o f the segm ent th at deletions 3.5 an d 7.6 have in com m on.

L G L2905

3.5

25 .6

7.6

C3

C759

2082

LGL1134

LGL1101

33.1
t(X;13) . / -

Figure 2 Deletion map of the CHM locus at Xq21.2. Deleted segments are given as horizontal
bars. The location of the X-chromosomal breakpoint in a female with an X;13 translocation
and choroideremia is shown as a broken bar at the bottom. The exact physical distances be
tween the anonymous probes is not known, except for pJ7.6A/plbD5 and pJ15/pJll/pJ60
which have been linked by genome walking (Cremers et al., 1989b, 1990c).
 CHOROIDEREMIA 307

2. Preparativefield inversion gel electrophoresis (FIGE)

Em ploying FIGE analysis o f control DNA, the DNA probes dem arcating the criti
cal region for CHM, i.e. p lb D 5 an d pJ60, were shown to hybridize to the same
625kb Sfil fragm ent indicating th at at least part o f the CHM gene was located on
this fragm ent (Van de Pol et al., 1990). High m olecular w eight DNA from a cell
line that contained only the hu m an X chrom osom e in a ro d e n t background was
digested with Sfil an d subjected to FIGE. DNA was extracted from agarose slices
containing the 625 kb Sfil fragm ent, digested with EcoRI an d subcloned into the
lambdaZAP vector (Van de Pol et al., 1990). From this library, 7 clones were iso
lated which derived from the correct 625 kb Sfil fragm ent. Two o f these probes,
i.e. pZ l72 and p Z ll, are depicted in Figure 2. pZ172 was located centrom eric to
plbD5 an d p Z ll was positioned telom eric to pJ60.

3. Deletion breakpoint cloning

As indicated above, the centrom eric b reakpoint o f the deletion in p atien t 7.6 was
located proxim al to p lb D 5 . C onventional S outhern analysis using a centrom eric
ju n ctio n probe showed th at the deletion junction was situated on a 10.5 kb EcoRI
fragm ent (C rem ers et al., 1990a). This fragm ent was gel-purified from genom ic
DNA o f p atien t 7.6 an d cloned into a lam bda vector. Using the centrom eric ju n c
tion clone, several clones were isolated, one of which was analysed in m ore detail.
T he deletion ju n c tio n was located on a 0.9 kb H indi II fragm ent which in norm al
genom ic DNA detected the expected 7.5 kb EcoRI fragm ent (pJ7.6A) and a 8.5
kb EcoRI fragm ent (pj7.6B) spanning the distal deletion breakpoint. pJ7.6B is
located distal to the ju m p clo n es an d to p Z ll (Figure 2), an d is located on a dif
feren t Sfil fragm ent (C rem ers et al., 1990a).
Em ploying the new probes, six additional deletions were fo u n d in patients with
CHM (Figure 2) which enabled us to locate p a rt o f the CHM gene to a 50 kb re
gion flanked by p j l l an d p Z ll. Recently, two balanced translocations were found
in females showing choroiderem ia (Kaplan et al., 1989; Siu et al., 1990). In both
translocations, the breakpoints on the X -chrom osom e were situated in band
Xq21.2, while the autosom al breakpoints were at 7 p l4 and 13pl2, respectively.
A part from CHM, both females showed prim ary am enorrhoea. T he X;13 translo
cation breakpoint could be positioned betw een pJ60 an d p j l l , in the critical re
gion for the CHM gene (Figure 2; C rem ers et al., 1989b). M erry et al. (1990)
isolated a ju m p in g clone in the vicinity of p jl 1 which also detected the X;13 trans
location breakpoint. S outhern blotting has indicated th at the X;7 translocation
breakpoint is located m ore distally (C. Philippe et al., unpublished data).

ISOLATION OF THE CHM GENE

Cloning o f CHM cDNAs

Em ploying two ju m p clo n es from the critical region for CHM, i.e. p j l l an d pJ60
(Figure 2), hum an genom ic DNA phage clones were isolated, which together
308 FRANS P. M. CREMERS AND HANS-HILGER ROPERS

sp an ned a region of approxim ately 45 kb. Single and low copy probes were subse
quently tested for evolutionary conservation by hybridization to S o u th ern blots
containing DNAs from a variety of vertebrates. Using one o f the probes that
detected hom ologous sequences in e.g. m ouse and chicken, eight overlapping
cDNAs were isolated from a adult hum an retinal cDNA library (C rem ers et al.,
1990c). R escreening of the adult retina, a fetal retina, and a fetal brain cDNA
library with DNA probes from the 5 and 3 ends o f the consensus cDNA resulted
in th e isolation o f a nu m b er o f d ifferent clones, together spanning 4.7 kb o f the
cDNA including a poly(A) tail indicative o f the 3 end. An overview of the most
relevant CHM cDNA clones is given in Figure 3; the sequence o f the ORF is given
in Figure 4. T he consensus cDNA contains an ORF o f 1257 bps encoding a
polypeptide o f 419 am ino acids. At the 5 end, no in-frame stopcodons have been
fo u n d indicating that the gene m ight be longer.
M erry et al. (1992) independently isolated a CHM cDNA clone from an adult
h u m an retin a library which contains a sequence similar to the above cDNA, T120,
except for the presence o f a 190 bp inversion flanked by short inverted repeats at
the 5 end. T he co rrect o rientation of the 5 sequence could be deduced from the
sequences o f the closely related m ouse CHM an d hum an CHML cDNAs (C rem ers
e ta l., 1992).

T8-
T2-
T 45 -(A)n
T110
T120 -

Xhol EcoRI Ncol Hindlll Ncol Hindlll

EcoRI
fr a g m e n ts 7 .5 4 .5 1 2 .5 2 .6 4 .0 1.0 7 .0
(kb)

Figure 3 cDNA a n d e x o n m ap o f th e c h o ro id e re m ia g e n e. S chem atic re p re se n ta tio n o f th e

o v erla p p in g cD NA clo n es c o rre sp o n d in g to th e C H M g e n e w ith th e re stric tio n m ap o f th e
consen su s cD NA se q u e n c e show n in th e m id d le. T h e c o d in g re g io n is in d ic a te d by an o p e n
box. cDNA clo n es T2 a n d T 8 w ere iso lated fro m a h u m a n re tin a cDNA lib ra ry (C rem ers
e t al., 1990c), clo n es T 45 a n d T 1 10 from a h u m a n fetal b ra in library, T 120 fro m a fetal re tin a
cD NA library. C lo n e T45 co n ta in s a poly (A) se q u e n c e indicativ e o f th e 3 e n d o f th e c o rre
sp o n d in g mRNA, T h e o p e n re a d in g fra m e e n c o d e s a p re d ic te d p o ly p e p tid e o f 419 am in o
acids.
 CHOROIDEREMIA 309

C T G C T G T A T T C T C G AG G A T T A C T A A T T G A T C T T C T A A T C A A A T C T A A T G T T A G T C G A T A T G C AG A G T T T A A A A A T A T T A C C A G G A T T C T T 90
L L Y S R G L L I D L L I K S N V S R Y A E F K N I T R I L 30
><
GCATTTCGAGAAG GA CG A G TGG A A CA G GTTCC G TG TTCC AG A G CA G ATG TCTTTA ATA G CA A A CA AC TTAC TATG G TAG A A A AG CG A A TG 180
A F R E G R V E Q V P C S R A D V F N S K Q L T M V E K R M 60
><
C T A A T G A A A T TT C T T ACA TTTTG TA TGG A A TA TG A G A A A TA TC C TG A TG AA TA TAA A G G ATA TG AA G A GA TCA C ATTTTA TG A A TATTTA 270
L M K F L T F C M E Y E K Y P D E Y K G Y E E I T F Y E Y L 90

A A G A CT C A A A A A TT A A .CC C C C A A C C T C C A A T A T A T T G T C A T G C A T T C A A T T G C A A T G A C A T C A G A G A C A G C C A G C A G C A C C A T A G A T G G T 360
K T Q K L T P N L Q Y I V M H S I A M T S E T A S S T I D G 120

CTC A A A G CTA CCA A A AA CTTTCTTCACTGTCTTG G G CG G TATG G CA A CA CTCCATTTTTG TTTCCTTTA TA TG GCCA A GG A G AA CTCCCC 450
L K A T K N F L H C L G R Y G N T P F L F P L Y G Q G E L P 150
><
CAGTGTTTCTG CAG G A TG TG TG CTG TG TTTG GTG G A ATTTA TTG TCTTCG CCA TTCA GTA CA G TG CCTTG TAG TG G ACA AA G A ATCCA G A 540
Q C F C R M C A V F G G I Y C L R H S V Q C L V V D K E S R 180

A A A T G T A A A G C A A TTATA G A TC A GTTTG GTC AG A G A ATA A TCTCTG A GC A TTTCC TCG TGG A G G AC AG TTA CTTTCCTG A G AA C ATG TG C 630
K C K A I I D Q F G Q R I I S E H F L . V E D S Y F P E N M C 210
>< ><
T C A C G T G TG C A A T A C A G G C A G A T C T C C A G G G C A G T G C T G A T T A C A G A T A G A T C T G T C C T A A A A A C A G A T T C A G A T C A A C A G A T T T C C A T T 720
S R V Q Y R Q I S R A V L I T D R S V L K T D S D Q Q I S I 240
><
T T G A C A G T G CC AG CA G A G GA A CC AG G A AC TTTTG CTG TTCG G GTC ATTG A G TTA TG TTC TTCA AC G ATG A CA TG CA TGA A A GG C AC CTA T 810
L T V P A E E P G T F A V R V I E L C S S T M T C M K G T Y 270

TTG GTTCA TTTG A C TTG C A C A TC TTC TA A A A C A G C A A G A G A A G A TTTA G A A TC A G TTG TG C A G A A A TTG TTTG TTC C A TA TA C TG A A A TG 900
L V H L T C T S S K T A R E D L E S V V Q K L F V P Y T E M 300
><
G A G A T A G AA A A TG A A CA A G T A G A A AA G CC AA G A A T T C T G T G G G C T C T T T A C T T C A A T A T G A G A G A T T C G T C A G A CA T C A G C A G G A G C T G T 990
E I E N E Q V E K P R I L W A L Y F N M R D S S D I S R S C 330
><
TA TAA TG ATTTA CC ATC C A AC G TTTA TG TC TGC TC TG GC C C AG A TTGTG G TTTA G G A AA TG ATA A TG C A GTC AA A C A G G C TG AA A C A C TT 1080
Y N D L P S N V Y V C S G P D C G L G N D N A V K Q A E T L 360

TT C C A G G A A A T C T G C C C CA A TG AA G A TTTC TGTC CCC CTCC AC CA A ATC CTG AA G A CA TTATC CTTGA TG G AG A CA G TTTA C AG CC A GA G 1170
F Q E I C P N E D F C P P P P N P E D I I L D G D S L Q P E 390

G C T T C A G A A T C C A G T G C C A T A C C A G A G G C T A A C T C G G A G A C T T T C A A G G A A AG C A C A A A C C T T G G A A A C C T A G AGG A G T C C T C T G A A T A A 1260
A S E S S A I P E A N S E T F K E S T N L G N L E E S S E * 419

Figure 4 S eq u e n ce o f th e p ro te in c o d in g reg io n o f th e C H M cDNA a n d o f th e p re d ic te d p ro

tein p ro d u c t (in o n e -le tte r am in o acid c o d e ). E x o n -e x o n b o u n d a rie s a re in d ic a te d above th e
n u c le o tid e seq u en ce; ex o n s a re given a t th e rig h t.

Cloning o f Part o f the CHM Structural Gene

T he genom ic organization o f the central portion and 3 en d of the CHM gene

could be inferred by com paring cDNA sequences and their genom ic co u n ter
parts (Figures 2 and 3). T he CHM gene spans a m inim um of 12 exons, 9 o f which
have been subcloned into plasm id vectors from phage, cosm id and YAC clones
spanning the CHM gene (van d en H u rk et al., 1992; E.Bogerd et al., unpublished
data). To ascertain the relationship betw een the cloned cDNA an d the CHM
gene, genom ic DNAs of eight patients with CHM carrying subm icroscopic dele
tions o f the Xq21 band were reexam ined with a single copy cDNA fragm ent
which spans exons A3-B4 (Figure 3). In all patients, the deletions h ad rem oved at
least p art o f the ORF (Figure 5). Also, by em ploying different parts of the cDNA
as probes, we could locate the X -chrom osom al breakpoint o f the t(X;13) associ
ated with CHM in a fem ale, to in tro n sequences separating exons C and D (Fig
ure 2; C rem ers et al., 1990c). These findings provide convincing evidence that
this cDNA is p art o f the CHM gene.
It is notew orthy th at M erry et al. (1992) were unable to find deletions with the
CHM cDNA probe in 34 u n related patients. T he m arked difference between these
results an d those of C rem ers et al. (1990b,c), who found 11 deletions in 80 pa
tients with classical CHM, m ight be explained by fo u n d er effects resulting in a rel
atively low nu m b er of different CHM m utations in the A m erican population.
310 FRANS P. M. CREMERS AND HANS-HILGER ROPERS

kb
- - 12.5

- 7.5
b

- 4.5

T1E 0.5

Figure 5 S o u th e rn blo t analysis o f EcoRI d ig ested DNAs fro m several p a tie n ts with classical
CHM . T h e cDNA p ro b e T 1E0.5 en co m p asses ex o n s A3, B l / 2 , B3, a n d B4 w hich a re lo cated
o n E coR I fra g m e n ts o f 7.5 kb, 4.5 kb, 12.5 kb, a n d 2.6 kb, respectively (F ro m C re m e r s e t a l.,
1990c, by p erm issio n o f Nature).

Expression Pattern o f the CHM Gene

N o rth e rn blot analysis o f RNA from several hum an cell lines and tissues using
CHM cDNA clones revealed th at the CHM gene is no t only expressed in ocular
tissues or ocular derived cell lines, bu t also in various cells o f non-ocular origin,
e.g. HeLa, lym phoblast cells and fibroblasts (Figure 6, C rem ers et al., 1990c;
M erry et al., 1992). Yet clinical symptoms are confined to the eye. This finding is
n o t u n p reced en ted , however. Gyrate atrophy (GA), a choroidal disease with clini
cal similarity to choroiderem ia, is also caused by a deficiency o f an ubiquitously
expressed protein, the enzyme o rn ith in e am inotransferase (Valle and Simell,
1989). A part from ocular symptoms resulting from the typical sharply dem ar
cated, circular areas o f chorioretinal degeneration, m ost patients with GA are
 CHOROIDEREMIA 311

asym ptom atic. Proxim al m uscle weakness which is seen in less than 10% of the
patients is the only extraocular m anifestation o f this disease (Valle an d Simell,
1989).

4"
> X*

T1 EN0.6

4 M > I

pAct 1

F ig u re 6 N o rth e rn b lo t analysis o f RNA fro m several h u m a n cell lines a n d tissues usin g cDNA
clo n e T 1 . a. B lot c o n ta in in g RNA fro m H eL a, two re tin a l cell lines (H E R RC2 a n d H E R X C 2 ),
an EBV -im m ortalized ly m p h o b lasto id cell lin e (L C L1154), as well as h u m a n c h o ro id /re tin a l
p ig m e n t e p ith e liu m a n d re tin a sc re e n e d w ith clo n e T lE N 0 .6 .b . H y b rid izatio n o f a h a m ste r
actin cD NA c lo n e (pA ct-1). T h e p o sitio n s o f th e 18S a n d 28S rib o so m al RNA b a n d s a re in d i
cate d (F ro m C re m ers e t a l., 1990c, by p e rm issio n o f Nature).
312 FRANS P. M. CREMERS AND HANS-HII.GER ROPERS

MUTATION SPECTRUM

N o rthern Analysis

As shown in Figure 6, the CHM gene is expressed at a low level in lymphoblasts.

N o rth e rn blot analysis using total mRNA isolated from EBV-immortalized B-cells
showed th at CHM mRNA was absent, or p resen t at greatly red u ced levels, in 8 out
o f 11 patients (F.P.M.C., unpublished data). M erry et al. (1992) used the reverse
transcriptase-polym erase chain reaction (RT-PCR) technique em ploying exon-
prim ers an d observed red u ced o r absent levels of CHM mRNA in 25 out of 34
patients. This kind of analysis, using RNA isolated from EBV im m ortalized cell
lines has several pitfalls. Firstly, expression o f the CHM gene in B-cells o r B-cell
derived cell lines m ight differ considerably from the expression o f the CHM gene
in ocular tissues o f the respective patients. Secondly, the expression of the CHM
gene varies am ong B cell lines derived from healthy controls thereby com plicat
ing the quantitation o f mRNA levels in patients with CHM (M erry et al., 1992).
Thirdly, the highly hom ologous CHML gene on chrom osom e 1, m ight interfere
with both N o rth e rn blot an d RT-PCR analysis o f the CHM gene. T he high
sequence identity betw een CHM and CHML necessitates the use o f very specific
p rim ers for RT PCR.

SSCP Analysis

Forty-five CHM patients an d an equal n u m b er o f healthy individuals from the

W estern E uropean population were screened for nucleotide sequence variation
in exons B3-E em ploying the single strand conform ation polym orphism (SSCP)
technique. Significantly altered electrophoretic m obilities were observed in
eleven patients but in none o f the healthy individuals. D irect DNA sequencing
revealed five small deletions and 3 substitutions in the exon sequences and 3
m utations altering splice sites (Figure 7). All m utations result in stopcodons at
the site o f m utation or ju st dow nstream because of a fram eshift (Van den H urk
et al., 1992; Sankila et al,, 1992; Schwartz et al., 1993). No missense m utations
have yet been found.
T he m ost frequently occurring m utation found so far is the CHM*SALLA m u
tation, w here a splice d o n o r site ju st dow nstream o f exon C is inactivated through
the insertion o f a single n ucleotide (Sankila et al., 1992). This m utation cosegre-
gates with the disease phenotype in the large Salla pedigree com prising m ore than
60 living patients and 100 carriers descending from a fo u n d e r couple b o rn 13 gen
erations ago (Sankila et al., 1987). D etailed analysis o f mRNA isolated from a B cell
line o f one o f the patients using RT-PCR indicated that the m utation results in two
ab e rra n t cDNA products both o f which give rise to stopcodons.
An overview o f the small m utations is given in Figure 7. It is of n ote that two pa
tients (COP-E an d 2086), carry the same m utation, i.e. a four base deletion in
exon C. P atient COP-E is a Danish patient, whereas 2086 is an u n related patient
 CHOROIDEREMIA 313

from S outhern Germany. T he d eleted sequence (TGTT) is present in tandem

which may indicate th at this m utation is due to a replication slippage event. Also,
there is an ap p aren t clustering o f m utations in exon B4; 4 m utations were ob
served in a stretch o f 97 bps. U p to now, only 610 bps of the ORF have been inves
tigated for small m utations. This segm ent corresponds to approxim ately half of
the cloned CHM cDNA an d to approxim ately one-third of the anticipated total
length o f the ORF. T he m ouse cDNA hom ologue o f the CHM gene as well as a
highly hom ologous gene from chrom osom e 1 show open reading fram es which,
at their 5 ends, are 173 an d 236 am ino acids longer respectively (see below).
H ence, it is assum ed th at approxim ately 35% o f the CHM ORF is still missing. Tak
ing into account that, u n d e r the conditions used, the SSCP technique only reveals
80% o f all m utations (M ichaud et al., 1992) the observed percentage o f small m u
tations am ong non-deletion CHM patients (11/45 = 24%) suggests a relatively
even distribution o f m utations th ro u g h o u t the ORF and the p ro m o ter o f the CHM
gene. So far, small m utations o r larger rearrangem ents like deletions and translo
cations could be d etected in approxim ately 35% of patients. T ogether with the re
sults from mRNA analysis, these findings provide convincing evidence for the
candidate gene being identical with the choroiderem ia gene.

(ATG) TAA

CHM cDNA

CHM exons

2084 CC G
100 bp
1.2 -------- del(A)
I-------- 1
2.1 ----------- G T

17 .1 ------------- - C A

C O P -C -------------- C- A

C O P -D
3'ss A G

C O P -E /2 0 8 6 - - del(TGTT)

CHM *SALLA " 5 ss ins(T)

C O P -B --------------------------------------- del(TT)

10.1 3'ss G -* A

F ig u re 7 N o n sen se m u ta tio n s in th e CH M g en e . U p p e r half: g e n e se g m e n t th a t has b e e n in

vestigated fo r sm all m u ta tio n s by em p lo y in g th e SSCP te c h n iq u e . Low er half: m u tatio n s o b
serv ed a n d le n g th o f th e re su ltin g tr u n c a te d p ro tein s.
314 FRANS P. M. CREMERS AND HANS-HILGER ROPERS

CLONING OF THE MURINE HOM OLOGUE AND A HUMAN AUTOSOMAL

HOM OLOGUE FROM CHROMOSOME 1

Em ploying a cDNA clone from the 5 e n d o f the CHM gene (T8; Figure 3), a
cDNA corresponding to the m urine CHM gene, mCHM, was isolated. T he
mCHM cDNA sequence is m ore com plete than the consensus hum an CHM
cDNA sequence; it contains 173 additional am ino acids at its 5 end. Overall,
mCHM an d hCHM show 88% nucleotide an d am ino acid sequence identity, illus
trating th eir high degree o f evolutionary conservation. W hen conservative am ino
acid replacem ents are disregarded, mCHM displays 97% similarity to hCHM
(C rem ers et al., 1992; Figure 8).

hC H M L M A D N L P T E F D W IIG T G L P E S IL A A A C S R S G Q R V L H ID S R S Y Y G G N W A S F S F S G L L S W L K E Y Q Q N N D IG E E S T W W Q D L IH E T E E A IT L R 90
mCHM M E Q - L - N ---------L - S 16

h CHML KKD E T IQ H T E A F S Y A S Q D M E D N V E E IG A L Q K N P S L G V S N T FT E V L D S A L P E E S Q L S Y F N S D E M PA K H T Q K SD T E IS L E V T D V E E SV E K E K 1 8 0
mCHM S K --------- V - V - C ----------- LHKD-------A ---------------- A S V M -A Q A A A E A -E A A -A T E A A E A A E A -E A A C L P T A -E S T R S C -L P A - Q S Q 1 0 4

hCHML, YCGDKTCMHT V SD K D G D K D E SK ST V E D K A D E P IR N R IT Y S Q IV K E G R R F N ID L V S K L L Y S Q G L L 1 D L L IK S D V S R Y V 2 5 7
mCHM C M -P E S S PQ V N D AE - G E K E TQ S - A K S E Q S S E I L P K - Q - N T E T -K K V --------------1 ------------------------------------- R ----------------------- N---------A 1 9 4
hCHM ------R --------------- N------A 21

hCH M L E FK N V T R IL A F R E G K V E Q V P C S RA D V FN SK ELTM V EKRM LM K FLT F C L E Y E Q H PD E Y Q A FR Q C S F S EY L K T K K L T PM L Q H FV L H SIA M T S 3 4 7

mCHM ---------1 --------------------- T ------------------------------------Q --------------------------------------V -------D G K -Y E E T T ---------------- Q---------------- Y ----------------------- 2 8 4
hCH M --------- 1 --------------------- R ------------------------------------Q --------------------------------------M------ KY--------- K G Y E E I T -Y ----------- Q ---------------- Y I - M ---------------- 1 1 1

hC H M L E SS C T T ID G L N A T K N F L Q C L G R F G N T P F L F P L Y G Q G E 1 PQ G F C R M C A V F G G IY CLR H K V Q CF W D K E SG R C K A 11D H F G Q R IN A K Y F I V E 4 3 7
HiCHM - T T S S - V -------K------ K ---------------- Y ---------------------------------L C -------------------------------------- S -------L --------------R K ---------V - Q ----------- I S - H - V I - 3 7 4
hCHM - T A S S ----------- K--------------H --------- Y ---------------------------------L C --------------------------------------S -------L --------------R K --------------Q ----------- I S E H - L 2 0 1

hC H M L D S Y L S E E T CSN V Q Y K Q IS R A V L IT D Q S IL K T D L D Q Q T S I L I V P P A E P G A CA V RV TELC S STMTCMKDT Y L V H L T C S S S K T A R E D L E S W K 5 2 7

mCHM --------------N -------G------ R ----------------------- G - V P - S ------ V ------ T A E - S - S F --------- 1 -------------------------- G ------------------ M----------------------- R Q 4 6 4
hCH M -------F P - N M R -------R ----------------------- R - V ---------S -------1 -------T A E-------T F ---------1 -------------------------- G ------------------ T ------------------------------ Q 2 9 1

h CHML K L F T P Y T E T E IN E E E L T K P R L L W A L Y F N M R D S S G IS R S S Y N G LPSN V Y V C SG PD C G LG N EH A V K Q A E T L F Q E IF P T E E F C P P P P N P ED 1 1 6 1 7
mCHM -------------------I - A E N - Q V E -------1 ---------------------------- D ------ D C D-------------------------------N -------DN Q-------I V K - C - N - D -------A ------------------ 5 5 4
hC H M -------V ---------M E N -Q V E -------1 ---------------------------- D ---------C D---------------------------------------- DN---------------------------- C - N - D ---------------------------- 3 8 1

hC H M L F D G D D K Q P E A P G T N N W M A K L E SS E E SK N L E S PE K H L Q N 6 5 6
mCHM L -------S S Q - V S E S S V I P E T N S - T P K T V - G D S - E P S E 592
hCH M L -------S L --------- S E S S A I P E - N S - T F K T G N L - E S S E 419

Figure 8 A lig n m e n t o f th e h u m a n C H M L am in o acid se q u e n c e w ith m o u se C H M a n d h u m a n

C H M seq u en ces. D ashes in d ic a te id e n tic a l a m in o acid residues. T h e n u m b e rs at th e rig h t co r
re s p o n d to th e am in o acid resid u es in th e resp ectiv e seq u en ces (A d ap ted fro m C rem e rs e t
al., 1992, by p e rm issio n o f Hum. Mol. Genet.).

In an attem p t to isolate sequences from the 5 end o f the hCHM cDNA, a

mCHM cDNA clone was used as a probe to screen a hum an fetal brain cDNA li
brary. A m ong six phage recom binants identified, four were partly overlapping but
different from the hCHM cDNA consensus (C rem ers et al., 1992). T he consensus
cDNA o f these new clones contains an ORF encoding 656 am ino acids an d is des
ig nated hCHM L which stands for hum an choroiderem ia-like. T he nucleotide se
qu en ce and the deduced am ino acid sequence o f hCHM L was com pared with
hCHM sequences. Am ino acid residues 237-655 o f hCHM L show 76% identity and
95% similarity to hCHM; the nucleotide sequences are 80% identical (Figure 8).
N o rth ern analysis has shown th at the CHML gene is expressed in the same tissues
as th e CHM gene, although at d ifferent a n d generally lower levels (F.P.M.C., u n
published data). O ne o f the hCHM L cDNA clones was hybridized to a panel o f 32
hum an-m ouse o r hum an-ham ster cell lines containing various different hum an
 CHOROIDEREMIA 315

chrom osom es. These studies showed th at the hCHM L gene resides on chrom o
some 1. In addition, hCHM L subclones were hybridized to S outhern blots with
DNAs from three somatic cell hybrids carrying defined segm ents o f chrom osom e
I. In this way, hCHM L could be assigned to lq42-qter (C rem ers et al., 1992;
F.P.M.C., unpublished data). Recently, the gene for an o th er form o f retina degen
eration, U sher syndrom e type 2 (U SH 2), has been localized in approxim ately the
same chrom osom al interval by linkage analysis (K im berling et al., 1990; Lewis
et al., 1990). U sher syndrom e consists o f retinitis pigm entosa with hearing loss and
is in h erited as an autosom al recessive condition (C hapter x x ). T he chrom osom al
region containing the USH2 locus a n d hCHM L is estim ated to com prise no m ore
than 2% o f the hum an genom e. This co-localization, together with the clinical
similarities betw een CHM an d USH2, renders hCHM L a prom ising candidate
gene for this disorder. M ore accurate physical m apping o f hCHM L and closely
linked flanking m arkers o f USH2, as well as hCHM L segregation studies in USH2
families, are n ee d ed to substantiate this hypothesis. Final p ro o f for the identity of
hCHM L a n d USH2 may com e from the identification o f m utations in the hCHML
gene in patients with the USH2 phenotype. It is o f note, however, that so far,
screening o f 10 patients with USH2 has failed to detect gross rearrangem ents or
point m utations in the hCHM L gene (T. J. R. van de Pol et al., u n p ublished d a ta ).

THE CHM PROTEIN MAY BE A COMPONENT OF A GERANYLGERANYL

TRANSFERASE

Many m em brane-associated eukaryotic proteins contain a covalently b o u n d far-

nesyl or geranylgeranyl g roup attach ed in th io eth er linkage to cysteine residues
n ear the CO O H term ini (Farnsw orth et al., 1990; Rilling et al., 1990). This m odi
fication facilitates p ro p e r binding to specific m em branes and is essential for the
activity o f the relevant proteins. At least three different protein prenyltransferases
have been identified in cytosolic extracts o f anim al tissues. O ne of these, a Rab
geranylgeranyl transferase (designated Rab GGT) isolated from rat brain, con
sists of two com ponents, A and B, o f 95 kD and 90 kD respectively. T he latter o f
these is com posed o f two subunits (Seabra et al., 1992a,b). Six tryptic peptides
from com p o n en t A show alm ost com plete identity to the m ouse hom ologue of
the CFIM protein, strongly suggesting th at co m ponent A is the rat hom ologue of
the CHM protein.
This im p o rtan t finding sheds new light on an earlier observation th at the CHM
and CHML polypeptides show weak b u t significant hom olog)' to Rab3A (or smg
p25A) GDP dissociation inhibitor (GDI), a bovine brain protein which inhibits the
exchange of b o u n d GDP by GTP on the small ras-like GTP-binding protein Rab3A
(Matsui et al., 1990; Fodor et al., 1991). CHML shows two regions of approxim ate
ly 50 am ino acid residues which are 50% identical to bovine Rab3A GDI (Crem ers
et al., 1992). Because bo th Rab3A GDI an d Rab GGT interact with Rab3A, their
316 FRANS P. M. CREMERS AND HANS-HILGER ROPERS

sequence hom ology may indicate that these proteins have sim ilar binding do
m ains for this small G TP-binding protein.
Assuming that CHM and Rab GGT are identical, there are several rem aining
questions related to the pathogenesis o f choroiderem ia, as follows.
1. Both CHM (Rab GGT) and Rab3A are expressed in a variety o f tissues, includ
ing brain. Why then, is the defect lim ited to the choroid and retina?
2. W hat is the role of the CHML gene product? Does it geranylgeranylate the
same or a n o th er class o f small GTP binding proteins? In view o f its high de
gree of similarity with CHM, is it possible that CHML expression m odulates
the CHM phenotype? Is there a CHML hom ologue in mice and rats?
3. Rab3A is tho u g h t to regulate vesicle tran sp o rt in brain synapses (Fischer von
M ollard et al., 1990; Mizoguchi et al., 1990). C ould Rab3A or a related gera-
nylgeranylated GTP binding protein be involved in m em brane turnover in
ro d o u ter segments? In accordance with this hypothesis, the fundus ab n o rm al
ities observed in early stages o f the disease suggest that lesions first occur in or
n ea r the retinal pigm ent epithelium (W aardenburg, 1942; Pam eijer et al.,
1960; M cCulloch, 1969; H am m erstein et al., 1979; H am m erstein and Bohm,
1985).
F uture studies will concentrate on the identification of Rab G G T/C H M gera-
nylgeranylated small GTP binding proteins in the retina, RPE, an d choroid. The
subcellular expression o f these m olecules as well as the CHM gene p ro d u ct will be
studied by im m unohistochem ical techniques. Also, a b etter u n d erstan d in g of the
pathological processes in choroiderem ia may be achieved by the construction of
a m ouse m odel via insertional inactivation o f the m ouse CHM gene. Eventually,
com plete un d erstan d in g of the pathological processes underlying choroiderem ia
may provide a basis for rational therapy.

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Siu, V. M., G onder, J. R., Ju n g , J. H., Sergovich, F. R., an d Flintoff, W. F. (1990) C h oroiderem ia
associated with an X -autosom al translocation. Hum Genet, 84, 459-464.
Tabor, A., A ndersen, O., L undsteen, C., N iebuhr, E., an d S ardem ann, LI. (1983) Interstitial dele
tion in th e critical re g io n o f the long arm o f the X chrom osom e in a m entally re tard e d boy
an d his no rm al m other. Hum Genet, 64, 196-199.
Valle, D. and Simell, O. (1989) T he h y p erornithinem ias. In The Metabolic Basis O f Inherited, Disease
I, C. R. Scriver, A. L. B eaudet, W. S. Sly, a n d D. Valle, eds. (pp. 599-627.New York: McGraw-
hill, Inc).
Van de Pol, T. J. R , C rem ers, F. P. M., B rohet, R. M., W ieringa, B., and Ropers, H.-H. (1990) Der
ivation o f clones from the ch o ro id erem ia locus by preparative field inversion gel electro
phoresis. Nucleic Acids Res, 18, 725-731.
Van d en H urk, J. A. J. M., Van de Pol, T. J. R., Molloy, C. M., B runsm ann, F., R uther, K., Z renner,
E., Pinckers, A. J. L. G., Pawlowitzki, I. H ., Bleeker-W agemakers, E., W ieringa, B., Ropers,
H-H., and C rem ers, F. P. M. (1992) D etection an d characterization o f p o in t m utations in the
ch o ro id erem ia candidate gene by PCR-SSCP analysis and d irect DNA sequencing. Am J Hum
Genet, 50, 1195-1202.
W aardenburg, P. J. (1942) C ho rio id erem ie als E rbm erkm al. Acta Ophthalmol, 20, 235-274
Yang, H.-M., L und, T., N iebuhr, F,., N_rby, S., Schwartz, M., and Shen, L. (1990) A deletio n panel
of the long arm o f the X chrom osom e: subregional localization o f 22 DNA probes. Hum
Genet, 85, 25-30.
 13. NORRIE DISEASE

A. A. B. BERGEN1, W. BERGER2, Z.-Y. CHEN3, P. J. DIERGAARDE1, E. M. BLEEK

ER-WAGEMAKERS1, E. M. BATTINELLI3, M. WARBURG4, H.-H. ROPERS2,
I. CRAIG3
1The Netherlands Ophthalmic Research Institute, P.O. Box 12141, NL-1100 AC Amsterdam,
The Netherlands
University Hospital Nijmegen. Department of Human Genetics,
P.O. Box 9101,6500 HB Nijmegen, The Netherlands
3University of Oxford. Department of Biochemistry. Genetics laboratory.
South Park Road, Oxford OX 13QU, UK
4Gentofte Hospital, Department of Ophthalmology, Division of Paediatric Ophthalmology and
Handicaps, 40 Sognevej, DK-2820 Gentofte, Denmark

CLINICAL FEATURES

N orrie disease (NDP) was d elineated by W arburg (W arburg, 1961; 1966) who
subsequently fo u n d th at patients with this disorder had been m entioned by the
Danish ophthalm ologist to the Royal Institute for the Blind, G ordon N orrie
(N orrie, 1927). As N orrie was the first to recognise the genetic origin o f the dis
order, W arburg decided to nam e the disease after him . T he condition has been
observed in all ethnic groups. Since the disease is X-linked it is m anifest in boys
only. Two affected fem ales have b een observed (I. Craig, personal com m unica
tion). It is probably the m ost com m on disorder with bilateral retrolental vascular
masses.

Ophthalm ological findings

T he m ain pathology o f NDP is congenital o r very early bilateral blindness due to

opacities b eh in d the lens presenting in eyes of norm al size. T he patients are born
at full term an d n eed no oxygen treatm ent. W ithin the first m o n th o f life, a white
or yellow vascular m em brane is seen b e h in d the lens, and by ultrasonography this
is recognised as a totally detached retina. In rare cases, the d etach m en t appears
in the second eye only after some m onths an d a progressive cystic schisis is then
observed in the p osterior pole of this eye (I ^ure 1 ). T he cyst soon changes into a
total detachm ent. Most affected boys are blind from the detachm ent, b u t some
perception o f light may rem ain for a couple o f years. In the beginning the lens is
clear, and elongated ciliary processes are seen b eh in d it (Figure 2 ). T he affected
eyes have shallow an terio r cham bers, an d may in rare cases develop glaucom a.
After a few years the lens becom es cataractous, the cornea becom es opaque, and
the eyes begin to shrink (Figure 3). At that stage m icrophthalm os may be erro n e
ously suspected (W arburg, 1966; 1975). T reatm ent with vitrectom y or peeling the
d etached retinal m em branes has b een tried bu t was in no case successful
(Liberfarb et al., 1985; Enyedi e ta l., 1991).
322 A. A. B. BERGEN ET AL.

Figure 1 D elayed re tin a l d e ta c h m e n t in NDP. Progressive cystic schisis in th e p o ste rio r p o le

in th e less affe c te d se c o n d eye o f a N o rrie disease p a tie n t (C ).

Figure 2 E lo n g ate d ciliary p rocesses (arro w ), w hich can be seen b e h in d a cle a r len s (L ).
 NORRIE DISEASE 323

F igure 3 P hthisis o f th e g lo b e in NDP. T h e sh rin k in g o f th e eye, a n d opacity o f th e c o rn e a car

b e seen in this N D P p atie n t.

Histology

Sometim es the retrolental masses are m istaken for retinoblastom a and the eye is
enucleated. Histological exam ination shows that the detached retina contains
m alform ative rosettes, m any large retinal vessels, haem orrhage, gliosis, and optic
atrophy. Hyperplasia of the retinal pigm ent epithelium was also observed
(W arburg, 1966; A ndersen and W arburg, 1961; L iberfarb et al., 1985; Blodi and
H unter, 1969). In ultram icroscopical sections (Liberfarb et al., 1985) the in n er
and o u ter neuroblastic cells are undifferentiated.

Systemic signs

T he patients are usually healthy an d have a norm al life span. Many are very intel
ligent; in m ost cases procreation is norm al. D uring their lifetime, about one third
of the patients develop progressive neurosensory hearing im pairm ent. This may
begin in childhood o r as late as in the 6th decade. O ne q u arter o f those affected
show dem entia or autistic signs which may develop at any time between the age of
2 and 60 years. In som e cases the psychiatric im pairm ent may be a com plication
o f the com bined visual and auditory com m unicative disorder, while in others the
blind m en an d boys show autistic signs even when hearing is still norm al. In a few
324 A. A. B. BERGEN' ET AL

families, the affected blind males had systemic m alform ations, seizures, and
severe m ental retardation (Bleeker-W agemakers et al., 1988; De la C hapelle et al.,
1985; D onnai et al., 1988; Zhu et al., 1989). Microcephaly, large ears, p ro m in en t
nasal bridge, and thin u p p er lips have been described (D onnai e ta l., 1988). O ne
p atien t (Zhu et al., 1989) was autistic and self abusive, he had growth failure,
abnorm al sexual m aturation, hypotension, paroxysmal irregularity o f respiration,
flushing, atonic seizures, m yotonic contractions and hyper-reflexia w ithout spas
tic paresis. H e was unable to talk and walk. A D utch p atien t (Bleeker-W agem ak-
ers et al., 1988) had cutaneous hyperkeratosis and microcephaly, cryptorchidism
an d penis hypoplasia. Brain stem audiography showed sensory hearing loss. A
Danish patient recently re p o rted was mildly m entally re ta rd ed an d lived in an
o p en institution. His only extraocular m alform ations were m icropenis and cryp
torchidism . He was childish and had very little confidence in the people aro u n d
him o r in himself. In these atypical patients, deletions or o th er rearrangem ents
involving the NDP locus were d em onstrated by m olecular genetic exam ination
(B erger et al., 1992a; C hen et al., 1992b). It is conceivable th at th eir com plex
p henotype is due to the involvem ent o f o th e r genes which have n o t yet been
identified. An X /autosom al translocation was also found in some o f the N orrie
p atients (O hba and Yamashita, 1986; M cM ahan e ta l., 1991).

C arrier phenotype

In general, fem ale carriers are com pletely healthy, O phthalm ological an d audio-
logical exam inations are usually norm al (W arburg, 1966; 1975; Parving and
Schwartz, 1991) and both carriers of deletions and o f o th er m utations have had
children. Two fem ale carriers m anifesting signs o f the disease have been
observed. For one of these cases evidence has been fo u n d supporting a m odest
inactivation bias (70:30) in favour of expression of the defective allele (I. Craig,
personal com m unication).

Pathogenesis

U ltrastructural studies (Enyedi et al., 1991) showed that the in n er and o u ter neu-
roblastic layers of the retina were u n differentiated. This may explain the lack of
coaption o f the retinal pigm ent epithelium and the n euroretina, leading to con
genital detachm ent. Most o f the clinical signs are secondary, nam ely the persis
tence o f the prim ary vitreous, the elongation o f the ciliary processes, the cataract
an d the shrinkage with resulting corneal opacities. T he progressive hearing
im pairm ent an d the freq u en t autistic signs may be in terp re ted as a progressive
neurological disorder o f unknow n character.

D ifferential diagnosis

Diagnosis is sim ple if there are o th er affected males in the family. W hen only a
single person is affected, the diagnosis can only be tentatively established. The
 NORRIE DISEASE 325

differential diagnosis depends on the time when the patient is first observed. In
the neonate, it includes retinoblastom a, retinopathy of prem aturity, persistent
hyperplastic prim ary vitreous (PHPV), oculo-palato-cerebral dwarfism (Frydm an
et al., 1985), the osteoporosis-pseudogliom a syndrom e (Saraux an d Frezal, 1967),
X-linked prim ary retinal dysplasia (G odel and G oodm an, 1981) which is probably
identical with X-linked familial exudative vitreo-retinopathy, trisomy 13, and
incontinentia pigm enti. In this respect, it is of particular interest to note th at the
locus for X-linked exudative vitreo-retinopathy is localized to the same Xp region
as NDP, and, recently, a m utation in the NDP gene has been found in affected
m em bers o f an X-linked exudative vitreo-retinopathy family (C hen et al., in
p ress).
T he patients with NDP are usually b o rn at term and n eed no oxygen, which
excludes retinopathy o f prem aturity. T h eir disorder is bilateral while PHPV is
unilateral, except in the autosom al recessive oculo-palato-cerebral dwarfism char
acterised by m icrocephaly, hypotonia, bilateral leukocornia, an d cleft lip-palate.
X-linked retinal dysplasia and X-linked falciform folds m anifest as a falciform
retinal fold, and carriers of X-linked retinal dysplasia may show p eripheral folds;
in NDP the affecteds are m ale - n o t fem ales as in incontinentia pigm enti. In chil
dren, the differential diagnosis com prises infections, X-linked congenital cataract,
X-linked m icrophthalm os and autosom al d om inant familial exudative vitreoretin-
opathy (FEVR). C ataract an d small eyes are n o t congenital in NDP, and FEVR is
rare in children, in addition to which one of the relatives may show the retinal
vascular anom alies o f FEVR (von Nouhuys, 1991). In adults, the differential diag
nosis com prises xerophthalm ia an d m icrophthalm os o r phthisis due to a different
aetiology.
Clearly, m olecular genetic exam ination will soon facilitate the differential diag
nosis.

Penetrance and Incidence

M ore than 300 m ale patients have b een described worldwide, (W arburg, 1961;
1966; 1975; Blodi et al., 1969; N ance et al., 1969; Lomickova an d Raska, 1969; Mor-
eira-F ilho an d N eustein, 1979; B leeker-W agem akers 1981; Jo h n sto n et al., 1982;
Bleeker-W agem akers et al., 1985; L iberfarb et al., 1985; Phillips et al., 1986; Hill
et al., 1987; Kivlin et al., 1987; Esakowitz et al., 1988; Gal et al., 1988; H arenda de
Silva et al., 1988; Curtis et al., 1989; N adol et al., 1990). P enetrance was com plete,
b u t expression was variable except for congenital blindness which is always
present. T he incidence is no t known. Phillips (Phillips et al., 1986) calculated a
m utation rate o f 3.9 x 10'6 in the Scottish population, but ascertainm ent was p ro b
ably n o t com plete.
326 A. A. B. BERGEN ET AL.

T ab le 1 L o d sco res b e tw e e n N D P a n d DXS7

Author zmax 0 max

Gal et al. (1985a) 3.50 0.00

Bleeker-Wagemakers (1985) 3.81 0.00
Warburg etal. (1985) 4.10 0.00
Kivlin etal. (1987) 5.42 0.00
Hill etal. (1986) 5.60 0.00
Ngo et al. (1988) 7.58 0.05
Curtis et al. (1989) 7.99 0.05

CHROMOSOMAL ASSIGNMENT OF TH E NDP LOCUS

Linkage

As early as 1965, W arburg em ployed the classical X chrom osom al Xg blood group
m arker in an attem p t to establish linkage with NDP (W arburg, 1965). No linkage
could be found between NDP and the Xg blood group system. T he same results
were re p o rted within a Brazilian N egro sibship (M oreira-Filho a n d Neustein,
1979). A loose a n d unreliable linkage was fo u n d betw een NDP an d the glucose-6-
pho sphate dehydrogenase m arker in an A m erican N egro family (Nance et al.,
1969). In D utch and Danish families, Gal et al. (1985a) n o ted close linkage o f
NDP with an anonym ous RFLP m arker, L I .28 (DXS7) (Bakker et al., 1983) from
the X p ll.4 - p ll.3 region. After this rep o rt, extensive linkage studies were per
fo rm ed on N orrie families from all over the w orld by several research groups.
T h e results from these studies clearly assigned NDP to the proxim al Xp n ear
o th er regionally assigned eye disease loci (I *ure 4). T he accum ulated LOD
scores are sum m arized in Table 1. A recom bination between NDP and DXS7 was
re p o rted in only one family (Ngo et al., 1988). S ubsequent analysis o f the same
family with an RFLP detected by a hum an o rn ith in e-8 -am in o tran sferase (OAT1)
cDNA probe (Ngo et al., 1989, L afreniere et al., 1991b), a polym orphic CA-repeat
at the 5'-end o f the MAOB gene (Sims et al., 1992) and the DXS426 CA-repeat
(Lindsay et al., 1992) suggested the genetic order: Xpter-(DXS7,MAOB)-NDP-
(DXS426,OATL)-Xcen (Figure 5).

Cytogenetics

T he first physical confirm ation for the localization o f NDP in b an d X p l 1.4 on the
sh o rt arm o f the X chrom osom e came from a pap er by O h b a and Yamashita in
which they described a fem ale infant NDP p atien t carrying a balanced X;10 trans
location (O hba an d Yamashita, 1986). T he expression o f the m utated gene in
this fem ale could be explained by the fact th at in an X-autosome translocation
th e translocated X chrom osom e is preferentially activated (T herm an et al.,
1974). M ore recently, a second X-chrom osom al ab erratio n associated with NDP
 NORRIE DISEASE 321

Xp2i .1 Xpter
RP3
[
ND X p 1 1.4

CSNBX
RP2 X p 1 1.3

WAS

IP

Cen

F igure 4 G en etic d iso rd ers a n d an o n y m o u s DNA p ro b e s assigned to th e p ro x im al X p. Dis

o rd e rs o n p ro x im a l Xp: RP2, RP3; R etin itis P ig m en to sa types 2 a n d 3. N D P; N o rrie disease.
CSNB1; C o n g e n ita l S tatio n ary N ig h t B lin d n ess type 1. WAS; W iskott-A ldrich Syndrom e. IP:
In c o n tin e n tia P ig m en ti. T h e DNA p ro b e s o n th e rig h t w ere a rra n g e d in intervals d em a rca ted
by ch ro m o so m a l b re a k p o in ts (D ierg a a rd e e t al., 1989; L a fre n ie re e t al., 1991).

was published in which a familial pericentric inversion (X) (p ll.4 -q 2 2 ) was found
in males affected with NDP. Fem ale carriers o f this inversion showed no clinical
symptoms o f NDP (M cM ahan e ta l., 1991).

Regional assignm ent o f DNA probes to the proxim al Xp

Im p o rtan t progress towards the m apping an d isolation of the NDP gene came
from the cloning and ord erin g o f genes and anonym ous DNA probes to defined
X -chrom osom al intervals. O ne o f the first m arkers, L I.28 (DXS7) (Bakker et al.,
1983), was assigned to the proxim al Xp by linkage analysis in D uchenne M uscular
Dystrophy families (W ieacker et al., 1984). Subsequently, a large n u m b er o f m ark
ers and genes have been localized to the proxim al Xp region using linkage analy
sis, deletion m apping, in situ hybridization an d by m apping probes in hum an
ham ster cell hybrids containing parts o f the hum an X chrom osom e (Lafreniere
e ta l., 1991; Davies et al., 1991) (Figure 4).
328 A. A. B. BEROEV F.T Al

L I.2 8 MAOB
ploA6 pX59 MAOA de l 2 cpXr31 8

tel cen
----- 1 J ----------------- 1
-------

-^ 1

R ecom bination event = N o t precisely m a p p e d b re a kp o in t

-------- = Deleted ge n om ic D N A
= Retained ch ro m o so m a l D N A I 1 = O b lig a te NDP region

Figure 5 G e n e tic a n d physical lin e m a p p in g oi th e NJJP locus. Top line: Relative o rd e r of

an o n y m o u s D N A m ark ers sp a n n in g th e N DP reg io n . B roken line: physical d istan ce u n k n o w n .
L ine 1: re c o m b in a tio n events b etw een closely lin k ed m ark e rs a n d NDP. (N go e t al., 1988;
Sims et al., 1991). D eletions: 2; G al e t al., 1986, 3; d e la C h ap elle e t al., 1985, 4; F o rre st e t al.,
1987, 5; Z hu e t al.. 1989, 6 ; Sim s e t al., 1992, 7; B erg en et al., 1992.

PHYSICAL FINE-MAPPING OF THE NDP GENE.

Deletion studies

In 1985, two research groups sim ultaneously re p o rted physical evidence for close
proxim ity of the NDP gene to the DXS7 locus (Gal et al., 1985b; 1986; de la
C hapelle et al., 1985). In each case the DXS7 locus was found to be deleted, in a
D utch an d in a Finnish m ale with atypical features of NDP respectively. Co-dele
tion o f the N orrie gene to g eth er with the DXS7 locus could explain the NDP p h e
notype of these patients. T he physical length o f the deletions was n o t known, but
as no changes could be found in high resolution chrom osom e banding experi
m ents, the deletion in the D utch patient could n o t be m uch larger than about 3
m illion bp (Gal e ta l., 1986).
 NORRIE DISEASE 329

Subsequently, two additional subm icroscopic interstitial deletions encom pass

ing the DXS7 and the NDP locus were described (Forrest et al., 1987; D onnai et
al., 1988; Zhu et al., 1989). F u rth er m olecular analysis revealed th at these four
NDP-associated deletions included several regionally assigned anonym ous DNA
m arkers and the hum an m onoam ine oxidase A an d B loci (MAOA, MAOB) (Bach
et al., 1988; Hsu et al., 1988; Lan et al., 1989) (Figure 5).
Since the MAOA an d MAOB genes were absent in the DNA o f these four pa
tients, and because o f the role o f biogenic am ines in neu ro n al developm ent
(Lauder, 1985; Lam et al., 1981) an d retinal ueurotransm ission (O sborne, 1981),
they were initially considered to be im plicated in NDP. However, enzyme, m etab
olite and DNA studies in several classic NDP patients did no t reveal any MAOA or
MAOB related abnorm alities, an d these genes were excluded as candidate genes
for NDP (Sims et al., 1989).
T he discovery in 1991 o f subm icroscopic interstitial NDP-deletions which did
no t include DXS7 (Sims et al., 1992), MAOA o r part of the MAOB loci (Bergen et
al., 1993), assigned the NDP locus unam biguously proxim al to the MAOB locus.
By this time, the physical o rd e r Xpter-(DXS228,DXS77)-DXS7-(MAOA, MAOB)-
NDP-DXS742-Xcen could be established on the basis o f d ifferen t NDP deletion in
tervals (Figure 5).

Long range restriction mapping

After the discovery of the first subm icroscopic X p-deletions associated with NDP,
inform ation on the relative gene m arker o rd e r in the proxim ity of NDP came
from yet an o th er source: long range restriction analysis.
D iergaarde etal., (1989) assigned the probes plA a6 (DXS228), pX59 (DXS77),
L I.28 (DXS7) and M AOA/B to a region o f 1500 kb, as they all hybridized to an
identical BssHII restriction fragm ent o f this size. In the same study, the distal
b reakpoint of the D utch deletion (Gal et al., 1985b) was m apped within a 400 kb
Sail fragm ent shared by pX59 (DXS77) an d L I.28 (DXS7), but which could not
be identified with M AOA/B. F u rth erm o re, with different com binations o f single
and double digests using a set of restriction enzymes, the physical o rd e r DXS228-
DXS77-DXS7-(MAOA/B, NDP) was suggested (D iergaarde et al., 1989). Refine
m en t and extension o f this m ap cam e from physical m apping experim ents using
YACs corresponding to the L I.28 a n d MAOA/MAOB loci (C hen et al., 1992a,
Sims et al., 1992), and from genom ic pulsed-field gel electrophoresis (PFG) stud
ies with a novel probe, cpXr318 (DX S742), which is localized ju st proxim al to the
N orrie disease locus (Bergen et al., 1993). The resulting o rd e r o f m arkers and
physical distances betw een them were in general agreem ent with the data ob
tained from deletion studies (Figure 5).
330 A. A. B. BERGEN ET AL.

THE CLONING AND CHARACTERISATION OF THE CANDIDATE

NDP GENE

Isolation o f the candidate cDNA

By 1992, linkage analysis, deletion studies an d long range PFG restriction m ap

ping reduced the obligate NDP gene region to approxim ately 150 kb proxim al to
the MAOB locus. This m apping inform ation paved the way for the positonal clon
ing o f the gene im plicated in NDP,
Several research groups set out to survey the obligate NDP region in detail. O f
p articular im portance was the availability o f a large YAC o f 640 kb corresponding
to the DXS7 locus (Larin et al., 1991) an d which encom passed at least p art o f the
NDP locus (C hen et al., 1992a; Sims et al., 1992). T he g roup o f B erger et al.,
(1992a) cloned 350 kb spanning the obligate NDP region into a set of 14 overlap
ping cosmids. In parallel, C hen et al. (1992b) were able to isolate the region as a
160 kb Bsshll-Sstll YAC fragm ent. Subsequently, these cosmids as well as the entire
160 kb YAC-fragment were em ployed as probes to look for genom ic abberations in
classic NDP patients (non-deleted for established m arkers), an d to isolate hom ol
ogous cDNAs from retina-specific (both fetal and adult) and brain-specific librar
ies.
Using a cosm id, G8, from the NDP region Berger et al. identified at least three
new m icrodeletions sm aller than 20 kb associated with ND. Five retinal and brain
cDNAs hom ologous to G8 were shown to be evolutionarily conserved an d to be
specifically expressed in retinal tissue (B erger et al., 1992a). These overlapping
cDNAs form ed a sequence o f 1.9 kb including a 3' poly A tail. T he sequence con
tains two overlapping reading fram es o f 474 and 417 bp, respectively, o f which
probably only the first encodes a protein (B erger et al., 1992a).
Simultaneously, C hen et al. (1992b) used the 160 kb YAC fragm ent to isolate
four retin a specific cDNAs which were partially or com pletely deleted in the p re
viously described NDP m icrodeletion patients. O ne o f these clones, FR2, also d e
tected very small deletions in five o u t o f 20 un related classical NDP patients
surveyed. N o rth e rn analysis with FR2 as a probe revealed specific expression in
brain o f two RNA transcripts o f about 1.7 a n d 2 kb. Analysis of the sequence ob
tained from the longest cDNA clone hom ologous to FR2 provided strong evidence
for an open reading fram e o f 399 bp (C hen et al., 1992b).

T he structure o f the NDP gene

B erger e ta l. (1992b) and M eindl e ta l. (1992) established the intron-exon struc

ture o f the N orrie disease candidate gene by sequencing. T he genom ic size of the
gene is 28 kb in length, an d it contains 3 exons of approxim ately 201-221, 381
and 1246-1249 bp separated by introns o f approxim ately 16 and 9 kb (C hen et
al., 1993) (I jures 6 and 7). In genom ic DNA, the 5' region of the gene is inter
ru p te d by the first intron with the result that the first exon is entirely untrans-
 NORRIE DISEASE 331

Xcen Xpter
G8------------------------------------------------------------ -V -
M8---------
Al0-*~ ---- --- - ------------------------
kbp j 3.7 12.5| 9.5 J 5.4 j2jlj 10 J 4 (1.3J

promoter exon 1 exon 2 exon 3

-370 1 209 210 417 590 591 815 1836

-42 ATG TGA 1819
TATAT AATAAA

F ig u re 6 T h e ex o n -in tro n -stru ctu re o f th e N o rrie disease g e n e as p u b lish e d elsew here (B erg
e r e t al., 1992c; M ein d l e t al., 1992). T h e co sm id clo n es u sed fo r se q u e n c e analysis are given
by th e co d es M 8 , G8 a n d A10. EcoRI re stric tio n sites a re in d ic a te d by arrow s a n d fra g m e n t
sizes in kb. O p e n arrow s in d ic a te x o n c o n ta in in g g en o m ic frag m en ts. T h e p ro te in co d in g
p a rt o f th e e x o n s is given as h a tc h e d boxes, th e p ro m o te r re g io n as d o tte d , a n d th e 3' a n d 5'
flan k in g re g io n s as o p e n boxes. P ositions o f th e TATA box, th e start a n d sto p c o d o n as well
as th e p o ly ad en y latio n signal c o rre sp o n d to seq u e n c e s p u b lish e d e a rlie r (B e re e r e t al., 1992a;
M ein d l e ta l., 1992).

B E EB EBBEB B B E EBE
U III I
exon 1
1 ... S &=
Cen Tel
13545

8838

3883

6446 a H M H I

12316

F igure 7 (a) G en o m ic restrictio n m a p o f th e N D P g e n e a n d th e lo catio n s o f th e th re e exons

a c co rd in g to C h e n e t al. (1992c; a n d u n p u b lish e d resu lts). T h e th re e o v erlap p in g lam bda
p h a g e clo n es c o n ta in in g th e N D P g e n e a re d e p ic te d above th e m ap. Below th e re strictio n
m ap, th e re g io n s d e le te d in th e m ic ro d e le tio n p a tie n ts a re in d ic a te d (o p e n bars d eleted ;
filled bars: p re se n t; h a tc h e d bars: p recise e n d p o in ts n o t d e te rm in e d ). In (b) th e g en o m ic
stru c tu re a cc o rd in g to C h e n e t al. (1992b, c; a n d u n p u b lish e d results) o f th e N D P g en e is
show n. T h e n u c le o tid e se q u e n c e s o f th e re g io n s flan k in g th e th re e ex o n s (b o ld a n d u n d e r
lin ed ) a re show n. P o te n tia l TATA-boxes a re u n d e rlin e d . T h e sta rt o f th e first ex o n s has b e e n
ta k e n as c o in c id e n t w ith th e lo n g e st cD NA clo n e (F R 7).
332 A. A. II BERGEN F.T Al

AAATTCAGAT ATTCATTGGC CTCTTATTAG TTCCATAATA CCATTAAAAA

AGAAAGAAAG AAAGAAACTT CCTCGCCCTT GTTCTCGTAC GCTGTTCCCA

TCGTAAGATG CTCCGTGGAA GGGAGCCGAG CGGTGGGCAG AGGCTGAGTC

CCCGATAACG AGCGCCTCAC ATTTCCGTGG CATTCCCATT TGCTAGTGCG

CTGCTGCGGC CGCACGCCTG ATTGGATATA TGACTGCAAT GGCACTTTTC

CATTTGACAT TCTTC EXON 1 r2 0 1 b o > TAAG GTAAGCAGGA

AAACAAG CGC TGCCGGCTTG ------1 6 k b ------ ACCCAATTCG GTTACGTTGT

TGCCAGAACA ACATGTTTAA TCTTTAACAT GGGTTCAAAC TATTCTTGGC

CCTAGGAACA TGGACTTCAG CAATTAAAGT CAACATGTGC TTCCATTAAA

CCATTGTGTC CACCTCCAAA TGGTTATAAA TATCTAGCAT AAGCTATGGG

AGTTGGGGTG GAATGGATGA CAGCCTTTGC TAATGACCCT CTAGAAACCA

ATATTCTCCT CTCAAAATAA CATGGAAAAA TTCTACTTAA TACCTCCTGG

CTTCCATTAG TGGTTCTGGG TAAATAATTC TGGGGAAAGT AATTTCTGTT

TTCATTCCAG CTGT EXON 2 <380 b o I AAAG GTAAGACCAA

GGTCTCTGTG AGGAGAGCAT ------8 k b CCCACAGATA ACCTCCTTTT

CTTTCCCCAG ATGG EXON 3 (1 2 5 7 bo) TC AAATTTGACT

GTTAGTATTT
Figure 7 (b)

lated. The ATG start codon lies within exon 2, and the stop codon as well as the
polyadenylation signal are located in exon 3 (Figures 6 an d 7). T he open reading
fram e of 399 bp encoding a p rotein of 133 am ino acids, is com pletely contained
within exons 2 an d 3. W ithin the first intron, a canonical TATAAAT sequence was
identified 185 bp 5' to the start o f the second exon. This structural organisation
would allow an alternative start of transcription, suggesting that expression o f the
NDP een e may be reeulated by the activity of two in d ep en d e n t p ro m o to r regions
(C hen e ta l., 1993).

M utation analysis and diagnostic im plications

C onvincing evidence for the involvem ent of the isolated candidate gene in the
etiology o f N orrie disease has com e from th e detection o f point m utations in
patients. Berger et al., (1992b) described the identification o f small sequence
alterations in 10 out of 12 non-deletion patients with classical NDP. F urtherm ore,
m utation analysis established the diagnosis in 2 out o f 5 patients, in whom the
clinical diagnosis o f NDP was suspected. Sim ilar results were re p o rted by M eindl
et al., (1992) and C hen et al., (1993). Most o f the m utations described so far give
 NORRIE DISEASE 333

rise to an am ino acid substitution (7 ou t o f 15). In 6 patients, m utation events

predict a tru n cated p rotein. Only o n e m utation was found to affect a splice site,
while one o th er m utation extends the predicted polypeptide by 127 am ino acids.
It is o f interest to note that 6 o u t o f these m utations alter o r generate a cysteine
codon which may indicate that cysteine residues are particularly im portant for
the function of the protein.
F urtherm ore, these studies prove th at m utation screening is a powerful diagnos
tic tool in clinically uncertain, sporadic cases. C om parison between the type o f m u
tation and the clinical phenotype has failed to reveal any obvious correlation
(Berger et al., 1992b). A part from the typical ophthalm ological signs, both m ental
changes and progressive deafness were seen in several patients with different point
m utations.

Expression o f the NDP gene

N o rth e rn analysis of mRNA derived from hum an glioma, glioblastom a, an d n eu

ral cells reveals strong hybridisation to a transcript o f 1.8 kb. Also, o th er tran
scripts with d ifferent sizes have been observed from N o rth ern analyses (I. Craig,
personal com m unication; C hen et al., 1992b; Berger et al., 1992a).
Using PCR prim ers designed for specific am plification of cDNA (RT-PCR), ex
pression is found in fetal eye, fetal an d ad u lt brain, fetal lung and adult muscle.
No expression is fo u n d in fetal liver, adult liver cells o r cultured liver cells (C hen
et al., 1993). Thus, the expression o f the NDP gene is no t only confined to the eye
o r brain. Analysis o f the tissue expression pattern may h elp to u nderstand the dis
ease m anifestation in typical N orrie patients.

T he function o f the predicted protein

An extensive DNA an d p rotein sequence com parison betw een the hum an NDP
gene and related genes from the NBRF and Swiss protein database revealed sig
nificant hom ology with C -term inal cysteine-rich dom ains shared by a family of
mucin-like proteins an d von W illebrand factor (M eindl et al., 1992; C hen et al.,
1993). Many m ucins are secreted proteins and some are integral m em brane p ro
teins an ch o red at a m ucin-like, glycosylated dom ain. No glycosylation sites are
identified in the NDP gene product. However, an N -term inus hydrophobic region
of the NDP p ro d u ct contains features o f a signal sequence containing a basic
region, a hydrophobic "core" an d a m ore polar region (C hen et al., 1993). A weak
hom ology between this N -term inal dom ain and the rat-horsefall protein supports
the secretory status of the NDP gene p ro d u c t (M eindl et al., 1992). Prediction of
signal sequence cleavage sites based on the m eth o d o f G. von H eijne (1986)
defined a signal peptide o f 24 am ino acid residues with a 0.744 probability
(B erger et al., unpublished data). T he same region also exhibits hom ology with
two im m ediate early genes; the p h o rb o l ester-repressive an d v-src-inducible Cyr61
gene identified in m urine fibroblast (M eindl et al., 1992; C hen et al., 1993).
334 A. A. B. BERGEN ET AL.

Based on the observed hom ologies, it can be postulated th at the NDP gene is
involved in a pathway that regulates neural cell differentiation and proliferation.
Also, because o f the potential o f cysteine rich regions to form disulphide bonds,
it can be postulated th at the function o f the protein may concern interaction with
o th er growth factors im p o rtan t in neurological developm ent.

O utlook

T he precise function of the NDP gene rem ains to be established. D irect inform a
tion on the role, tissue specificity and localization of the protein can com e from
studies using specific antibodies raised against the in vitro synthesized gene p ro d
u ct (M arston, 1986). T he isolation o f the m ouse gene and the construction of
transgenic m ice or o th er anim al m odels should provide a m ore detailed insight
into the function o f the N orrie disease gene.
At present, direct m utational analysis for coding changes in the second and the
th ird exons is possible, while SSCP (O rita et al., 1989) will assist in rapid screening
for additional lesions outside these regions.
T he occurrence o f small DNA deletions in 20% o f the NDP patients an d the
identification of p o in t m utations in 73% o f the cases offers the possibility o f reli
able DNA diagnosis in 93% o f all cases (B erger et al., unpublished results).
Finally, it is encouraging to note that the small size and the secretory status of
th e N orrie gene translation product raises perhaps the possibility o f therapeutic
approaches in som e instances.

ACKNOWLEDGEMENTS

We thank T. P ut for technical assistance in preparing this m anuscript.

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h e re d ita ire a tran sm issio n a u to so m a l recessive. Annale Ocul. 200, 12411252
Sim s K. B., O zelius L., C orey T., R in e h a rt W. B., L ib e rfa rb R., H a in e s J., C h e n W. J., N o rio R.,
S ankila E., d e la C h a p e lle A., M u rp h y D. L., G u s e lla J ., B reak efield X .O . (1989) T h e N o rrie
disease g e n e is d istin c t fro m th e m o n o a m in e oxidase g e n es. American Journal o f Human Genet
ics 45: 4 2 4-434.
Sim s K. B., L eb o R. V., B e n so n G., S halish C , S h u b a c k D., C h e n Z. -Y., B ru n s G., C raig I.W., Gol-
bus M. S., B reak efield X. O . (1992) T h e N o rrie disease g e n e m ap s to a 150 kb re g io n o n c h ro
m o so m e X p l 1.3. Human Molecular Genetics 1, 2: 83 -8 9 .
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Hurnangenetik 25, 1-16.
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v itre o re tin o p a th y . American Journal o f Ophthalmology 11, 34-41.
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75 7-772.
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age d a ta . Acta Genetics (Basel) 15, 103.
338 A. A. B. BERGEN ET AL.

W arb u rg M. (1966) N o rrie 's disease: a c o n g e n ita l progressive o c u lo -a co u stic o -ce rcb ra l d e g e n e r
a tio n . Acta Ophthalmology (C o p e n h ) 89 [S uppl] ,1-147.
W arb u rg M. (1975) N o rrie 's disease. D iffere n tia l d iag n o sis a n d tre a tm e n t. Acta Ophthalmology
(C o p e n h ) 53, 21 7 -2 3 6 .
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H . H . (1984) T ow ard a c o m p le te lin k a g e m a p o f th e h u m a n X c h ro m o s o m e : re g io n a l assign
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 14. X-LINKED JUVENILE RETINOSCHISIS

ALBERT DE LA CHAPELLE, TUNA ALITALO and HENRIK FORSIUS

Department of Medical Genetics, University of Helsinki, and Folkhalsan Institute of Genetics,
Helsinki, Finland

INTRODUCTION

Retinoschisis is a descriptive term indicating a splitting o f the retin a into two lay
ers an d is often subdivided into congenital and acquired forms.
T he differential diagnosis o f congenital retinoschisis includes a diverse g roup of
disorders. Gorlin an d K nobloch (1972) distinguished 6 different genetic syn
drom es in which juvenile retinal d etach m en t occurs, including juvenile retin
oschisis. In m ost textbooks, the lists o f conditions to be considered in patients with
retinal d etach m en t are long. Two m ain developm ents now m ake fu rth e r subdivi
sions of the retinoschisis syndrom es easier than before. First, im proved clinical
m ethods allow distinct syndrom es to be delineated. Second, the genetic m apping
o f several congenital eye disorders provides a m eans of genetic classification.
T he subject o f this review is X-linked juvenile retinoschisis (MIM 312700, McK
usick, 1990), a disease th at belongs to the vitreoretinal dystrophies. We shall use
the designation RS ad o p ted by the H um an G ene M apping W orkshops (McAlpine
et al., 1991). A ccording to this classification RS indicates the gene an d RS the dis
ease phenotype; for simplicity this distinction is n o t always necessary. While the
first reports suggesting th at RS may reside in the distal p art of the short arm o f the
X chrom osom e appeared over 20 years ago (Eriksson et al., 1969; Race and
Sanger, 1975) its m ore precise localization has been accom plished in the last 5-10
years. This has paved the way for detailed linkage studies, carrier detection, and
pre- an d postnatal diagnostics w ithin families. Thus the concept of a disorder well
defined both by clinical and genetic criteria is em erging.

EPIDEMIOLOGY

As reviewed by Alitalo (1990), the first description o f RS probably ap peared in

1898; however its birth as a distinct entity occurred as late as the m iddle of this
century (M ann and McRae, 1938; Sorsby et aL, 1951; Jager, 1953). Already in
their textbook published in 1974 Franceschetti et al., listed 218 patients with RS
described in the literature. Most patients were from W estern E uropean popula
tions. C om bined clinical an d genealogical studies revealed a strikingly high prev
alence in a small district in southw estern F inland w here over 100 patients
probably descended from a com m on ancestor (Vainio-Mattila et al., 1969). In
1973 Forsius et al. described 183 cases. Later, as m ore patients with RS were diag
340 ALBERT DE LA CHAPELLE. ET AL.

nosed in Finland (over 300 today) the co n cep t em erged that RS is m ore preva
len t in F inland than elsewhere in the w orld (Forsius an d Eriksson, 1980; Norio,
1981).
T he idea o f an uneven geographic o r ethnic distribution now needs to be re-ex
am ined. In the past, case re p o rtin g has do m in ated the literature on RS m aking it
difficult to assess its frequency an d distribution. However, it is obvious th at RS oc
curs in m ost parts o f the world and in d ifferen t ethnic groups such as Am erican
Blacks (C onstantaras et al., 1972; Ide an d Wilson, 1973), Chinese (Zhang, 1984),
Jap anese (Kawano e ta l., 1981; M inato, 1991), Indonesians (D eutm an, 1971) and
East E uropeans (Janaky an d B enedek, 1990; Nizankowska and Wozny, 1991).

PREVALENCE

From the m edical an d sociological points o f view, the prevalence (the n u m b er of

living affected individuals p er total n u m b er o f individuals in the population) is
an im p o rtan t figure. This is distinct from the number of mutations, th at is, the nu m
b er o f unrelated affected individuals, families or pedigrees in a given population.
An exam ple o f a discrepancy between the two figures com es from Finland, where
the prevalence exceeds 1: 17,000 (over 300 patients in a population o f 5 m illion).
However, as d eterm in ed by genealogy a n d haplotype analysis, as few as 2 m uta
tions (one in the Pori district, one in the O ulu district) may account for the
m ajority o f all patients in Finland (Alitalo et al., 1991a). C orresponding data are
n o t available from o th er countries. However, RS should no longer be regarded as
rare. Already in 1953 Jag e r in the N etherlands h ad seen 25 patients. K raushar
(1972) described 40 patients seen in Boston, USA; D eutm an (1977) working in
the N etherlands re p o rted having seen over 100 cases, a n d V erdaguer (1982) had
seen 48 patients in Chile. M ore re cen t com prehensive reports from France
describe 60 patients (T urut et al., 1989; C astier et al., 1990) and 22 patients from
7 u n re la ted families, (Kaplan et al., 1991), respectively. In a G erm an series 52
patients were described (K ellner et al., 1990). In M ichigan, USA, 56 affected
m em bers o f 7 families were recently re p o rte d (Sieving et al., 1990). These data
allow us to suggest that RS is m uch m ore com m on than previously thought, and
th at a geographically o r ethnically uneven distribution o f m utations worldwide
does n o t necessarily exist. Very high local prevalences such as the one in the Pori
region in Finland (Vainio-Mattila et al., 1969) an d C anada (Ewing and Ives, 1969;
C ondon et al., 1986), obviously exist in stable populations. These are usually the
result o f a fairly recen t (<20 generations) fo u n d e r having by chance transm itted
the m utation to num erous offspring.
Few reports address the question of the actual prevalence o f RS. In an effort to
d eterm in e the prevalence o f the main hereditary retinal dystrophies P uech et al.
(1991) studied the occurrence o f these disorders in a defined population o f 4 mil
lion people in n o rth e rn France during 1972 to 1989. For RS the prevalence figure
was 1 in 28,092. As a com parison, the corresp o n d in g figure for typical retinitis
 X-LINKED JUVENILE RETINOSCHISIS 341

pigm entosa was 1 in 4,225 and for choroiderem ia 1 in 177,454. Even though the
authors attem pted to correct for incom plete ascertainm ent, the above figures
should be m inim um estimates.
In a sim ilar study in central Sweden, Skoog et al. (1990) en c o u n tered 9 patients
with RS am ong a total o f 487 patients referred to an opthalm ological centre for
suspected or obvious hereditary degenerative o r dystrophic eye disorders. While
no estim ates of prevalence were offered, the same series com prised 38 patients
with retinitis pigm entosa and 2 patients with choroiderem ia, respectively. These
data are com patible with the data from n o rth e rn France an d indicate that RS in
d eed accounts for a relatively large p ro p o rtio n o f these disorders. Taken together
with the Finnish prevalence figure o f 1 in 17,000 (u n corrected for incom plete as
certainm ent) we tentatively conclude that the prevalence o f RS is between 1 in
15,000 an d 1 in 30,000, o r even higher. At face value this m eans, for instance, that
there are over 30,000 RS patients in E urope an d over 300,000 in the world. T he
condition is certainly underd iag n o sed for many reasons, including the m ildness
o f symptoms in some cases, an d the difficulty ophthalm ologists have in m aking the
diagnosis. In the study by Skoog et al. (1990) for instance, only 4 o f the 9 RS pa
tients had been correctly diagnosed by the referring ophthalm ologists.

CLINICAL FEATURES

Pathogenesis

According to A rden et al. (1988), RS is to be considered as a disorder o f retinal

developm ent ra th e r than a dystrophic process. T he prim ary defect may be in
M uller cells, as suggested by electrodiagnostic findings. T he first sign of the dis
ease appears to be the abnorm al developm ent o f the cortical vitreum that is m an
ifest at birth and so m ust have already begun in the fetal state. Progression results
in a slow an d gradual deterio ratio n o f retinal structure an d functions.
In 1968 Yanoff an d collaborators p e rfo rm ed a histopathological exam ination in
a 4-year-old affected boy. A large cystic cavity was found betw een the equator and
the ora serrata. This cavity was the result o f the internal lim iting m em brane and
p art o f the in n erm o st retinal tissue having split away from the rem ain d er o f the
sensory retina. Sections from the inferior calotte showed early schisis in the nerve
fibre layer. U ltrastructural findings led C ondon et al. (1986) to the hypothesis that
defective M uller cells produce the filam ents that accum ulate extracellularly within
the re tin a and secondarily lead to retinoschisis.

Anatomical features

In m ild cases com prising 60% of all patients changes occur in the m acular area
only. In this site a wheel-like cystic form ation about the size o f the optic disc is
seen (Figure 1). T he changes at the m acula are probably congenital but are diffi
cult to differentiate from their surroundings before the age of about 2 years.
342 ALBERT DE IA CHAPELLE, ET AL.

F igure 1 R e tin o sc h isis in a n 11-year-old b o y sh o w in g typ ical cystic fovea.

A radiation of superficial retinal tissue is som etim es seen outside the cystic area
in young subjects. A round the age of 40, the highly typical an d readily distinguish
able central schisis flattens out, atrophies an d becom es m ore difficult to recogn
ise. In aged patients, the pigm ent layer disappears so that the disease can no
lo n g er be differentiated from circum scribed m acular d egeneration (Figure 2). In
ad d ition to the m acular changes, m oderately severe cases show sem itranslucent
vitreous veils in the lower tem poral perip h ery (Figure 3). These veils consist of d e
tached portions o f the superficial layer o f the retina, and contain retinal blood ves
sels. O ften a dem arcation line is seen at the b o rd e r o f the schisis (Figure 4),
although this may also occur in retinal detach m en t unassociated with schisis. The
 X-LINKED JUVENILE RETINOSCHISIS 343

F igure 2 R e tin o sc h isis in a 2 7-year-old m a n s h o w in g a n e le v a te d s u p e rfic ia l le a f o f th e re tin a ,

a so -c alled v itre o u s veil.

veils can rem ain un ch an g ed for decades. Parts o f them may float freely in the
deg en erated vitreous body, bu t as a rule they ap p ear elevated from the ocular
fundus and form c o h e ren t sheets which seem to start from apparently norm al
retinal tissue close to the optic disc. In the periphery, the veil again has contact
with the fundus. In young subjects up to a few years o f age, the veil can form a
large cyst in which holes gradually appear, and the cyst collapses. In adults, the
holes often increase in size and finally only rem nants of the veil aro u n d blood
vessels may be seen. Sorsby et al. (1951) were the first to describe this p h en o m e
non. In severe cases, the veil may form a roll below the optic disc. Increasing atro
phy including degeneration o f the pigm ent layer occurs in the attached p art of
the retina. Up to m iddle age, garland-like glistening form ations nearly always
344 ALBERT DE LA CHAPELLE, ET AL.

F ig u re 3 R e tin o sc h isis in a 6 9-year-old m a n s h o w in g d is a p p e a r a n c e o f th e fo v eal cysts le a d in g

to a p ic tu r e in d is tin g u is h a b le fr o m s e n ile m a c u la r d e g e n e r a tio n .

o ccur in the perip h ery of the fundus, which otherw ise appears norm al. D rusen of
the optic disc have been described, as well as whitish streaks along the blood ves
sels and pigm ented areas; in extrem e cases, the entire retina an d choroid are dis
organised. Patients with these features can be totally blind from birth.
Some investigators have re p o rted follow-up observations of several years' d u ra
tion beginning in patients at the age o f a few m onths. Forsius et al. (1990) followed
40 patients for an average o f 22 years. An initial high bullous detachm ent is fol
lowed by hole form ation in the wall, after which the balloon-shaped detached ret
ina flattens out. Conversely, even in affected individuals as old as 50-60 years,
retinoschisis w ithout holes has been observed by some investigators.

Visual acuity and refraction

In young subjects, visual acuity can be nearly norm al. Typically, the disease is no t
d etected until the first school year when a vision o f 0.5 to 0.7 (2 0 /4 0 -2 0 /3 0 ) is
found. However, occasionally the acuity can be very low at this stage. Am ong our
own 300 cases, two boys were nearly totally blind from birth.
 X-LINKED JUVENILE RETINOSCHISIS 345

F ig u re 4 R e tin o sc h isis in a 6-year-old b o y s h o w in g b o th c e n tra l a n d p e r ip h e r a l c h a n g e s . A

la rg e h o le is s e e n in th e a b la te d r e tin a l l e a f a t th e b o tto m .

U p to 40-50 years o f age the vision is stable bu t th en gradually deteriorates, as

shown in large p atien t series (Ewing a n d Ives, 1969; Forsius et al., 1973). T he vari
ability o f this p aram eter is exem plified by patients in whom good visual acuity can
be preserved up to 60 years o f age (K ellner et al., 1990). T he disease rarely results
in total blindness.
Patients are usually hyperopic a n d in addition can be astigmatic, som etim es
quite rem arkably so. Myopia is rare.

Colour vision

Secondary dyschrom atopsia is the ru le in m ore advanced cases. Disturbances

both in tritanopic an d re d -g re e n vision have been dem onstrated. T he abnorm al
ities have been attrib u ted to dysfunction o f the o u ter sensory retin a occurring
secondarily to changes in the in n er sensory retina (Helve, 1972; M inato, 1991).

Electro-oculography, electroretinography, dark adaptation, fluorescein angiography

Except in advanced cases, electro-oculography usually shows a slight reduction of

norm al values according to Tanino et al. (1985).
346 ALBERT DE LA CHAPELLE, ET AL.

O n electroretinography, the a-wave is norm al in light-adapted eyes b u t subnor

mal in dark-adapted eyes. Likewise the b-wave shows norm al values in light-adapt
ed eyes an d som ewhat subnorm al values at lower light intensities (K ellner et al.,
1990). In severely affected eyes the electroretinogram s are abnorm al as a result of
dysfunction o f the M uller cells.
T he final threshold in dark adaptation is 1-2 log units above norm al. (Miyake
et al., 1981) T he visual fields correspond to the area o f retinal in v o lv em en t.
A ccording to Krause et al. (1970), fluorescein angiography is norm al in young
patients with an intact pigm ent layer an d with changes only in the m acular area.
In old subjects, the pigm ent layer disappears and large choroidal vessels ap p ear in
the macula. In cases with both central and p erip h eral changes, epithelial dam age
involving pigm ent occur both centrally a n d peripherally o f the vascular veil.

Com plications

B leeding in the retina an d vitreous body have been re p o rted in up to 25% of all
cases. These often constitute the cause o f the first consultation with an o p h th al
m ologist. In large series studied as p art o f family investigations, the incidence is
m uch lower, e.g. 4.7% according to K ellner et al. (1990). In our series o f 300
mostly familial cases, the figure is less than 10%. H aem orrhages occur both in the
cavity o f the schisis and in the vitreous body an d are probably caused by increased
splitting o f the ablated layer. In advanced cases, neovascularisation has been fre
quently observed in the p erip h ery bu t can also occur in the optic nerve (Pearson
a n d ja g g e r, 1989). Blood vessels can be seen in both the ablated layer and in the
p art o f the retin a th at rem ains attached to the fundus. T he bleeding is mostly
quickly resorbed b u t som etim es requires vitreous surgery (Greven et al., 1990).
T rue, full thickness retinal d etach m en t is re p o rted at highly varying frequencies
u p to 20% d ep en d in g on w hether the series em anate from hospital reports or
from studies on families. For instance, in family studies D eutm an (1971) found
retinal d etach m en t to be rare an d we have observed only 5 cases ou t o f 300. In
these cases a hole has ap peared even in the in n er retinal layer. T reatm ent is the
same as for regular retinal detachm ents a n d the results are acceptable.

T herapy

No therapy to cure the degenerative condition is known. T he rare tru e retinal

d etachm ents should be treated by conventional surgical techniques. Large
in traocular bleeding may require vitrectomy. P eripheral schisis rarely progresses
to the central area b u t may need photocoagulation. Prophylatic photocoagula
tions were frequently used earlier bu t are hardly beneficial as progression o f an
elevated leaf is rare. In the last decade it has been observed that prophylactic
p hotocoagulation can itself cause detachm ent; it should be ab an d o n ed (Castier
et al., 1990; Kellner et al., 1990). T he indications for vitrectom y have been
reviewed by Schulm an et al. (1985).
 X-LINKED JUVENILE RETINOSCHISIS 347

Differential diagnosis

ReUnoschisis is som etim es not noticed at all. It is often m isdiagnosed as heredo-

d egeneration o f the m acula, o r as retinal periphlebitis. In the aged the central
schisis is nearly im possible to distinguish from senile m acular degeneration.
O th er types o f sim ilar retinoschisis exist. T he m ost com m on one, senile retin
oschisis, has a d ifferent age distribution an d seldom poses differential diagnostic
difficulties.
C entral retinoschisis may have autosom al d om inant inheritance (D eutm an et
al., 1976; Yassur et al., 1982). Autosom al recessive central retinoschisis has also
been re p o rted (Forsius et al., 1978; Lewis et al., 1977; Noble et al., 1978; Zografos
and Faggioni, 1980). Several syndrom es display retinoschisis as one com ponent.
A com bination o f central and perip h eral retinoschisis is seen in W agner disease
(Wagner, 1938), which has autosom al d o m in an t heredity. This syndrom e is fu rth e r
characterized by myopia, hem eralopia, extinguished electroretinogram , an d cata
ract. A variant of W agner disease was studied by Yamaguchi and H ara (1989). O th
er disorders som etim es posing diagnostic difficulties are congenital retinal folds,
an d N orrie disease (Godel and G oodm an, 1981). Stickler disease may have the
same symptoms and heredity b u t in addition jo in t abnorm alities occur (Stickler et
al., 1965). Pedigrees published by Yassur et al. (1982) (autosom al dom inant RS)
and Shimazaki an d Masakazu (1987) probably rep resen t diseases belonging to the
same family as RS.
T he G oldm ann-Favre type of hered itary idiopathic retinoschisis is character
ised by vitreous degeneration with liquefaction and the form ation of preretinal
strands and cords, perip h eral an d central retinoschisis, pigm entary and degener
ative changes of the retina, hem eralopia with abolition o f the electroretinogram ,
progressive decrease in visual function, lens opacities, an d autosom al recessive h e
redity (Favre, 1958). A radiating stellate p attern in the m acula may occur in this
disorder (Nasr et al., 1990).

Penetrance and expressivity

The p enetrance in males appears to be 100%. As described above, expression is

quite variable. T h at this is in large p a rt due to factors o th er than the type of
m utation in the RS gene is shown by exam ples o f highly d ifferent severity within
pedigrees and even within sibships. We have seen brothers with phenotypes rang
ing from slight cystic m acular changes to alm ost com plete blindness.

Phenotypes o f heterozygotes

Most authors hold that even careful ophthalm oscopy o f the central and perip h
eral retin a does n o t disclose changes in RS gene carriers that can be distin
guished from those seen in norm al w om en of the same age (e.g. V ainio-M attila
et al., 1969; D eutm an, 1971). However, occasional reports o f pathological foveal
348 ALBERT DE LA CHAPELLE, ET AL.

findings such as m ottling and gliosis in the p erip h ery have appeared (Ewing and
Ives, 1969; Kaplan et al., 1991). In several carrier wom en, a consistent distur
bance o f retinal function was detected by A rden et al. (1988), who exam ined the
relationship o f cone flicker threshold to changing light intensities o f the back
g ro u n d at a single retinal site.
It may in d eed be th at some heterozygous wom en can display phenotypic effects
o f the gene m utation they carry. However, these changes are n eith er typical
en o ugh n o r do they occur in a sufficient p ro p o rtio n o f carriers so as to provide
reliable carrier detection by conventional clinical investigation.
A small nu m b er o f affected fem ales have been described (Forsius et al., 1962;
U chino and Shimizu, 1976; H am aguchi et al., 1989). These are wom en who are
hom ozygous as a result o f the m ating o f an affected m ale and a heterozygous fe
male. As expected, their phenotype resem bles th at o f affected males. T he same
was tru e o f a 45,X fem ale with RS (H om m ura et al., 1982).

MOLECULAR STUDIES

Linkage studies in retinoschisis

T he first attem pts to m ap the RS gene were m ade by typing RS families for the Xg
blood group. These linkage studies suggested that RS m ight be located at a m ea
surable distance from the Xg locus (Eriksson et al., 1969; Ives et al., 1970; Race
an d Sanger, 1975; Boman et al., 1976; Forsius and Eriksson, 1980). Subsequently
W ieacker et al. (1983) used a polym orphic DNA m arker (RC8) to study two RS
families which had also been typed for Xg. T h eir linkage results with RC8 (Zmax
= 1.74 at max = 0.15) gave additional support for the localisation of the RS gene
on the distal part o f Xp, although no definite location could be ascertained. O ne
o f the two RS families was fu rth e r analyzed with additional m arkers by Gal et al.
(1985). These prelim inary two-point linkage studies, re p o rted in an abstract, sug
gested that RS is also linked to DXS43, DXS16, an d DXS85.
All two-point linkage studies betw een RS and anonym ous RFLPs published to
date (with m axim um lod score values >3) are in agreem ent that RS is closely linked
to m arkers DXS16, DXS43, DXS207, DXS9, DXS274, and DXS41 on Xp22T-p22.2
(Table 1). With the exception o f one family in the initial study by W ieacker et al.,
(1983), the m arker locus DXS9 has shown no recom binations with RS in the fam
ilies studied (Alitalo et al., 1988; G ellert et al., 1988; Sieving et al., 1990). In addi
tion, no recom binations have been found between RS and DXS197 (Zmax = 4.82
at 0 max = 0.00; Alitalo et al., 1991a). T he results o f one study ap p ear divergent,
b u t all the lod scores re p o rted are sm aller than 3 (G ellert et al., 1988). W hether
the families described by W ieacker et al. (1983) and G ellert et al. (1988) rep resen t
d ifferent eye disorders, genetic heterogeneity in RS, o r indicate greater distances
betw een RS and the m arkers DXS9, DXS16, DXS43, and DXS41 than is suggested
by the o th er studies, rem ains to be resolved. Incidentally, when com paring the re
sults of various two-point linkage studies, it is good to rem em ber th at although sig
nificant lod scores can be o btained between various pairs of m arkers by two-point
 X-LINKED JUVENILE RETINOSCHISIS 349

linkage analysis, the confidence intervals ten d to be wide (e.g. Alitalo et al.,
1991b). Thus, while two-point linkage analysis is a good m ethod for establishing
linkage, the genetic distances o b tained should be in terp re ted with caution. This
applies especially w hen m axim um lod score values are close to or sm aller than 3
an d the n u m b er o f inform ative m eioses is small.

7 A
Table 1 Two-point linkage data ( max at max) between RS and six markers

DXS43 Confidence DXS207 Confidence DXS 9 Confidence Reference*

Zmax e max interval z max 0 max interval z max e max interval

13.87 0.02 0.00-0.06 5.25 0.05 0.01-0.15 3.61 0.00 0.00-0.10 1.

4.98 0.00 NA 2.93 0.00 NA NA NA NA 2.
1.36 0.08 NA NA NA NA 2.66 0.00 NA 3.
5.91 0.00 NA 4.17 0.00 NA NA NA NA 4.
7.37 0.05 0.01-0.16 NA NA NA 4.93 0.00 0.00-0.12 5.

DXS 16 Confidence DXS274 Confidence DXS41 Confidence Reference

zmax 0 max interval zmax 0 max interval max 0 max interval

12.60 0.06 0.02-0.13 9.02 0.05 0.01-0.14 9.60 0.06 0.02-0.13 1.

1.41 0.00 NA NA NA NA 4.09 0.00 NA 2.
0.07 0.40 NA NA NA NA 0.00 0.50 NA 3.
2.42 0.00 NA 3.28 0.08 NA 1.16 0.18 NA 4.
7.81 0.07 0.02-0.18 NA NA NA 9.47 0.07 0.02-0.16 5.

*1. Alitalio e ta l., 1987, 1988, 1991a; 2. Dahl e ta l., 1988,3. G ellert et al., 1988; 4. Kaplan et al., 1991; 5.
Sieving e t al., 1990
N A =not available

M ultipoint linkage analysis provides a m ore effective an d quantitative m eans of

answering the question o f the m ost probable location o f the RS gene with respect
to the m arkers. Two in d e p e n d e n t studies have localised the RS gene betw een the
m arkers DXS43 and DXS41 (Alitalo et al., 1988; Sieving et al., 1990). T he m ulti
point results presented in an abstract by van Schooneveld et al. (1989) are in
agreem ent with this localisation. Only the study by G ellert et al. (1988) is in dis
agreem ent. Estimates for the distance betw een DXS43 and DXS41 vary from 10
cM to 15 cM (Page et al., 1990; Sieving et al., 1990; Alitalo et al., 1991a; Benham
and Rowe, 1992). T he genetic m ap aro u n d the RS locus has been fu rth e r im
proved by m ultilocus linkage studies in an expanded database including 31 Finn
ish RS families and a set o f 40 norm al CEPH families (Figure 5; Alitalo et al.,
1991b). T he refined linkage m ap is in agreem ent with o th er linkage m aps from
the area with slight differences in distances betw een loci (Drayna an d White, 1985;
Donis-Keller et al., 1987; Brown et al., 1988; Econs et al., 1990; Page et al., 1990;
Sieving et al., 1990; T hakker et al., 1990; Davies et al., 1991). W ith the help o f m ul
tipoint linkage studies, the location o f the RS gene has been refined to between
the m arkers DXS274 an d (DXS43,DXS207) with odds of 1.8xl04:l favouring this
350 ALBERT DE LA CHAPELLE, ET AL.

cM Locus

F igu re 5 E s tim a te d d is ta n c e s (cM ) b e tw e e n m a rk e rs o n X p. T h e d is ta n c e s a r e c o m p ile d fro m

five s e p a r a te se v e n -p o in t analyses. T h e o r d e r o f th e m a rk e rs D X S 207, D X S43, a n d D X S 9 w ith
re s p e c t to e a c h o th e r, as w ell as th e o r d e r o f th e m a rk e r s D X S 274 a n d D X S41, h a s n o t b e e n
e s ta b lis h e d (d a ta ta k e n fr o m A lita lo 1990; A lita lo e t al., 1 9 9 1 b ).

location over the second best location (Figure 6 and Table 2). It has b een estim at
ed that the genetic distance between the flanking m arkers is 7 cM and th at the RS
gene is located 2 cM proxim al to (DXS43,DXS207). T he exact o rd e r of DXS9 and
DXS197 relative to RS o r the m arkers DXS43 and DXS207 has n o t been deter
m ined due to lack of recom binations.

Diagnostic procedures

Based on all available linkage data, it is now possible to predict which m em bers of
RS families are carrier fem ales and to diagnose the syndrom e pre- an d post-
natally. Since close linkage (with Zmax > 3) betw een RS and m arkers in the
Xp22.1-p22.2 region has been observed in all families rep o rted , there is no
strong evidence for genetic heterogeneity o f RS. T he closest distal m arkers
DXS43 and DXS207 are about 2 cM from the RS gene, and the closest proxim al
 X-LINKED JUVENILE RETINOSCHISIS 351

DXS207 DXS92
Locus DXS 16 DXS43 RS DXS274 DXS41

Xpter Xcen

Distance 0.02 0.02 0.05 0.03

(theta)
Figure 6 M a p o f c h r o m o s o m e X p sh o w in g lo c a tio n s o f six lin k e d m a r k e r lo c i a n d th e RS
g e n e u n d e r th e b e s t-s u p p o rte d g e n e o r d e r (T a b le 2 ).

m arker DXS274 is about 5 cM from the gene (Alitalo et al., 1991a). Because it is
known that linked m arkers flanking the disease locus provide m uch m ore diag
nostic reliability than linked b u t non-flanking m arkers, we have used these three
flanking m arkers in carrier an d prenatal diagnosis. In a few cases it has also been
necessary to screen the families with the m arkers DXS16 and DXS41, because the
diagnostic utility o f linked RFLPs is som etim es lim ited by a lack of heterozygosity
at the m arker loci in fem ale carriers. We have p erfo rm ed risk calculations using
the risk-calculadon option o f the MLINK program from the LINKAGE package of
com puter program s (L athrop et al., 1984). M icrosatellite repeats isolated from
cosm id clones correspondin g to the m arker loci DXS207 an d DXS274 are now
available, greatly im proving the inform ativeness o f these m arkers (Biancalana et
al., 1992; Rowe et al., 1992) an d thus reducing the n eed to use additional m ark
ers in diagnostics.
In diagnostic work, the population structure a n d available genealogical data
should be taken into consideration. Finland provides an exam ple. First, allele fre
quency studies (on 12 m arkers from Xp) in un related Finnish individuals have
shown that the observed heterozygosity value for DXS207 deviates significantly
from the value found in o th er populations, thus m aking DXS207 less inform ative
in Finnish families than elsewhere (Alitalo et al., 1991a). C orresponding data on
a new m icrosatellite rep eat are n o t yet available. Second, m ost o f the known RS
patients com e eith er from South West F inland an d can be traced to one super-ped
igree, o r from N orth C entral Finland a n d can be traced to a few pedigrees. H ap
lotype analysis with the m arkers DXS207, DXS43, DXS197, DXS274, and DXS41
have shown that patients from South West Finland have a haplotype association
which differs from the haplotype association found in the patients from N orth
C entral Finland. Both haplotypes differ from the m ost frequently observed haplo
type in norm al chrom osom es, which is shared by bo th groups (Alitalo et al.,
1991a). These data can som etimes be useful in genetic counselling; m oreover they
favour the hypothesis that the m utations in the two groups arose independently.
We have p erfo rm ed 17 analyses to d eterm in e carriership an d one for prenatal
diagnosis. T he results do n o t show any discrepancies with clinical an d genealogical
evidence regarding phenotype.
352 ALBERT DE LA CHAPELLE, ET AL.

Pointers fo r future studies

With the aim o f cloning and characterizing the RS gene, ongoing an d future
studies will first attem pt to refine the genetic m ap o f the region. A nu m b er of
new m arkers have been m apped close to DXS43 an d DXS41, although these
m arkers have no t yet been used in linkage studies o f RS. Econs et al. (1990)
m ap ped GLR close to DXS43 (Zmax = 5.40 at 0max = 0.00 ), and m arkers
DXS365 an d DXS257 have been localised betw een the m arkers DXS43 and
DXS41 (Davies et al., 1991; Browne et al., 1992; Econs et al., 1992). In addition,
m arkers DXF22S4/S5 an d 1 6d/E are located close to DXS43 (Davies et al., 1991;
Rowe et al., 1992). New m arkers isolated from the Xp22.1-p22.2 region are pres
ently being m apped (B enham and Rowe, 1992; Biancalana et al., 1992).
T he next step is the physical m apping o f RS, th at is, the delineation o f a contig
uous stretch o f DNA an d the localisation o f RS within it. This will require the
building of con tigs with the aid of yeast artificial chrom osom es and cosmids. Phys
ical m apping of the RS region is in progress (Alitalo et al., 1992) with the aim of
o rd erin g the m arkers DXS9, DXS197, DXS43, DXS207 and GLR with respect to
each other, and allowing new m arkers closer to the RS gene to be isolated.

SUMMARY AND CONCLUSIONS

X-linked juvenile retinoschisis is caused by a m utation in the RS gene on the dis

tal h alf o f the short arm o f the X chrom osom e. We estim ate the prevalence to be
approxim ately one in 15,000 to 30,000, th at is, h ig h er than previously estim ated.
W hile there are geographical regions with very high prevalences such as in Fin
land an d Canada, the frequency o f in d e p e n d e n t m utations may n o t be unequally
distributed. T he disease is a developm ental defect o f the cortical vitreus being
m anifest at birth. Its m ain feature is p o o r eyesight that occurs in all affected
m ales (penetrance 100%). Expression is highly variable (from alm ost total blind
ness to a slight reduction in visual acuity) betw een a n d even within families and
sibships. T h ere is little w orsening o f eyesight up to 40-50 years of age w hereafter
d eterio ratio n occurs, probably as a result o f the degeneration o f several retinal
structures and deterioration o f related functions. H eterozygotes have norm al
vision an d have no or m inim al recognisable signs of disease.
T he RS gene has been m apped by linkage analysis to an interval o f less than 10
cM betw een cloned polym orphic m arkers. Definitive flanking m arkers are
DXS207 a n d DXS43 on the distal, and DXS274, DXS4I and DXS92 on the proxi
mal side. U ntil recently this region has been relatively poor in m apped m arkers
an d genes. Newly g enerated m arkers o f high inform ativity in conjunction with
yeast artificial chrom osom e- a n d cosm id-m ediated building o f contigs will eventu
ally allow RS to be placed on a physical m ap o f the region. This in tu rn will make
it possible to clone and characterise the RS gene. T he structure an d function of
the gene an d the characterisation of its p ro tein p ro d u ct and m utations are needed
 X-LINKED JUVENILE RETINOSCHISIS 353

to provide leads regarding the pathogenesis of RS, and ultimately, even therapy
and prevention.

ACKNOWLEDGMENTS

O u r RS studies have been su pported by grants from the Academy of Finland, the
Sigrid Juselius F oundation, the Emil A altonen Foundation, the Jenny and Antti
W ihuri F oundation an d the Finnish C ultural F oundation. We thank the RS
patients an d families for their enthusiastic participation. Dr T iina Alitalo is pres-
endy at the D epartm ent o f Genetics, Case W estern Reserve University, Cleveland,
O hio, USA.

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 15. CLINICAL AND GENETIC HETEROGENEITY OF
USHER SYNDROME

W. J. KIMBERLING, M. WESTON AND C. MOLLER

Boys Town National Research Hospital, Omaha NE, 68131 and
University of IJnkoping, Linkoping, Sweden

INTRODUCTION

U sher Syndrom e (U S )1 is an autosom al recessive disorder characterized by h ear

ing loss and retinitis pigm entosa. Von Grafe (1858) was the first to n ote the simul
taneous occurrence o f deafness with retinitis pigm entosa. T he heritable n ature of
U sher syndrom e was recognized early by Liebreich (1861) b u t U sher (1914) was
the first to em phasize its recessive p attern o f inheritance an d he is the one after
whom the disorder has been nam ed. T h e clinical heterogeneity o f U sher syn
drom e wras n o ted by several early researchers (Bell, 1933). H allgren's 1959 m ono
graph, however, was the first large study on U sher syndrom e an d he clearly
p o in ted out that at least two distinct types o f U sher syndrom e existed. Only ju st
recently has the underlying genetic heterogeneity been d etected using linkage
analysis and these findings indicate th at the heterogeneity o f U sher syndrom e
may be m ore extensive than first thought, in that five o r m ore types may exist
each with a d ifferent responsible locus. It is the purpose o f this p aper to give the
read er an u pdate on the progress o f th e identification o f the genes involved and
to speculate on the im plication o f these findings.

CLINICAL CONSIDERATIONS

U sher Syndrome Phenotypes

T he clinical heterogeneity o f U sher syndrom e has b een recognized for several

years (N uutila, 1970; M erin et al., 1974; Fishm an, 1979) . H allgren (1959) studied
a large series of U sher syndrom e patients in Sweden an d b ro u g h t atten tio n to the
fact that two different clinical types ap p eared to exist. T he first, U sher type 1, was
th o u g h t to be characterized by a p ro fo u n d hearing loss, vestibulocerebellar
ataxia, and retinitis pigm entosa. T he second, U sher type II, presents with a

jThe nomenclature of Usher syndrome used is as follows: US refers to the syndrome and
the types are indicated by USla, USlb, etc, and are used to refer to the clinical syndrome;
USH refers to the corresponding locus, so that USHla is responsible for the clinical suptype
USla.
360 W. J. KIMBERLING, ET AL.

Table 1 Genetic and Clinical Classification of Usher syndrome

Gene Localization Hearing Vestibular Visual

US I USH1A 14q31-ter Severe to profound Absent Retinitis

(rare) hearing loss vestibular pigmentosa with
USH1B 11 q 13-14 involving all responses. diagnosis usually
(common) frequencies (Deaf). in the preteenage
USH1C 11 p 13-14 Congenital. years
(Acadian)

US II USH2A lq 32-41 Sloping m oderate to Normal Diagnosis often

(common) profound hearing vestibular made later, after
loss (Hard o f responses. puberty.
USH2B Unknown hearing).
(rare) Congenital.

US III USH3 Unknown Progressive hearing Hypoactive Not well

(rare) Loss. May have to normal differentiated from
norm al hearing at vestibular types I and II
birth reflexes

m ilder hearing loss, norm al vestibular reflexes, and retinitis pigm entosa. In fact,
type I patients are often described as d ea f and going blind while type II patients
are described as going blind with hearing im pairm ent. T he distinction between
the two is dram atic and th ere is seldom reason for confusing the two types. T he
two phenotypes seldom , if ever, occur to g eth er in the same family. This observa
tion suggested th at d ifferent loci were involved, a fact which was established once
the gene localizations were found. In fact, so far 5 different loci have been identi
fied, 3 o f which produce the type I phenotype and two which cause the type II
symptoms. T he genes and their corresponding phenotypes are listed in Table 1.
T he genes for U sher type lib and type III rem ain unlocalized. O th e r rarer, and
clinically unusual, types may exist but th eir relationship to types I and II is as yet
unclear.

HEARING LOSS IN USHER SYNDROME

A udiologic evaluations are an im portant p a rt o f the clinical w orkup o f the patient

suspected to have U sher syndrom e. T he severity o f involvem ent is highly corre
lated with genetic type. Typical audiogram s for type I an d II patients are p re
sented in Figure 1 for the purposes o f com parison. T hree patients o f each type
are shown and all were drawn from known linked U sher type I (1 lq) an d II (lq )
families. Type I patients have a profound hearing loss, have poor speech, are sel
dom h elp ed by hearing aids, an d prefer com m unication through signing . Type
II patients show a sloping loss which is m ilder in the low frequencies. Bilateral
 CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 361

hearing aids frequently benefit the U sher type II patient; som etim es the patient,
w hen fitted with bilateral hearing aids, hears an d speaks well enough th at the
hearing im p airm en t is often n o t noticeable. O ne characteristic of U sher syn
drom e has been the consistency of the audiom etric profile within subtypes. T he
extent o f the variability for both types is quite lim ited. Any deviations away from
the expected sloping audiogram , when occuring w ithout reasonable explanation,
is reason to suspect a typical type I o r II diagnosis. F urtherm ore, the hearing loss
is symmetric an d o f the same approxim ate ex ten t in both ears (M oller et al.,
1989).
Progressive hearing loss has been observed in some families with U sher syn
drom e an d may re p resen t a type III, alth o u g h its o ccu rren ce had been generally
accepted as rare. (G orlin, 1979). However, there are now several reports th at sug
gest that progressive hearin g loss may be m ore com m on than expected. For exam
ple, M erin et al. (1974), re p o rted observing a slowly progressive h earing loss in
26% of their series o f patients. Kum ar et al. (1984) observed significant differenc
es in speech discrim ination betw een type II patients with similar hearing deficits
and suggested that these differences could be due to progressive losses in som e pa
tients. Type III may be m ore freq u en t in Scandinavian countries since two o f the
rep o rts showing a high frequency are in Finnish populations (Karjalainen et al.,
1989; Nuutilla, 1970). O u r observations, however, indicate that type III U sher syn
drom e with progressive hearing loss am ong U sher type II patients is n o t com m on
in a large series o f families from the USA an d Sweden (Moller, 1989). It may be
th at the progressive loss seen by o th e r investigors is due to the norm al process of
ageing and is n o t an effect o f the gene. A nother possibility is th at the progression
o f hearing loss a p p a ren t in some type II patients is really a progressive loss in com
m unication skills due to the gradual loss in visual abilities an d which affect lip-
reading skills. N onetheless, the possibility that type III actually exists and is due to
allelic variation at o n e o f the known U sher loci m ust be considered. In fact, M erin
et al. (1974) postulated th at type III was simply a variant of type II. W hether type
III is linked to one of the known chrom osom al locations of the 4 USH loci would
answer this question b u t the possibility rem ains to be investigated.
T here has been little discussion o f the question o f progressive hearin g loss in
U sher syndrom e type I. It is generally assum ed th at a total or p ro fo u n d hearing
deficit is present at birth. T h ere have been, however, a few unpublished accounts
of residual hearing detectable in infancy by auditory evoked brain stem responses
(ABR) which disappear quickly. A lthough speech com m unication is generally not
useful for type I patients, there is variability an d one could postulate th at this is
due to the presence o f hearin g in som e patien ts. So-called co rn er audiogram s are
frequently seen in type I patients; these may be due to eith er simply the sensation
of vibration or, in some cases, to the presence o f a very m inim al residual hearing
reflex presum ably in the cochlear apex. It may be that, in type I U sher syndrom e,
the cochlea develops norm ally u ntil ab o u t the 7 -9 th m onth o f gestation and then
starts on a rapidly progressive d eterio ratio n which is usually com plete by birth.
This hypothesis is attractive because it makes cochlear dam age progressive just like
362 W. |. KIMBF.RLING, ET AL.

Usher Type I Audiograms

Hoomg WvWmJB

I s
I2S 260 SCO 1000 2CCC 0 8000

Usher Type II Audiograms

Figure 1 A u d io g ram s o f b o th U sh er I an d II p atien ts. T h e to p th re e p an els show a p ro fo u n d

h e a rin g loss ch aracteristic o f type I; th e arrow s in d ic a te th a t th e h e a rin g th re sh o ld is below
d e te c tio n . T h e b o tto m th re e show th e slo p in g a u d io g ra m typically seen w ith type II.

the retinitis pigm entosa. In term s of a m echanism for the neurosensory dam age,
it is m ore palatable intellectually to have both systems act in an abiotrophic m an
n e r ra th e r than having the hearing deficit ap p ear developm ental an d the re tin o p
athy being degenerative. Knowledge th at a progressive co m ponent exists, for
eith er types I o r II, will be im p o rtan t in the evaluation o f strategies to eith er re
store o r prevent the loss o f hearing in U sher syndrom e. Obviously, if the hearing
loss is progressive, then we may have an opportunity to in terfere with the deterio
ration so long as the time o f the onset o f the deterioration is known.
A uditory evoked emissions are consistent with the degree o f hearing loss in both
type I an d II patients. A uditory evoked brain stem (ABR) responses are, as expect
ed, absent in type I patients. In type II patients the results indicate that the hearing
im p airm en t is due solely to a p erip h eral lesion involving the cochlea (M oller et
al., 1989).
 CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 363

VESTIBULAR DEFICIT

T he second im p o rtan t distinguishing feature betw een types I and II U sher syn
drom e is the presence or absence o f vestibular responses. T he vestibular system
in type I U sher patients is virtually non-functional. Occasionally, an affected type
I individual will be seen with m inim al vestibular responses, b u t this is extrem ely
rare. This deficiency is m anifest in the adult as a non-progressive ataxia in the
form o f a swaying and swinging gait. T he ataxia is entirely o f perip h eral origin
an d there is no evidence to indicate an additional cerebellar com ponent. T he
vestibular deficit appears to be congenital, or at least occurs at a very early age,
since U sher I children are late to sit a n d walk. N one of the patients (M oller et al.,
1989) in one large well studied series walked before the age o f 18 m onths. In fact,
walking age is a good predictor o f the US subtype, since few type II children are
delayed in walking for as long.
T he postural system in hum ans relies on in p u t from three sources: vestibular, vi
sual, an d som atosensory. While the patients have com pensated well for the vestib
ular deficit, postural stability is aggravated because o f the progressive visual loss
due to RR T he result is th at U sher I patients are m ore posturally unstable than ex
pected based upon experience with o th er patients with hearing o r visual loss
alone. Type II patients, however, are essentially n orm al with regard to the vestibu
lar system. T here have b een some rep o rts of m ild vestibular abnorm alities, such as
hypo- o r hyperactivity, bu t it is unclear w hether these findings are w ithin the range
o f norm al. W hether there are vestibular deficits present in U sher II rem ains to be
definitely established.
Sophisticated vestibular studies, including rotary chair, bitherm al calorics, dy
nam ic an d posturography, are often necessary to d eterm in e if the "ataxia" of
USH1 is central or vestibular in origin and to determ ine the degree to which the
visual loss is affecting postural control. Visual im pairm ent com plicates, but does
n o t invalidate, the otoneurologic exam ination. T he RP affects the corneo-retinal
potential which is the basis o f electro-nystagm ography (ENG); the gain of the re
sponse m ust som etim es be increased an d the test p erfo rm ed in the dark since
even dim light will decrease the signals. If the ataxia is no t due to a labyrinthine
defect, then the diagnosis o f U sher syndrom e type I should be questioned. The
vestibular phenotype is highly co rrelated with the severity o f hearing loss. T he
finding o f a norm al vestibular response with p ro found deafness o r absent vestibu
lar responses with the sloping type II audiom etric profile should alert the re
searcher th at the diagnosis is n o t certain. Such patients may rep resen t new
subtypes o f U sher syndrom e or could be due to as yet unrecognized allelic varia
tion at one o r the o th er known loci.
364 W. J. KIMBERLING, ET AL.

Figure 2 B
 CLINICAL AND GENETIC HETEROGENEITY OF LSHER SYNDROME 365

Figure 2 D

F igure 2 P h o to g ra p h s o f th e fu n d i o f 4 in d e p e n d e n t p a tie n ts with U sh e r sy n d ro m e show ing

th e ra n g e o f involvem ent. T h e first two p a n e ls a re fro m type 1 p a tie n ts a n d th e o th e r two a re
fro m type II p atien ts. See C o lo u r P late VI a t th e back.
366 W. J. KIMBERLING, ET AL.

Visual Phenotype

Fundus changes are typical for patients with retinitis pigm entosa and currently
there is no recognized p attern o f fundus involvem ent that is correlated with any
specific subtype. In some cases the pigm entary abnorm alities are m inim al an d in
others they are extensive. Bone spicule form ation is com m on. A set o f typical fun
dus photographs are illustrated in Figure 2 for both type I an d II patients. How
ever, there are prelim inary indications o f fundoscopic: differences related to
genetic subtype, at least for type II. O ne family with U sher type II unlinked to
lq32-41 was found to have a m ilder course o f RP which had been described as RP
sine pigmento. Some bone spicule form ation was noted in the oldest affected sib
ling. O n the o th er hand, RP sine pigmento has been found in both type I a n d type
II persons and is believed to rep resen t an early stage o f the retinal deterioration
com m on to all RP (Heckenlively, 1988).
T he m ost freq u en t foveal change described by Fishm an (1979) is th at o f an atro
phic appearing lesion which was seen in 31% o f the patients. An additional 11%
showed a cystic-like foveal lesion associated with leakage from the perifoveolar ar
teries.
Several studies have shown th at electroretinography (ERG) m ight be subnorm al
as early as 2 to 3 years o f age, before abnorm alities can be seen functionally o r on
fundoscopic exam ination (M erin, 1974; De Haas, 1970; D avenport, 1978). Both
scotopic and photic responses are involved. Responses extinguish totally as the d e
g en eration progresses. T he visual evoked responses also show decreased am pli
tudes and longer latencies (Abraham , 1977). T here is no general consensus about
the severity o f the RP as a function o f subtype, bu t it has been suggested th at type
I may have a m ore severe visual involvem ent (Fishman, 1979). In type I patients,
cone an d ro d responses are usually u ndetectable by the m iddle o f the second de
cade and often in the first. Many type II patients have a recordable, b u t depressed,
cone response (Fishman, 1979). In one type II family with four affected brothers,
the gradual decline o f the ERG over two decades has been docum ented. While
electrophysiological studies provide the earliest and m ost reliable diagnosis o f re
tinitis pigm entosa, they are n o t necessary in all cases. An absent o r dim inished
ERG in at least one affected sibling is a necessary condition for the diagnosis of
U sher Syndrom e in a family.
R estricted visual fields are an integral p a rt o f the syndrom e. Im p airm en t o f p e
rip h eral vision may n o t be noticed until the teenage years b u t is probably present
early in life. Generally, visual field abnorm alities are n o t associated with scotom as
in type I patients. Several type II patients have shown a ring scotom a which may
eith er be a characteristic o f the specific type II genotype o r could simply reflect
the m ilder course of the disease for some type II patients. Searching nystagmus is
uncom m on in U sher syndrom e and the retinal d egeneration is uniform ly progres
sive from the p eripheral regions to the central.
N ightblindness is a concom itant to retinitis pigm entosa. Most patients have a
history o f nightblindness that dates back to childhood. T he nightblindness is par
 CLINICAL AND GENETIC LIETEROGENEITY OF USHER SYNDROME 367

ticularly evident in type I patients w here disorientation is m ore severe in the

evening and n ight because o f the com bined visual and vestibular im pairm ent. T he
U sher I p atien t may ap p ear especially clumsy in low light an d bum p into things
an d fall. This may lead to an earlier recognition o f the RP which could be inter
p re te d incorrectly as relating to differences in severity between types I an d II.
O th er ophthalm ologic com plications are cataracts a n d Coat's disease. H allgren
(1959) considered cataracts as p art o f the syndrom e. W hile posterior subcapsular
cataracts occur in ab o u t 30% -60% o f U sher patients, the general consensus now
is th at the cataracts are a secondary result of the RP. This hypothesis is supported
by the observation th at cataracts occur at about the same frequency in all types of
retinitis pigm entosa an d are n eith er characteristic n o r m ore freq u en t in U sher
syndrom e.
While total blindness in n o t com m on, the severity of the visual defect restricts
the patient's mobility. For m ost U sher patients, significant visual loss is due to ei
th e r posterior subcapsular lens opacities o r the developm ent o f a foveal lesion.
T he foveal lesion can cause significant central vision loss. While the degree of
hearing loss has been observed to correlate with the degree o f perip h eral field
loss, th ere is no association o f h earin g im pairm ent with central vision loss
(Fishman et al., 1983; von A lberth et al., 1958). T he discrepancy betw een auditory
and visual involvem ent was also n o ted by o th er investigators (de Haas et al., 1970).
These findings suggest, bu t do n o t prove, th at clinical and genetic type may n o t be
correlated with the presence o r absence o f foveal lesions a n d /o r cataracts.
It should be recognized that the audiologic profile and vestibular responses,
while the m ost reliable indicators o f clinical type, are relatively non-specific and,
except for progressive versus non-progressive hearing loss, hold little hope for fur
th e r subclassification. Visual phenotype, on the o th er han d seems m ore variable
and may be used to d eterm in e if any o f the 5 genetic subtypes can be classified on
the basis o f ophthalm ologic param eters. For exam ple, is the fundoscopic appear
ance different betw een types? Specifically, does U sher syndrom e lib have a differ
en t ophthalm ologic picture than Ila? Are cataracts o r foveal lesions m ore freq u en t
in one type o r another? Because o f the extensive genetic heterogeneity, answers to
these questions may prove invaluable in a priori assignation o f families to different
subtypes and thus assist in the identification an d cloning of the responsible genes.

Inter- and Intrafam ilial Variability

U sher types I an d II are distinct phenotypes and only rarely do the two occur in
the same family. For th at reason, it was hypothesized th at the two were genetically
distinct as well, a fact subsequently established by linkage analysis. T here is little
variability in expression within sibships for type I patients but greater intrafam il
ial variation in bo th hearin g an d visual involvem ent has been observed for type II
(Fishman, 1979; H allgren, 1959). T he origin o f the variation in hearing acuity of
type II patients has n o t been rigorously investigated. Certainly, one m ight expect
som e o f the variation to be due to the d ifferent loci known to be involved. O n the
368 W. J. K1MBERLING, ET AL,

o th e r hand, some o f the variation could be due to different m u tan t alleles at

eith er of the type II loci.

Mental R etardation a n d /o r O ther CNS Dysfunction.

T he existence of psychiatric disturbance a n d m ental retardation has been the

subject of discussion with regard to U sher syndrom e. H allgren originally n o ted a
high frequency o f m ental deficiency (24%) in his series. A lthough H allgren was a
psychiatrist, this could have rep resen ted a bias of ascertainm ent or, m ore likely, a
sim ple m isinterpretation o f the available records. Few of the patients were actu
ally exam ined by Dr. H allgren and m uch o f the results d e p e n d ed u p o n an inter
p retation o f the existing m edical records. F urtherm ore, with U sher syndrom e
type I, the coexistence o f both vestibular dysfunction (leading to a walking age of
18 m onths o r greater) and speech delay could easily lead to a m isdiagnosis of
m ental deficiency. W hatever the cause, the association has not b een no ted by
m ost o th er investigators (N uutila, 1970; Fishm an et al. 1983; M oller et al., 1989)
with the exception of Lindenov (1945). M erin et al. (1974) designated U sher
patients with m ental retardation (MR) as type IV. Several investigators have ques
tioned the existence of type IV (Nuutila, 1970; Fishm an et al., 1983; M oller et al.,
1989) which has som etim es been re ferred to as H allgren syndrom e. A lthough the
literature is confused and confusing with regard to the nosology o f type IV /
H allgren syndrom e, there is little do u b t th at retardation is an uncom m on aspect
of all types of U sher syndrom e. T he occasional cases w here it does occur may rep
resent a contiguous syndrom e where the U sher gene is deleted along with adja
cent DNA an d the MR is the result n o t o f a defective U sher gene bu t of
abnorm ality in neighboring genes. Also, th ere are a n u m b er of o th er disorders
associated with hearing im pairm ent and retinitis pigm entosa which do include
MR (e.g. L aurence-M oon and B ardet-B iedl syndrom es). Variation o f the p h en o
type o f these disorders could possibly lead to their inclusion in a series o f suppos
edly U sher syndrom e patients.
Various psychiatric disorders have been associated with U sher syndrom e. This
includes schizophrenia-like disorder, atypical psychosis, an d re c u rre n t depression
(U sher 1914; H allgren, 1959; V ernon, 1962; N uutila, 1970; Karjalainen; 1983).
H allgren (1959) estim ated the highest frequency o f m ental disturbance (>20%)
and while subsequent studies did no t confirm such a high prevalence, U sher syn
d ro m e patients are undoubtedly at a h ig h er risk for psychiatric disturbance than
the norm al hearing, norm al seeing population. Two different etiologies have
b een hypothesized as the cause o f this association. O ne hypothesis is th at it is part
of the Syndrome and reflects a pleiotropic effect of the U sher gene. T he pleiotro-
pic theory would have been m ore reasonable had the putative association between
m ental deficiency and U sher syndrom e been up h eld by subsequent studies. T he
second, an d m ore likely, hypothesis is th at the psychiatric dysfunction is due to
stress an d is a reaction to the loss of both o f the two m ajor senses. This prem ise is
su p p o rted by the finding th at patients who have gone blind or deaf have often
 CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 369

been re p o rted to experience tem porary reactive psychosis. F u rth erm o re, the
schizophrenia-like symptoms seen in U sher syndrom e are also tem porary, a fea
ture n o t seen in true schizophrenia.
However, there is now accum ulating evidence th at there may be a cerebral le
sion associated with U sher syndrom e. Bloom e ta l. (1983) found indications o f cer
ebellar atrophy by CT scan on 6 o f 12 U sher patients. F u rth er evidence o f CNS
changes has been found by o th er investigators (Koizumi et al., 1988; Schaefer et
al., 1992). T he findings are relatively non-specific and indicate both cerebellar
an d cerebral anom alies. In one study, sim ilar anom alies were NOT seen in d eaf or
RP controls (Schaeffer et al., 1992) suggesting that the brain anom alies are no t
secondary to the sensory deficit. T he brain anom alies were seen in both type I and
II patients although they ap p eared to be m ore freq u en t in type II, which is som e
what surprising in th at type II has the m ilder sensory deficit. T he cerebellar and
cerebral anom alies have no t yet been associated with any clinical neurologic prob
lem.

Congenital Anomalies And O th er Associated Defects

C ongenital anom alies are n o t p art o f U sher syndrom e. Occasionally, congenital

anom alies have been no ted in association with U sher syndrom e. However, no
clear pattern o f dysm orphology has em erged. In fact, it is likely that whenever
m ajor or m inor anom alies are found in conjunction with U sher syndrom e, they
occur eith er by chance or are the indication o f a different syndrom e. O ne m ight
expect to find a few legitim ate cases o f U sher syndrom e with anom alies a n d /o r
m ental im pairm ent as the result o f a contiguous deletion of the U sher gene and
its neighbors. Such a finding would be extrem ely useful in attem pts to clone the
U sher gene(s). U nfortunately, this has never been found.

THE GENETICS OF USHER SYNDROME

P attern O f Inheritance

It has long been recognized th at U sher syndrom e is inherited as an autosom al

recessive disorder with full pen etran ce. B oughm an and Fishm an (1983) showed
that the segregation o f U sher syndrom e did n o t deviate from th at expected for an
autosom al recessive. A re cen t study by S hugart et al. (1991) showed that the seg
regation ratio is 25% for bo th type I and II an d that the frequency of sporadic
cases did not exceed th at expected for recessive disorders o f the same frequency.
F urtherm ore, the pen etran ce was n o t significantly d ifferent from 1.0. Thus there
is no evidence to suggest th at any fraction of U sher syndrom e is due to a non-
genetic etiology.
X-linked inheritance was considered in one type II family (D avenport et al.,
1978) because of four affected brothers, but this family was later shown to be
370 W. J. KIMBERLING, ET AL.

linked to lq32-41 m arkers. B oughm an observed a slight excess of U sher syndrom e

males in a population study of retinitis pigm entosa in Louisiana. This observation
was n o t m ade in earlier studies (H allgren, 1959; Bell, 1933) n o r in subsequent
studies (Kim berling et al., 1989; Shugart et al., 1992).

Prevalence and Population D istribution

It has been estim ated th at U sher type I represents 90% of U sher syndrom e in the
U nited States, with type II accounting for nearly 10%, and o th er types accounting
only about 1%. However, Fishm an's g ro u p re p o rted 71 ou t o f 106 patients with
type II (Piazza, 1986). A N orw egian study o f 28 cases were distributed as: type
1 - 1 4 cases; type 1 1 -1 0 cases; an d o th er - 4 cases (G rondahl, 1986; G rondahl and
M joen, 1986)). O ur own series has alm ost 50% type II cases both from the USA
an d Sweden (unpublished d a ta ). We believe U sher type II is at least as com m on
as type I. However, type II patients are n o t usually ascertained in schools for the
d eaf because their hearing loss is n o t severe enough to place them there. Schools
and program s for the blind an d ophthalm ologic clinics have a relatively higher
p ro p o rtio n of type II patients com pared to ascertainm ent from schools for the
deaf.
T he frequency of U sher syndrom e has been estim ated at 3.0/100,000 in Scandi
navia (H allgren, 1959; N uutila, 1970) a n d at 4.4/100,000 in the U nited States
(B oughm an et al., 1983). T he prevalence o f U sher syndrom e am ong d eaf individ
uals has b een re p o rted to range from 0.6 to 28% (Vernon, 1969). Conversely, the
frequency of deafness in the retinitis pigm entosa population is estim ated to range
betw een 8.0 an d 33.3%. Overall, there are ab o u t 16,000 deaf an d blind people in
the U nited States, o f which m ore than h alf are believed to have U sher syndrom e.
Most o f these will have U sher type I. M ost U sher type II individuals, with a m ilder
h earing loss, may n o t be n o ted in surveys which have focused on the profoundly
deaf. Piazza et al. (1986) postulated that the frequency of type II is at least as great
as th at for type I.

U sher Type I

Localization of Usher Type I to Chromosome 14q

T he U sher syndrom e type I gene was first localized to chrom osom e 14q by a link
age with m arker D14S13 in fifteen families (Kaplan et al., 1992). A linkage was
suggested by the fact th at for the total sam ple the lod score m axim ized at a
recom bination fraction (9 ) o f 0.20 with lod score (Z) o f 0.83 (C hapter 1). How
ever, the test for genetic heterogeneity was significant, assum ing linkage, at the
5% level suggesting the possibility th at two or m ore d ifferent genes m ight be
involved. Ten o f the 15 families showed linkage to the 14q m arkers and 8 o f these
cam e from the same region known as Poitu-C harentes in France bu t there was no
evidence o f consanguinity. W hen these families were pooled, the two point lod
 CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 371

score went to 3.78 at 9 = 0 with D14S13. Based upon this location, Kaplan et al.
(1992) hypothesized th at the USH gene may be involved in an abnorm ality o f cil
iary structures of the retina, vestibule, and cochlea. T he sensory cilium o f the
rods and cones separate the o u ter segm ent of stacked discs containing the visual
pigm ent from the in n er segm ent an d it is reasonable to expect th at defective cilia
could lead to deg en eratio n of the o u te r segm ent of the retina. However, vestibu
lar and cochlear hair cells do not have tru e sensory cilium with the characteristic
9+0 m icrotubules. Vestibular hair cells have kinocilia with 9+2 m icrotubules an d
are missing the dynein arm s fo u n d in m otile cilia. D uring developm ent, the
cochlear hair cells possess a transitory kinocilium that may be responsible for
organizing the developm ent of the stereocilium characteristic o f the cochlear
hair cells. Thus, a defective kinocilium could lead to defective hair cells and
abnorm alities of hearing an d balance. Because o f the transitory n atu re o f kinoci-
lium developm ent, it w ould be expected that any hearing or vestibular abnorm al
ities would be congenital but the sensory cilia o f the eye are req u ired for norm al
retinal m aintenance th ro u g h o u t life an d a defect would be m ore likely to cause a
progressive deterio ratio n (H u n te r et al., 1986).
P h o to recep to r and sperm axonem e abnorm alities had been previously ob
served in U sher syndrom e (H u n te r e t al., 1986); nine o f the 10 U sher patients
studied had type II. In a subsequent report, B arrong et al. (1992) also observed
abnorm al connecting cilia in a type II patient. These abnorm alities are no t seen
in the general population of X-linked and autosom al retinitis pigm entosa patients
with norm al hearing. Studies o f axonem e structure o f nasal cilia showed abnor
malities associated with a group o f RP patients including U sher syndrom e bu t the
results were n o t clearly broken down into diagnostic group so it is h ard to draw
any conclusions. Sperm m otility an d structure has also been found to be abnorm al
in U sher patients (H u n te r et al., 1986; N uutila, 1970). Kaplan et al. (1992) point
out th at the gene controlling h e a rt position in the m ouse has been m apped to a
region hom eologous to hum an 14q32 an d was identified by a recessive situs inver
sus m utation, iv. T he hum an hom ologue o f the m ouse iv gene should lie exactly
between the hum an alpha-1 antitrypsin gene an d the heavy chain im m unoglobu
lin which is where the U sher gene was m apped. T hese are intriguing observations.
T here is confusion ab o u t which types o f U sher syndrom e actually have abnorm al
cilia an d m ost cases re p o rted have b een type II and n o t clinically equivalent with
the type I phenotype localized to 14q. A ciliary defect makes sense for type I U sher
syndrom e since bo th the vestibular a n d cochear functions are im paired. However,
the re p o rted ciliary abnorm alities mostly occur in type II individuals. F u rth er in
vestigation into this p h en o m en o n is w arranted and, now that the d ifferent types
can be differentiated from each o th e r at the m olecular level, the research can be
directed towards defining the differences between types. It is unreasonable, al
though no t impossible, to expect th at all types o f U sher syndrom e share a ciliary
defect. T he expectation is th at one o r m ore types would have a different biologic
basis. If this is fo u n d to be the case, th en ciliary o r axonem al structure could be
372 W. J. KIMBERLING, ET AL.

an additional m eth o d to distinguish betw een types w ithout p rio r reliance on link
age groups.
O th e r investigators have been unable to confirm the localization to 14q in fam
ilies outside o f the original French g roup (Keats et al., 1992). This analysis includ
ed a large series o f families from the USA an d N o rth e rn Europe, notably E ngland
an d Sweden. This created some initial do u b t about the linkage and raised the
question th at it may have occurred by statistical chance. However, the fact th at 8
o f the 10 linked families com e from the same region in France supports the argu
m en t for linkage an d for the existence of a locus on chrom osom e 14. Definitive
evidence will com e when those families are shown n o t to be linked to the o th er
two regions now shown to contain the U sher gene. This locus has been given the
designation o f U SH la.

Linkage of Usher type I to chromosome 11

T h e discovery o f the linkage o f U sher syndrom e type I to chrom osom e 11 was

m ade alm ost sim ultaneously by two d ifferent investigators. T he 11 q linkage was
m ade originally with the m arker D11S527 in 19 families. T he m ultipoint lod
score reached a m axim um o f 4.75 at a position about 7 cendM organs cen tro
m eric to D11S527. T he gene localized h ere was designated U S H lb to distinguish
it from the one localized to 14q. F u rth er analysis o f the data indicated th at the
USH1 gene was flanked by the m arkers DI 1S527 an d DI 1S35. However, the odds
o f placem ent of the U S H lb gene to within this flanking region was n o t at the tra
ditional 1000:1 level. T he m ap o f chrom osom e 11 is given in Figure 3 which
shows the m ost recent evidence for localization o f the U sher lb gene to the
11 q 13-14 region. R ecent results (unpublished data) indicate th at the U S H lb
g ene lies between the m arkers D11S527 and D11S416. This is a 2 tolO centiM or-
gan interval o f DNA
T h ere was a slight possibility th at the gene could be located between D11S527
an d FGF3 o r even m ore centrom eric than FGF3. This raised the possibility that
FGF3 could be the U sher lb gene. Induced m utation in FGF3 p roduced abnorm al
ities o f both the ear and eye in mice (M ansour et al., 1992) but these abnorm alities
were prim arily structural and n o t at all like those observed in U sher syndrom e.
F u rth erm o re, FGF3 shows several crossovers with U sher type I thus reducing its at
tractiveness as a candidate gene. ROM1, a retinal m em brane protein gene which
is p art o f a p h o to rec ep to r specific protein family, is located proxim al to FGF3. It
show crossovers with U sher lb and hence can also be excluded as a candidate.
T he third localization o f U sher syndrom e was to chrom osom e 1 lp (Smith et al.,
1992). This linkage has so far been found only in families o f French Acadian de
scent. T he French Acadians m igrated to the bayous o f Louisiana from Q uebec
over 100 years ago and had, until the second world war, rem ained genetically iso
lated. Presum ably because of a fo u n d e r effect, U sher syndrom e has been fo u n d to
be at a high frequency in that population. In fact, so u th ern Louisiana may have
the highest frequency of U sher syndrom e in the USA, perhaps even the world. T he
 CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 373

HBB* -
4.0
D11S875-
2.0
D11S569.
1.0
D11S926-
3.0
D11S902-
D11S899- 3.0

5.0
USHlc D11S928*
1.0
D11S915-
1.0
D11S904*
1.0
D11S916-

12

13.1
13.2
13.3
13.4
13.5
14.1 lUSHlb
14.2 | TYRand OMP1
14.3

21

22.1
22.2
22.3

23.1
23.2

23.3

24

25

Figure 3 Map of chromosome 11 which shows the most recent evidence for localization of the
Usher lb gene to the 11 q l3-14 region.

Acadians were originally from the N orm andy area in France and there is no evi
dence th at they share ancestry with the families from Poitu-C harentes showing
linkage to 14q. It was originally th o u g h t that the use o f the Acadian isolate avoided
the com plications o f heterogeneity a n d allowed the pooling of data across sibships
w ithout m ixing types I and II. However, it was subsequently discovered that both
types I and II occur in the population. A lthough type I appears to be the m ost prev
alen t and type II may have a frequency approxim ately equal to th at in the non-Ac-
374 W. J. KIMBERLING, ET AL.

adian USA population, the frequency distribution in this population needs to be

established with full attention to the clinical differences betw een types.
T he linkage found by Sm ith's group was between U sher syndrom e an d the
m arkers D11S419 and D11S875. T he lod score m axim ized at about 5.0 and indi
cated with a high degree o f confidence th at the U S H lc gene is located between
the m arkers D11S875 an d M fdl32. Physically, this region corresponds to 11 p 13-
p l4 .
It is interesting that the two regions 11 p 13-p 14 an d 11 q 13-q 14 are paralogous
an d share an evolutionary ancestry (L undin, 1993). Tyrosinase is on 1 lq and there
is a tyrosinase-like (tyrosine hydroxylase) gene on l i p . Also, potassium channel
genes are found in both regions. This raises the question w hether U S H lb and lc
are loci coding for related products. Given the paralogy o f the two regions, this
would n o t be surprising an d m eans th at once eith er of the two genes are cloned,
th en the o th er should be detectable because of probable hom ologous DNA seg
ments.
T he U S H lb locus lies close to the region where the m ouse m utation known as
shaker-1 (sh-1) is found. Sh-1 causes deafness with vestibular disturbances. No re tin
itis pigm entosa has been observed (K. Steele, personal com m unication). N one
theless, sh-1 rem ains high on the list as a possible m ouse hom ologue for U sher lb.
Sh-1 is closely linked to the olfactory m arker protein (OM P1). OMP1 and sh-1 are
n o t the sam e, since no m utations have been found in OMP1 which co rrespond to
the defect seen in shaker-1 mice. However, OMP1 was a attractive candidate for
U sher syndrom e given the evidence cited above about the possible abnorm alities
in nasal cilia seen in some U sher patients. T he role o f OMP1 is no t known and it
is tem pting to speculate that it may have a function in nasal ciliary developm ent.
OMP1 has been shown to have a dinucleotide polym orphism an d was found to
have crossovers with U sher type lb families, so that it is excluded as a candidate.
T he fact th at there are at least 3 different genes for U sher type I considerably
com plicates the problem o f refining th eir localizations well enough to ensure
gene identification an d cloning. U sher type lb is the m ore freq u en t type am ong
people of presum ed n o rth e rn E uropean ancestry. Types la and Ic have yet to be
found outside o f the populations in which they were originally described. Howev
er, as m uch as a 15% rate o f heterogeneity could exist in the USA sample a n d still
give a non-significant chi-squared for heterogeneity. Thus a considerable fraction
o f cases o th e r than type lb could exist in the population w ithout o u r having the
m eans to d etect them reliably
F uture research will focus on two aspects. First, there is the question o f possible
clinical differences between the three types. Will ciliary abnorm alities be found in
one type an d no t in the o th e r types? Are th e ophthalm ologic findings consistent
across subtypes? Does one type have a m ore freq u en t association with the presence
of foveal lesions a n d /o r cataracts? Are th ere any significant differences in the ERG
or progression o f retinal degeneration? Are there any hearing or vestibular differ
ences (some type I patients ap p ear to benefit from hearing aids)? It is critical to
define now the clinical differences betw een the subtypes o f U sher type I in o rd e r
 CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 375

to have ways o f a priori dividing families used in fine gene m apping o f U S H la, lb,
or lc.
F u rth er refin em en t o f location will d ep en d upon the observation of critical
crossover events that will bisect an d red u ce the region o f flanking m arkers. If a
critical crossover is actually d ue to the inappropriate inclusion o f a type Ic family
in a type lb cohort, then the assessment o f location may be off by a considerable
physical distance. If the investigation is lim ited to the Acadians, th ere is little
chance o f contam ination by no n -U sh lc families. However, until we know the true
frequencies o f the no n -U sh lb families in the general population, the possibility of
e rro r will always be present fo r U S H lb. Two ways to m inim ize this effect is first to
develop clinical criteria th at allow subclassification o f the U sher type I families
w ithout resorting to m olecular data. T he second is to type all three sets of m arkers
from 11 q , l i p , an d 14q and to include only those which show definite non-linkage
to two of the three loci.

U sher Type II

Linkage of the. Usher Type II Gene to lq

U sher syndrom e type II was localized to chrom osom e lq32 (Kim berling, 1990;
Lewis, 1990). T he gene has subsequently been shown to be flanked by the m ark
ers D1S237 and D1S229. T he m ost recen t m ap o f chrom osom e lq is shown in Fig
ure 4 along with the putative location o f the U sher type II gene. T here is some
am biguity in the co rrespondence betw een the CEPH collaborative study m ap and
the second generation chrom osom e 1 m ap (W eissenbach et al., 1992), so th at the
exact position of the m arkers betw een D1S245 and D1S227 m arkers is provi
sional. D1S237 and D1S229 are definitely between D1S70 and D1S81 (u n p u b
lished data). T he best estim ate o f the physical location of the U sher II gene is in
Iq32-lq42. Localization o f the U sher II gene is narrow ed to ab o u t 4 centiM or-
gans. This region am ounts to about 4 M egabases o f DNA.
T here are few genes in th at region th at rep resen t true candidates. An autosom al
hom ologue of the X-linked choroiderem ia gene, known as hCHM L, has been lo
calized to lq. This gene shows hom ology with a bovine p rotein which regulates the
G D P/G T P exchange o f GTP binding p ro tein smg 25A. It is expressed in the chor
o id /re tin a l pigm ent epithelium an d several o th er tissues (C hapter 12). It is not
known w hether this gene is expressed in the cochlea. hCHM L has been localized
to lq 3 1 -lq te r an d hence m ust be considered a logical candidate for U sher syn
drom e.
Two o th er genes in the region are transform ing growth factor (32 (TGFB2) and
the hom eobox gene HB24 (HLX1). Both genes m ap betw een D1S81 and D1S70,
which places them in close proxim ity to the U sher II gene. Both TGFB2 an d HLX1
have been localized to lq41. TGFB2 is a m em ber o f a polypeptide family which
regulates growth an d cellular differentiation; it is expressed in a wide variety o f tis
sues. HLX1 has been fo u n d to be expressed in hem atopoietic tissues an d has been
376 W. J. KIMBERLING, ET AL.

D1S53

REN
CR1/CR2/DAF
12
11 D1S70
11 D1S245
D1S205
12 D1S217 J TGB2
D1S237 J
21 USH2a
22
D1S229
| HLX2
23 D1S227
24 PPOL
D1S8!
25

D1S48
31 D 1SI 78

32

D1S103
41

42

43

44

Figure 4 M ap o f ch ro m o so m e 1 show ing th e p ro b a b le lo catio n o f th e U sh er II g e n e in th e

Iq 3 2 -lq 4 2 reg io n .

p roposed as a regulator o f hem atopoietic differentiation. HLX1 expression was

also found in the fetal brain. It seems unlikely that eith er o f these genes are the
U sher II gene b u t until they are localized m ore accurately o r until their functions
are b etter understood, they rem ain legitim ate considerations.
T he gene for poly (ADP-ribose) polym erase (PPOL) is localized in the region
betw een D1S70 an d D1S81. Its exact location relative to the G enethon (Weissen
bach et al., 1992) m arker set is n o t yet known bu t it seems likely to lie outside the
D1S237-D1S229 segm ent. N onetheless, th ere is some evidence that it m ight som e
how be involved in U sher Syndrom e. Lym phoid an d fibroblast cells from patients
with U sher syndrom e have been im plicated in being m ore sensitive to DNA-dam-
aging agents (Robbins et al., 1984). O ne p atien t treated with cytotoxic agents was
re p o rte d to have an accelerated course o f retinitis pigm entosa which was hypoth
esized as being due to sensitivity of the retinal cells to the DNA dam aging p ro p e r
ties o f the chem otherapeutic agent. If these speculations are true, it w ould imply
 CLINICAL AND GENETIC LIETEROGENEITY OF USHER SYNDROME 377

th at the U sher gene affects eith er DNA repair o r synthesis which provides a clue
towards the selection o f candidate genes. U nfortunately, these studies do n o t well
characterize the type o f U sher syndrom e. In fact, the case re p o rted by B lanchet et
al. (1992) h ad an atypical RP which was n o t diagnosed until the 6th decade and
an optic atrophy n o t typical o f U sher syndrom e. T here was no m ention o f vestib
ular responses. Clearly, studies of hypersensitivity to X -irradiation o r similar agents
need to be repeated with atten tio n to the clinical an d m olecular subtype. PPOL
(or ADPRT) activity is in d u ced by single strand breaks in DNA an d is req u ired for
cellular repair. It has been suggested as the gene for eith er Fanconi's anem ia or
xeroderm a pigm entosa. A gene involved in the com plem entation of the defect in
group A x ero d erm a pigm entosa cells has b een localized to lq42-lqter. T he PPOL
gene was localized lq 4 2 by in situ hybridization an d it is interesting th at a second
hybridization peak was observed on 14q24, close to the localization o f U S H la.
O ne can only speculate ab o u t the possibility of contiguous deletions involving
both the U sher II gene an d PPOL giving rise to the observation o f increase chro
m osom e breakage in some type II patients.
A potential m ouse m odel for U sher syndrom e type II has recently b een discov
ered by C hang et al. (1993). T he R BF/D nJ albino m ouse carries a recessive m uta
tion, rd-3, which causes a retinal d egeneration similar to retinitis pigm entosa.
E lectroretinogram s an d retinal appearance are both suggestive o f hum an type RP.
T here is noth in g particularly notable ab o u t the RP seen in U sher II patients which
would suggest that U sher II an d the rd-3 m ouse are the same. T he RP phenotype
is too non-specific. However, the rd-3 gene m aps to 10 centiM organs distal to the
alkaline phosphatase gene on m ouse chrom osom e 1. This region shows consider
able hom ology with hum an chrom osom e I. T he region w here the rd-3 m ouse gene
would lie is theoretically close to the position w here the m ouse hom ologue for the
U sher II gene should lie. T h ere was no re p o rt o f w hether these m ice are hearing
im paired.

Discovery of USH2 Heterogeneity

O ne family with U sher type II was observed to have at least 3 double crossovers in
the lq 3 2 region betw een m arkers D1S70 an d D1S81 if th eir USH2 gene were
located in th at segm ent. A form al heterogeneity analysis using O tt's HOM O G
program was statistically significant w hen this family was included in an anlysis of
a large sam ple o f U sher type II families. T he significance disappears w hen the
family is rem oved. T he affected family m em bers had audiogram s typical o f U sher
type II along with norm al vestibular function. T he ERG was dim inished to absent.
T here was a history o f nightblindness an d visual fields were restricted. Thus, a
diagnosis o f U sher syndrom e h ad been given to the family. However, it was no ted
th at the retinitis pigm entosa was described as sine pigmento an d one ophthalm olo
gist had observed m inute white flecks in the retina. In retrospect, the retinal
findings are subtly d ifferent suggesting th at there is a good chance th at this type
o f family may be identified p rior to m olecular genetic testing.
378 W. J. KIMBERLING, ET Al

T he family did no t show linkage to the lq32 - q41 region containing the U sher
type II gene. N or does the U sher syndrom e in the family show linkage to m arkers
on chrom osom es 11 q, l i p , o r 14. This establishes the fact that a second locus for
U sher type II exists which is n o t located on lq32-41 and that the gene is no t an
allele at any of the o th er 3 U sher I loci. This new gene has been given the desig
nation USH2b, leaving USH2a to refer to the original lq locus. T he frequency of
U sher lib relative to Ila is n o t known, although it m ust be relatively less com m on
inasm uch as it has been seen only once in a series o f over 30 type II families.

CONCLUSIONS

U sher syndrom e was first described as h earin g im pairm ent with retinitis pigm en
tosa. With time, investigators realized that at least two clinical types existed and
th at they could be consistently an d reliably distinguished from each other. How
ever, linkage analysis showed that a m inim um of five d ifferent genes are involved,
th ree for type I an d two for type II. T h ere are no clear clinical differences
betw een the d ifferent subtypes within each type.
T he next stage o f U sher research is focused on identifying and cloning the re
sponsible genes. Saturation m apping o f the critical regions by chrom osom e walk
ing m ight be necessary to accom plish this task. Saturation m apping o f the
chrom osom e region will eventually pro d u ce a series o f "contigs" o f overlapping
clones th at collectively span the region o f interest, which hopefully will involve
only 1 M egabase or less. G enetic heterogeneity will rem ain a problem until the
genes are identified and sequenced. For exam ple, a family with two children af
fected with a separate type o f U sher syndrom e would show non-double crossovers
in the critical region 25% o f the tim e th at th eir m arkers are inform ative: for three
affected children, this frequency would be 6.25%. Most U sher families are small
with only 1 affected and in m ost of those cases it will be im possible to determ ine
which subtype o f U sher syndrom e is present by linkage analysis. W hen a family has
two or m ore affected m em bers, the probability that linkage can rule o u t a subtype
is related to the n u m b er o f affected children. At the p resen t time, the diagnosis of
o ne subtype o r the o th er by m olecular testing m ust rely solely on exclusion of the
alternate types. Clinical differences betw een the subtypes may exist an d the possi
bility o f being able to differentiate one type from an o th er needs to be fu rth e r in
vestigated, Resolution of the question o f clinical differences may help in the
selection o f families helpful in identifying the U sher genes.
M eanwhile, until the U sher genes are found, DNA m arkers close to the U sher
gene can be used in genetic counseling. Given tight linkage and an inform ative
family with one previously affected child, early diagnosis is possible for subsequent
children. T he m arkers showing tight linkage could also be used to d eterm in e
which of the unaffected relatives are carriers. U nfortunately, effective screening of
spouses who m arry into a family will not be possible until the ap propriate U sher
g ene is cloned.
 CLINICAL AND GENETIC HETEROGENEITY OF USHER SYNDROME 379

Ultimately, the goal o f genetic research is to develop a treatm ent for U sher syn
drom e, however, the underlying defect causing it is unknow n. Most biochem ical
and physiologic studies have cen tered on the study o f retinitis pigm entosa an d the
results have been disappointingly negative in term s of elucidating the basic defect.
We would expect that the gene th at causes U sher syndrom e does so by an effect
on a substrate that is essential for som e com p o n en t of in n er ear function, balance,
and visual ability. D eterm ination o f the function o f the gene would fu rth e r o u r u n
derstanding o f the cellular an d biochem ical bases for these sensory abilities. An
u n d erstan d in g o f the underlying pathologic process occurring from gene to p h e
notype is an im p o rtan t step towards finding an effective m eth o d o f rem ediation
a n d /o r prevention. G ene cloning a n d its subsequent characterization may be the
only approach that could lead to the discovery o f the basic defect o f U sher syn
drom e.
C onsidering the fact that U sher syndrom e is the m ajor cause o f d ea fn ess/b lin d
ness, it is im p o rtan t to pursue research into the cause (s) o f U sher Syndrom e in the
hope th at som eday an effective rem edy may be possible. A bout 1 ou t o f every
20,000 individuals is affected with U sher syndrom e. T he b u rd e n caused by the loss
o f the two m ost vital hum an senses is trem endous. It isolates patients from the
m ainstream o f society, resulting in severe psychological stresses and reduces their
ability to act independently. T h e answers to many intriguing questions regarding
U sher syndrom e will no t be available until the genes are cloned. An un d erstan d
ing o f the etiologies o f U sher syndrom e will provide critical inform ation about the
underlying m echanism s o f function a n d expression o f the genes.

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 16. MARFAN SYNDROME

PETROS TSIPOURAS AND MICHAEL W. KILPATRICK

Department of Pediatrics, University of Connecticut Health Center.; Farmington, CT 06030,

USA

SUMMARY

In 1896 M arfan (M arfan, 1896) described the m usculoskeletal m anifestations

(dolichostenom elia) characteristic o f the condition that presently bears his
nam e; som e doubts rem ain to date as to w hether the original p atien t was actually
affected with the M arfan syndrom e (MFS). T he ocular abnorm alities associated
with the MFS were identified in the 1940s (Lutm an and Neel, 1949) an d the car
diovascular com plications began to receive attention when studied by McKusick
in the 1950s (McKusick, 1955). T he diagnosis o f MFS is clinical and based on well
established criteria (B eighton et a I., 1988). Clinical variability o f expression
within families b u t also betw een families is a hallm ark o f the disorder. T he aetiol
ogy of MFS rem ained elusive until recently when several m utations in the fibril
lin gene on chrom osom e 15 were identified.

CLINICAL MANIFESTATIONS AND NATURAL HISTORY

MFS is a systemic disorder o f connective tissue which prim arily involves the m us
culoskeletal, cardiovascular and ocular systems (Pyeritz and McKusick, 1979).
MFS has been re p o rte d in d ifferent races an d ethnic groups (McKusick, 1972).
T he prevalence o f MFS has b een estim ated to be approxim ately 46 p er 100,000
population in the U nited States (Pyeritz an d McKusick, 1979).

M usculoskeletal M anifestations

D olichostenom elia (long and narrow fram e) is the characteristic skeletal abnor
mality observed in MFS. Tall stature, decreased u p p er to lower segm ent ratio,
arm span in excess o f height, dolichocephaly, an d arachnodactyly o f fingers and
toes are some o f the m anifestations directly associated with dolichostenom elia
(Pyeritz an d McKusick, 1979) (Figure 1). In m ost individuals affected with MFS
the distal bones o f the extrem ities tend to be longer. Thus, a decreased ratio
(approxim ate value o f 0.85) o f u p p e r segm ent to lower segm ent is frequently
found in adults with MFS. T he face is long an d narrow, the palate is high-arched,
384 PETROS TSIPOURAS AND MICHAEL W. KILPATRICK

Figure 1 In div id u al a ffected w ith M arfan sy n d ro m e . P anel A: asym m etric p e ctu s c a rin a tu m
a t th e age o f 14 years. P an el B: sam e p a tie n t a t th e age o f 29 years.

prognathism a n d crowding o f the m axillary an d m andibular teeth are frequently

observed. Scoliosis o r kyphoscoliosis, whose age of onset is during childhood,
occurs in 30 to 60% of individuals with MFS. T he kyphoscoliosis is severe in the
infantile form o f MFS. T horacic kyphosis is associated with pulm onary deficien
cies including reduced vital capacity an d residual volum e (Magid et al., 1990).
T he spinal canal is often enlarged in d ep th or width due to the ectasia o f the n eu
ral foram ina an d sacral erosion. This feature, which is term ed "dural ectasia", has
been observed by com puted tom ography and it is of considerable diagnostic
value (Pyeritz et al.. 1988). T he thoracic deform ities, pectus excavatum or pectus
carinatum , p resen t in MFS, result from excessive longitudinal growth o f the ribs.
T he presence of a pectus deform ity in a child m akes it im perative to consider the
diagnosis o f MFS. T he "asthenic" habitus frequently observed in individuals
affected with MFS is due partly to m uscular underdevelopm ent an d also to a sig
nificant decrease o f the subcutaneous fat. Some patients appear to have a facial
expression rem iniscent o f a m yotonic facies. Ligam entous laxity and generalized
jo in t hyperm obility are com m on in MFS. Pes planus, genu recurvatum , re cu rre n t
dislocation o f hips an d o th er jo in ts result from the redundancy and weakness of
jo in t capsules, ligam ents, tendons and fasciae.
 MARFAN SYNDROME 385

Cardiovascular M anifestations

Overall, in the majority o f infants an d children, m anifestations related to multi-

valvular incom petence predom inate. This contrasts with the m ajority o f adults in
whom the m anifestations are prim arily from the aorta.
Dissecting aneurysm o f the ao rta is the m ost serious and potentially fatal cardiac
m anifestation in MFS; it develops post-natally an d reflects the underlying pathol
ogy o f the tunica m edia (McKusick, 1955). Dilatation o f the aorta usually precedes
the developm ent of the aneurysm a n d it is found in approxim ately 50% o f chil
dren and 70 to 80% o f adults. T he dilatation is confined eith er to the area aro u n d
the sinuses of Valsalva or it is generalized. T he generalized aortic dilatation is usu
ally associated with h ig h er m orbidity (R om an et al., 1988). Both types of aortic di
latation b reed true in families, suggesting the presence o f distinct phenotypic sub-
types (Devereux, personal com m unication, 1992; Tsipouras, unpublished data).
N ot all affected individuals with MFS develop aortic dilatation an d a small percen t
age o f individuals with unequivocal MFS m aintain norm al aortic diam eters into
their sixties. T he dilatation o f the ao rta could extend to the thoracic or abdom inal
segm ents an d occasionally to m edium size arterial branches such as the internal
o r external carotid arteries o r even intracranial branches. E nlargem ent of the pul
m onary artery is seen in MFS albeit at a m uch lower frequency com pared to the
aorta.
Aortic regurgitation is a com m on a n d usually progressive com plication of MFS.
It is relatively uncom m on in childhood, bu t its frequency in adults has been esti
m ated to be u p to 70%. Aortic regurgitation occurs only rarely in individuals af
fected with MFS but having norm al-sized aortic roots. A positive correlation
betw een aortic ro o t diam eter an d aortic regurgitation has been observed by Lima
e t al. (1985). T he acceleration o f aortic ro o t enlargem ent described in some pa
tients with aortic regurgitation reflects the causative relationship between the two
phenom ena.
T he M arfan syndrom e is the leading cause o f dissecting aortic aneurysm s in in
dividuals u n d e r the age o f 40 years. A lthough a dissecting aneurysm m ight be the
first presenting m anifestation in MFS, it is usually superim posed on underlying di
latation of the aortic ro o t and frequently of the ascending aorta as well (Pyeritz et
al., 1981). Most aortic dissections in MFS arise in the aortic ro o t o r proxim al as
cending ao rta and ex ten d distally th ro u g h a variable portion o f the aorta and its
branches, but dissections can also begin in m ore distal aortic segm ents (Roberts
and H onig, 1982; Crawford, 1983; Larson an d Edwards, 1984).
Approxim ately 60 to 70% o f persons with MFS exhibit m itral valve prolapse and
in a m uch sm aller fraction tricuspid valve prolapse is also observed (Pyeritz and
McKusick, 1979). T he m itral cusps a n d chordae tendinae may be re d u n d a n t re
sulting in m itral regurgitation.

O cular m anifestations

Ectopia lends, alm ost always bilateral, is a clinical hallm ark of MFS (Lutm an and
Neel, 1949; M aum enee, 1981). Only 50 to 80% o f individuals with MFS have this
386 PETROS TSIPOURAS AND MICHAEL W. KILPATRICK

abnorm ality. T he ciliary zonules, when visualized with a slit-lamp, appear re d u n

dant, atten u ated and frequently broken. T h e lenses are norm al in shape an d size
(M aum enee, 1981). T h e lens displacem ent is usually supero-nasal. T he observa
tion of iridodonesis indicates the presence of lens dislocation. Myopia is found in
a large n u m b er of individuals affected with MFS (M aum enee, 1981). S pontane
ous retinal d etach m en t occurs with an unusually high incidence in MFS; it is also
an im p o rtan t com plication o f the extirpation o f the lens.

M iscellaneous m anifestations

S pontaneous pneu m o th o rax occurs in approxim ately 10% of individuals with

MFS (Wood et al., 1984). Apical bullae, congenital pulm onary cysts an d em phy
sem a are some ra rer pulm onary findings. C utaneous striae distensae in the pecto
ral, deltoid, an d lum bar areas are frequent. Inguinal, fem oral an d incisional
hernias occur ra th e r com m only in m en with MFS. Finally, the voice o f individuals
with MFS is som etim es high pitched with a characteristic tim bre (McKusick,
1972).

Infantile M arfan Syndrome

A lthough infants and young children were am ong the initial patients in whom
MFS was docum ented, recognition o f the disorder in early life is n o t always an
easy m atter. O n the o th e r hand, certain infants present with a distinct set of clini
cal m anifestations which has led to the suggestion o f the existence o f a distinct
entity, term ed infantile or neonatal MFS. A characteristic aged facial appearance
due to deep-set eyes, high arched palate, arachnodactyly, pectus deform ities, flex
ion contractures, pes planus, multivalvular involvement, aortic ro o t dilatation
and ectopia lends are am ong the phenotypic m anifestations described (Morse et
a l . 1990).

INHERITANCE

T he autosom al dom inant m ode o f transm ission was clearly dem onstrated for the
first tim e by Weve in 1931. Since then, scores of pedigrees have been re p o rted
which clearly dem onstrate autosom al d o m in an t inheritance (Tsipouras et al.,
1992). A bout 80% of individuals docu m en ted to have the MFS by reasonably
strict criteria in h erited the gene from a parent; in the rem aining 20% the condi
tion arises as a result of new m utations. P aternal age effect on m utation has been
d em onstrated in the MFS . A ccording to o n e study, the average age o f fathers of
isolated cases was seven years greater than the m ean p atern al age in the popula
tion (M urdoch et al., 1972a). Most infants with the severe infantile form o f MFS
are isolated cases; for some o f these infants the possibility o f homozygosity has
been raised (Chem ke et al., 1984, Schollin et al., 1988).
 MARFAN SYNDROME 387

MOLECULAR BASIS

B rief overview

Over the years several m olecules o f the extracellular m atrix o f the connective tis
sue had been considered as potential candidates in the aetiology o f MFS. Am ong
them were the d ifferent types o f fibrillar collagens, elastin, hyaluronic acid, beta-
glucuronidase, elastase an d decorin (Tsipouras, 1990). In 1986, Sakai et al., using
m onoclonal antibodies raised against microfibrils, identified a 350 kDa glycopro
tein for which they coined the term fibrillin. Indirect im m unofluorescence stud
ies on the skin and cultured derm al fibrobasts of norm al individuals and
individuals affected with MFS provided the first strong evidence o f a structural
p rotein abnorm ality in M arfan syndrom e (Godfrey et al., 1990, H ollister et al.,
1990) (Figure 2). In parallel, a consortium o f investigators attem pted to localize
the gene responsible for M arfan syndrom e by positional m apping. An exclusion
m ap was constructed that elim inated nearly 75% o f the hum an genom e as a likely
location o f the MFS gene (B lanton et al., 1990). This led to the subsequent
assignm ent o f the MFS gene to chrom osom e 15 (15ql5-q23) (K ainulainen et al.,
1990; Dietz et al., 1991a; Tsipouras et al., 1991).

F igu re 2 In d ire c t im m u n o flu o re sc e n c e o f h y p e rc o n flu e n t d e rm a l fib ro b last c u ltu re s sta in e d

w ith m o n o c lo n a l a n tib o d y to fibrillin. T h e slides w ere c o u n te r sta in ed w ith p ro p id iu m io d id e
to visualize th e cell n u clei. P an el A: d e rm a l fibroblasts fro m a n o rm a l c o n tro l. N o te th e n o r
m al m eshw ork o f elastin-associated m icrofibrils. (C o u rtesy o f Dr. M aurice G o d frey a n d Mr. J a
son Cisler, U niversity o f N ebraska)
388 PETROS TSIPOURAS AND MICHAEL W. KILPATRICK

Figure 2 P an el B: d e rm a l fibroblasts fro m an in d iv id u al a ffected with M arfan sy n d ro m e. N o te

th e sparsity o f th e im m u n o sta in a b le m icrofibrils. (C ourtesy o f Dr. M aurice G odfrey an d
Mr. Ja s o n Cisler, U niversity o f N ebraska)

In view o f the im m unohistological evidence im plicating fibrillin in MFS, several

groups o f workers attem pted to isolate the gene coding for this protein. T he sub
sequent m apping o f a gene for fibrillin to the same chrom osom e interval as MFS
thus designated this gene as a strong candidate in the aetiology of MFS.
G enetic linkage studies in m ultiplex families using intragenic m arkers for the
fibrillin gene on chrom osom e 15 (FBN1) clearly showed genetic linkage o f MFS
to the fibrillin locus (m axim um lod score o f Z=25.6 at 9=0.00) (Lee et al., 1991,
Dietz et al., 1991b; Tsipouras et al., 1992). G enetic analysis strongly indicated the
absence o f locus heterogeneity in MFS (Tsipouras et al., 1992). However, Boileau
et al.(1991), re p o rted a family in which the phenotype segregated discordantly
with the FBN1 gene and eight o th er chrom osom e 15 m arkers.

The Fibrillin gene and its product

T he term m icrofibril was originally used to identify m orphologically sim ilar extraj
cellular m atrix structures displaying a diam eter o f less than 20nm and lacking the
characteristic 67nm banding periodicity o f interstitial collagen "'fibres (Low,
1962). Currently, the m icrofibrils are divided into two classes according to their
average diam eter, the larger o f the two classes being the lOnm m icrofibrils also
 MARFAN SYNDROME 389

re ferred to as the elastin-associated m icrofibrils (Cleary an d Gibson, 1983).

Im m unohistochem ical studies have identified m icrofibrils in the suspensory liga
m ent, pleura, perichondrium , periosteum , m eninges, aorta, cartilage, tendon,
m uscle and m any o th e r tissues (Figure 3). T he com plete m acrom olecular com po
sition o f the m icrofibrils is as yet unclear, and has been difficult to d eterm in e
because o f the highly insoluble n atu re of the m atrix aggregate. T he m ajor p ro
tein co m p o n en t appears to be fibrillin. However, four o th er proteins, with m olec
ular weights o f 78kDa, 70kDa, 31kDa and 25kDa, have also been identified
(Gibson et al., 1989; M addox et al., 1989).

Figure 3 E le ctro n m ic ro g ra p h s o f m icro fib rils (m ag n ificatio n 69000x) in d ev elo p in g elastic

fibers in b o vine lig a m e n tu m n u c h a e a t 210 days o f g estatio n al age. T h e elastin d ep o sits a m o n g
th e m icrofibrils are black d u e to e n b loc stain in g o f th e tissue w ith ta n n ic acid. P a n el A: cross-
section. (C ourtesy o f Dr. E laine C. Davies a n d Dr. R o b e rt P. M echam , W ash in g to n U niversity
S chool o f M edicine)

Fibrillin itself is an acidic glycoprotein with an estim ated m olecular mass of

350kDa. T he am ino-acid sequence o f the fibrillin m olecule has been deduced
from the cloned cDNA sequence (Lee et al., 1991; Maslen et al., 1991). T he pro
tein has an unusually high cysteine co n ten t of 14%, one third o f which has the po
tential to participate in disulphide bonding. In vitro studies suggest that fibrillin
is synthesised in a precursor form . Following secretion, the protein is converted to
its m ature form by proteolysis p rio r to assembly (Milewicz et al., 1992).
390 PETROS TSIPOURAS AND MICHAEL W. KILPATRICK

Figu re 3 P an el B: lo n g itu d in a l section. (C ourtesy o f Dr. E laine C. Davis a n d Dr. R o b e rt P. Me-

ch am , W a sh in g to n U niversity S chool o f M ed icin e)

T he fibrillin m olecule is characterised by the presence o f a com m on six cysteine

re p eat and a less freq u en t eight cysteine re p e a t (Lee et al., 1991; Maslen et al.,
1991). T he structure o f the six cysteine re p eat is rem iniscent o f the epiderm al
grow th factor (EGF) peptide (Sakai et al., 1991). EGF-like motifs are distributed
widely in n atu re and are found in m olecules as diverse as m am m alian LDL recep
tor an d the D rosophila hom eotic gene p ro d u c t N otch (Davis, 1990). W ith respect
to m atrix proteins, it has been proposed th at the EGF-like repeats may serve as lo
calised signals for cell growth and differentiation (Engel, 1989).
From the regions of the FBN1 gene that have been characterised to date, it ap
pears that the genom ic organisation m irrors the m odular arran g e m e n t o f the p ro
tein p ro d u ct (Lee et al., 1991) (Figure 4). A transcript 10 kb in size has been
identified in cultured derm al fibroblasts a n d sm ooth muscle cells (Lee et al.,
1991).
A surprising outcom e o f the cloning of the fibrillin gene was that fibrillin is ge
netically heterogeneous, with evidence for an o th e r fibrillin gene on chrom osom e
5 FBN2. Cross-hybridization of a clone for FBN1 to a genom ic library identified
clones which m apped to chrom osom e 5 (5q23-31) (Lee et al., 1991). C om parison
of the genom ic DNA sequence of regions o f FBN2 an d FBN1 suggest the two genes
have a sim ilar in tern al structure (Lee et al., 1991). A lim ited am o u n t o f cDNA se
quence data has allowed a partial am ino-acid sequence to be ded u ced for the
FBN2 gene pro d u ct (Lee et al., 1991; Maslen et al., 1991).
Prelim inary evidence suggests FBN2 may be expressed in cultured derm al fibro
blasts (Lee et al., 1991). However, to date no distinct FBN2 gene p ro d u ct has been
d etected an d the function o f this protein is unclear. G enetic analysis in families
with congenital contractural arachnodactyly, a condition phenotypically sim ilar to
MFS (Beals an d H echt, 1971), has established genetic linkage o f this condition to
 MARFAN SYNDROME 391

FBN2 (Lee et al., 1991; Tsipouras et al., 1992). It is possible to speculate th at the
am elioration o f the clinical m anifestations o f congenital contractural arachnodac-
tyly with age may be the consequence o f a developm ental switch involving the
FBN2 an d FBN1 genes.

F ig u re 4 D iag ram m atic re p re se n ta tio n o f th e F ib l5 g e n e show ing th e EGF-like re p e a ts a n d 8-

cysteine rep eats. T h e p o sitio n s o f th e seven p o in t m u ta tio n s a n d two d e letio n s id e n tifie d to
d a te in MFS p a tie n ts are in d ic a te d by vertical arrow s. T h e o n e le tte r am in o -acid ab b rev iatio n
is u sed to identify am in o -acid ch an g e s c o n s e q u e n t to th e p o in t m u tatio n s.

M arfan Syndrome and m utations in the Fibrillin gene

Analysis o f fibrillin biosynthesis in cultured derm al fibroblasts from individuals

with MFS has shown defects in fibrillin synthesis, secretion a n d incorporation
into the extracellular m atrix (Milewicz et al., 1992). As has been shown to be the
case in o th er dom inantly in h erited disorders the defective gene pro d u ct appears
to interfere with the function of the wild-type protein at many d ifferent levels.
F u rth er insight into the relationship betw een the structure and function o f the
fibrillin m olecule is beginning to em erge from the m olecular approach, which has
facilitated the identification o f several distinct m utations in the fibrillin gene. Ini
tial attem pts to delineate m utations in the fibrillin gene in individuals affected
392 PETROS TSIPOURAS AND MICHAEL W. KILPATRICK

with MFS revealed no gross deletions o r rearrangem ents, an d to date only one ge
nom ic deletion has been identified (K anulainen et al.,1992). T he deletion results
in the loss o f 366 bases o f the fibrillin mRNA which consequently produces a tru n
cated protein. This sh o rten ed protein appears to be synthesised in norm al
am ounts, secreted efficiently from the cell, and assem bled into microfibrils. T he
m icrofibrils thus produced are structurally an d functionally abnorm al.
Seven p o in t m utations (five missense an d two nonsense) have so far b een iden
tified and characterised (Dietz et al., 1991b, 1992): four o f the missense m utations
result in cysteine substitutions an d one in the substitution o f an arginine (Dietz et
al., 1992) (Figure 4). As substitution o f a cysteine residue would be expected to
d isru p t local folding an d possibly extracellular protein-protein interactions, the
consequence o f such a m utation can be readily envisaged. Similarly the arginine
substitution, which occurs in an EGF-like repeat, m ight be expected to alter the
local secondary structure an d thus affect cysteine-m ediated bonding. O ne o f the
nonsense m utations leads to the production o f a fibrillin m olecule which lacks the
last 116 am ino acid residues and is apparently n o t secreted from the cell (Kainu-
lainen et al., 1992). Im m unofluorescence studies of fibroblasts from the individual
with this m utation reveals red u ced am ounts o f extracellular fibrillin (K ainulainen
et al., 1992). T he o th er nonsense m utation, however, results in a tru n cated m ole
cule which, although apparently synthesised in reduced am ounts, is secreted and
does participate in fibrillogenesis. (K ainulainen et al., 1992). Thus even a small
am o u n t o f structurally abnorm al fibrillin is able to elicit a clinical phenotype, thus
dem onstrating the potential for fibrillin m utations to have a d o m in an t negative ef
fect.
W ith the exception o f the originally re p o rte d missense m utation which was
fo und in two unrelated isolated cases o f MFS all the m utations re p o rted to date
have been unique (Dietz et al., 1991b). In each case, screening o f a large nu m b er
o f un related individuals affected with MFS for the presence o f the m utation has
proved fruitless. It is also o f interest to n o te th at the m utations identified to date
are distributed th ro u g h o u t the entire length o f the gene, with no evidence o f clus
tering o f m utations in particular regions o r corresponding protein dom ains.

ANIMAL MODEL

An anim al m odel has been provided by a h e rd of cattle recently re p o rted to be

affected by a disorder seemingly analogous to MFS (Besser et al., 1990). T he
affected anim als display long thin limbs, jo in t laxity, ectopia lentis an d aortic dila
tation an d aneurysm . Autopsy on a calf which died suddenly at age 16 m onths
identified the cause of d eath to be aortic ru p tu re resulting in haem opericardium
an d cardiac tam ponade. Electron m icroscopy revealed that the tunica m edia of
th e affected aorta displayed thin an d irregular elastic lam inae. Im m unofluores-
cent staining o f skin biopsy m aterial using fibrillin m onoclonal antibodies did not
distinguish affected calves from norm al. However, it was possible to distinguish
 MARFAN SYNDROME 393

affected anim als from norm al by reduced derm al staining for elastin (Besser
et al., 1990).
A lthough the n atu re o f the genetic defect is as yet unknow n, it is possible that
this MFS-like condition will provide an excellent m odel for fu rth e r study and char
acterisation of the precise m olecular pathology o f MFS.

CLINICAL MANAGEMENT

Individuals affected with MFS often require m ultidisciplinary m anagem ent by a

team o f m edical specialists. From the clinical point o f view, the m ost serious and
potentially fatal com plications o f the condition are the cardiovascular ones.
D eath in adults affected with MFS is m ost com m only due to aortic root disease, in
which progressive dilatation results in eith er aortic regurgitation or aortic dissec
tion an d ru p tu re. T he m edian age of d eath in MFS in the only detailed study was
the m id 40s; m ore than 95% o f deaths result from cardiovascular com plications
(M urdoch et al., 1972b). T he cardiological exam ination schedule depends on
the severity o f the cardiovascular com plications and in individuals with m ild fea
tures, such as m itral valve prolapse an d m inim al aortic dilatation, annual exam i
n ation including an echocardiogram is sufficient. However, if aortic dilatation
and regurgitation are significant, m ore freq u en t follow-up is necessary. In addi
tion to echocardiography, com puted tom ography (CT) and m agnetic resonance
im aging (MRI) have been used for the evaluation o f the ao rta in MFS. A direct
relationship has been observed betw een aortic ro o t dim ension an d com plications
of the MFS. Aortic regurgitation is virtually non-existent in MFS patients whose
aortic root dim ension is 3.6 cm or less, b u t occu rred uniform ly in patients with
an aortic root dim ension of 6.0 cm o r greater, according to one study (Pyeritz et
al., 1981a). Patients in whom aortic dilatation extended into the ascending aorta
were m ore likely to have aortic regurgitation and appeared to be at greater short
term risk o f progressive dilatation necessitating surgery than patients in whom
the aortic dilatation was lim ited to the sinuses o f Valsalva (Rom an et al., 1988).
Pyeritz et al. (1983) dem onstrated th at propanolol therapy results in a reduction
of the rate of aortic dilatation in affected children an d adults. However, the b en e
ficial effects o f long term beta-blockade o r negative inotropy on the aorta are dif
ficult to ascertain because they have n o t been subjected to the scrutiny o f a
controlled d ru g trial. It is therefore prem atu re to propose treatm en t o f all indi
viduals with the MFS until m ore inform ation is available.
D uring the last 10 years, the developm ent o f im proved surgical techniques for
repair o f the dilated o r dissected aortic ro o t with sim ultaneous replacem ent o f the
aortic valve has been of great im portance. An elective rep air o f an aorta, the ro o t
diam eter of which is 6 cm o r less, is preferable to em ergency repair. A m odified
Bentall p ro ced u re with a variety of grafts is used (Bentall and DeBono, 1968). A
10 year survival rate o f m ore than 75% has b een re p o rted in one study (G ott et al.,
1986). T h e occurrence o f dissection p rio r to surgery or the presence o f aneurysm s
394 PETROS TSIPOURAS AND MICHAEL W. KILPATRICK

in m ultiple aortic segm ents is associated with a worse postoperative prognosis.

Crawford an d Coselli (1988) re p o rted a 10 year survival of 56% am ong the latter
group. R eplacem ent o f the aortic or m itral valves is a freq u en t procedure in MFS
patients. In children with m itral regurgitation, reconstruction of the m itral valve
ra th e r than its replacem ent is often feasible (Shumway et al., 1988).
Scoliosis is a freq u en t orth o p ed ic com plication in MFS (Pyeritz an d McKusick,
1979; Viljoen and B eighton, 1990). Its age o f onset varies from early childhood to
adolescence. Conservative treatm ent by bracing o r surgical correction with differ
en t types o f fixation and rodding can be used successfully (Birch an d H erring,
1987; W inter, 1990). Pectus deform ities are present in m ore than two-thirds o f in
dividuals with MFS. Surgical correction is n o t w arranted for cosm etic reasons only.
O th er orth o p ed ic problem s such as re c u rre n t jo in t dislocations an d subluxations,
are m anaged according to established o rth o p ed ic principles.
R egular ophthalm ic evaluation is an im p o rtan t aspect o f the m anagem ent o f in
dividuals with MFS. Im provem ent of im paired visual acuity due to m yopia is
achieved easily with conventional means. Removal o f a dislocated lens by surgery
should be considered only if satisfactory visual acuity cannot be achieved (Behki
et al., 1990; Peyman et al., 1979).
Pregnancy m ight pose a considerable risk to wom en affected with MFS (Pyeritz
et al., 1981b; Beighton, 1982). In general, wom en with an aortic ro o t diam eter
g reater than 4 cm (as m easured by echocardiography), valvular disease o r any ev
idence of cardiac decom pensation are at g reater risk, while those with m inim al
cardiac findings tolerate pregnancy well (Pyeritz, 1981). T he co n tin u ed use of
beta-blockers during pregnancy rem ains controversial.
A lthough the majority o f individuals affected with MFS cope well with their con
dition, som e require psychological counselling as part of their m anagem ent. In
that respect they do no t differ from any person affected with a chronic an d p o ten
tially debilitating condition. T h e N ational M arfan F oundation in the U nited States
and sim ilar lay groups in o th er countries provide valuable psychological and em o
tional support and dissem inate inform ation to their m em bers and o th er interest
ed individuals.

FUTURE DIRECTIONS

T he recen t identification o f fibrillin as the m olecule m utated in MFS heralds a

new era in the un d erstan d in g o f this disorder. T he possibility now exists to d eter
m ine fully the m olecular pathology o f MFS and, in the process, to begin to
unravel the com plexities o f the m icrofibrillar system. It can be envisaged th at the
elucidation o f the full structure of the FBN1 gene will m ake it possible to begin to
d eterm in e the organisation and regulation of this gene. In this regard, the possi
bility rem ains th at genes flanking the FBN1 gene are also involved in the path o
genesis o f MFS. T he fu rth e r identification o f fibrillin m utations in patients
affected with MFS will allow a genotype-phenotype correlation to be m ade. In
 MARFAN SYNDROME 395

addition to providing clues as to the relationship between structure and function

for the various regions o f the fibrillin m olecule, this may m ake possible the g en o
typic diagnosis, both prenatally an d postnatally, o f MFS. It will also be im portant
to d eterm in e the full ex ten t o f the fibrillin gene family, the relationship between
the various m em bers o f this family a n d the developm ental role o f the fibrillins.
T he identification o f fibrillin m utations in individuals with MFS has already be
gun to provide some clues as to the role played by fibrillin in m icrofibril assembly
an d function. T he fu rth e r d eterm in atio n of the m olecular com position o f mi
crofibrils along with the isolation an d characterization o f the genes for these p ro
teins an d the investigation o f the involvem ent o f the various m icrofibrillar
com ponents in the pathology o f MFS-like disorders will advance o u r un d erstan d
ing o f the entire m icrofibrillar array. In the same way, the co n tin u ed delineation
of the precise m olecular pathology o f MFS-like disorders may lead to im prove
m ents in both the diagnosis and trea tm e n t of this group of conditions.
It may also be timely to begin to speculate as to the possibility o f gene therapy
for MFS. A lthough gene therapy for a d o m in an t condition such as this requires
specific repression o f the m u tan t allele ra th e r than sim ple provision o f an absent
gene product, the intensity o f investigation of the fibrillin gene may provide the
necessary background for progress in this area. A recom binant m ouse m odel, con
structed by hom ologous recom bination into the FBN1 gene, will definitely be
com e the organism o f choice to test gene therapy o f MFS.

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 17. LOWE OCULOCEREBRORENAL SYNDROME

ICHIRO OKABE1 AND ROBERT L. NUSSBAUM12

1Howard Hughes Medical Institute Laboratory; 2Department of Genetics
University of Pennsylvania School of Medicine, 422 Curie Boulevard,
Philadelphia, PA 19104, USA

INTRODUCTION

T he Lowe oculocerebrorenal syndrom e (OCRL), (Me Kusick #309000), first des

cribed in 1952 (Lowe, Terrey, an d M acLachan, 1952), is an X-linked disorder
characterized by congenital cataract, m ental retardation and renal tubular dys
function. Several h u n d re d cases have been described since the initial re p o rt but
the exact incidence o f this d isorder is n o t known. T he prim ary biochem ical
defect has been variously ascribed to abnorm alities in proline uptake (States,
Palm ieri, an d Segal, 1982) m itochondrial function (G obernado et al., 1984;
Moraes et al., 1991), an d proteoglycan synthesis (Yamashina et al., 1983; Yokoi et
al., 1981; Fukui et al., 1981). T he OCRL locus was first m apped to Xq25-26 by
linkage (Silver, Lewis, an d N ussbaum , 1987; W adelius et al., 1989; Reilly et al.,
1988; Reilly, Lewis, and Nussbaum , 1990) an d cytogenetic m ethods (H odgson et
al., 1986; M ueller et al., 1991) and then isolated by a positional cloning strategy
(Okabe et al., 1992; A ttree et al., 1992). Striking hom ology of the OCRL gene to
an enzyme, platelet inositol 1,4,5 tris phosphate-5-phosphatase (Ross et al.,
1991), suggests th at the prim ary defect in this disorder may be d eran g ed inositol
phosphate m etabolism . C u rre n t work now focuses on d eterm in in g the exact
function of the p ro d u ct o f the gene for OCRL and the pathogenesis of the p h e
notype. T he clinical, laboratory, and genetic features o f OCRL have been
recently reviewed (C harnas an d Nussbaum , 1994).

CLINICAL FINDINGS

Incidence and Inheritance

OCRL is an X-linked disorder th at occurs nearly exclusively in males. T he disease

is rare bu t appears to be ubiquitous am ong various nationalities an d races (Korn-
feld et al., 1975; A nanthakrishna and Biswakumar, 1977; Banerjee, Allen, and
McKee, 1982; Yamashina et al., 1983; M cSpadden, 1991). Several females have
also been re p o rte d (Svorc et al., 1967; Sagel, Ores, an d Yuceoglu, 1970; H arris et
al., 1970; Lythgoe and Ramsey, 1973; Cyvin, W eidem ann, and B athen, 1973;
Jo h n so n an d Hiles, 1977; Yamashina et al., 1983). In some o f these females, how
ever, only a po rtio n of the OCRL phenotype is present while in others, congenital
400 ICHIRO OKABE AND ROBERT L. NUSSBAUM

m alform ations an d b irth defects no t seen in males with OCRL m ake the diagno
sis o f OCRL uncertain. O ne fem ale p atien t was re p o rted with a deletion o f m ito
chondrial DNA, which raises the possibility th at a m itochondrial genocopy for
OCRL may exist (M oraes et al., 1991), b u t h er phenotype was that o f a progres
sive m ultisystem disease only partially overlapping with OCRL. Finally, th ere are
two fem ales re p o rte d in the literature in which clear-cut OCRL has arisen de novo
d u e to balanced X /au to so m e translocations involving the Xq25-Xq26.1 region of
the X chrom osom e (H odgson et al., 1986; M ueller et al., 1991). These patients
have an OCRL phenotype indistinguishable from that o f hemizygous affected
males because the translocations d isru p ted the OCRL locus on the translocated
X chrom osom e an d im peded random X inactivation such th at the X chrom o
som e n o t involved in the translocation is inactivated preferentially.

O cular Findings

Bilateral congenital cataracts are a m ajor sign o f OCRL, present in 100% of

affected males. A bnorm al lens developm ent begins at 7-9 weeks as a disordered
m igration o f the em bryonic lens epithelium (Ginsberg, Bove, an d Fogelson,
1981). Lens opacities have been detected clinically in utero in some cases as early
as 20-24 weeks (Endres et al., 1977; G aary et al., 1993). G laucom a develops in
ab out 50% o f patients, usually during the first year o f life, bu t may develop as late
as the second o r third decade (M cSpadden, 1991). C orneal keloid form ation
develops in approxim ately 25% o f the patients, on average at 5 years o f age but
with a range from 5 m onths to 18 years (M cSpadden, 1991; Cibis et al., 1982).
Keloids are often stable an d cause no visual difficulty but, if extensive an d cen
trally located, may interfere with vision a n d can be the m ajor cause o f visual dis
ability after age 5 years in patients whose cataracts a n d glaucom a have been
successfully m anaged. However, som e degree of dim inished acuity is present in
all OCRL patients that is n o t explainable by cataracts o r keloids and may result in
strabism us (20%) and nystagmus (69%) (M cSpadden, 1991). Overall, about 15%
o f OCRL patients over 5 years old are blind.
Fem ale carriers can be diagnosed by characteristic lens opacities using the slit-
lam p exam ination (Tongue, 1972; G ard n er and Brown, 1976; Cibis et al., 1986;
Reilly et al., 1988; W adelius et al., 1989). In Figure 1A are shown the m ost com m on
an d typical opacities o f fem ale carriers. They are punctate, white to gray in color,
vary in size from m icrons to several m illim eters, and are distributed in radial or
wedge-shaped clusters in the cortex layer bu t n o t in the nucleus o f the lens (Cibis
et al., 1986; Reilly et al., 1988). T he distribution of opacities ju st outside the n u
cleus suggests that the opacities develop very early in life while the m ore superfi
cial opacities m ust arise in adu lth o o d (Cibis, T ripathi and Tripathi, 1990). These
opacities are usually g reater than 15 in n u m b er and may be in the hundreds. A few
fem ale carriers show a single subcapsular dense cataract in the posterior pole
(Tongue, 1972) (Figure IB ). This form o f cataract may be present congenitally
and becom e clinically m ore ap p a ren t with age, thus requiring surgical therapy. Al-
 LOWE OCULOCEREBRORENAL SYNDROME 401

F igu re 1 Slit lam p e x a m in a tio n o f fem ale c a rrie rs o f Lowe's Syndrom e. In p a n e l A is show n
th e typical p a tte rn o f h u n d re d s o f m ic ro p u n c ta te , grey o p acities d istrib u te d in a rad ial distri
b u tio n in th e lens. In p a n e l B is a single d e n se p o ste rio r c a ta ra c t as see n in som e O CR L h e t
erozygotes. (C ourtesy o f R. A. Lewis, M.D.)
402 ICHIRO OKABE AND ROBERT L. NUSSBAUM

though som e controversy exists in the literature concerning the specificity and
sensitivity o f the lenticular opacities as a m ethod o f carrier detection (G ardner
and Brown, 1976), reliance on strict diagnostic criteria for both appearance and
distribution of the opacities m akes carrier detection by slit lam p exam ination a re
liable an d sensitive technique (Cibis et al., 1986; Reilly et al., 1988).

Systemic Findings

Growth and development

Average birth weight and length o f the patients with OCRL are well within nor
mal range b u t the rate o f growth begins to decline by 3 m onths o f age. By 18
m onths, weight and height are below norm al range, an d continue to fall. T he
growth curve in the patients does n o t ap p e ar to plateau at 16 to 18 years o f age as
it does in norm al m en. Growth continues on into early adulthood b u t m ean final
weight and height are less than the third percentile o f norm al values for m en
(M cSpadden, 1991; C harnas et al., 1991).
A wide range o f intellectual ability are rep resen ted in OCRL patients (M cSpad
den, 1991; Kenworthy, Park, and C harnas, 1993); 63% have m oderate to severe
m ental retadation, 33% have a mild to bord erlin e form , with a few patients dem
o n strating intelligence within the norm al range. T hough m ental developm ent is
generally delayed, the developm ental abnorm ality is stable, in contrast to the p ro
gressive course seen in degenerative disorders.
Some behavior problem s are troublesom e, especially self-abusive behavior, non-
cooperative an d violent behavior, and eccentric, autistic-like behavior. These be
havior problem s are both m ost prevalent an d m ost severe in the 5-13 age group,
and ten d to decrease in late adolescence a n d adulth o o d (M cSpadden, 1991; Ken
worthy, Park, and C harnas, 1993).

Neuromuscular signs

H ypotonia with decreased m uscle mass and decreased d eep ten d o n reflexes are
n o ted in m ost patients. H ypotonia becom es a p p a ren t from a few m onths o f age
but is n o t progressive an d tends to im prove in association with general growth.
T he deep ten d o n reflexes are present at birth but disappear by 1 year o f age
(Abbassi, Lowe, and Calcagno, 1968). Significant nerve o r m uscle pathology is
absent and suggests th at the hypotonia a n d areflexia are o f central nervous sys
tem origin (B anerjee, Allen, an d McKee, 1982; C harnas et al.,1988).
Seizures occur in 50% o f OCRL patients (C harnas, 1989). All three m ajor types
o f seizure have been reported: febrile, p etit mal, and grand mal. T he average age
o f first seizure for these three types of seizure is 2 years, 5 years, 9 years o f age re
spectively. T he grand mal type is the m ost com m on seizure (M cSpadden, 1991).
As in the general population, severe, early onset seizures are a p o o r prognostic
sign for intellectual developm ent and seizure control.
 LOWE OCULOCEREBRORENAL SYNDROME 403

Renal dysfunction

Fanconi Syndrom e (proxim al tubular acidosis, am ino aciduria, phosphaturia,

and proteinuria) is a principal co m p o n en t of OCRL. T he renal tubular dysfunc
tion, in contrast to the cataracts, is n o t always present at birth an d may require a
few weeks to m onths to becom e apparent. Renal tubular dysfunction is quite vari
able in its severity or clinical significance, and may n o t require m edical interven
tion (C harnas et al., 1991). T he acidosis is due to bicarbonate wasting (Schoen
and Young, 1959: Richards et al., 1965) an d leads to the failure to thrive, recur
ren t infections, and m etabolic collapse seen in early case reports when the dis
ease was poorly recognized an d u n treated . W ater resorption is also defective, as
reflected in elevated 24 h o u r volum es and low urine osmolality (C harnas et al.,
1991). A m inoaciduria is generalized bu t variable, ranging from ju st above the
u p p er lim it of norm al to 15 times above the u p p er limit o f norm al (Payton et al.,
1989; C harnas et al., 1991), b u t the p attern o f am inoaciduria is no t diagnostic of
OCRL (Manz, Bremer, an d B rodehl, 1978). P roteinuria is very frequently seen
b u t the am ount of u rin ary losses an d age of onset are both highly variable. Pro
teinuria is usually present in infancy b u t can first occur later in childhood. W hen
present, protein losses can be substantial (1.3810.77 g m /m 2 /d , norm al <0.1 g /
m 2 /d ) . Glycosuria is generally not a feature of the renal tubular dysfunction seen
in OCRL. T he renal Fanconi syndrom e, once established, is usually stable.
H yperphosphaturia is also freq u en t but variable, an d may lead to osteom alacia,
renal rickets, an d pathologic fractures if untreated. In approxim ately half o f pa
tients, serum phosphate levels are norm al but the fractional excretion of phos
phate is elevated. T ubular phosphate wasting often worsens with age (C harnas et
al., 1991), and may require p h osphate supplem entation orally o r by nasogastric
tube (Redfield et al., 1991). N ephrolithiasis an d nephrocalcinosis may occur in
OCRL (Schaper an d H orstm ann, 1963) because of hyperphosphaturia an d hyper-
calciuria, bu t high urinary volum e may be partially protective. G radual loss o f cre
atinine clearance reflecting progressive renal failure is a feature o f OCRL in the
second an d third decades of life. T he average age of onset o f renal insufficiency
and its severity is, however, not clearly defined.

Skeletal signs

M usculoskeletal com plications in OCRL can result eith er as a prim ary feature of
the disease or as secondary consequences o f renal and neurom uscular abnorm al
ities o f the illness, i.e., Fanconi Syndrom e a n d hypotonia. Tenosynovitis and
arthritis or arthropathy are freq u en t an d appear to be prim ary com plications of
OCRL. Jo in t involvem ent can p resen t as n o n te n d e r jo in t swelling involving both
small and large joints, focal nodules on the finger, or bilateral p lan tar masses. On
occasion these masses becom e painful an d require resection (C harnas and Gahl,
1991). It seems likely th at these changes are the m anifestation of abnorm al,
excessive growth o f fibroblasts from periarticular tissue or tendons. Secondary
404 ICHIRO OKABE AND ROBERT L. NUSSBAUM

com plications o f OCRL include jo in t hyperm obility due to hypotonia or contrac

tures resulting from decreased m ovem ent. Rickets is noted in about h alf o f the
patients. A bout h alf of the patients have had at least one fracture because o f dif
fuse osteopenia (M cSpadden, 1991). O steopenia can be w orsened by u n treated
renal phosphate wasting, but is alm ost always present, with variable severity
despite adequate phosphate replacem ent, an d appears to be a specific m anifesta
tion of OCRL. Bone fracture occurs mainly at the arm s and legs and increases
with age. Scoliosis is also com m on am ong th e older patients.

Other features

T he onset an d progression of puberty is generally norm al although cryptorchid

ism is com m on (C harnas an d Gahl, 1991). P eritubular fibrosis an d azoosperm ia
presum ably reduce fertility (Matin an d Sylvester, 1980). OCRL patients lack a
strong sexual drive in com parison with o th e r adult males with similar intellectual
com prom ise.
D ental "blue dom e" cysts occur during prim ary tooth eru p tio n an d double rows
of teeth caused by delayed shedding of prim ary teeth with sim ultaneous presence
of prim ary an d p erm a n en t teeth have b een re p o rted (M cSpadden, 1991).

Prognosis

Severe renal insufficiency a n d dehydration o r in te rc u rre n t infection (C urtin,

Joyce, and Ballin, 1967; M atin and Sylvester, 1980; Tripathi, Cibis, and Tripathi,
1980) are the m ain cause o f death in patients with OCRL. Many patients are
known to have lived beyond adolescence. T he m ean age o f the 82 patients known
to the Lowe's Syndrom e Association is 13 years 2 m onths, with a range o f 1 year 7
m onths to 41 years 3 m onths (C harnas et al., 1991).

Pathological Findings

Ophthalmic pathology

Lenses are small, flattened, an d discoid. T h ere is a diffuse cataract with no differ
entiation o f cortex and nucleus, and p ro n o u n ced degeneration in the posterior
p o lar region (C urtin, Joyce, and Ballin, 1967; Tripathi, Cibis, an d Tripathi, 1980).
T he an terio r subcapsular epithelial hyperplasia an d nodular excrescences of the
lens capsule are present; however, these are no t necessarily specific for OCRL
(Z im m erm an and Font, 1966; C urtin, Joyce, and Ballin, 1967; Tripathi, Cibis, and
Tripathi, 1980; Tripathi, Cibis, an d T ripathi, 1986). An anom alous a n terio r cham
b er angle with an terio r displacem ent o f rudim entary ciliary processes, which
could contribute to glaucom a, m ight be secondary to a m icrophakia in which
zonular fibres drag the ciliary body anteriorly (Zim m erm an and Font, 1966; Cur
tin, Joyce, and Ballin, 1967; Tripathi, Cibis, and T ripathi, 1980; Cibis, Tripathi,
 LOWE OCULOCEREBRORENAL SYNDROME 405

and T ripathi, 1990). T he small islands o r folds o f retinal tissue, which m ight also
be dragged anteriorly by the zonules,are p resen t in pars ciliaris retinae (Zim m er
m an an d Font, 1966; C urtin, Joyce, an d Ballin, 1967; Garzuly et al., 1973). O th er
wise, the retina including optic nerve, sclera, and choroid appears unrem arkable
in the light m icroscopic exam ination (C urtin, Joyce, and Ballin, 1967; Garzuly et
al., 1973).

Renal Pathology

H istological exam ination by light m icroscopy is usually norm al in very young

infants but dilatation, atrophy, loss of b rush border, and accum ulation o f protein-
aceous m aterial in the tubular lum en appear within the first few m onths of life
(Schoen an d Young, 1959; Richards et al., 1965; W itzleben et al., 1968; Van Acker
et al., 1967). These tubular abnorm alities have been docu m en ted in a n u m b er of
studies, including som e in which serial biopsies from the same patients were
exam ined, and affect predom inantly the proxim al tubules, with some involve
m ent of H enle's loop, distal tubules, an d the conducting system occasionally
cited as well. G lom eruli are frequently norm al in young children, even when
proxim al tubular lesions are present. A fter the first few years o f life, however,
glom erular lesions can be seen. T hese include thickening o f basem ent m em
branes, focal fibrosis, and sclerosis. By electron microscopy, swelling of m itochon
dria in renal tubules was re p o rted in a 22 m onth old who showed no glom erular
abnorm alities (Schoen and Young, 1959). W hen the same child was biopsied 7
years later, distinctive g lom erular changes including podocyte fusion and base
m ent m em brane thickening were ap p a re n t (W itzleben et al., 1968). O ne re p o rt
o f severe electron m icroscopic abnorm alities in glom eruli and proxim al tubules
was based on m aterial from a fem ale p atien t who did no t satisfy cu rren t clinical
diagnostic criteria for OCRL (Sagel, Ores, an d Yuceoglu, 1970).

Central nervous system pathology

A variety of pathological findings in the brain have been re p o rted (Richards et

al., 1965; C hutorian an d Rowland, 1966; Matin a n d Sylvester, 1980; Banerjee,
Allen, and McKee, 1982). T he m ost consistent change in the brain is a loss o f or
defective form ation o f myelin. Such changes are n o t seen in all cases, vary in dis
tribution an d extent, an d are nonspecific since similar abnorm alities have been
no ted in o th er m etabolic disorders such as m aple syrup u rine disease, homocysti-
nuria, an d phenylketonuria. M ultiple tiny cysts w ithout inflam m atory changes
have also b een described in the cerebral white m atter bu t ap p ear to be of no clin
ical significance (Dem mer, W ippold, an d Dowton, 1992).
406 ICHIRO OKABE AND ROBERT L. NUSSBAUM

BIOCHEMICAL FINDINGS

Previous studies o f OCRL patients and cells have disclosed a nu m b er o f potential

abnorm alities. Increased proline uptake in fibroblasts from one p atien t was
re p o rte d (States, Palm ieri, an d Segal, 1982). Since one o f the m ajor pathways of
p roline m etabolism is incorporation into collagen, Palm ieri et al. (1985) exam
ined collagen form ation and fo u n d that collagen production was substantially
d ecreased in cultured skin fibroblast from patients. T he m echanism an d signifi
cance o f these findings rem ain unclear.
M etabolic abnorm alities of glycosaminoglycans(GAGs) have been re p o rted be
ginning in 1969 (Suschke and M urken, 1969). Elevated excretion o f urinary GAGs
was seen in patients with OCRL. However, the results were conflicting, an d diffi
cult to reproduce. Akasaki et al. (1978) re p o rted increased low sulfated chon-
droitin-4-sulfate (C4S) excretion in the urine. Hayashi et al. (1978a;1978b) have
shown that low sulfated C4S an d heparan sulfate (HS) were elevated in the urine
o f the p atien t with elevated excretion o f urinary GAGs. This was verified by Kieras
e ta l. (1984) and Wisniewski e ta l. (1984) except that they found HS excretion was
w ithin the norm al range. However, Yokoi et al. (1981) re p o rted a decrease in the
u rinary excretion o f HS with no elevation o f urinary GAGs in patients.
Given the findings in urine, GAGs synthesis an d sulfation have been studied in
cu ltured skin fibroblasts from OCRL patients. O n one hand, D onnelly et al.
(1984) an d H a rp er et al. (1987) showed th at OCRL fibroblasts synthesized nor
mally sulfated GAGs and did no t express defects in sulfation o f GAGs. Yokoi et al.
(1982) re p o rted undersulfation o f HS b u t no t CS and DS in fibroblasts o f OCRL
patients. Yamashina et al. (1983) re p o rted undersulfation of GAGs confined to
ch on d ro itin sulfate (CH) and derm atan sulfate (DS) with HS n o t affected, an d this
undersulfation o f GAGs was attrib u ted to depleted active sulfate (adenosine 3 -
ph osphate 5 -phosphosulfate; PAPS) pools in OCRL fibroblasts, reportedly result
ing from elevated levels o f a nucleotide pyrophosphatase (NPPase) activity de
grading PAPS (Fukui et al., 1981; Yoshida et al., 1982; Yamashina et al., 1983).
They w ent on to show that the NPPase activities in heterozygotes were in term ed i
ate between patients and norm al individuals (Yamashina et al., 1983) and the ele
vated NPPase activities in fibroblast from patients resulted from increased
am ounts o f the enzyme p rotein (H orie et al., 1988). Elevation of NPPase activities
in patients was verified by one o th er g roup (H arp er e ta l., 1987). NPPase, however,
was elim inated as the prim ary locus for OCRL by the finding th at the NPPase gene
m aps to 6q22-q23 (Funakoshi et al., 1992). Thus, the occurence and significance
o f abnorm alities o f GAGs as prim ary defects for OCRL still rem ain obscure.
T here are several reports about m itochondrial abnorm alities in OCRL (Payton
et al., 1989; Ores, 1970; Sagel, Ores, an d Yuceoglu, 1970; G obernado et al., 1984;
M oraes et al., 1991). Some o f these rep o rts were female patients with phenotypes
th at overlapped partially with OCRL. All h ad norm al chrom osom e constitutions
an d showed m orphological m itochondria abnorm alities (Sagel, Ores, and Yuceo
glu, 1970), or m itochondrial DNA deletion (Moraes et al., 1991). G obernado et
 LOWE OCULOCEREBRORENAL SYNDROME 407

al. (1984) showed a defect in oxidative phosphorylation in m itochondria in a male

patient with OCRL. F u rth er studies are n ee d ed to determ ine w hether th ere is a
reproducible defect in m itochondrial function in X-linked OCRL but it appears
th at m itochondrial DNA alteration o r deletion may p roduce partial genocopies of
the OCRL phenotype.

MOLECULAR STUDIES

Gene M apping

Linkage studies o f OCRL were first re p o rted in 1982 (H ittner, C arroll, and
Prchal, 1982) which ru led ou t close linkage of OCRL to eith er the Xg blood
g roup (Xp22.3-pter) or to glucose-6-phosphate dehydrogenase (Xq28). Using
restriction fragm ent length polym orphism s (RFLPs) on eight X chrom osom e
loci, Silver et al. (1987) analyzed fo u r large families with OCRL and identified
two loci, DXS10 at Xq26 an d DXS42 at Xq24-26, which were tightly linked to
OCRL and supported a m ap assignm ent to Xq24-q26. Evidence for placing
OCRL at Xq24-q26 also cam e from two fem ale patients with an X;3 translocation
b reakpoint at Xq25 (H odgson et al., 1986) and an X;20 translocation breakpoint
at Xq26.1 (M ueller et al., 1991). DXS42 and DXS10 have been shown to flank the
Xq25 breakpoint by analysis o f som atic cell hybrids g enerated from this t(X;3)
p atient (Reilly, Lewis, an d Nussbaum , 1990). No recom bination was found
between OCRL and the proxim al m arker DXS42 in two studies with a com bined
lod score o f 11.8 (Reilly, Lewis, a n d Nussbaum , 1990; Wadelius, Fagerholm ,
Pettersson, an d A nneren, 1989).

Positional Cloning

Nelson et al. (1991) isolated yeast artificial chrom osom es whose hum an inserts
spanned the breakpoint o f a fem ale patient with an X;3 translocation. Using
these yeast artificial chrom osom es as probes, a candidate cDNA, OCR1.-1, was iso
lated which is in te rru p te d by both the t(X;3) an d t(X;20) breakpoints (O kabe et
al., 1992; A ttree et al., 1992). G enetic evidence th at OCRl^-1 is the gene for OCRL
relies on the discovery of m utations in the gene in patients with the disease. T he
transcript o f this gene is absent in both fem ale OCRL patients with X;autosom e
translocations an d is absent or ab e rra n t in size in some u n related m ale patients
with no detectable re arra n g em en t (Attree et al., 1992). D irect sequencing of
reverse transcribed mRNA from m ale patients with detectable message has
revealed p oint m utations leading to prem ature term ination or missense m uta
tions as well as an exon skipping m utation an d a three base deletion of a single
am ino acid (Leahey, C harnas, an d Nussbaum , 1993).
T he function of OCFLL-1 is unknow n. O ne clue, however, is th at the protein p re
dicted to be encoded by OCR!. I has 71% similarity to hum an platelet inositol poly
408 ICHIRO OKABE AND ROBERT L. NUSSBAUM

phosphate-5-phosphatase (Ross, Jefferson, Mitchell, a n d Majerus, 1991). These

data suggest th at OCRL may be an in b o rn e rro r of inositol phosphate m etabolism
(Irvine, 1992). T he role o f inositol phosphate is well established as an intracellular
m essenger for calcium signalling and protein kinase C activation which in tu rn
regulate cellular processes including m etabolism , secretion, contraction, neural
activity, and cell proliferation (B erridge, 1993). A bnorm alities in inositol m etabo
lism have been proposed to have a role in the pathogenesis o f cataracts in galac
tosem ia (C am m arata, Tse, a n d Yorio, 1990), an d diabetic com plications such as
cataracts, p eripheral neuropathy, and nep h ro p ath y (G reene, Lattim er, an d Sima,
1987). T he dim inution o f sulfated proteoglycan concentration is frequently ob
served in hum an and anim al diabetic states (Epstein, 1989). Thus abnorm alities
o f GAGs in OCRL may be a secondary d eran g em en t caused by im paired inositol
m etabolism . However, it should be stressed th at the involvem ent o f inositol phos
phate m etabolism in the pathogenesis o f OCRL rem ains an unproven hypothesis
until the tru e function of OCRf.-1 is d eterm in ed and a definitive biochem ical d e
fect in inositol m etabolism is dem onstrated in OCRL.

THERAPY

C u rre n t therapies for OCRL are mainly sym ptom atic treatm ent, such as extrac
tion o f cataracts, m anagem ent of glaucom a, bicarbonate replacem ent for renal
tubular acidosis, anticonvulsant drugs, o rth o p ed ic care for bone fracture, physi
cal rehabilitation, a n d special education. P hosphate a n d vitamin D supplem enta
tion may also be n eed ed to control rickets. Identification o f the gene for OCRL
facilitates the u n d erstanding of m olecular pathogenesis, and will contribute to
the developm ent of new strategies for treatm en t as well as accurate carrier detec
tion an d prenatal diagnosis.

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American Medical Association, 196, 96104.
 18. CRYSTALLIN GENES AND CATARACT

MIREILLE CARTIER1, IAP-CHEE TSUI1'2, SARAH P. BALL3

and NICOLETTE H. LUBSEN4

1Department of Genetics, Research Institute, The Hospital for Sick Children, 555 University
Avenue, Toronto, Ontario M5G 1X8, Canada
2Department of Molecular and Medical Genetics, University of Toronto, Toronto,
Ontario M5S 1A8, Canada
3Department of Biologica l Sciences, Washington Singer Laboratories, University of Exeter,
Exeter EX4 4QG, United Kingdom,
4Department of Molecular Biology, University of Nijmegen, Toernooiveld,
6525 ED Nijmegen, Netherlands

ABSTRACT

Crystallins constitute over 90% o f the water-soluble proteins in the ocular lens.
They are subdivided into d ifferent classes according to their m olecular proper
ties. Early biophysical studies indicated that crystallins are im p o rtan t for confer
ring, as well as m aintaining the transparency o f the lens. M ore recent inves
tigations have focused on the isolation and characterization of the genes encod
ing these proteins; these studies have resulted in the identification of m utations
associated with a n u m b er o f cataracts in m ammals, thus strengthening the
involvem ent o f the crystallins in lens transparency. Moreover, the phenotype o f at
least one o f these m utants suggests th at crystallins are also im p o rtan t for p ro p er
lens developm ent.

INTRODUCTION

Crystallins are the m ajor soluble proteins in the eye lenses o f vertebrate animals.
Most of the crystallins are contained in the fibre cells which occupy the bulk of
this m ulticellular organ (] gure 1; for a review o f em bryology and m orphology of
the lens, see Zigman, 1985). T he u n iq u e spatial arran g em en t and short range
ord er of these m olecules are tho u g h t to be im p o rtan t for the m aintenance of the
rem arkable transparency an d refractive properties o f the lens (Delaye and Tar-
dieu, 1983).
T he functional im portance of crystallins is also reflected by their evolutionary
conservation and their role in disease conditions. Loss o f lenticular transparency,
com m only known as cataract, can be due to a variety o f reasons, such as aging,
traum a, o r m etabolic im balance, but at least two congenital form s have already
been shown to be due to m utations in crystallin genes (see below). Since cataracts
414 MIREILLE CARTIER ET AL.

zone of
primary fibre cell
anterior lens fibres differentiation
lens vesicle epithelium

secondary
lens fibres

Figure 1 L en tic u lar d e v e lo p m e n t a n d grow th. D ev e lo p m e n t o f th e lens is in itia te d fro m a

len s vesicle w h ere th e cells from th e a n te rio r layer will fo rm th e c u b o id al e p ith elial cells a n d
th e cells fro m th e p o ste rio r re g io n will e lo n g a te to fo rm th e p rim a ry lens fibres. G row th of
th e len s will c o n tin u e th ro u g h o u t life, only fro m th e e q u a to ria l ep ith elial cells w hich will
slowly m ig rate tow ards th e p o ste rio r a re a o f th e len s a n d d iffe re n tia te in to fibre cells. T h e se
new fib re cells, called th e seco n d a ry fibre cells, will th e n p u sh th e p rim a ry fib re cells to th e
c e n te r o f th e lens, w h ere they will fo rm th e n u c leu s o f th e lens.

range from slight, dust-like opacities which do no t disturb the vision to com plete
opacification o f the lens, som etim es accom panied by loss o f the norm al anatom y
o f the organ, a careful analysis o f these in h erited cataracts should help in u n d er
standing the various roles o f crystallins in cataractogenesis.
D ifferent classes of crystallins have been described for a n u m b er o f vertebrates
and, m ore recently, for some invertebrate species. Most of the initial knowledge
ab o u t crystallins was derived from the calf, simply because of their accessibility.
T h re e m ajor classes of crystallins, a , p an d y, were originally described. They
are distinguished on the basis of their chrom atographic properties, with the high
m olecular weight fraction being the a-crystallins, the interm ediate m olecular
w eight fraction, the f$-crystallins and the low m olecular weight fraction, the y-crys-
tallins. A lthough the proteins contained in each of the th ree fractions are ra th e r
h eterogeneous, they are antigenically closely related. As we now know, the com
plexity of the crystallin proteins is not only due to the variety of genes that encode
these polypeptides, bu t is also caused by post-translational m odifications o f the
p roteins themselves (for a review of the crystallins, see H arding and Dilley, 1976;
B loem endal, 1981, 1985; Piatigorsky, 1984; Lindley et al., 1985; Wistow and
Piatigorsky, 1988; B loem endal and de Jong, 1991).
While a-, P- and y-crystallins can be found in alm ost all vertebrate lenses,
u nique "taxon-specific" crystallins have been identified for many species (for re
view, see de Jo n g et al., 1989; Piatigorsky an d Wistow, 1989, 1991). For exam ple,
lenses from birds and some reptiles contain a d ifferent class o f proteins called the
 CRYSTALLIN GENES AND CATARACT 415

5-crystallins. In hum ans, there is no evidence for crystallins o th er than the a-, (3-
and "/-cr ystallius.
T he various crystallin species are n o t uniform ly distributed within the lens; a
specific concentration grad ien t for each protein is established through tem poral
and spatial regulation o f gene expression as well as post-translational modifica
tions. T here is, however, a general increase o f p rotein concentration towards the
cen ter (nucleus) o f the lens, prim arily due to the increasing concentrations o f |3-
an d y-crystallins. As a consequence, the refractive index increases towards the core
o f the lens, com pensating for the changing curvature of the lens (Fernald and
W right, 1983). In addition, since the term inally differentiated fibre cells are never
lost from the lens, but ju st m igrate towards the lens center while gradually losing
their nucleus an d subcellular organelles, older proteins are located at the nucleus
o f the lens and newly synthesized ones are located in the o uter cortex region. T he
o lder crystallins in the lens nucleus cannot, therefore, be replaced a n d m ust last
the lifetim e of the organism . H ence, crystallins are very stable proteins, able to re
sist d en atu ratio n by oxidation, UV radiation (sunlight) as well as racem ization, all
processes th at occur with age.
In the following sections, we provide a discussion on each of the three m ajor
classes of crystallins found in the hum an lenses and o u r c u rre n t knowledge on the
involvem ent of these proteins in cataractogenesis.

GENERAL PROPERTIES OF THE CRYSTALLINS AND THEIR GENE

FAMILIES

a-Crystallins

T he a-crystallins are polypeptides o f about 20 kDa found as large aggregates with

a m olecular weight aro u n d 800 kDa. T he a-crystallins are encoded by two genes,
the aA and the aB gene, which, respectively, specify proteins with an acidic (aA2)
and basic (aB2) isoelectric point. These two prim ary gene products share about
60% am ino acid identity. Both polypeptide chains can be phosphorylated, giving
rise to two o th er species, a A l an d a B l, respectively (Spector et al., 1985). Fur
th er m odifications o f these proteins may also occur th ro u g h deam idation
(Voorter et al., 1987), N -term inal acetylation (H oenders an d B loem endal, 1967),
O -linked N-acetylglucosam ine (R oquem ore et al., 1992), C-term inal degradation
(Stauffer et al., 1974; van Kleef, Nijzink-Maas and H oenders, 1974), and racem
ization (G roenen et al., 1990), leading to a variety o f a-crystallin aggregates in
the aging lens.
In spite o f many years o f study, the tertiary structure of a-crystallin rem ains u n
known. O n the basis o f in tern al sequence similarities, as well as the e x o n /in tro n
distribution of the gene, it has b een suggested that a-crystallin is a two dom ain
p rotein (Wistow, 1985). T he n atu re o f the a-crystallin aggregates found in the lens
is also unclear, with som e suggesting th at it is an o rd ered layered oligom er
416 MIRE1LLE CARTIER ET AL.

(Tardieu et al., 1986), while others propose that the protein has a m icellar struc
ture (Walsh, Sen and C hakrabarti, 1991; Rad lick and Koretz, 1992).
T h e a-crystallins may also have o th er functions than that o f being an a b u n d a n t
structural p rotein o f the lens. They show sequence hom ology to the small heat
shock proteins (Ingolia and Craig, 1982). In fact, aB-crystallin has been found in
o th er tissues than the lens an d its synthesis can be induced by stress (such as heat
shock and incubation in Cd+2) in cultured fibroblasts (Bhat and N agineni, 1989;
Dubin, Wawrousek and Piatigorsky, 1989; Klemenz et al.. 1991). This protein also
accum ulates in the brains o f hum ans suff ering from A lexander's disease (a degen
erative neurological disorder characterized by the presence of R osenthal fibres or
g ran u lar inclusions within astrocytes) an d in the brains o f scrapie-infected ham
sters (Duguid, Rohwer and Seed, 1988; Iwaki et al., 1989). M ore recently, a-crys
tallins have been found to have chaperone activity (Horwitz, 1992), raising the
possibility that they may stabilize o th er crystallins in the lens.

Genomic organization

As best dem onstrated in the ham ster, the two a-crystallin genes have the same
genom ic structure; both have three exons an d the introns are located at equiva
len t positions in the coding sequence, suggesting a close evolutionary relation
ship between these two genes (Quax-Jeuken et al., 1985b; van d en Heuvel et al.,
1985). In hum ans, a partial sequence has been obtained for the aA gene (McDe-
vitt et al., 1986) and a com plete sequence for aB (D ubin et al., 1990). T heir
stru ctu re is similar to th at o f the ham ster genes. In addition, the hum an A-crystal-
lin gene is localized to chrom osom e 21 region q22.3 (Table 1 and Figure 3; Quax-
Jeu k e n et al., 1985c; Hawkins et al., 1987) and aB-crystallin to chrom osom e 11,
betw een bands q22.3 and q23.3 (Table 1 and Figure 3; B rakenhoff et al., 1990b).

Expression pattern

D uring developm ent of the lens, at least in rodents, a-crystallins are the first crys
tallins to appear. They are p resen t in the lens vesicle before form ation o f the pri
m ary lens fibre cells (McAvoy, 1978; van Leen e ta l., 1987a, b). T he synthesis o f a-
crystallins com m ences mainly in the epithelial cell layer and early stages o f fibre
cell elongation. In the rat, the levels o f both aA- and aB-crystallin mRNAs are rel
atively constant at all ages o f growth and developm ent (Aarts, Lubsen and
Schoenm akers, 1989; V oorter et al., 1990), suggesting an even distribution o f a-
crystallins in all cells. Relatively little is known about the developm ental xegula-
tion o f a-crystallin gene expression in hum ans, except that aA-crystallin mRNA
can be detected in the fetal lens (B rakenhoff et al., 1990a).
 CRYSTALLIN GENES AND CATARACT 417

T ab le 1 L o ca tio n o f th e h u m a n crystallin g en es

Crystallin Gene Chromosomal References

gene symbol '* location

aA CRYA1 21q22.3 Quax-Jeuken et al.. 1985c

Hawkins et al., 1987

aB CRYA2 1lq22.3-23.1 Ngo etal., 1989

Brakenhoff et al., 1990b

pAl /A3 CRYBla 1 7qll.2-ql2 Law et al., 1986

Sparkes et al., 1986

PA2 unassigned unm apped

PA4 CRYBA4a 22q l 1.2-13.1 van Rens, Geurts van Kessel

and Bloemendal, 1991

PBl unassigned probably 22 N.H.L., unpubl.

pB2 CRYB2A3 22q l l . 2- q l2 Sparkes et al., 1987

Hogg et al., 1987
Hulsebos et al., 1991

PB3 CRYB3a 22q l 1,2-ql 2 Hulsebos et al., 1991

yA-F CRYG1-6 2q33-q35 Tsui et al., 1985

Willard et al., 1985
den D unnen et al., 1986
Shiloh et al., 1986

ys CRYG8 3 van Rens et al,, 1989

a The symbols for the p-crystallins may be changed in the near future to better reflect the name of the
protein: PA1/A3 would be CRYBA3/A1, PB2 would be CRYBB2 (and the pB2 pseudogenc: CRYBB2P1)
and pB3 would be CRYBB3.

[3-Crystallins

T he [3-crystallins ap p ear in the lens as heterogeneous aggregates ranging from

dim ers (PL) to octam ers (Ph )- T he calf lens contains seven prim ary P-crystallin
translation products which can be divided into th ree basic proteins, known as
PBl, pB2 and PB3, and four acidic chains, PA1, PA2, PA3 and PA4 (Berbers et al.,
1982). T he same n u m b er of species probably exist in the hum an lens on the basis
o f a sim ilar two-dim ensional gel separation p attern o f em bryonic hum an lens
418 MIREILLE CARTIER ET AL.

proteins, b u t an unequivocal assignm ent cannot be m ade (W.W. de Jong, per

sonal com m unication).
P art o f the difficulty in the identification of the different p-crystallin species is
d ue to post-translational m odifications o f these proteins. A portion of the N-termi-
nal arm from both (3B1 an d pB2 can be rem oved by proteolytic cleavage (Berbers
et al., 1983; McFall-Ngai et al., 1986; Takem oto et al., 1987). T he glutam ine resi
dues in the N -term inal arm s o f (3A3, PB2 and pB3 are substrates for transam ina
tion, in which the N -term inal arm of PBl (or the C-term inus of aB-crystallin)
serves to donate the e-amino groups from its lysine residues (Lorand, Parameswa-
ran and Velasco, 1991; G roenen, B loem endal and d e jo n g , 1992). In some anim al
species, phosphorylated PB2 can be found (Kleinian et al., 1988). Further, oxida
tion, cross-linking and deam idation o f P-crystallins occur with age, contributing to
a com plex elution profile consisting o f eight (Zigler, Horwitz and Kinoshita, 1980)
o r m ore (Thom son an d Augusteyn, 1985) com ponents in isoelectric focusing, i.e.
gel electrophoresis.

Protein structure

T h e stru ctu re o f the calf pB2 dim er has been solved by X-ray crystallography (Bax
et al., 1990). T he core of each m olecule contains four antiparallel P-sheeted (P~
b arrel) structures known as "Greek key motifs, organized into two similar
dom ains o f two motifs each (Figure 2). T he dom ains are flanked by a N- and a C-
term inal arm . T he two dom ains are separated by a connecting peptide in an
ex tended conform ation, such that the N -term inal dom ain o f one pB2 chain inter
acts with the C-term inal dom ain of the second PB2 chain and vice versa. All
known p-crystallin sequences can be m odeled in the same tertiary conform ation
and it is generally assum ed that all p-crystallins fold into the same four m otif/tw o
dom ain structure. The m ost distinct feature for individual P-crystallin m olecules
is located at their N- an d C-term inal arms. In addition to sequence variations, the
N -term inal arm s differ significantly in length (from about 55 am ino acid residues
in pBl to 15 am ino acid residues in PA1). T he C-term inal arms, varying between
13 an d 18 am ino acid residues in length, are only found in the basic P-crystallins
(for review, see Lubsen, Aarts an d Schoenm akers, 1988).

Genomic organization

T h e com plete set o f calf P-crystallin sequences has been derived from cDNA
clones (G orin and Horwitz, 1984; Q uax-Jeuken et al., 1984; Hogg et al., 1987; van
Rens et al., 1991a). PA1 an d pA3 are encoded by the same mRNA, using the same
reading fram e, bu t with d ifferent initiation codons, resulting in a pAl polypep
tide sh o rter than pA3 by 17 am ino acid residues (Quax-Jeuken et al., 1984). This
double initiation o f the p A l/A 3 mRNA has been preserved in evolution and is
fo u n d no t only in calf, but also in chicken and in m an (Hogg et al., 1986; Peter
son an d Piatigorsky, 1986). Further, on the basis of cDNA hybridization patterns,
 CRYSTALLIN GENES AND CATARACT 419

Figure 2 (A) F ig u re 2 (B)

F ig u re 2 In te ra c tio n s b etw een th e d iffe re n t d o m a in s o f 7- a n d p-crystallins. (A) d ep icts th e

in tra m o le c u la r in te ra c tio n s o f d o m ain s II a n d IV o c c u rrin g in a y-crystallin m o lecu le, w hile
(B) illustrates th e sam e d o m ain s, fro m two p-crystallin m olecules, in te ra c tin g in a n in te rm o -
le c u la r fash io n . T h ese re p re se n ta tio n s are free in te rp re ta tio n s o f th e d a ta p re s e n te d by B lu n
d ell e t al., 1981 a n d Bax e t al., 1990.

orthologous m em bers o f the calf p-crystallin gene family probably exist in hum an
(van Rens e ta l., 1991b).
T he structure o f the p-crystallin genes has been derived from representative
m em bers in hum an, rat, a n d m ouse (Inana et al., 1983; den D u nnen et al., 1985b;
Hogg et al., 1986). A lthough the paralogous P-crystallin genes no longer share suf
ficient sequence identity to recognize each o th er in hybridization experim ents,
the genes are clearly evolutionarily related. A typical P-crystallin gene contains six
exons. T he first exon o f the pR l gene is non-coding, whereas the first exon o f the
PA3/A1 gene contains the start codon for the PA3 protein. T he rem ainder o f the
gene structure is rem arkably sim ilar am ong all P-crystallin genes, with the second
exon coding for the N -term inal arm an d each o f the 4 following exons (exons 3 to
6) encoding a G reek key motif. In addition, for the basic chains, exon 6 also en
codes the extension o f the C -term inal arm .
Because o f the similarity in gene structure, it is generally believed th at all P-crys-
tallin genes were derived from a single ancestor sequence. D uplication of this an
cestral gene an d subsequent divergence would have led to the proto-acidic and
proto-basic P-crystallin genes which would then have been duplicated fu rth e r to
form the 6-m em ber gene family seen today. In m an, all P-crystallin genes except
the pA 3/A l and possibly the PA2 are located on chrom osom e 22 (Table 1 an d Fig
ure 3; Law et al., 1986; Sparkes e t al., 1986; H ogg et al., 1987; H ulsebos et al.,
1991). T hree o f the genes, pB2-l, pB2-2 and pB3, have been m apped within a dis
tance o f 120 kb (Bijlsma et al., 1991), but the pB2-2 sequence probably co rre
sponds to a pseudogene (B rakenhoff et al., 1992). It is o f interest to note that only
a single copy o f the PB2 gene can be fo u n d in the rat, although th at gene is also
closely linked to pB3 (Aarts et al., 1987). Moreover, chrom osom e duplication has
420 MIREILLE CARTIER ET AL.

Figure 3 Ideogram o f the hum an chrom osom es showing the Giemsa banding patterns. T he
locations of the m ajor genes expressed in the lens and loci responsible for dom inantly inher
ited congenital cataracts are indicated. S tandard gene nom enclature according to H um an
G ene M apping 11 (McAlpine et al., 1991) is used. T he gene symbols used are as listed in Ta
bles 1 and 2, except for CRYB2 which, here, represents the whole cluster of (i-crystallin genes
m apped to chrom osom e 22: (3A2, [3B2, pB3 an d (3B4 an d MIP, which is the symbol for the ma
jo r intrinsic protein o f the lens fibre m em brane.
 CRYSTALLIN GENES AND CATARACT 421

b een proposed as a m echanism for P-crystallin gene duplication; the close prox
imity o f the PA1/A3 gene to the von R ecklinghausen neurofibrom atosis locus
(NF1) on chrom osom e 17 (Fain et al., 1989) an d the co-existence o f the pR2 and
PB3 genes and neurofibrom atosis 2 (NF2) on chrom osom e 22 (H ulsebos et al.,
1991) are tho u g h t to be m ore than coincidental.

Developmental regulation of gene expression

T he developm ental regulation o f the P-crystallin genes has been well docu
m ented for the chicken (Ostrer, Beebe an d Piatigorsky, 1981; H ejtm ancik et al.,
1985) and rat (Aarts, Lubsen an d Schoenm akers, 1989). T he latter study shows
th at the pBl and pB3 genes are both active d u rin g early lens form ation an d that
their respective transcripts reach th eir m axim um levels aro u n d birth. In contrast,
the PB2 mRNA only accum ulates after birth b u t rem ains p ro m in en t at least up to
one year o f age. T he PA3/A1 gene shows an interm ediate p attern o f expression,
as its transcript is m ost ab u n d a n t betw een one an d three m onths after birth. Rel
atively little is known ab o u t the developm ental profile for the hum an p-crystallin
genes. T he p attern may be sim ilar to th at o f the rat, however, because hum an PB3
is also expressed early and PB2 is p resen t in later stages (B rakenhoff et al., 1992).
Based on the mRNA profile, it may be assum ed that there is a differential gradi
en t of p-crystallin species across the lens. For exam ple, the nucleus is expected to
contain relatively high levels of PBl and PB3 whereas the cortex would include
prim arily PB2. C onsistent with this assum ption and the fact that the form ation of
PH requires the presence o f pB l, th ere is a clear shift of pH in the rat lens nucleus
to p in the cortex (Siezen, Anello an d Thom son, 1986).

y-Crystallins

T he y-crystallins are found in the low m olecular weight fraction o f the soluble
proteins from the lens. T he 6-7 m onom eric proteins o f about 20 kDa included in
this family can be divided into two branches. T he older branch com prises a single
protein called ys, which is p resen t in all vertebrate species exam ined thus far.
This protein was once tho u g h t to be a m onom eric P-crystallin (thus previously
nam ed Ps), mainly because its N -term inal residue is acetylated, as is the case for
the P-crystallins, but n o t for the o th er y-crystallins. C loning and sequencing stud
ies, however, revealed th at the coding sequence and e x o n /in tro n organization of
the ys gene are m ore related to those o f the y genes than those o f the P genes
(Quax-Jeuken et al., 1985a; van Rens et al., 1989). T he o th er branch o f the y-crys-
tallin gene family contains u p to six closely related proteins (whose genes are
clustered to g e th e r), which are known as yA to yF. In contrast to the a - , P- an d y s-
crystallins, these yA to yF-crystallins are n o t present in all vertebrate species;
while these low m olecular weight proteins can be found at varying levels in fish,
frogs and m am m als, they are absent in birds an d som e reptiles.
422 MIREILLE CARTIER ET AL.

Protein structure

T he y-crystallins also contain fo u r highly symmetrical G reek key motifs. T he over

all structure is sim ilar to th at of the P-crystallin, as m ight be expected from their
hom ology at the prim ary sequence level. In fact, the three-dim ensional structure
of the bovine y-crystallin was the first crystallin structure solved by X-ray crystal
lography (B lundell et al., 1981). T he m ajor difference between the |3- an d y-crys
tallin structures is th at the P-crystallin dom ain interactions are interm olecular,
while in the y-crystallins the dom ain interactions are intram olecular (Figure 2).
As a consequence, y-crystallins are m onom ers, while the p-crystallins dim erize. A
fu rth e r difference between P- an d y-crystallins is the lack o f N- an d C-term inal
arm s in y-crystallins. T he high in tern al sym m etry a n d com pact shape o f the y-
crystallins, together with the placem ent o f surface ion pairs, contribute to the sta
bility o f these m olecules, which can w ithstand dehydrated environm ents. The
y-crystallins are, therefore, b etter suited for the less hydrated, hard lenses of
ro dents than the softer, m ore hydrated lenses o f hum ans and birds.

Genomic organization

T he y-crystallin genes are highly conserved in structure an d sequence; each con

tains 3 exons, with the first exon encoding 3 am ino acid residues an d the two
rem aining exons each specifying a dom ain o f two G reek key motifs (M oorm ann
et al., 1983; Lok et al., 1984). At least six y-crystallin genes have been described
for each m am m alian species exam ined. In ra t and m ouse, five genes (yA, yB, yC,
yD an d yE) are tandem ly linked within a 50 kb DNA segm ent, while the sixth
gene (yF) is located at least 25 kb upstream (M oorm ann et al., 1985; Q uinlan et
al., 1987; den D unnen et al., 1989). T he various gene m em bers in this family
share betw een 75 and 90% sequence identity within the coding region, resulting
in easy detection of all genes by cross-hybridization.
T he hum an genom e also contains six highly related genes (yA-yF), clustered on
the long arm o f chrom osom e 2, region q33-q35 (Table 1 and Figure 3; d en D un
n en et al. 1985a; Meakin, B reitm an an d Tsui, 1985). T he less related ys gene is lo
calized on chrom osom e 3 (Table 1 an d Figure 3). T he relative positions o f the yB,
yC, y D an d yE genes have been established on a 40 kb DNA segm ent b u t the exact
locations for yA and yF in the gene cluster have yet to be determ ined. It is o f in
terest to note th at the hum an yE an d yF genes bo th contain an identical in-frame
stop codon at the same position within the second exon (Meakin, B reitm an and
Tsui, 1985). In addition, the yF gene lacks an upstream TATA box which is
th o u g h t to be im p o rtan t for transcription; the gene also has num erous o th e r m u
tations dow nstream from the prem ature stop codon. A lthough these two genes are
considered as pseudogenes, a low level o f y E transcript has been d etected (see be
low) . A segm ent o f the y F gene is also fo u n d to be duplicated within the gene clus
ter; this sequence (yG) is non-functional (B rakenhoff et al., 1990a).
 CRYSTALLIN GENES AND CATARACT 423

Evolution of the Y-crystallin genes

T he y s gene is p resen t in virtually all vertebrate species exam ined. It is probably a

direct descendant o f a gene re cru ited by the lens in early vertebrate evolution
(C hang an d C hang, 1987; van Rens, de Jo n g an d B loem endal, 1991). T he o th er y-
crystallin genes are present in m am m als, fishes an d frogs, b u t absent in birds and
h ig h er reptiles. Sequence analysis shows, however, that the m am m alian y-crystal-
lin genes are m ore related to each o th er than to any o f the genes in fishes and
frogs, suggesting th at the y-crystallin g en e family evolved separately in the differ
en t evolutionary lineages. T he yA-yF genes are thus specific to mammals.
As the burst o f gene duplications th at produced the m am m alian yA-yF-crystal-
lin genes occurred recently, these genes are still closely related and clustered to
gether. From sequence com parisons, it has fu rth e r been speculated th at the six
y-crystallin genes (yA-yF) were already p resen t in the com m on ancestor of m an
and rodents, b u t that, in the prim ate lineage, fu rth e r reshuffling o f this gene fam
ily has taken place. H ence, the hum an yF gene could be a re cen t duplicate o f the
yE gene ra th e r than being the hom olog (ortholog) o f the ro d e n t yF gene, which
itself becam e partially deleted to form yG in the hum an genom e (B rakenhoff et
al., 1990a). It is possible th at gene conversion also plays a role in the evolution of
these y-crystallin sequences (den D unnen, Lubsen an d Schoenm akers, 1986; Aarts
et al., 1988).
Loss of functional gene sequences as well as decreased levels o f expression (see
below) have occu rred after the divergence o f prim ates from rodents. These m od
ifications may be related to the fact th at d u rin g hom inoid evolution a softer lens
with accom m odative capacity has apparently been favoured, an d a soft lens is
probably incom patible with a high level of y-crystallins (the same argum ent can be
p u t forw ard to explain the lack of y-crystallins in the soft and flat bird lenses). Loss
o f expression of y-crystallins would, thus, have been an acceptable m utation in the
prim ate lineage.
Based on the structures o f (3- and y-crystallins (both have four sim ilar motifs or
ganized into two dom ains) it may be argued th at these two gene families are evo-
lutionarily related. Close exam ination of the gene structure an d DNA sequences
indicates th at the two families are probably related only th ro u g h a com m on ances
tral sequence encoding a single "Greek key" motif. It has been proposed th at sep
aration o f the P- an d y-crystallin families occu rred after the form ation o f a dom ain
structure o f the protein and before the form ation of the two-domain structure
found today (for review, see Lubsen, Aarts an d Schoenm akers, 1988). In this as
sum ption, the two motifs in the ancestral dom ain sequence were separated by an
intron which was retained in the p-crystallin genes, b u t lost before duplication of
the dom ain to form the y-crystallin gene. It is also possible, however, th at the p~
and y-crystallins actually evolved in d ependently from the single ancestral m otif se
quence.
424 MIREILLE CARTIER ET AL.

Regulation of gene expression

All y-crystallins are specific to the lens; they are located prim arily in the nucleus
region, presum ably to provide the dehydrated environm ent required. In rodents,
the precise spatial an d tem poral regulation o f individual y-crystallin genes has
been well d o cum ented and, as expected, m ost o f the genes are expressed at early
stages o f developm ent (M urer-O rlando et al., 1987; van Leen et al., 1987ab;
Goring, B reitm an and Tsui, 1992). T ranscription of the yA to yF genes is
restricted to the term inally differentiated fibre cells and can be readily detected
in day 12 em bryonic lenses w hen the prim ary fibre cells begin to form . T he
steady state level o f mRNA for m ost y-crystallin mRNAs increases gradually and
reaches a peak at aro u n d day 10 after birth. In addition, the yD, yE and yF are
relatively m ore ab u n d a n t than the o th er species, whereas the yB gene rem ains
active long after the o th ers have been shut down. A sim ilar expression p attern is
fo und in the calf, w here yB is the p ro m in en t cortical y-crystallin (Slingsby and
Croft, 1973; Slingsby an d Miller, 1983).
T h ere are th ree to four times less y-crystallins in the hum an lens than in the ro
d e n t lens (Siezen et al., 1988; T hom son an d Augusteyn, 1985). In addition, al
though the hum an yA, yB, yC and y D genes are transcriptionally active, only the
latter two yield ab u ndant mRNA and p rotein (Russell et al., 1987; Siezen et al.,
1987; B rakenhoff et al., 1990a). T he yC an d yD genes are relatively active in the
fetal lenses bu t are differentially shut-off after birth. At 22 m onths, only the yD
mRNA can be detected an d it rem ains present till at least 10 years o f age (B raken
h o ff et al., 1990a). T he yE gene is transcribed at a low level; nevertheless, a small
am ount o f its predicted (truncated) protein product has been detected in the h u
m an lens (N.H.L., u n p u b l.). T he yF gene lacks a TATA box and is probably silent
(M eakin, B reitm an an d Tsui, 1985).
T he exceptional position o f the ys gene in the y-crystallin gene family is also re
flected by its expression pattern: in the ra t lens, the ys gene is active only after
b irth (Aarts, Lubsen and Schoenm akers, 1989). However, although the develop
m ental expression o f ys has not been followed in m an, it is known that the tran
script o f this gene is p resen t in a 9 week old hum an fetal lens (N.H.L., unpubl.).

CATARACTS

Since lens opacity can occur for many reasons, it is impossible to define a com
m on pathway o f lens disorganization leading to the various form s o f cataracts.
Nevertheless, num erous studies have described the m orphological differences
betw een the norm al an d cataractous lens (see review by B erm an, 1991). T he gen
eral features of a cataractous lens, aside from the opacity, may include the pres
ence o f vesicles, lens fibre swelling, gaps betw een the fibre cells which may be
filled with liquid o r debris, and loss of m em brane integrity.
 CRYSTALLIN GENES AND CATARACT 425

At the biochem ical level, cataractous lenses generally display high m olecular
weight proteinaceous aggregates as well as some proteolysis products, unbalanced
concentrations o f ions such as calcium , sodium and potassium and loss o f glu
tathione an d soluble crystallins. T he high m olecular weight protein aggregates are
th ought to be caused by the unfolding o f the various crystallins, leading to the ex
position of hydrophobic groups which may then interact together.
Some o f the high m olecular weight protein aggregates p resen t in the hum an
cataractous lens have been isolated an d fractionated, to reveal peptides of m olec
ular weights aro u n d 43 kDa, 20 kDa an d 10 kDa (Spector et al., 1979; Garner,
G arner and Spector, 1979). While the 43 kDa p rotein seems to be an extrinsic
m em brane protein, the 20 kDa is th o u g h t to be a crystallin and the heterogeneous
10 kDa fraction is com posed o f y-crystallin (as d eterm in ed by im m unological re
action) (Takem oto, Straatsm a and Horwitz, 1989). It has been postulated that
such 10 kDa fragm ents may occur from cleavage at the connecting peptide be
tween the two dom ains o f the p rotein. Indeed, this 10 kDa fragm ent, which also
accum ulates with age, has been identified as the C-term inal dom ain o f yD (Srivas-
tava, Srivastava an d Silney, 1992). F urtherm ore, in a study o f 13 cataractous lenses,
10 were fo u n d to contain y-crystallin proteins in close association with the m em
b rane (all ten cataracts presenting nuclear sclerosis), while 4 displayed (3-c,rystal-
lins associated with the m em brane (two o f which had no nuclear involvem ent) and
n o n e showed a-crystallin association with the m em brane (Kodam a and Takem oto,
1988). From this study, it may be concluded that y-crystallins can play a role in the
cataractogenic process, especially in cases of nuclear cataracts. Such a role may be
attrib u ted to th eir high concentration of thiol groups on the surface of the m ole
cule, which may be oxidized, leading to a destabilization o f the protein with con
com itant unfolding, a n d /o r participate in interm olecular disulfide bonds with the
m em brane.
All o f these changes, which have b een studied mainly in senile cataracts, point
to sites for potential m utations in in h erited cataracts, such as the crystallin genes
and the m em brane proteins including the pum ps and channels responsible for
the osm otic balance. F urtherm ore, o th e r genes known to play a role in lens devel
op m en t may be im plicated in hered itary congenital cataracts.

Human Inherited Cataracts

In h erited cataract is an im p o rtan t cause of visual handicap in childhood account

ing for 20% of blindness in children u n d e r 15 years, in the U nited Kingdom. The
majority of these conditions are genetically determ ined. A lthough some of them
are associated with o th e r ocular abnorm alities, or as part o f a systemic syndrom e,
most occur as isolated abnormality. T h ere are m any different phenotypic form s
o f in h erited cataracts an d m orphological variation may be observed even within
families, reflecting the heterogeneity o f the underlying genetic defects an d the
com plexity o f lens developm ent (H arding and Dilley, 1976). Autosom al dom i
426 MIREILLE CARTIER ET Al..

n a n t transm ission is the m ost com m on m ode o f inheritance, but autosom al reces
sive an d X-linked form s are also known to occur.

Morphologies

In h e rite d congenital cataracts frequently exhibit a m orphology described as pul

verulent, ie. an accum ulation o f dustlike spots. In pulverulent cataracts, the opac
ities occur in the interm ediate layer o f the m ature lens, in the secondary fibre
cells. T he in n er nuclear region may be clear in zonular pulverulent cataracts or
op aque in nuclear pulverulent cataracts, while the o u ter cortex of the lens is gen
erally clear. As a result, many nam es ap p ear in the literature, including zonular,
lam ellar o r perinuclear cataract, central o r zonular pulverulent cataract, diffuse
o r total nuclear cataract. In addition, various m odes o f inheritance have been
attrib u ted to these cataracts, suggesting th at they may result from d ifferent
genetic defects.
O th e r com m on form s o f in h erited congenital cataracts may have an an terio r
p olar location in the lens. T he opacity is often dense an d conical in shape and lo
cated eith er overlying the an terio r epithelial layer o f an otherw ise n orm al lens or
within the epithelial cell layer of the cortex an d may be associated with nuclear
opacities. P osterior polar locations are rare.
H om ogeneously opaque lenses, o r total diffuse congenital cataracts, usually rep
resent the e n d p o in t in the progressive process o f opacification, w hen it is too late
to d eterm in e the p attern o f cataract m orphology' in the im m ature state. These cat
aracts, therefore, probably include m any d ifferent genetic forms. In anim als, it has
b een shown th at the size o f the lens d u rin g em bryonic developm ent has a signifi
cant effect in controlling the overall size o f the eye (Zwaan, 1979). Since many
types o f congenital cataract result in re ta rd ed growth o f the lens, m icrophthalm ia
(reduced size of the eye) is inevitable.

Genetic factors

Lens opacification may result directly from a b e rra n t functions expressed in the
lens itself, such as alteration o f structural proteins (e.g. crystallins) o r proteins
th at serve to pro tect the lens from dam age and to preserve clarity o f the lens
m atrix (e.g. glutathione S-transferase). Alternatively the defect may lie in a m eta
bolic pathway resulting in accum ulation an d deposition o f insoluble m aterial in
the lens (e.g. galactokinase activity).
In general, it is anticipated th at a gene defect causing a disruption o f the struc
tural proteins would have a d om inant phenotype w hereas an enzymatic defect
m ight be recessive. T herefore cataracts caused by defects in the crystallins an d ma
j o r intrinsic proteins of the lens fibre cells may be predicted to exhibit dom inant
p atterns o f inheritance, since the presence o f a m utant p ro d u c t is likely to result
in loss o f lens clarity. O n the o th er hand, proteins involved in protecting the lens
from dam age are likely to rem ain effective even at reduced levels o f activity, at least
 CRYSTALLIN GENES AND CATARACT 427

during early life, an d so congenital cataracts resulting from m utations in these

genes will show recessive m odes o f inheritance. It is possible, however, that in some
patients cataracts acquired in later life may be due to a partial deficiency in activity
o f these protective proteins (sem i-dom inant).

Linkage studies

T he genetic linkage approach may be used to identify the basic genetic defect
underlying the various in h erited lens opacifications in hum ans. So far, all o f the
gene m apping studies on in h erited congenital cataract loci have considered only
those exhibiting a d o m in an t m ode o f inheritance. T he genetic loci known to
cause dom inantly in h erited congenital cataracts are listed in Table 2 and local
ized on Figure 3. T he possible genetic heterogeneity an d the small family size
re n d e r the m apping approach m uch less attractive in the case of the recessive
form s o f cataracts.

T ab le 2 Positive lin k a g e d a ta fo r d o m in a n tly in h e r ite d cata racts

Description Locus Linkage Lod score References

of cataract symbol

Zonular, CAE FY(lq21-q25) +3.78 at 0 = 0.0 Renwick and Lawler,

pulverulent 1963
(Coppock)

Zonular, (CAE) 1QH (1 q 12) +2.70 at 6 = 0.0 Conneally et al.,

pulverulent FY(lq21-q25) +0.05 at 0 = 0.2 1978
(Coppock)

Posterior, CCDN HP ( 16q22.1) +2.11 at 0 = 0.1 Richards et al.,

polar 1984

Zonular, central CTM HP (16q22.1) +8.31 at 0 = 0.05 Eiberg et al.,

stellate (M arners) 1988

Embryonal, CCL CRYG (2q33-35) +7.58 at 0 = 0.0 Lubsen et al.,

nuclear, pulverulent 1987

In 1963, Renwick an d Lawler investigated a six generation pedigree, originally

described by N ettleship (1909), segregating for a congenital, zonular, pulverulent
cataract, otherw ise known as a C oppock cataract. Strong evidence was obtained to
show close linkage betw een the cataract locus (CAE) and the Duffy blood group
locus (FY) on the proxim al long arm o f chrom osom e 1 (Iq21-q25) (Figure 3), with
a m axim um lod score of 3.78 at a recom bination fraction o f zero (m ale an d fem ale
data c o m b in ed ).
428 MIREILLE CARTIER ET AL.

Conneally et al. (1978) investigated seven families with d om inant congenital bi

lateral cataracts for linkage with 19 m arker loci. While the data were equivocal,
o ne o f the families (Family 1) with a zonular pulverulent cataract, sim ilar to that
studied by Renwick and Lawler (1963), provided a suggestive lod score (2.70 at a
recom bination fraction o f zero) for linkage to the heterochrom atic region on the
proxim al long arm of chrom osom e 1 (1QH) (Figure 3). T he tests for linkage be
tween the cataract an d FY were inconclusive in the same family, however, with m ax
im um lod score of 0.05 at a recom bination fraction o f 0.2. Nevertheless, it rem ains
possible that this cataract is caused by a m utation allelic with the CAE locus. T he
lack o f evidence for linkage with the chrom osom e 1 m arkers in the o th er families
probably reflects genetic heterogeneity in dom inantly in h erited congenital cata
racts.
Strong evidence for linkage was dem onstrated betw een the locus responsible for
M arner's cataract (CTM), a zonular, central stellate cataract and the haptoglobin
locus (HP) on chrom osom e 16 (region q22.1, Figure 3) (Eiberg et al., 1988). The
m axim um lod score obtained was 8.31 at a recom bination fraction o f 0.05. This
same family, originally described by M arner (1949), had already been tested for
linkage with o th er m arkers, resulting in a suggestion of linkage to the P blood
g roup locus (Mohr, 1954), b u t the data was questioned by Renwick an d Lawler
(1963) due to possible mistyping o f the P blood group. A linkage has also been
suggested betw een a posterior polar cataract locus (CCDN) and HP, with m axi
m um lod score of 2.1 at a recom bination fraction o f 0.1 (Richards et al., 1984). It
is possible th at these two HP-linked cataracts are, although phenotypically distinct,
caused by allelic m utations. T he basic defects responsible for these cataracts have
yet to be defined.
Lubsen et al. (1987) re p o rted a tight linkage between the y-crystallin g en e clus
ter on chrom osom e 2 (q33-q35) (Figure 3) and the disease locus in a family with
a cataract m orphologically resem bling the C oppock cataract studied by Ren
wick an d Lawler (1963), except that this cataract was confined to the em bryonic
nucleus o f the lens. T he m axim um lod score was 7.58 at a recom bination fraction
o f zero, suggesting that this Coppock-like cataract (CCL) could be caused by a d e
fect in the y-crystallin locus. Prelim inary data show th at the cataract m ight be re
lated to overexpression o f the tru n cated yE polypeptide in the CCL patients
(N.H.L., unpubl.).

Chromosomal abnormalities associated with congenital cataracts

T h ere are at least two disease syndrom es w here cataracts appear frequently,
potentially as a result o f abnorm al crystallin gene expression. First, about 60% of
patients with Down's syndrom e, which is due to trisomy 21, suffer from cataracts.
It has b een postulated that lens opacity in Down's syndrom e may be due to the
over-expression of the aA-crystallin gene located on th at chrom osom e. Second,
neurofibrom atosis type II (NF 2) has been associated with posterior capsular cat
aracts (K aiser-K upfer et al., 1989). Since the NF2 locus is close to the cluster of
 CRYSTALLIN GENES AND CATARACT 429

p-crystallin genes on chrom osom e 22 an d since neurofibrom atosis occurs when

o n e copy o f this specific chrom osom al region is lost, it has been suggested th at
the appearance of these cataracts may be caused by a deletion o f one or m ore
p-crystallin gene(s).

Other gene mapping studies

While positive linkage data have b een d o cum ented in som e family studies, nega
tive findings an d inconclusive results have b een obtained in m any o th er studies.
H am m erstein and Scholz (1974) fo u n d no evidence for linkage between a he
reditary central cataract and 11 m arkers, including FY in a six generation family.
No linkage was detected betw een the locus responsible for an autosom al dom i
n a n t nuclear cataract an d 21 inform ative m arker loci in a large family studied by
H untzinger, W eitkamp an d Roca (1977).
In a study on a single large Italian family with dom inantly in h erited pulverulent
cataracts, there was no evidence for linkage of the locus responsible for the cata
racts to FY (Stabile et al., 1983).
B atem an et al. (1986) investigated a large family with dom inantly in h erited con
genital cataracts exhibiting considerable variation between affected m em bers of
the family, but with opacification o f the em bryonal nucleus as a com m on trait to
all the cataracts available for exam ination. Linkage was excluded conclusively with
11 genetic m arkers including FY an d HP. A fu rth e r 15 polym orphic loci yielded no
evidence for linkage.
B eaum ont et al. (1989) and Birdwood et al. (1992) investigated a large English
family with autosom al d o m in an t lam ellar cataracts for linkage with chrom osom e
Iq21-q23 m arkers and with the y-crystallin gene cluster. T here was no evidence for
linkage to either o f these regions.
These studies em phasize the high degree o f genetic heterogeneity underlying
dom inantly inherited cataracts. F uture studies on the m apping o f loci responsible
for in h erited cataracts should investigate the segregation o f alleles of the genes
identified as potential candidates for defects in lens clarity, the crystallins and m a
jo r intrinsic proteins o f the lens.

ANIMAL MODELS

Since lens opacity is a phenotype th at is readily recognizable, it is perhaps no t sur

prising that there are many anim al m odels for in h erited cataracts. In addition to
the various cataractous mice (N akano, Deer, CAT-Fraser, Em ory), exam ples also
exist in the rats, guinea pigs, and dogs (reviewed in Gelatt an d Das, 1984; Zigler,
1990). R esearch with anim al m odels offers num erous advantages including the
ease with which large am ounts o f lens m aterial can be obtained, the ability to
carry out extensive linkage studies an d the possibility to test the role o f different
430 MIREILLE CARTIER ET AL.

m utations in the cataractogenic process through the production o f transgenic

animals.
We will lim it the discussion to three anim al models: the Pliilly m ouse, the Elo
m ouse an d the 1 3 /N guinea pig, mainly because they are the ones for which the
corresponding m utations have been defined at the m olecular level. In all three
cases, the m utation is found to be located in a crystallin gene.

T he Philly Mouse

T h e Philly m ouse suffers from a dom inantly inherited, progressive cataract which
appears approxim ately 15 to 30 days after birth, dep en d in g on w hether the ani
m al is homozygous or heterozygous for the m utation (Kador et al., 1980; C arper
et al., 1982). T he cataract first appears as a faint an terio r subcapsular opacity
aro u n d the lens sutures which, within 1 m onth, develops into a dense an terio r
subcapsular and nuclear cataract. In the m utant lens, the first cytologically
detectable aberration is the appearance of dense bodies in the an terio r portio n
o f the newly elongating fibre cells at one week after birth (Uga, Kador and Kuwa-
bara, 1980). This change is followed by the failure of the fibre cells to elongate as
well as a general swelling o f the lens cells. This swelling initially led to the conclu
sion that the Philly cataract was due to a defect in osm otic regulation.

Mutation in the [5B2-crystallin

A m arked decrease in the levels o f soluble (3- and y-crystallin proteins was n oted
in the m utant lens whereas the levels o f a-crystallins were apparently norm al
(Piatigorsky, K ador and Kinoshita, 1980). In addition to this general decrease of
soluble crystallin levels, often observed in cataractous lenses, a specific p-crystal
lin related antigen ap p eared to be com pletely lost (Zigler, C arper an d Kinoshita,
1981). S ubsequent in vitro RNA translation studies showed that a 27 kDa P-crystal-
lin p rotein, now known as PB2, was no t p roduced from hom ozygous Philly lens
RNA (C arper et al., 1982). Sequence com parison o f the pB2 cDNA isolated from
b o th norm al and m utant lenses revealed th at the cDNA found in the Philly lens
carried a 12 nucleotide deletion at the 3' e n d o f its coding region (C ham bers and
Russell, 1991). This deletion would result in a m utant pB2 protein lacking 4
am ino acids at the end o f the fourth G reek key motif. C onsistent with the DNA
finding, a p rotein slightly sm aller than pB2 was in fact detected in the Philly lens
with a polyclonal antibody directed against PB2, b u t no t with a m onoclonal anti
body raised against the C-term inal p o rtio n of the norm al protein (N akam ura et
al., 1988). This result suggested that the m u tan t Philly phenotype may be caused
by the presence o f an altered PB2 polypeptide.
 CRYSTALLIN GENES AND CATARACT 431

Altered, Properties of the mutant f5B2-crystallin

In a norm al m ouse, the am ount o f [3B2 polypeptide is alm ost undetectable at 1-5
days after birth bu t increases m arkedly thereafter (C arper et al., 1982). This pat
tern of expression is consistent with the im m unocytochem ical localization o f the
pB2-crystallin to the newly elongating fibre cells in the neonates (Carper, Smith-
Gill and Kinoshita, 1986). Biochemically, the m utant pB2-crystallin differs from
its wild-type co u n terp art in two ways. First, whereas the wild type PB2 protein is
very heat-stable, the m u tan t protein is heat-labile (N akam ura et al.. 1988). Sec
ond, the m u tan t p rotein is found as high m olecular weight aggregates but the
wild-type protein is found as an octam er, in the pH fraction (Russell an d C ham
bers, 1990). Since failure o f the fibre cells in the cortical layers to elongate
appears to be an early sign o f the defect in the Philly lens, pB2-crystallin may be
im p o rtan t for norm al secondary fibre cell elongation.
T he Philly lens also differs from a n orm al lens in its phase-separation tem pera
ture (Tc), o r the tem p eratu re at which the lens cytoplasm moves from a transpar
ent state (single phase) to a condition of opacity (dual phases). For every lens, this
Tc varies with age, since the com position o f the lenticular cytoplasm is continually
changing. T he Tc o f the hom ozygous Philly lens is approxim ately 17C h ig h er than
that o f the wild type lens at day 1 after birth (Clark and Carper, 1987). Thereafter,
the Tc of the Philly lens varies in a fashion sim ilar to that o f the wild-type lens until
aro u n d day 27, when the Tc of the Philly m ouse suddenly soars, resulting in the
appearance o f a cataract. Meanwhile, the Tc o f the wild-type lens continues to de
cline. It seems probable th at the increase in Tc results from the lack o f heat stabil
ity an d the abnorm al aggregation p attern o f the m utant PB2 p rotein, bu t how this
alteration interferes with fibre cell elongation rem ains to be elucidated.

T he Elo Mouse

T he Elo (Eye lens obsolescence) m ouse carries a d o m in an t and fully p en e tran t

m utation that perturbs em bryonic lens developm ent an d results in m icro
p hthalm ia at b irth (O da, W atanabe an d Kondo, 1980). A bnorm al developm ent
of the m u tan t lens can be detected at day 12 in utero: while the posterior cells
elongate and fill the lum en in a n orm al lens, the central posterior cells fail to
elongate com pletely and leave a gap in the lum en of the Elo lens (O da et al.,
1980). In addition, these m u tan t fibre cells lack the eosinophilic granules nor
mally present an d contain num erous lysosomal bodies. These m alform ed fibre
cells subsequently necrose, collapse, an d detach from the lens capsule, resulting
in the form ation o f a lens one h alf to one fifth o f the norm al size at birth. In this
m u tan t lens, the an terio r epithelium , as well as the short equatorial fibre cells,
ap p ear norm al, suggesting th at the defect is lim ited to the elongating fibre cells.
432 MIREILLE CARTIER ET AL.

Cell autonomous defect

W hen eye rudim ents were taken from the Elo m ouse an d cultured in vitro, the
lens fibre cells failed to elongate properly, suggesting that the defect was not
caused by an extraocular com p o n en t (W atanabe et al., 1980). In addition, chi
m eric anim als derived from m ixing BALB/c and C 3H -Elo/+ em bryos were fo u n d
to have lenses with a spectrum o f phenotypes, ranging betw een wild type, n orm al
sized b u t opaque an d Elo-like. T he severity o f the lens defect was pro p o rtio n al to
the relative contribution o f lens fibre cells from the m u tan t strain (Yoshiki et al.,
1991). In fact, im m unohistological analysis showed that the necrotic fibre cells
originated from the C3H strain. These studies thus argued strongly that the Elo
phenotype was expressed in a cell autonom ous fashion.

Mutation in theyE-crystallin gene

T he Elo m utation is located on chrom osom e 1, close to the -crystallin gene clus
ter (yA to yF) (Masaki and W atanabe, 1989; Q uinlan et al., 1987). T he specific in
volvem ent o f the y-crystallin locus is fu rth e r suggested by the m arked reduction of
the general y-crystallin mRNA an d p rotein levels when com pared to those o f p-
an d y-crystallins (Masaki and W atanabe, 1988; Q uinlan et al., 1987). M ore recen t
ly, a single base pair deletion has been fo u n d in the yE-crystallin gene of the Elo
m ouse (Cartier, B reitm an and Tsui, 1992). T he deletion, which is located in the
m iddle of the last exon o f the gene, would result in a truncated polypeptide miss
ing the last G reek key m otif o f the norm al protein (replaced by 11 am ino acids
specified by a different reading fram e). T h e causal relationship between this dele
tion an d the Elo phenotype is fu rth e r su p p o rted by the genetic linkage data show
ing no recom bination between the two in 274 meioses. Since mRNA from the
m u tan t allele is readily detectable in the Elo lens, it seems probable th at the failure
o f the lens fibre cells to elongate is caused by the presence o f a putative tru n cated
polypeptide. T he cu rren t hypothesis, therefore, about the Elo phenotype is that
its small lens would be due to the presence o f a tru n cated yE polypeptide. Al
though the m olecular m echanism by which this m u tan t y-crystallin polypeptide
causes a small lens is unknow n, this result suggests a role for y-crystallins in fibre
cell differentiation.

13 /N Guinea Pig

T he 1 3 /N strain o f guinea pigs carries a bilateral, congenital nuclear cataract

m utation which is a dom inant and fully p en e tran t phenotype (Am sbaugh and
Stone, 1984; Stone and Am sbaugh, 1984). T he defect seems to be associated with
a taxon-specific lens protein, called the ^-crystallin which has, so far, only been
found in some hystrichom orph rodents a n d camels (H uang et al., 1987; G arland
et al., 1991). This 38 kDa protein is p resen t at high levels (up to 10% o f the solu
ble proteins) in norm al guinea pig lens and, in somewhat lower am ounts, in the
 CRYSTALLIN GENES AND CATARACT 433

liver and kidney. In the hom ozygous m u tan t guinea pig the protein is absent
from all three organs, while it is p resen t at h alf the norm al levels in the liver and
kidney o f the heterozygous anim al (H uang et al., 1990; Zigler an d H uang, 1988).
Thus, an association betw een the in h erited cataract condition and the absence of
^-crystallin has been postulated.

M utation in the ^-crystallin

M olecular analysis shows that the ^-crystallin gene in the cataractous guinea pig
harbors a deletion at the splice-acceptor site o f exon 7 (Borras et al., 1990;
R odriguez et al., 1991). As a result, the m ajor mRNA transcript from this gene
lacks the entire exon 7 sequence and, since this exon contains 102 base pairs
(bp), an internally deleted polypeptide is predicted. In addition, however, the
m utation has apparently activated a cryptic splice-acceptor site, giving rise to a
m inor mRNA species with a fram eshift caused by the insertion of a 20 bp intron
sequence in place o f the missing exon. T he presence of these putative m u tan t
polypeptides may explain the fully p e n e tra n t an d d om inant phenotype o f the cat
aract m utation. N either o f the m u tan t form s are predicted to form the tetram eric
structure postulated for the norm al p ro tein (Rao an d Zigler, 1992).

Enzymatic activity ofZ,-crystallin

The presence o f f^-crystallin in liver a n d kidney im plies an enzymatic function for

this protein. A distant relationship has in fact been no ted betw een ^-crystallin
and alcohol dehydrogenase (R odokanaki, Holm es an d Borras, 1989). T he C,-crys
tallin is characterized, however, by the absence of the oxidation-sensitive thiol
groups as well as the lack o f superficial segm ents typical o f alcohol dehydroge
nase. T he absence o f these sequences has apparently resulted in the loss o f an
alcohol dehydrogenase-specific enzymatic activity, while leading to an increased
stability for the lens protein (Borras, Persson an d jo rn v a ll, 1989). Recently, it has
been shown that ^-crystallin is active as an o th er enzyme, an N A D PITquinone oxi-
doreductase, which may serve to regulate NADPH oxidation or to detoxify quino-
nes in the lens (Rao, Krishna an d Zigler, 1992; Rao an d Zigler, 1991). It is of
interest to n ote that the tru n cated polypeptide found in the cataractous lens does
no t bind NADPH an d has no enzymatic activity (Rao an d Zigler, 1992), leading to
dram atically red u ced levels of NADPH levels in the m u tan t lens (Rao and Zigler,
1990). T herefore, while these data strongly imply a role for the m u tan t crystallin
in the cataractous phenotype, it is n o t known which defective property (or p ro p
erties) of this polypeptide is in fact responsible for the diseased state.

M utagen-Induced C ataract Mice

Studies on the genetic causes o f cataract have also been perfo rm ed with m ouse
m utants obtained after parental m utagenic treatm en t (Kratochvilova, 1981; Graw
434 MIREILLE CARTIER ET AL.

et al., 1986; Kratochvilova and Favor, 1988). Fifteen of the m utants o btained have
been tested for allelism an d found to segregate into seven d ifferent groups
(Kratochvilova and Favor, 1992). T he largest group com prises five alleles and,
based on m apping results from two o f the strains, the com m on im itation locus
appears to be located on chrom osom e 1, close to the y-crystallin cluster (J. Favor,
M.F. Lyon an d J. Loster, personal com m unication). These d o m in an t cataracts
may, therefore, be caused by m utations in the y-crystallin genes.

CONCLUSION

Crystallins are the m ain soluble proteins o f the eye lens. As such, they play a
m ajor role in the two specific properties o f this organ: accom odation an d trans
parency. It is, therefore, th o u g h t that th eir recru itm en t by the lens was due to
th eir appropriate biophysical characteristics. This view is fu rth e r stren g th en ed by
the fact th at different anim al species, with varying lenticular requirem ents, have
recru ited num erous proteins as crystallins to fit their particular needs. For exam
ple, rodents, which are n octurnal anim als, have evolved a hard, dehydrated and
spherical lens with high levels of y-crystallins. Meanwhile, birds, requiring high
accom m odative power, n eed a m ore flexible and hydrated lens, obtained by
replacing the y-crystallins with the 8-crystallins, which allow m ore hydration.
T he study of inherited cataracts has fu rth e r dem onstrated the im portance of
the crystallins for lenticular function, since fo u r m utations in crystallin genes have
been associated with lens disorders so far: the hum an y-crystallin cluster associat
ed with the Coppock-like cataract, the m urine pB2 and yE im plicated in the m u
tan t phenotypes of the Philly an d Elo m ice and the ^-crystallin m utated in the
cataractous N /1 3 strain o f guinea pigs. F urtherm ore, two o f these crystallin m uta
tions, the ones present in the m urine (JB2 an d yE genes, affect the fibre cell elon
gation process and may, therefore, im plicate these crystallin proteins in the
developm ent o f the lens. Such results are p art o f the growing data im parting a
function o th er than structural to the d ifferent crystallins. For exam ple, m ost tax-
on-specific crystallins were derived from enzymes and still possess an enzymatic ac
tivity which may be necessary for p ro p e r lens function. Also, as discussed above,
the a-crystallins have recently been fo u n d to possess some ch ap ero n e activity.
T h erefore, although the three-dim ensional structure and biophysical properties
of the crystallins are probably the main reasons for the recru itm en t o f these p ro
teins by the lens, their enzymatic activity may have also played an im p o rtan t role
in this process and may still be req u ired for the p ro p e r function o f the organ.
A nother p oint which should also be stressed is the advantage o f the genetic ap
proach to investigating the role of crystallins in cataractogenesis. In d eed , al
th ough biochem ical studies had im plicated these m olecules in the actual
lenticular opacity, it was never possible to d em onstrate w hether the crystallin in
volvem ent was a prim ary or secondary event. In the case o f in h erited cataracts,
however, if the m utation responsible for the disease lies in a crystallin gene, it is
 CRYSTALLIN GENES AND CATARACT 435

p ro o f that defective crystallins can be a prim ary cause o f cataract. T he d eterm in a

tion of which m utations cause lenticular opacities will be helpful in gaining a bet
ter un d erstan d in g o f the interactions of the d ifferent crystallins, both in healthy
an d diseased states. To that end, it will be im p o rtan t to screen the various crystallin
genes, in the different in h erited lenticular diseases, for potential m utations.
A lthough there rem ain unansw ered questions about the crystallins an d some of
th eir roles in the developm ent an d m aintenance of lenticular architecture and
function, there is no do u b t that these m olecules are vital for the p ro p e r function
ing o f this organ.

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 19. ANIRIDIA

ISABEL HANSON, TIM JORDAN AND VERONICA VAN HEYNINGEN

MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh
EH4 2XU, UK

INTRODUCTION

Phenotype

A niridia is a congenital in h erited disorder o f the eye which derives its nam e from
the noticeable iris hypoplasia seen in m ost cases. T he nam e is som ething o f a mis
n o m er since there is always som e iris tissue present although this may be so m ini
mal as to be invisible to the naked eye. T he nam e also draws attention to the m ost
dram atic feature o f the phenotype from the observer's point o f view, when in
reality the effects on the rest o f the eye are far m ore visually disabling for the
patient. T he associated eye an d visual defects include some or all o f the follow
ing: corneal opacification, cataract, lens dislocation, ciliary body hypoplasia,
foveal dysplasia, optic nerve hypoplasia, strabism us and nystagmus (H ittner,
1989). To this long list o f what ap p ear to be mainly prim ary disorders resulting
from m aldevelopm ent o f the eye an d from altered control o f eye m ovem ent by
the central nervous system, we m ust add the com plication o f glaucom a. Very
rarely this is actually p resen t at birth, indicating th at it may be caused by a pri
m ary dysgenesis o f the aqueous outflow system but m ore com m only it has its
onset in pre-adolescence o r later, w hen it appears to be due to obstruction o f the
drainage angle by adhesions between the lens and the trabecular m eshwork
(Margo, 1983). T he wide range o f tissues affected has justifiably led to aniridia
being described as a panocular disorder (Nelson et al., 1984).
O ne striking feature o f aniridia is the variable expressivity. T he phenotype can
vary dram atically betw een individuals even within the same family. For exam ple,
the iris hypoplasia ranges from a readily visible alm ost com plete absence o f the
iris, th ro u g h enlargem ent an d irregularity of the pupil m im icking a colobom a, to
small slit-like defects in the an terio r layer seen only on transillum ination with a slit-
lam p (Figure 1; H ittn er etal., 1980). T he m ildest phenotype is detectable as subtle
abnorm alities o f the iris an d m acula which can be seen only by fluorescein
angiography (H ittn er et al., 1992). T he effect o f aniridia on visual function is also
very variable. Some families have been re p o rted in which m ost affected m em bers
only had iris hypoplasia an d alm ost n orm al visual acuity whereas in o th er cases the
presence of various com binations of defects elsewhere in the eye has caused a se
vere reduction in acuity (Grove, Shaw and B ourque, 1961; Elsas et al., 1977; H itt
n e r et al., 1980). In addition, all aniridia patients are at risk o f developing
glaucom a. T he incidence o f this com plication has been variously re p o rted as be
tween 6% and 75% which may reflect differences in the selection o f patients stud-
446 ISABEL HANSON ET AL.

Figu re 1 V ariable expressivity o f a n irid ia in d iffe re n t m e m b e rs o f an affected p e d ig re e . A: vir

tually c o m p lete an irid ia. B: sm all a m o u n t o f iris visible. C: atypical iris co lo b o m a. D: typical iris
co lo b o m a. E: iris th in n in g with ro u n d p u p il. F: c o rn e a l p a n n u s w ith iris th in n in g a n d ro u n d
p u p il. {American Journal o f Ophthalmology, 1980, 89, 5 3 1 -5 3 9 ). T his figure was a g e n e ro u s gift
fro m Dr. H . M. H ittn e r a n d is p u b lish e d w ith p erm issio n fro m th e American Journal of
Ophthalmology. C o p y rig h t by T h e O p h th a lm ic P u b lish in g C om pany.
 ANIRIDIA 447

ied an d th eir ages at the tim e o f assessm ent (Nelson et al., 1984). T he glaucom a
o f aniridia is notoriously difficult to m anage and if n o t treated successfully will d e
stroy any residual vision an d can cause intractable ocular pain necessitating rem ov
al of the eye.

Genetics

A niridia is a relatively rare disease, with a population frequency o f 1:60,000 to

1:100,000 (Shaw, Falls a n d Neel, 1960; S hannon et al., 1982). A bout two thirds of
cases are familial, with highly-penetrant autosom al d o m in an t inheritance. T he
rem aining th ird are sporadic cases with no previous family history. Some of these
are new cases o f isolated aniridia, with no associated anom alies, which will show
the usual d o m in an t p attern o f in heritance in subsequent generations (Shaw, Falls
an d Neels, 1960). However, a considerable p ro p o rtio n o f these new m utations are
associated with cytogenetically visible deletions involving band p i 3 on the short
arm o f chrom osom e 11 (Riccardi et al., 1978; Francke et al., 1979). Most such
deletion cases have a constellation o f associated anom alies referred to as the con
tiguous gene syndrom e WAGR: a predisposition to Wilms' tu m o u r (a childhood
kidney tum our), aniridia, g en itourinary abnorm alities (particularly cryptorchid
ism an d hypospadias in boys) and often m ental retardation. Occasionally o th er
associated anom alies are found with aniridia; these rare syndrom es m erit special
attention to discover the heritability an d o th er allied risks (Nelson et al., 1984 ).
A niridia accom panied by cerebellar ataxia and m ental retardation is known as
Gillespie syndrom e (Gillespie, 1965; Nevin and Lim, 1990). This very rare syn
drom e has been re p o rte d in sibs with two unaffected parents, leading to the sug
gestion o f an autosom al recessive m ode o f inheritance (Gillespie, 1965; Francois,
Lentini and de Rouck, 1983; W ittig et al., 1988). However, consanguinity has never
been dem onstrated an d a single p a re n t to child transm ission has also been record
ed (Crawfurd et al,, 1979). It is th erefo re quite likely th at we are in fact dealing
with new dom inant m utations, and vertical transm ission is rarely seen because se
verely affected patients tend n o t to reproduce.

MAPPING OF THE ANIRIDIA LOCUS

The Aniridia Locus M aps to 1 lp !3

T he iris hypoplasia and associated ocular anom alies seen in aniridia provide no
simple im m ediate biological inform ation ab o u t the underlying functional defect.
However, the existence of a m utation can be used to identify the gene responsible
for these abnorm alities. D eterm ination o f the genom ic position o f the m utant
gene can lead us by positional clo n in g (Collins, 1992) to the aniridia gene.
Genom ic position can be ded u ced e ith e r from linkage studies in familial cases or
from the analysis o f cytogenetic (karyotypic) abnorm alities associated with the
448 ISABEL HANSON ET AL.

phenotype. Together, these approaches have provided strong evidence th at the

aniridia locus m aps to chrom osom e 11, b an d p l3 .

Cytogenetics

A key initial observation in m apping the aniridia gene cam e from cytogenetic
analysis o f patients with the WAGR syndrom e. T he visible constitutional intersti
tial deletions of the short arm o f chrom osom e 11 in these patients h ad a com m on
region o f overlap in band p l3 which suggested th at 11 p 13 contained a gene or,
m ore likely, a n u m b er of genes which, w hen hemizygous, caused the WAGR p h e
notype (Riccardi et al., 1978; Francke e ta l., 1979).
T he 11 p i 3 assignm ent was strongly su p p o rted by evidence from three pedigrees
in which aniridia segregated with a chrom osom al translocation. Simola et al.
(1983) described a family in which three affected m em bers from three genera
tions carried a balanced translocation t (4; 11) (q22;pl3). M oore et al. (1986) re
p o rted a family in which four affected m em bers spanning three generations
carried t( l 1;22) (p 13;q 12.2). Pettenati, Weaver and B urton (1989) described an af
fected fath er an d d au g h ter with t(5 ;l 1) (q l3 .1 ;p l3 ). Wilms' tum our was absent
from all three pedigrees, suggesting that Wilms' tum our and aniridia are caused
by separate loci within 11 p 13. T he chrom osom e 11 aniridia locus was designated
AN 2.

Linkage

A second approach taken to m ap the aniridia gene was th at of linkage, in which

affected pedigrees were tested for the segregation o f the disease with specific alle
les o f m ultiple polym orphic m arkers of known m ap position. In one large family
all affected individuals carried the same allele of the ACPI gene an d statistical
analysis of the p attern o f segregation provided evidence for linkage betw een anir
idia an d ACPI which m aps to 2p25 (Ferrell et al., 1980). T he chrom osom e 2 anir
idia locus was designated AN1. In an in d ep en d e n t study an o th er large kindred
was tested for segregation of aniridia with m arkers from 2p25 and 11 p i 3. Linkage
with 2p25 was excluded in this pedigree while linkage with 11 p i 3 was confirm ed
(M annens et al., 1989). It is quite possible for similar phenotypes to be caused by
m utations at d ifferent loci. However a re cen t study o f the chrom osom e 2-linked
family, including re-evaluation o f diagnoses and use o f additional m arkers from
2p and l i p , excluded linkage o f aniridia in this family with chrom osom e 2 and
confirm ed linkage with 11 p i 3 (Lyons et al., 1992). Since linkage an d cytogenetic
studies converged on the same small genom ic region, this has been the site of
intensive effort to identify the aniridia gene(s).

C haracterization o f the I l p l 3 WAGR Region

T h e l i p deletions in d ifferent WAGR patients are highly variable in extent, but

each m ust include the genes responsible for the phenotype. T herefore, by d eter
 ANIRIDIA 449

m ining at the m olecular level the proxim al (centrom eric) an d distal (telom eric)
limits of the deletions in m any patients, it was possible to define the smallest
region of overlap (SRO) o f the deletions in which the critical genes m ust lie (Fig
ure 2).

CC cc
C9 O .
< < < CD
5 5 5 5
x< cc
<
Q

15

HVBS1

FSHB
14
D11S16
495, AN
13 p5

WT
12
CAT
11
D11S9

Figure 2 Sim plified d e le tio n m a p o f th e WAGR re g io n o n th e sh o rt a rm o f ch ro m o sm e 11,

V ertical bars re p re s e n t p o rtio n s o f c h ro m o so m e 11 p re s e n t in each p a tie n t, as d e fin e d by th e
p re se n c e o r a b sen ce o f th e p ro b e s listed to th e rig h t. WT, W ilm s' tu m o u r locus; AN, a n irid ia
locus. T h e p h e n o ty p e o f e ach p a tie n t is show n in b rackets: W, W ilms' tu m o u r; A, an irid ia; G,
g e n ito u rin a ry a b n o rm alities; R. m e n ta l re ta rd a tio n . BAN a n d SOA a re d esc rib e d in C ouillin
e t al. (1989); DAR in Davis et al. (1988) a n d C o u illin e t al. (1989); MAX in Seaw right e t al.
(1989); a n d SIM O i n j u n ie n a n d M cB ride (1989).

At first the proxim al an d distal breakpoints o f the deletions were positioned us

ing the relatively low resolution technique o f chrom osom e banding (Riccardi et
al., 1978; Francke et al., 1979). H igher resolution was achieved by identifying
m arkers which m apped within the WAGR deletions. This m olecular deletion anal
ysis was accom plished eith er by isolating the d eleted chrom osom e 11 in a somatic
cell hybrid or by dosage studies in WAGR cell lines (Seawright et al., 1988; Porte-
ous et al., 1987; Gessler et al., 1989). Initially the m arkers used were gene products
such as enzymes an d cell surface m arkers; later however cloned genes an d batter
ies of random DNA probes isolated from genom ic sources en rich ed in chrom o
some 11 were predom inantly used.
450 ISABEL HANSON ET AL.

T he genes for catalase ( CAT) and the p-subunit o f follicle stim ulating h o rm o n e
(.FSHB), both o f which m ap to 11 p 13, were found to be deleted in many bu t not
all WAGR patients, placing them close to, b u t no t within, the SRO (Figure 2; Gla
ser et al., 1986; P orteous et al., 1987; Seawright et al., 1988). Cytogenetic com par
ison o f the extent o f deletions in patients lacking o n e m arker bu t n o t the o th er
revealed th at FSHB was distal to G1Y with the SRO betw een the two. T he CAT-FSHB
interval was subdivided into regions which m ust contain the Wilms' tu m o u r and
aniridia genes by d eterm in in g the positions of the breakpoints in key patients with
incom plete WAGR phenotypes. Since aniridia shows com plete penetrance, the
characterization o f the deletion in a p atien t with reduced catalase activity, Wilms'
tu m o u r and genitourinary abnorm alities, bu t not aniridia, placed the Wilms' tu
m our gene proxim al to the aniridia gene (Patient DAR, Figure 2; Davis et al.,
1988). T he precise position o f these breakpoints was defined using random DNA
m arkers. This approach pro d u ced a detailed consensus breakpoint m ap (Junien
and McBride, 1989).
O nce a high density of m arkers becam e available, the construction o f a long
range restriction m ap by pulsed-field gel electrophoresis (PFGE) was possible. T he
high resolution physical m ap o f the 11 p 13 region pro d u ced in this way showed for
the first tim e the distances, in kilobase pairs, between m arkers, and the positions
o f the key disease-associated chrom osom al rearrangem ents (Figure 3; C om pton et
al., 1988; Gessler an d Bruns, 1989; Rose et al., 1990). T he sites for rare cutting re
striction enzymes used for PFGE m apping also m ark the position o f regions which
are relatively rich in unm ethylated CpG dinucleotides. Such 'CpG island' sites are
often associated with the 5' ends o f genes, an d are in fact found upstream o f both
the Wilms' tu m o u r gene (Call et al., 1990; Gessler et al., 1990) an d the aniridia
gene (Ton e ta l., 1991).

Cloning o f candidate aniridia genes

In theory, disease-associated chrom osom al translocations are particularly valu

able as tools for positional cloning because the translocation b reakpoint often
occurs within the gene responsible for the disease phenotype (Gessler, Simola
an d B runs, 1989). For this reason, an obvious step towards the identification of
the aniridia gene was the characterization of the aniridia-associated transloca
tions. U pon m ore detailed m olecular analysis, the t (11 ;22) translocation was
shown to be associated with a deletion o f betw een 650kb an d 850kb o f DNA from
the 1.4Mb Notl fragm ent in 11 p i 3 (P atient DG-85, Figure 3; Davis et al., 1988;
C om pton et al., 1988; Rose et al., 1990). Since a stretch o f DNA o f this size could
theoretically encode several genes, the t (11 ;22) translocation was o f lim ited use
in identifying the precise location o f the aniridia gene. However, two m arkers
deleted in the t (11 ;22) translocation were shown to flank the t (4; 11) breakpoint
and used to position it 100-200kb from the proxim al en d o f the 1.4Mb Notl frag
m ent (P atient SIMO, Figure 3; Davis et al., 1989). A nother m arker m apping just
below the breakpoint was used to isolate overlapping DNA fragm ents from
 ANIRIDIA 451

WT1 PAX6
t# <#
p5 495 D11S16 FSHB

I \ I I
N N N N N

kb: 500 375 325 1400

SIMO (A) ............. . . .. ;,|------ - ' .................. ..................... > -...t :---------------:

SATO (WAR) [ ,, - ...."?' t

d a r (WG) >r t v r , .-g - T, Z Z ______________________ , . J.'Z1 S

d g -85 (A) i z^ a M c a a i

Figure 3 Physical m ap o f the m inim al WAGR region in band 11p i 3 constructed by pulsed
field gel electrophoresis (C om pton et al., 1988; Gessler and Bruns, 1989; Rose et al., 1990).
Sites for the infrequently cutting restriction enzyme Notl are shown (N ). Positions of some of
the probes used to construct the m ap are rep resen ted by vertical arrows. T h e breakpoints in
key patients which helped subdivide the m ap into regions containing the genes for Wilms'
tum our and aniridia are shown below the map. T he phenotypes of these patients are desig
nated as for Figure 2. DG-85 is described In Davis et al. (1988), C om pton et al. (1988), and
Rose et al. (1990); DAR in Call et al. (1990), and van Heyningen et al. (1991); SATO in Ton
et al. (1991); and SIMO in Gessler, Simola and Bruns (1989), Davis et al. (1989), an d Ton et
al. (1991). T he Wilms' tum our (WT1) an d aniridia (PAX6) genes are rep resen ted by horizon
tal arrows (Call et al., 1990; Gessler et al., 1990; Ton et al., 1991).

g e n o m ic lib raries a n d th u s to clo n e th e ju n c tio n fra g m e n t (P ro b e 495, F igure 3;

Gessler, Sim ola a n d B ru n s, 1989). N o sizeable d e le tio n a p p e a rs to have o c c u rre d
as b o th d e r(4 ) a n d d e r ( l l ) are d e te c te d by th e 350bp ju n c tio n frag m e n t. T his
fra g m e n t w hen h y b rid ised to g e n o m ic DNA fro m d iffe re n t v e rte b ra te species
d e te c te d c o n se rv e d fra g m e n ts in o th e r m am m als a n d ch icken. B ecause ex
p re sse d se q u en ces a re m o re highly c o n se rv e d betw een species, this zoo b lo t
a p p ro a c h has p ro v ed successful in id en tify in g g en es in g en o m ic DNA. W hen
se q u e n c e d , th e b re a k p o in t re g io n was fo u n d to c o n ta in a n o p e n re a d in g fram e
flan k ed by c o n sen su s splice sites, fe a tu re s w hich are c h a rac teristic o f a co d in g
ex o n o f a g en e. A lth o u g h th e b re a k p o in t locus is a stro n g a n irid ia c a n d id a te o n
th e basis o f p o sitio n , n o co g n a te cDNA clo n es o r se q u e n c e d a ta have yet b e e n
re p o rte d . T h e ro le o f this p ro p o s e d g e n e in a n irid ia rem a in s u n c e rta in b u t it is
now know n th a t th e ju n c tio n fra g m e n t is n o t d e le te d in a WAGR p a tie n t w ith clas
sical a n irid ia w hose distal b re a k p o in t is th e m o st c e n tro m e ric yet know n (P a tie n t
SATO, F ig u re 3; T on e t al., 1991).
M eanw hile a se c o n d c a n d id a te a n irid ia g e n e was isolated by T on e t al. (1991)
w ho w ere a p p ro a c h in g th e re g io n c o n ta in in g th e tra n slo c a tio n b re a k p o in t using
th e te c h n iq u e s o f c h ro m o so m e ju m p in g a n d w alking. T h e ir sta rtin g p o in t was a
p ro b e (p5, F igure 3) in th e 325kb Notl fra g m e n t im m ed iately p ro x im a l to th e
1.4M b Notl fra g m e n t know n to c o n ta in th e b re a k p o in t. W hile m oving tow ards th e
452 ISABEL HANSON ET AL.

breakpoint, they cloned the ju n c tio n o f the two AV//I fragm ents and since this po
tentially m arked a CpG island, it was decided to search for coding sequences in
this region. O ne genom ic fragm ent, conserved across species, was successfully
used to isolate cDNA clones from an 8.5d em bryonic m ouse library and a hum an
adult retinal library. T he m ouse cDNA was used in tu rn to isolate a cDNA from a
h u m an foetal eye library (Ton et al., 1991).
Sequencing of the cDNA clones revealed th at the candidate gene was a m em ber
of the Pax gene family. Pax genes are characterised by the presence of a m otif
known as a p aired box which encodes a 120-amino acid DNA-binding dom ain. T he
p aired box was first described in the Drosophila segm entation gene paired (Frigerio
et al., 1986; Bopp et al., 1986) and has since b een found in o th er developm entally
im p o rtan t Drosophila genes (B aum gartner et al., 1987; Bopp et al., 1989). Subse
quently, a family of paired-box containing genes have been described in m ouse,
m an, zebrafish and chicken (D eutsch an d Gruss, 1991). T he expression patterns
o f the Pax genes, as identified by RNA in situ hybridisation, an d the involvem ent
o f these genes in developm ental m utations in Drosophila, m ouse and m an has ar
gued for a key role for them in p attern form ation (Deutsch an d Gruss, 1991; Gruss
and Walther, 1992). T he vertebrate Pax gene family has been best characterised in
m ouse and so far contains eight m em bers (W alther et al., 1991; Gruss an d Walther,
1992). O n the basis o f predicted am ino acid sequence, the candidate aniridia gene
is the hum an hom ologue of th e m ouse Pax-6 gene; in d eed the predicted protein
products of the m ouse ( Pax-6) and hum an (PAX6) genes differ by only one am ino
acid. As well as the paired motif, the PAX6 gene also encodes a paired-type ho-
m eobox, an o th e r phylogenetically conserved m otif found in developm entally im
p o rta n t genes an d also known to encode a DNA binding activity (W alther and
Gruss, 1991; D eutsch an d Gruss, 1991).
T he candidacy of PAX6 as the aniridia gene was strengthened by RNA in situ hy
bridisation studies designed to d eterm in e the pattern of expression o f the PAX6
gene. This approach revealed that PAX6 was strongly expressed in the developing
eye, at a stage preceeding iris form ation, in both hum an an d m ouse em bryos (Ton
et al., 1991; W alther and Gruss, 1991). Interestingly, the dom ain o f expression in
th e eye was no t confined to the rim o f the optic cup which would ultim ately give
rise to the n eu ro ecto d erm al com ponent o f the iris, but also encom passed the n eu
ral retina, the lens and the overlying surface ectoderm which contributes towards
the cornea. All these structures can be affected in aniridia, and the in situ results
were therefore consistent with PAX6 being the aniridia gene. However, to show
th at a candidate gene isolated by positional cloning is really responsible for the
disease it is necessary to dem onstrate intragenic m utations in affected individuals.
T he first genetic evidence th at PAX6 was involved in eye developm ent cam e from
analysis of a m ouse m utation called Small eye.

Small eye : a m ouse m odel fo r aniridia

Small eye is a sem i-dom inant m utation affecting eye developm ent in the m ouse.
T h ree alleles have been described, two spontaneously occuring (Sey an d Sey^)ey)
 ANIRIDIA 453

an d one radiation induced (SeyH; G reen, 1989). T he m ost obvious phenotypic

feature o f heterozygous Small eye m ice is a reduction in eye size. A m ore detailed
exam ination o f the eyes o f Sey/+ heterozygotes may also reveal cataracts, small
vacuolated lenses, adhesions betw een the lens and cornea, and an abnorm ally
folded n eu ro retin a (H ogan et al., 1986). T he eyes o f S e y - ' / t an d SeyH/+ het
erozygotes often have colobom ata a n d are generally sm aller that those o f Sey/+
mice bu t otherw ise display a sim ilar range o f ocular anom alies (H ogan et al.,
1986; Theiler, V arnum an d Stevens, 1978).
Mice hom ozygous for the Sey allele have no eyes an d im perforate hypoplastic
snouts and die shortly after birth because they can n o t breathe while feeding (Fig
ure 4; H ogan et al., 1986; 1988). This im plicates the pro d u ct of the Small eye gene
no t only in eye developm ent but in the m orphogenesis of the frontonasal region
(H ogan et al, 1986; 1988). SeyDey an d SeyH hom ozygotes have n o t been observed
and are presum ed to die early in gestation. T he pleiotropic effects o f the Seyr^and
Sey m utations suggested th at these were deletions which affected a n u m b er of
genes req u ired for norm al growth an d viability in addition to eye developm ent. In
contrast, the relatively m ild phenotype associated with the Sey allele led to the pro
posal that this was m ost likely to be a point m utation (H ogan, H eth erin g to n and
L y o n ,1987).
No com parison was initially m ade betw een Small eye and aniridia because they
ap peared distinct at the gross phenotypic level. However, genetic m apping of the
Small eye m utation revealed that it fell into a linkage group on m ouse chrom osom e
2 which is syntenic with b an d 11 p 13 o f the hum an genom e (Siracusa et al., 1990;
van d er Meer-de Jo n g et al., 1990). It was then shown th at m arkers which are com
monly deleted in hum an WAGR patients are also deleted from mice carrying the
Sef*^ allele (Glaser, Lane an d H ousm an, 1990). This confirm ed that Seyr)ey was a
deletion m utation and, m ore im portantly, suggested that Small eye and aniridia
were genetically equivalent. F u rth er evidence to support this hypothesis included
the similarity betw een the phenotype o f Sey/Sey homozygotes an d th at o f a hum an
foetus, presum ed hom ozygous, b o rn w ithout eyes o r nose as the result o f an anir
id ia /a n irid ia m ating (H odgson and Saunders, 1980). In addition, the autosom al
sem i-dom inant m ode o f inheritance is com m on to Small ^ a n d aniridia, as is the
very high p en etran ce bu t variable expressivity o f the phenotype. Most recently, a
careful re-evaluation o f the eye phenotype in Sey>/+ heterozygotes has revealed for
the first time iris hypoplasia with a striking resem blance to th at seen in aniridia
(Jordan et al., 1992).

MUTATION ANALYSIS OF THE PAX6 GENE

Mouse m utations

Because the Small eye m ouse was such a plausible m odel for aniridia an d biologi
cal m aterial is m ore readily available from laboratory anim als than patients,
m utation analysis o f the Pax-6 gene was first carried o u t in the mouse.
454 ISABEL HANSON ET AL.

Figure 4 T h e Small eye m ouse: p h o to g ra p h s o f th e 15.5 day p o st co itu m m o u se em b ry o .

(a) wild type e m b ry o , + /+ . (b) h e tero zy g o te em b ry o , Sey/+. T h e eye is re d u c e d in size partly
as a re su lt o f th e sm aller lens, a n d th e o u tlin e o f th e o p tic cu p rim is irreg u la r, su g gesting delay
in th e c lo su re o f th e e m b ry o n ic fissure, (c) h o m o zy g o te em b ry o , Sey/Sey. N o visible eye is
fo rm e d a n d th e ab sen ce o f a nasal cavity alters th e sh ap e o f th e snout.
R e p rin te d w ith p erm issio n fro m Nature (H ill e t al., 1991, 354, 5 2 2 -5 2 5 ). C o p y rig h t 1991
M acm illan M agazines lim ite d .
 ANIRIDIA 455

Dosage analysis and interspecific backcrosses dem onstrated that Pax-6 was delet
ed in the Sey m ouse (Hill et al., 1991). A m ore im p o rtan t goal however was to
dem onstrate intragenic m utations o f Pax-6 in Small eye alleles which were predict
ed to be p oint m utations on the basis o f phenotype. At this stage little was known
about the genom ic organisation o f Pax-6 so the m ost feasible approach to m uta
tion analysis was th ro u g h mRNA. From the expression p attern it was clear th at Pax-
6 was m ost highly expressed in neural tissue in em bryos betw een 8 and 18 days ges
tation. T herefore RNA was extracted from the heads o f 15d em bryos, reverse tran
scribed, PCR-amplified and analysed for the presence o f p oint m utations by
chem ical cleavage o f m ism atch (Hill et al., 1991). This RNA-PCR approach re
vealed a single base change in the Sey Pax-6 gene which created a term ination
codon in the open reading fram e. T he predicted effect of this would be to tru n
cate the p rotein p ro d u ct before the hom eodom ain and thus to im pair the func
tion o f the Pax-6 protein (Figure 5b).
A dditional evidence th at Pax-6 is m u tated in Small eye came from analysis o f the
EN U -induced S e ^ m m utation (Hill et al., 1991). A lthough allelism of this m uta
tion with the Small eye locus had n o t b een form ally dem onstrated, genetic m apping
suggested that it m apped to the ap p ro p riate region o f chrom osom e 2, an d the
phenotype was virtually indistinguishable from that of Sey. RNA-PCR analysis of
Pax-6 in Sey revealed an unusually large Pax-6 transcript which was shown upon
sequencing to include an intron. Sequencing o f the genom ic DNA of SeyNeu
showed that the invariant G residue o f the splice d o n o r site was m utated, thus p re
venting intron excision from the mRNA. T he predicted effect o f this m utation
would be to term inate translation at a stop codon within the intron and thus to
truncate the protein before the serine-threonine rich C-term inal dom ain (Figure
5 b ). A lthough the function o f this dom ain is no t known, it is sim ilar in am ino acid
com position to the trans-activation dom ain o f the transcriptional regulators Oct-
1 and Oct-2 (Tanaka an d H err, 1990). Analysis o f Small rye alleles therefore provid
ed strong evidence for the involvem ent of Pax-6 in eye developm ent.

H um an m utations

T he analysis o f hum an aniridia m utations lagged beh in d that o f m ouse m utations

because the only m aterial available was lym phoblastoid cell lines which do no t
express PAX6 at high levels. Initially the hum an PAX6 gene was analysed at the
genom ic level on an exon-by-exon basis which first necessitated the elucidation of
the intron-exon structure o f the gene. T he genom ic organisation o f PAX6 was
found to be com plex with at least 10 exons spanning ab o u t 25kb o f the genom e
(Figure 5a; Jo rd a n et al., 1992). T h e m utation analysis strategy involved PCR-
amplifying a given exon from the genom ic DNA of a panel o f patients an d then
resolving the PCR p ro d u ct on a H ydrolink polyacrylam ide gel, w here h etero d u
plexes caused by annealing o f wild-type products with m u tan t products have an
altered mobility relative to hom oduplexes. This m eth o d can be used because
each p atien t is expected to have a norm al as well as a m utated copy o f PAX6 so
456 ISABEL HANSON ET AL.

(b) Paired box Homeo box S-T-rich domain

100bp

Sey (Mouse)

S e y '* 1(Mouse)

HZAMT (Human)

RUBAI (Human)

Figure 5 (a) P re lim in a ry g en o m ic o rg a n iz a tio n o f th e h u m a n P AX 6 g en e. E xons a re re p re

se n te d by rectan g les, w ith reg io n s e n c o d in g th e p a ire d d o m a in show n in black a n d reg io n s
e n c o d in g th e h o m e o d o m a in show n stip p led . Since th e n u m b e r o f ex o n s e n c o d in g th e 5' u n
tra n sla te d re g io n is u n c e rta in , as is th e p o sitio n o f th e tra n sla tio n in itia tio n c o d o n , know n ex
o n s a re arb itra rily d e sig n a te d by letters. E xon C e n c o d es a 42bp se q u e n c e w hich is
alternatively sp liced in to th e p a ire d box. E xon J e n c o d e s th e te rm in a tio n c o d o n (vertical
line) a n d th e 3' u n tra n sla te d re g io n (Jo rd an e t al., 1992).
(b) P A X 6/P ax-6m u ta tio n s in Small ^ a l l e l e s a n d h u m a n a n irid ia p atien ts. At th e to p is show n
th e stru c tu re o f wild-type PAX6/Pax-6 cD NA with th e p a ire d b o x (black), th e paired -ty p e h o
m eo b o x (stip p led ) a n d th e re g io n e n c o d in g th e se rin e -th re o n in e (S-T) rich d o m a in (T on et
al., 1991; W alth er a n d G russ, 1991). Below are illu strate d th e p re d ic te d effects o n th e cDNA
stru c tu re o f th e know n Pax-6 (Sm all eye) a n d PAX6 (an irid ia) m u ta tio n s (H ill e t al., 1991; J o r
d an e t al., 1992). H a tc h e d boxes re p re s e n t a lte re d o p e n re a d in g fram es b ey o n d th e m u ta tio n
site.
 ANIRIDIA 457

that m ism atched heteroduplexes are form ed when the region containing the
m utation is am plified. This approach was used successfully to d etect h etero d u
plexes of anom alous mobility in the PCR p ro d u c t derived from exon H o f one
aniridia patient. N ucleotide sequencing o f the PCR pro d u ct revealed a two nucle
otide insertion which changed the reading fram e and would be predicted to
cause chain term ination ju s t distal to the hom eodom ain (Patient HZAMT, Figure
5b;Jo rd a n e ta l., 1992).
A second strategy which has b een used to find PAX6 m utations is th at of nested
RNA-PCR, which is carried out essentially as described for the Small eye analysis but
with extra rounds o f PCR am plification to com pensate for the very low break
th ro u g h levels o f PAXdRNA prod u ctio n in the lym phoblastoid cell lines which
are the only available m aterial from the patients. Reverse transcription is followed
by two rounds o f PCR am plification using two ,PAX6-specific p rim er pairs, o n e nest
ed inside the o th er (Roberts et al., 1991). W hen this m ethod was applied to the
hom eobox region o f the gene in a panel of patients, an RNA-PCR product o f re
duced size was detected in addition to the norm al p ro d u ct in one patient. Se
quencing revealed that the short transcript did no t contain exon G, b u t no
deletion was detectable at the genom ic level. T herefore the m utation in this pa
tien t is m ost likely a point m utation o r sub-visible deletion at the DNA level, prob
ably at or n ea r to a splice site, which causes exon G to be spliced ou t o f the m ature
PAX6 mRNA product. T he predicted protein p ro d u ct o f this m u tan t allele would
term inate after the first third o f the hom eodom ain (Patient RUBAI, Figure 5b;
Jo rd a n et al., 1992). These analyses strongly suggest that PAX6 m utations cause
aniridia in at least som e patients.

CLINICAL SIGNIFICANCE

Im m ediate Clinical Applications

These early results o f PAX6 m utation studies open up the possibility o f genetic
counselling an d prenatal diagnosis, bo th o f which are often requested by aniridia
patients. T he frequency and spectrum of PAX6 m utations can now be assessed in
a large cohort o f patients. T he existence of w ell-docum ented deletion aniridia
patients whose phenotypes are clinically indistinguishable from familial cases,
who would be predicted to have p o in t m utations, suggests th at aniridia results
from .PAX6 haplo-insufficiency i.e. loss o f one active copy o f the PA X 6gene. Since
many different m utations could lead to loss of gene function, we expect to see a
wide spectrum of m utations both within the coding region an d in control regions
outside the transcribed sequence. This implies that each sporadic p atien t o r fam
ily will have to be assessed de novo before genetic counselling can be given. T he
way is already open for m utation analysis at the RNA level which will reveal point
m utations, small insertions o r deletions, or ab e rra n t splicing, all of which can
458 ISABEL HANSON ET AL.

lead to deleterious changes in am ino acid sequence (Figure 5b; Hill et al., 1991;
Jo rd a n et al., 1992).
In patients w here RNA-PCR analysis does n o t reveal a m utation, it is possible
th at one copy o f the PAX6 gene is n o t being expressed. This could result from a
deletion o f all o r p art o f the PAX6 gene or a m utation in a control region. N othing
is known at present about the elem ents which control PAX6 expression bu t these
will have to be defined, m ost probably in the m ouse, before m utation analysis is
possible. Loss o f expression o f one copy of PAX6 could be verified by searching in
the genom ic DNA of patients for heterozygosity at a transcribed site an d then
showing at the RNA level that only one allele is expressed. A fu rth e r possibility in
patients w here no PAX6 m utation is detectable is that they have m utations in a sec
o n d aniridia gene. T he existence o f a second gene is suggested by the t(4; 11)
breakpoint, which is associated with aniridia bu t which does n o t physically disrupt
PAX6 (Gessler, Simola and Bruns, 1989; Ton etal., 1991;Jordan etal., 1992). How
ever the aniridia in this family could ultim ately be explained by a position effect
w here the translocated portio n o f chrom osom e 4 silences PAX6 at a distance, p er
haps by altering chrom atin structure. Again, expressed heterozygosity would be
valuable in d eterm in in g w hether the copy o f PAX6 on the translocation chrom o
some is transcribed.
A nother clinically significant aspect of this work is the assessm ent o f the risk of
W ilm s tu m o u r in new borns with sporadic aniridia. In contrast to aniridia, both
Wilms' tu m o u r an d the associated g en itourinary abnorm alities show red u ced p en
etrance so that even sporadic cases of isolated aniridia may carry a deletion, pu t
ting them at risk o f developing nephroblastom a. D uring the m apping o f the
WAGR region, cosm id clones were isolated for WT1, PAX6 an d two intervening
m arkers (Fantes et al., 1992). These can be used in fluorescent in situ hybridisation
(FISH) analysis o f m itotic chrom osom es to d eterm in e w hether o r n o t a deletion
is present. If a deletion is indicated, assessm ent o f incipient Wilms' tu m o u r by re
nal u ltrasound should be carried ou t at regular intervals. We have validated this
technique retrospectively in several subm icroscopic WAGR deletions. In one u n
expected an d presum ably very rare case a m o th er with sporadic isolated aniridia
gave birth to a son with aniridia who later died as a result o f Wilms' tu m o u r which
arose in a horseshoe kidney. FISH analysis showed that both were deleted for WT1
and PAX6 (Fantes et al., 1992).

A role fo r PAX6 m utations in other eye disorders?

A great deal of phenotypic variability has been observed am ong aniridia patients,
even within families who are presum ably segregating for the same m utation (H itt
n er et al., 1980). This variability is particularly striking in the Small eye m ouse
where there are m arked differences in eye phenotype even within the same
in b red strain (Roberts, 1967). T he phenotypic heterogeneity is o f clinical im por
tance in assessing familial patterns o f inheritance an d the associated genetic risks
because some individuals are so mildly affected th at they may be m isdiagnosed
(H ittn er et al., 1980; Lyons et al., 1992). In view o f the heterogeneity am ongst
 ANIRIDIA 459

aniridia patients it is possible that PAX6 m utations are also responsible for o th er
disorders which are usually considered to be clinically distinct but may be part of
a wide phenotypic spectrum that encom passes aniridia. These include atypical
colobom a (Figure 1), early onset cataracts an d a range of an terio r segm ent
defects such as Peters' anomaly, all o f which have been observed in families segre
gating for aniridia an d which are consistent with the pattern o f PAX6 expression
in the eye (H ittn er et al., 1980; B eaucham p, 1978). It will be interesting to see
w hether PAX6 is m utated in these disorders and, if so, how the spectrum of m uta
tions com pares to that defined for classical aniridia.
In the majority o f aniridia patients n o neurological or brain abnorm alities have
been noted. It was therefore som ew hat surprising to find th at PAX6 is expressed
n o t only in the developing eye b u t also in the brain and neural tube (Ton et al.,
1991; W alther an d Gruss, 1991). However in cases of the rare Gillespie syndrom e,
aniridia is associated with cerebellar ataxia. M ight this phenotype also be account
ed for by m utation in PAX6? It may be significant in this respect th at in addition
to the eye the cerebellum is the m ajor expressing tissue in adult mice (Ton, Miwa
and Saunders, 1992).

THE ROLE OF PAX6 IN EYE DEVELOPMENT

Identifying the affected tissue

T he first attem pts to u n d erstan d the m olecular pathology o f aniridia began by

trying to identify which tissues are prim arily affected. To decide this, it was neces
sary to apply what was known ab o u t the em bryology of the eye. By taking the iris
hypoplasia as the typical feature o f the disorder an d identifying the tissues
involved in its em bryogenesis, two m odels were derived. T he two tissues believed
to contribute to the form ation o f the iris were the n eu ro ecto d erm and the m eso
derm , and the two m odels proposed were the n eu ro ecto d erm al and the m esoder
mal, the im plication being a prim ary defect in one o r o th er (M ann, 1957). The
posterior layer o f the iris arises from the an terio r rim o f the bilayered optic cup
which develops from n eu ro ecto d erm , while the an terio r layer is form ed from
what was then tho u g h t to be m esoderm bu t is now known to be mainly neural
crest-derived m esectoderm or ectom esenchym e (Johnston et al., 1979; Beau
cham p and Knepper, 1984). Looking at the affected ocular tissues th ro u g h o u t
the eye it becom es clear that they all receive an ectoderm al contribution. T he
corneal epithelium an d lens develop from surface ectoderm while p a rt o f the iris
and ciliary body, m ost o f the optic nerve and all o f the m acula develop from n eu
roectoderm . A lthough the surface ectoderm an d the n eu roectoderm can be dis
tinguished as two separate tissues, in the early stages o f developm ent (m id to late
gastrula through to n eural tube stage) they lie in very close proxim ity at a tim e
w hen the optic sulcus is first ap p earin g in the n eu ro ecto d erm and w hen the adja
cent surface ectoderm is first acquiring a lens form ing bias (Couly an d Le
460 ISABEL HANSON ET AL,

D ourain, 1988; Servetnick and Grainger, 1991). T he optic sulcus is the first m or
phological indication of the em ergence o f the prim ordial eye from the optic pla
code on the surface of the developing neural plate. It extends by invagination to
form the optic stalk, the en d o f which th en swells to develop the optic vesicle and
finally invaginates to produce the optic cup. T he posterior layer of the iris, the
epithelial layers o f the ciliary body and the whole o f the n eu ro retin a all develop
from the optic cup. T he surface ectoderm proceeds through acquisition o f a lens
form ing bias to specification of the lens placode. O n contact with the optic vesi
cle this placode invaginates to form a lens vesicle which eventually separates from
the surface ectoderm and moves into the optic cup which is form ing sim ulta
neously. T he corneal epithelium will form from the rem ains of the lens placode
(O'Rahilly, 1966; Kaufm an, 1979). It is therefore quite conceivable th at the pri
m ary defect in aniridia lies entirely within ectoderm and this idea is su p p o rted by
the discovery th at PAX6 is expressed in the rim of the optic cup, the developing
n euroretina, the lens and the corneal epithelium . It rem ains to be d eterm in ed
ju st what PAX6 is doing in this respect.

PAX6 affects form ation o f the lens

It is tem pting to speculate that PAX6 is som ehow involved in the induction o f the
lens. T he absence o f any functional Pax-6 transcripts i.e. in Sey/Sey m ice, results
in the absence o f lens form ation in the presence o f a correctly positioned optic
vesicle which fails to form an optic cup an d undergoes involution (H ogan et al.,
1986). Existing data show that lens vesicles can form in the absence o f the optic
vesicle an d that the optic vesicle is no t capable of producing lenses from n o n
specified surface ectoderm (M uthukkaruppan, 1965; Grainger, H enry and H en d
erson, 1988). These findings contradict the results o f earlier induction experi
m ents. T he m ost recent experim ents in this field point to the existence o f a
crucial interaction between the neural plate and the adjacent surface ectoderm
in o rd e r to achieve lens placode specification (H enry and Grainger, 1990). It is
interesting to note that m any of the so-called free lenses seen in earlier tissue
m anipulation experim ents i.e. lenses occurring in the absence o f an optic vesicle,
were found in association with the developing nose, since we now know that both
the olfactory bulb and the nasal placode express Pax-6 during developm ent
(Saha, Span and Grainger, 1989; Ton et al., 1991; W alther and Gruss, 1991).
These findings are evidence o f a critical role for Pax-6 in the form ation o f the
lens, perhaps by the early specification o f the lens placode. To u n derstand this
and o th er roles o f Pax-6 will require additional experim ents using both tradi
tional tissue m anipulation m ethods and m o d ern m olecular techniques. By assem
bling conjugates o f tissues from different stages of developm ent an d with
d ifferent degrees o f Pax-6 expression, it may be possible to define developm ental
windows an d tissue interactions. Somatic cell chim eras in which m ixed popula
tions o f Pax-6 expressing an d non-expressing cells are ju x tap o sed in the develop
ing em bryo will tell us w hether or no t the gene acts cell autonom ously o r if its
 ANIRIDIA 461

function can be rescued by the presence of adjacent norm al cells (H atta et al.,
1991). T he free lens evidence argues for the latter. It is to be h o p ed th at such
experim ents will also provide valuable insights into the m olecular events which
lie beh in d the norm al developm ent o f the eye.

W hat controls PAX6 and what are its targets?

A lthough aniridia can have serious consequences for affected individuals in

term s o f visual disability, from a developm ental point o f view the phenotype is
surprisingly subtle an d localized given that the dom ain o f expression of the PAX6
gene includes the developing eye, nose, forebrain and neural tube (W alther and
Gruss, 1991). T he phenotypes o f an irid ia patients and Sey/+ mice imply that the
eye is especially sensitive to a reduction in PAX6 levels and th at there is a thresh
old o f protein activity which m ust be exceeded for norm al developm ent to pro
ceed (Hill and van H eyningen, 1992). In Sey/Sey mice, which com pletely lack wild
type Pax-6 activity, the structures derived from the optic and nasal placodes fail to
m ature, but the brain and spinal colum n appear to be norm al (Hill et al., 1991).
It may be that neurological defects are present b u t have been overlooked because
they are so m uch less obvious than the abnorm al facial phenotype. Alternatively
there may be functional redundancy o f Pax gene expression such that absence o f
Pax-6 is com pensated for by o th er m em bers of the Pax family which are expressed
in overlapping dom ains d u rin g neural tube developm ent (Deutsch and Gruss,
1991).
If, as suggested above, Pax-6 p ro tein activity m ust exceed certain threshold lev
els to initiate developm ental pathways, the expression o f the Pax-6 gene m ust be
tightly regulated. Pax-6 contains two conserved DNA binding dom ains and is most
likely a transcriptional regulator which will itself control the expression of down
stream target genes. At present, n o th in g is known about these interacting genes
an d gene products, b u t one approach to identifying them is to study the expres
sion o f o th er developm ental regulators known to be expressed in overlapping tem
poral an d spatial dom ains. Am ong these are Hox-7 and Hox-8 (msh-like hom eobox
genes believed to define dom ains within the developing eye, M onaghan et al.,
1991); Emx-1 a n d Emx-2 (hom eobox genes related to the Drosophila empty spiracles
gene, Sim eone et al., 1992); XANF-1 (a Xenopits hom eobox gene expressed in the
an terio r neural folds, Zaraisky et al., 1992); an d the motch gene (encoding a trans
m em brane protein believed to be involved in cell-cell signalling, Del Amo et al.,
1992). In addition, it will be very interesting to look for alterations in Pax-6 expres
sion in m ice with o th er eye m utations such as aphakia (ak), colobom a (Cm), mi
crophthalm ia defective iris (midl), eye lens obsolescence (Elo), dysgenic lens (dl),
and cataract an d small eye (Cts) (G reen, 1989). By looking at the expression of
these genes in the Small eye m ouse a n d the expression of Pax-6 in these m utants it
may be possible to d etect a hierarchy o f interactions. A nother m ore d irect m ethod
to identify dow nstream targets involves incubating the Pax-6 p rotein with linkered
462 ISABEL HANSON ET AL.

genom ic DNA fragm ents an d PCR-amplifying the b o und fragm ents to create a li
brary o f Pax-6 recognition sequences (Kinzler and Vogelstein, 1989).
A lternative splice form s o f transcriptional regulators may be im p o rtan t in the
control o f target genes. For exam ple, alternative splice form s affecting the struc
ture o f the zinc finger DNA binding region o f WT1, the Wilms' tum our predispo
sition gene, can alter its binding specificity, thus raising the possibility of
in teraction with m ore than one physiological dow nstream target (Bickm ore et al.,
1992). A lternative splicing in the paired box o f PAX6, which results in the inser
tion o f an additional fourteen am ino acids into this DNA binding dom ain, has
been dem onstrated in m ouse, zebrafish an d m an, b u t its functional significance
rem ains to be dem onstrated (W alther an d Gruss, 1991;Jo rd a n etal., 1992;Puschel
et al., 1992). RNA in situ hybridisation using probes specific for the alternate splice
form s may reveal spatial or tem poral heterogeneity in their expression.

CONCLUSION

T he isolation o f the PAX6 gene and the subsequent discovery o f m utations in

PAX6 in aniridia patients illustrates the power of positional cloning techniques to
identify the gene underlying a hum an disease. A lthough the prospects o f devel
oping a treatm en t for aniridia lie in the distant future there are many im m ediate
benefits from the identification o f the aniridia gene. G enetic counselling o f anir
idia patients will now include the possibility o f pre-natal diagnosis, while in atypi
cal cases genetic counselling may be available for the first time. In addition, the
elucidation o f the role o f PAX6 in aniridia and Small eye, the m ouse m odel, will
yield fascinating insights into the norm al developm ent o f the eye.

NOTE:

T he com plete genom ic organisation o f the hum an PAX6 gene has now been
d ed u ced (Glaser, W alton an d Maas, 1992). Intragenic PAX6 m utations have been
characterised in eight additional aniridia patients (Glaser, W alton and Maas,
1992; H anson e ta l., 1993)

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 20. MITOCHONDRIAL DNA MUTATIONS
AND THE EYE

MICHAEL D. BROWN, MARIE T. LOTT AND DOUGLAS C. WALLACE*

Department of Genetics and Molecular Medicine, Emory University School of Medicine, 403
Dental Building, 1462 Clifton Road, Atlanta, GA 30322, USA

O phthalm ic dysfunction is one o f the most comm on clinical observations associated with mitochondri
al DNA (mtDNA)-based hum an disease. Ocular pathology in these diseases includes optic atrophy, pig
mentary retinopathy, retrochiasmal visual loss, and myopathy involving the extraocular muscles and
the eyelids. The wide spectrum of symptoms involving the eye demonstrates the dependence of the
ophthalmic system on mitochondrial energy production.

OXIDATIVE PHOSPHORYLATION (OXPHOS)

All tissues d ep e n d on chem ical energy in the form o f adenosine triphosphate

(ATP) for cell function an d m aintenance. T he vast majority o f cellular ATP is
produced by the m itochondrial oxidative phosphorylation (OXPHOS) system
which is com prised o f five m ulti-subunit enzyme com plexes (Com plex I-V)
within the m itochondrion in n er m em brane. Four of these enzyme com plexes,
N A D H :ubiquinone oxidoreductase (Com plex I), succinate:ubiquinone oxi-
doreductase (C om plex II), ubiquinokcytochrom e c oxidoreductase (Com plex
HI), an d cytochrom e c oxidoreductase (C om plex IV), are key com ponents o f the
electron transport chain which transfers electrons from NADH + H + and FADH2
to oxygen and concurrently translocates protons from the m itochondrial m atrix
to the in n er m em brane space at Com plexes I, III an d IV. Thus, the process o f
electron transfer generates an electrochem ical gradient across the m itochondrial
in n er m em brane which is utilized by the fifth enzyme com plex, ATP synthase
(Com plex V), to synthesize ATP in the m atrix by condensation o f ADP and inor
ganic phosphate. In the rate lim iting step, m atrix ATP is exchanged for cytosolic
ADP by the nuclear-encoded adenine nucleotide translocator.

MITOCHONDRIAL DNA AND OXPHOS

M itochondrial DNA is essential for aerobic m etabolism as it codes for 37

OXPHOS genes including 13 respiratory chain polypeptides, two rRNAs and 22
tRNAs (Figure 1). F unctional respiratory chains are assem bled from both m ito
chondrial and nuclear gene products: Com plex I contains seven (ND1, 2, 3, 4, 4L,

^ C o rre s p o n d in g a u th o r
470 MICHAEL D. BROWN ET AL.

5 and 6) m tDNA-encoded subunits o u t o f approxim ately 39 total polypeptides;

C om plex III contains one (cytochrom e b) m tDNA-encoded subunit o u t of
approxim ately 10 total polypeptides; Com plex IV contains three (COI, II an d III)
mtDNA-encoded subunits ou t of 13 total polypeptides; an d C om plex V (ATP syn
thase) contains two (ATPase 6 and 8) mtDNA-encoded subunits o u t o f 12 total
polypeptides (Wallace, 1992; Shoffner and Wallace, 1990). Thus, ATP production
is coordinately controlled by two physically distinct genetic systems.

D-Loop Region
12s
rRNA
0/16*#

r 16s
rRNA

Complex I genes
(NADH dehydrogenase)

Complex III genes

(ubk|ulno!:cytochronie c oxidoreductase)

Complex IV genes
(cytochrome c oxidase)

Complex V genes
(ATP Synthase)

Ribosomal RNA genes

Transfer RNA genes

;ND4L

KCOIIIStgS
W M r

K ATPase8

Figure 1 T h e h u m a n m ito c h o n d ria l DNA. T h e H (heavy)-strand is show n as th ick e r o u te r

circle. B oth o rig in s o f DNA rep lic a tio n (Oj^ a n d O l ) a n d b o th m a jo r p ro m o te rs (PH a n d PL)
a re in d ica ted . tRNAs d e p ic te d inside th e circ u la r g e n o m e are tra n sc rib e d fro m th e L (light)-
stra n d , as is th e N D 6 gene.
 MITOCHONDRIAL DNA MUTATIONS AND THE EYE 471

Structurally, the hum an mtDNA is a closed, circular m olecule o f 16569 nucle

otide pairs (np) located within the m itochondrial m atrix (A nderson et al., 1981).
It is norm ally present in thousands o f copies p er cell an d com prises roughly 1% of
total cellular DNA. T he two strands o f the mtDNA show a non-random nucleotide
distribution as the heavy strand (H -strand) contains a h ig h er p ro p o rtio n of gua
nine nucleotides com pared to the com plem entary light strand (L-strand) (Figure
1). T he H -strand contains the m ajority o f the coding inform ation as 12 protein-
coding, 14 tRNA, an d both rRNA genes are found on this strand of the m itochon
drial chrom osom e. T he hum an mtDNA is organized as a highly com pact genom e
as it contains no introns or repetitive DNA spacer sequence elem ents characteris
tic of nuclear DNA (nDNA) (Attardi, 1985). A single m ajor non-coding region, the
displacem ent loop (D-loop), is p resen t in the m itochondrial genom e and contains
m any o f the sequence elem ents essential for mtDNA replication and transcription.
MtDNA replication is a bidirectional, asynchronous process involving two ori
gins separated by two-thirds o f the genom e (Clayton, 1991). H -strand synthesis be
gins replication and is initiated in the D-loop by an RNA prim er p roduced by the
L-strand prom oter. H -strand replication proceeds clockwise aro u n d the circular
m olecule, displacing the original H -strand, until the L-strand origin is exposed.
The L-strand origin consists o f a conserved stem-loop structure w'ithin a tRNA-rich
region o f the mtDNA (Wong an d Clayton, 1985). L-strand replication then pro
ceeds counter-clockwise, using the displaced original Ll-strand as a tem plate. MtD
NA replication occurs independently o f nuclear DNA replication.
MtDNA transcription occurs bidirectionally from two m ajor prom oters which
initiate the synthesis o f large, polycistronic RNAs from both strands o f the mtDNA
(Clayton, 1991). These prom oters, PL (L-strand prom oter) and PH (H -strand p ro
m oter) , are separated by approxim ately 150 nucleotide pairs in the D-loop region
and are in d ep en d e n t transcriptional units. M ature mRNAs an d rRNAs (12S and
16S) are pro d u ced by RNAse-P-like processing o f (RNAs, which p unctuate the p ro
tein-coding an d rRNA genes (Figure 1). M itochondrial mRNAs are distinct from
nuclear-derived mRNAs in that they are not capped at the 5' en d n o r do they con
tain 5' untranslated sequences (Attardi, 1985; M ontoya et al., 1981). Further, m ost
m itochondrial mRNAs lack 3' un tran slated sequences except for polyadenylation,
which frequently com pletes translational term ination codons (UUA, UGA, UAA )
through the addition o f 3'-term inal ad e n in e nucleotides (Ojala et al., 1981). Ex
ceptions are the COI, COII, and the ND5 mRNAs which encode com plete term i
nation codons and have 3' un tran slated sequence prior to polyadenylation
(Attardi, 1985; Ojala et al., 1981).
M itochondrial mRNAs are translated within the m atrix using m tDNA-encoded
ribosom al RNAs (12S an d 16S) and tRNAs. T he universal genetic code has been
slightly m odified in m itochondria, preventing both efficient cytosolic translation
of m itochondrial mRNAs an d gene transfer betw een the organelle an d the nucle
us (Anderson et al., 1981; Wallace, 1982). For exam ple, in hum an m itochondria,
the UGA codon is used for tryptophan (instead o f a term ination codon), the AGA
472 MICHAEL D. BROWN ET AL.

and AGG codons are term ination codons (instead o f arginine codons), a n d the
AUA codon is used for m ethionine (instead of isoleucine).

THE UNIQUE GENETICS OF MTDNA

Five features o f the hum an mtDNA distinguish mtDNA genetics from nuclear
genetics (Wallace 1992a,b). First, m itochondria are m aternally in h erited (Giles
et al. 1980; Case and Wallace 1981). At fertilization, the egg contains greater than
105 m itochondria while the sperm contribution is less than 0.1% o f zygote
mtDNA (Gyllensten et al., 1991). Thus, mtDNAs are in h erited m other-to-daugh
ter, m eaning that pedigrees o f mtDNA diseases with m ultiple affected family
m em bers will show exclusive m aternal transm ission o f the phenotype. Second,
mtDNAs random ly segregate to d au g h ter cells u p o n mitosis o r meiosis (replica
tive segregation) (Wallace, 1986). T he high copy nu m b er o f mtDNA genom es
allows for the presence of both m u tan t an d wild-type mtDNAs (heteroplasm y)
within an organelle or cell. At cell division, the p ro p o rtio n o f m u tan t an d wild-
type mtDNAs stochastically segregate to d au g h ter cells and, over time, the p o p u
lation o f mtDNAs within a cell can drift towards eith er p u re (hom oplasm y)
m u tan t or wild-type genom es. Since the m agnitude o f an OXPHOS deficiency is
frequently co rrelated with the fraction o f m u tan t mtDNAs within a tissue, h etero
plasmy due to replicative segregation is an im portant feature o f mtDNA-based
disease. T hird, phenotypic expression o f an mtDNA defect is d e p e n d e n t on a tis
sue-specific OXPHOS threshold. Each tissue in the body depends on OXPHOS
for energy production to a d ifferent extent. For exam ple, central nervous system
tissue and skeletal muscle are am ong the m ost highly oxidative tissues in the body
and therefore are highly reliant on OXPHOS. Disease results when the ATP-gen-
erating capacity o f an organ drops below a m inim um OXPHOS threshold neces
sary for tissue m aintenance. Thus, clinical m anifestation o f an OXPHO S defect is
a p ro d u ct o f the n ature o f the mtDNA m utation, the fraction o f m u tan t mtDNA
genom es within a tissue, and the O XPHO S requirem ents of the affected tissue.
F ourth, ATP production within a tissue naturally declines with age (T rounce et
al., 1989; Yen et al., 1989). This process parallels an increase in som atic mtDNA
dam age in post-m itotic tissue with age (L innane et al., 1989; Cortopassi an d Arn-
heim , 1992; Corral-Debrinski et al., 1992). Oxygen radicals are produced at rela
tively high concentrations at the m itochondrial in n e r m em brane as a result of
respiratory chain redox activity, causing the mtDNA to accum ulate oxidative
dam age at a rate 16 times higher than nuclear DNA (R ichter et al., 1988). Thus,
the age-related decrease o f OXPHOS capacity may be co rrelated with the age-
related increase in mtDNA dam age. This p h en o m en o n would help explain why
some mtDNA diseases are expressed in mid-to-late life an d progress with age.
Fifth, new mtDNA m utations are fixed at a rate 10-20 times hig h er than are
m utations occurring in nuclear-encoded OXPHO S genes (N eckelm ann et al.,
1987; Wallace et al., 1987). Because mtDNA OXPHOS genes are essential for aer
obic m etabolism and subject to equivalent selective pressure as nuclear-encoded
OXPHOS genes, deleterious mtDNA m utations are expected to arise with signifi
cant frequency.
 M ITOCHONDRIAL DNA MUTATIONS AND THE EYE 473

MtDNA MUTATIONS AND OPHTHALMIC DISEASE

Because the retina, optic nerve and extraocular m uscles controlling eye move
m en t are am ong the m ost A T P-dependent tissues in the body, it is n o t surprising
that those mtDNA p o in t m utations an d deletio n s/d u p licatio n s which im pair
OXPHOS capacity often result in o cular dysfunction. T he m ost com m on o p h
thalm ic m anifestations o f mtDNA diseases include optic neuropathy, pigm entary
retinopathy, an d ophthalm oplegia with ptosis (Table 1).

Table 1 M tD N A m u ta tio n s a n d eye disease

MtDNA
Mutation Phenotype Ocular involvement

I. Point Mutations
A. Missense Mutations
1. Electron transport chain LHON Optic neuropathy
(Complexes I-IV)
2. ATP synthase N ARP/Leighs Pigmentary retinopathy
(Complex V) Disease
B. tRNA Mutations
1. Lysine MERRF Occasional optic atrophy, pigmentary
retinopathy
2. Leucine MELAS Occasional optic atrophy, pigmentary
retinopathy
3. Leucine MMC None
4. Leucine MM None
5. Isoleucine FICM None

II. Rearrangements
A. Deletions
1. Spontaneous CPEO O phthalm oplegia/Ptosis, occasional
pigmentary retinopathy
KSS Ophthalm oplegia/Ptosis, pigmtary
retinopathy
Pearsons Can progress to KSS
2. Heritable KSSa ophthalm oplegia/ptosis, bilateral
cataract formation
Diabetes plusb None
B. Duplications Variable Ophthalm oplegia/Ptosis, pigmentary
retinopathy

a Autosomal dom inant transmission

b Maternally inherited
LHON, Leber's hereditary optic neuropathy; NARP, neurogenic muscle weakness, ataxia and retinitis
pigmentosa; MERRF, myoclonic epilepsy and ragged red fiber disease; MELAS, mitochondrial enceph
alopathy, lactic acidosis and stroke-like symptoms; MMC, mitochondrial myopathy and cardiomyopa
thy; MM, mitochondrial myopathy; FICM, fatal infantile cardiomyopathy; CPEO, chronic progressive
external ophthalmoplegia; KSS, Kearns-Sayre syndrome; Pearson's, Pearson's m arrow /pancreas syn
drome; Diabetes plus, diabetes mellitus and hearing loss.
474 MICHAEL D. BROWN ET AL.

MtDNA P oint M utations and Ophthalm ic Disease

Pathogenic mtDNA p o in t m utations have been found in both tRNA genes and
protein-coding genes (Wallace, 1992a,b). T ransfer RNA m utations are associated
with severe m itochondrial m yopathies such as Myoclonic Epilepsy an d Ragged-
Red Fiber disease (MERRF1, an A to G transition at n p 8344 an d a T to C transition
at n p 8356 in tRNALys) (Shoffner et al., 1990; Zeviani et al., 1992); M itochondrial
Encephalom yopathy, Lactic Acidosis an d Stroke-like symptoms, (MELAS, an A to
G transition at nucleotide pair 3243 in tRNALeu) (Goto et al., 1990a); M itochon
drial Myopathy and Cardiom yopathy (MMC, an A to G transition at nucleotide
p air 3260 in tRNALeu) (Zeviani et al., 1991); an d Fatal Infantile Cardiom yopathy
(FICM, an A to G transition at nucleotide pair 4317 in tRNAIie) (Tanaka et al.,
1990). T ransfer RNA m utations usually alter nucleotides conserved betw een vari
ous species an d result in a protein synthesis defect. Despite the heterogeneity of
neuro-m uscular symptoms associated with these diseases, ophthalm ic involve
m en t is relatively uncom m on an d generally lim ited to occasional optic atrophy or
pigm entary retinopathy (MERRF an d MELAS) or retrochiasm al visual loss
(MELAS).
In contrast, diseases attrib u ted to mtDNA missense m utations feature o phthalm
ic involvem ent (Table 1). Both Leber's H ereditary O ptic N europathy (LHON)
an d N eurogenic Muscle Weakness, Ataxia a n d Retinitis Pigm entosa (NARP), are
caused by mtDNA p o in t m utations in OXPHO S polypeptides.

Leber's Hereditary Optic Neuropathy (LHON)

T he 1988 discovery o f an mtDNA m utation associated with a large n u m b er of

LHON pedigrees dem onstrated that an mtDNA p o in t m utation could cause
h u m an disease (Wallace et al., 1988). LH O N is a disorder o f young adults that
results in bilateral loss o f central vision due to optic nerve atrophy (Newman,
1991). T he average age o f onset in LHON patients is 27 years o f age a n d disease
progression is rapid, as blindness occurs in one eye an d is usually followed by loss
o f vision in the o th er within three m onths time (Newman, Lott, an d Wallace,
1991). Loss o f vision is usually the only clinical m anifestation o f LLION, although
cardiac abnorm alities (preexcitation syndrom es, prolonged QT internal) or
o th er neurological symptoms do ap p ear in occasional LH O N pedigrees (New
m an and Wallace, 1990; Newman, 1991; O rtiz et al., 1992).
T hree im p o rtan t features o f LH O N expression an d inheritance are evident
from pedigree analysis. First, pedigrees with m ultiple affected individuals show
strict m aternal inheritance (Figure 2), an inheritance p attern consistent with the
cytoplasmic transm ission o f the mtDNA. Second, disease expression is variable as
n o t all individuals harboring a hom oplasm ic LHON m utation m anifest clinical
symptoms and singleton cases are n o t uncom m on. T hird, males are m uch m ore
frequently affected than females as up to 80-90% o f Caucasian LH O N patients are
 M ITOCHONDRIAL DNA MUTATIONS AND THE EYE 475

AAA -AiA A A AGE 24

<TAT AG E 24

III
AGE 15

IV
AG E 17

Figure 2 M a tern ally -in h erited L H O N p e d ig re e . P ed ig ree shows in c o m p le te p e n e tra n c e a n d

m ale p re d ile c tio n o fte n se n n in L H O N fam ilies. A ge o f o n se t is in d ic a te d . Individuals d e
p ic te d have th e 14484 a n d 15257 L H O N m u ta tio n s a n d are h o m o p lasm ic.

male. Thus, although mtDNA m utations have a prim ary etiological role in the dis
ease, o th er factors m ust influence LHON expression. Such factors likely include
additional genetic determ inants, sex-limiting physiological determ inants, and u n
specified environm ental determ inants.
T he genetics o f LH O N have proven to be com plex and heterogeneous as twelve
mtDNA p o in t m utations have now b een associated with LHON (Brown et al.,
1992a; Johns, N eufeld and Park, 1992) (Table 2 ). Eleven o f these are missense m u
tations in genes contributing to respiratory Com plexes I and III. T he twelfth m u
tation alters the translational term ination codon o f the gene encoding
cytochrom e c oxidase subunit I (C om plex IV). These m utations appear to im pair
respiratory chain function to d ifferent degrees, a variation reflected in the risk
each m utation represents in causing LHON. Risk can be estim ated on the basis of
four criteria: (1) n ature o f the am ino acid change, (2) w hether o r n o t a m utation
can cause blindness by itself, (3) the expressivity o f the disease in LHON pedi
grees, an d (4) the pen etran ce o f the m utation in the general population. Amino
acid substitutions are eith er conservative o r radical replacem ents and, if function
ally significant, usually alter evolutionarily conserved am ino acids. Expressivity is
d eterm in ed by the p ro p o rtio n o f b lind individuals in a hom oplasm ic pedigree and
by the pro p o rtio n o f affected males within a pedigree. Since males are m ore com
m only affected than females, m ore severe LH O N m utations m ight be expected to
affect an increased p ro p o rtio n of females. T he p enetrance of a m utation in the
population involves three param eters, heteroplasm y (indicative o f a new m uta
tion), the occurrence o f a m utation on a single versus m ultiple mtDNA haplo-
types, and the frequency with which a m utation is found in unaffected controls.
Based on these criteria, m utations associated with LHON can be classified as high,
m oderate, o r low-risk LH O N m utations.
476 MICHAEL D. BROWN ET AL.
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 M ITOCHONDRIAL DNA MUTATIONS AND THE EYE 477

Hi.gh-nsk LHON mutations

Individuals harboring mtDNA m utations at nps 3460, 4160 an d 11778 are at the
greatest risk for expressing LHON. All of these m utations cause non-conservative
am ino acid substitutions a n d alter am ino acids which are conserved am ong many
species. These m utations do no t occur together in LHON pedigrees and have not
been d etected in unaffected controls, indicating that they are sufficiently delete
rious by themselves to cause blindness. Further, pedigrees harb o rin g these m uta
tions have a relatively high p ro p o rtio n o f affected individuals.
T he 4160 (ND1 gene, Com plex I) m utation appears to be the m ost severe LHON
m utation as roughly 80% o f the m em bers o f the single 4160-positive LHON p edi
gree are blind and nearly one-half o f the affected individuals are female (Howell
et al., 1991a). Atypical o f m ost LH O N pedigrees, additional neurological symp
toms such as dysarthria, ataxia, an d severe infantile encephalopathy are prom i
n e n t in this family. Further, the analysis o f OXPHO S enzyme activity from platelet
m itochondria revealed a m arked reduction in Com plex I specific activity in all
family m em bers assayed (Parker, Oley an d Parks, 1989).
The 3460 (ND1 gene, C om plex I) a n d the 11778 (ND4 gene, C om plex I) m uta
tions appear to be the n ex t m ost severe LHON nucleotide substitutions (Wallace
et al. 1988; H ouponen et al. 1991; Howell et al. 1991b). C om plete mtDNA se
quence analysis o f probands carrying eith er the 3460 m utation o r the 11778 m u
tation has revealed no o th e r mtDNA m utations which ap p ear to play a p rom inent
pathogenetic role in disease expression (Wallace et al., 1988; Brown et al., 1992a).
Also, OXPHO S biochem ical deficiencies have been detected in individuals har
boring these m utations. M ajander an d colleagues (1991) re p o rted an 80% reduc
tion in the rotenone-sensitive electron transfer activity o f Com plex I in 3460-
positive patients (M ajander et al., 1991). A com parable reduction in Com plex I
enzyme activity has n o t been observed in 11778-positive patients, however reduced
oxidation o f Com plex I-linked substrates has been detected with polarography
(Larsson et al., 1991; M ajander et al., 1991). Finally, the 11778 m utation is found
on strikingly d ifferent mtDNA genotypes, having been detected in African, Asian
an d Caucasian LHON pedigrees. Thus, this m utation has occu rred m ultiple dif
feren t times an d each occu rrence associated with LHON, providing convincing ev
idence th at the 11778 m utation is a prim ary causal LHON m utation (Singh et al.,
1989).

Moderate-risk LHON mutations

LHON m utations at nps 3394, 7444, an d 15257 appear to im part an interm ediate
risk for the expression of LHON. All th ree m utations have some characteristics of
the high-risk LHON m utations such as the radical substitution of highly con
served am ino acids and a relatively high fraction o f pedigree m em bers who
develop optic nerve atrophy. However, each m utation has been detected at a low
frequency in the unaffected control population and these LHON m utations are
478 MICHAEL D. BROWN ET AL.

frequently found linked to o th er LH O N m utations. Thus, the np 3394, 7444 and

15257 m utations may rep resen t a relatively high risk when associated with o ther
mildly pathogenic mtDNA m utations and a relatively low risk when no t associated
with the appropriate mtDNA genotype.
T he hom oplasm ic np 3394 (NDl gene, C om plex I) m utation changes a highly
conserved tyrosine to a histidine in the N -term inal en d o f the NDl polypeptide
(Brown et al., 1992a; Johns, N eufeld an d Park, 1992). This m utation has only been
fo und in association with low-risk LHON m utations. For exam ple, when proband
mtDNA from a large 3394-positive pedigree was com pletely sequenced, only the
low-risk m utations at n p 4216, 13708 an d 14484 were detected (Brown et al.,
1992a). No high-risk LHON m utation was fo u n d in the genom e. However, all af
fected m em bers of this pedigree were m ale and the m utation has been detected
in nearly 1% o f unaffected controls, indicating that the 3394 m utation may be less
severe that the 3460, 4160 an d 11778 m utations. T he predilection o f affected
males in this pedigree suggests the involvem ent o f an X-linked nuclear allele
which influences LHON expression. Such a "two-hit" m odel involving a mtDNA
m utation(s) and an X -chrom osom al m utation (s) has been proposed to partially
account for the variability o f disease expression observed am ong LHON pedigrees
(Bu and Rotter, 1991) , although linkage d ata supporting an X-linked susceptibility
locus has been equivocal (Vilkki et al., 1991; Sweeney et al., 1992). Thus, the n p
3394 m utation may be a risk-factor for LHON in association with o th er mtDNA
a n d /o r nuclear m utations.
T he hom oplasm ic 7444 (COI gene, C om plex IV) m utation is u n iq u e am ong
LH O N m utations for three reasons: (1) it alters the translation term ination codon
o f an mtDNA gene, (2) it results in the len g th en in g o f a m tDNA-encoded polypep
tide, and (3) the m utation im pairs C om plex IV activity (Brown et al., 1992c). This
n ucleotide substitution changes the AGA stop codon of the cytochrom e oxidase
su bunit I (COI) gene to an AAA lysine codon, causing a three am ino acid exten
sion of the protein. This structural alteration is easily detected by SDS-PAGE o f
35S-Met labelled m itochondrial proteins a n d results in a 40% reduction in Com
plex IV specific activity. Like the np 3394 m utations, the n p 7444 m utation has
been found with o th er LHON m utations a n d in 1% o f the unaffected population.
Also, disease expression is sporadic in np 7444-positive pedigrees with males m ost
com m only affected. Thus, the n p 7444 m utation may constitute an LHON risk fac
to r when in the appropriate genetic environm ent.
Perhaps the best exam ple o f a m oderate-risk LHON m utation whose pathoge
nicity is influenced by o th er mtDNA m utations is the hom oplasm ic np 15257 (cy
tochrom e b gene, Com plex III) m utation. This m utation alters a highly conserved
aspartate residue in cytochrom e b which is located n ear an invariant hem e-bind
ing histidine residue (Johns an d B erm an, 1991; Brown et al., 1992b). T he np
15257-mutation has been found in at least 16 LHON families and is found at a very
low frequency (0.3%) in the control population. Pedigrees harboring this m uta
tion show that the disease is m oderately p e n e tra n t and m ost o f the affected indi
viduals are m ale. H ence, the np 15257 m utation has many features characteristic
 MITOCHONDRIAL DNA MUTATIONS AND TH E EYE 479

of a high-to-m oderate risk LHON m utation, yet, in some cases, this m utation alone
may be insufficient to cause LHON.
T he n p 15257 m utation is alm ost invariably found with a specific subset o f lower-
risk mtDNA LHON m utations. MtDNA sequence an d restriction endonuclease di
gestion analysis has indicated that patients harboring the np 15257 m utation have
very sim ilar mtDNA haplotypes which include the frequently-observed low-risk
LHON m utation at np 13708 (ND5, gene, Com plex I), and the lower risk LHON
m utations at nps 15812 (cytochrom e b gene, C om plex III) and 5244 (ND2 gene,
C om plex I) (Brown et al., 1992b). Such sim ilar mtDNA genotypes cluster upon
phylogenetic analysis, dem onstrating a LHON mtDNA lineage which is defined by
the linked n p 13708 + 15257 m utations. T he np 15812 m utation is fo u n d in rough
ly one-half o f the individuals with the 13708 + 15257 genotype an d the 5244 m uta
tion has been fo u n d in a single individual with the 13708 + 15257 + 15812
genotype. As this lineage sequentially accum ulated these four m utations, the nu m
ber o f unaffected individuals decreased and the pro p o rtio n of blind individuals in
creased. Blindness occu rred in the lineage only after the occurrence o f the 15257
m utation. Thus, the n p 15257 m utation is the m ost pathogenic mtDNA m utation
in this lineage, bu t in some cases the risk for developing LHON is apparently in
creased with the presence of additional mtDNA m utations. T he additional m uta
tions in teract synergistically with the 15257 m utation to decrease OXPHOS
efficacy below an energetic threshold necessary for optic nerve function (Johns
and B erm an, 1991; Brown et al., 1992a,c).

Lozv-risk LHON mutations

Six mtDNA m utations appear to im part a low-risk for LHON expression: nps
4216 (N D l), 4917 (ND2), 5244 (ND2), 13708 (ND5) and 14484 (ND6) in Com
plex I genes and np 15812 in cytochrom e b o f Com plex III (Johns and B erm an,
1991; Brown et al., 1992a,b; Johns, N eufeld and Park, 1992). These m utations can
alter poorly o r highly conserved am ino acids and can be found in statistically sig
nificant frequencies in LH O N families. However, they can also be found in rela
tively high frequencies in unaffected controls, the exceptions being the
heteroplasm ic 5244 m utation which has several high-risk LHON m utation char
acteristics (Table 2) b u t has only been found in one 15257-positive LHON patient
an d no controls, an d the 14484 m utation which also has n o t been found in con
trols. Both of these m utations may therefore rep resen t a m ore significant risk for
developing LHON, bu t m ore data is necessary to draw this conclusion. In gen
eral, however, the low-risk LHON m utations probably cannot cause LHON as sol
itary etiological agents, bu t instead are either non-pathogenic mtDNA
polym orphism s linked to a LHON-causing mtDNA genotype o r contribute to dis
ease expression as a m utation of low pathogenicity which interacts synergistically
with m ore severe mtDNA m utations. Some of these m utations can be found asso
ciated with various o th er m ore severe LHON m utations and som e are specifically
associated with a second LHON m utation, such as the np 15812 m utation which
has only been fo u n d linked to the m ore severe np 15257 m utation in LHON
patients.
480 MICHAEL D. BROWN ET AL.

T he genetic heterogeneity observed in LH O N suggests th at optic nerve death

results from a generalized electron tran sp o rt im pairm ent as all LHON m utations
affect m tDNA-encoded subunits o f the m itochondrial electron tran sp o rt chain.
Because the LH O N m utations are o f varying severity, each represents a certain risk
for disease expression. Thus, LLION expression can be th o u g h t o f in term s of
probability: the m ore severe the m utation, the greater the risk for blindness and
m aternal transm ission of the phenotype. Conversely, less severe m utations im part
a lower risk for blindness and ap p ear m ore frequently as sporadic LHON cases.
Further, risk appears to be influenced by additional genetic and nongenetic fac
tors which eith er increase or decrease the probability of LHON expression.

Neurogenic Muscle Weakness, Ataxia, and Retinitis Pigmentosa (NARP)

Pigm entary retinopathy is a p ro m in en t clinical feature o f NARP, a second p h en o

type due to a mtDNA missense m utation (Table 1). NARP results from a hetero-
plasmic T to G transversion at n p 8993 o f the ATPase 6 (C om plex V) gene (H olt
et al.. 1990). This m utation changes a highly conserved leucine to an arginine in
the p ro to n channel of ATP synthase, thus inhibiting ATP synthesis (H olt et al.,
1990; Tatuch et al., 1992). Like LHON, NARP is m aternally transm itted and
p en etran ce is incom plete in an affected family. Unlike LHON, however, disease
symptoms often vary m arkedly am ong affected family m em bers.
Rapid intrafam ilial segregation o f heteroplasm ic mtDNAs accounts for the vari
ation in p en etran ce and expressivity seen in NARP pedigrees (Shoffner et al.,
1992; Tatuch et al., 1992) (Figure 3). Individuals harboring between 80-90% m u
tan t mtDNAs in their blood are at high risk for developing NARP while family
m em bers with less than 80% m utant mtDNAs are at a lower risk for NARP and may
rem ain asym ptom atic. NARP symptoms can range from retinitis pigm entosa plus
m ild neurological problem s in mildly affected patients to true retinitis pigm en
tosa, m uscle weakness, ataxia and generalized seizures in severely affected pa
tients. Individuals harboring greater than 90% m u tan t mtDNAs are at risk for
developing the m ost severe m anifestation o f this genetic defect: Leigh's disease.
Leigh's disease is a fatal infantile disorder characterized by psychom otor retard a
tion, brainstem abnorm alities and elevated blood lactate levels. Using both a re
striction endonuclease digestion assay and S outhern blot analysis, Tatuch et al.
(1992) were able to show in one Leigh's patient, that the brain, kidney, liver and
cu ltured fibroblasts all contained greater than 95% abnorm al mtDNAs (Tatuch et
al., 1992). Thus, the NARP phenotype represents a continuum o f symptoms, the
severity of which correlates with the p ro p o rtio n of m utant genom es in p atien t tis
sue.
T he genetics o f LHON an d NARP illustrate an im portant feature o f mtDNA-
based diseases: the variability in g en o ty p e/p h e n o ty p e relationships. LHON is the
singular clinical m anifestation o f m ultiple mtDNA m utations, acting in various
com binations and affecting three different OXPHOS enzyme com plexes, while a
 M ITOCHONDRIAL DNA MUTATIONS AND THE EYE 481

>6 26
n *
% Mutant mtDNA 0 0 86 >95 >95 >95 >95
Age (years) 2 14 11 1

RP + nd + + +
Leigh's - + - - +
Optic atrophy - - - - +
Migraine + - - - -
MR - + + + +
Ataxia - nd + + nd
ADD . nd + + nd

Figure 3 M tDNA g e n o ty p e re la tio n sh ip to clinical p h e n o ty p e in a p e d ig re e h a rb o rin g th e AT-

Pase 6 (NARP) m u ta tio n a t n u c le o tid e 8993. N o te Variable expressivity re su ltin g fro m th e ra p
id seg reg atio n o f th e 8993 m u ta tio n w ith in th e family. RP, re tin itis p ig m en to sa; MR,
p sy ch o m o to r re ta rd a tio n ; ADD, a tte n tio n deficit d iso rd e r; n d , n o t d e te rm in e d .

single, heteroplasm ic ATPase 6 point m utation can result in a m arkedly different

phenotype, even within the same pedigree.

MtDNA R earrangem ents and the Eye

Large-scale mtDNA deletions an d partial duplications are also associated with

hum an disease involving the eye (Table 1) (Wallace, 1992a). Pathogenic dele
tions are far m ore com m on than are duplications and account for a significant
fraction o f m itochondrial m yopathies, a h eterogeneous group of diseases hall
m arked by the accum ulation o f structurally a n d biochem ically abnorm al m ito
chondria in muscle. Both types o f rearran g em en ts are causally associated with
ocular myopathy, observed clinically as C hronic Progressive E xternal O p h th al
m oplegia (CPEO) o r the m ore severe disorder, Kearns-Sayre Syndrom e (KSS).
CPEO is characterized by ophthalm oplegia (paresis o f extraocular muscles lim it
ing eye m ovem ent), ptosis (droopy eye lids) and m itochondrial myopathy. In
addition to CPEO, KSS patients may m anifest pigm entary retinopathy, elevated
levels o f blood lactate a n d /o r CSF p rotein, ataxia, cardiac conduction abnorm al
ities, dem entia, and hearing loss. O th e r less com m on ophthalm ologic m anifesta
tions include corneal opacities, cataracts, and optic atrophy. Pearson M arrow /
Pancreas syndrom e has also been associated with mtDNA rearrangem ents. Pancy
topenia an d pancreatic dysfunction are the hallm arks o f this often fatal
482 MICHAEL D. BROWN ET AL.

Minor Deletion Arc Major Deletion Arc

10.4 kb deletion

Figure 4 P ath o g e n ic h u m a n m tD N A d e letio n s. D eletio n s typically o c c u r w ithin e ith e r th e

m ajo r o r m in o r arcs as d e fin e d by th e o rig in s o f DNA re p lic a tio n (O jj a n d O l ). D eletio n s
w hich e lim in a te O j , su ch as th e 10.4 kb d e le tio n , are ra re . A 5 kb d e le tio n associated with
C E O P a n d KSS is th e m ost c o m m o n p a th o g e n ic m tD N A d eletio n .

ch ildhood disease. Those children who survive the early stages o f Pearson Mar
ro w /P ancreas syndrom e often progress to KSS.

MtDNA Deletions

P athogenic mtDNA deletions can be subdivided based on inheritance patterns.

Pedigrees exhibiting familial transm ission o f deletions are rare an d such dele
tions usually have novel genetic features associated with them which perm it
 MITOCHONDRIAL DNA MUTATIONS AND THE EYE 483

inheritance. Spontaneous deletions are com paratively m uch m ore com m on and,
as a group, share certain general genetic characteristics.
Spontaneously-occurring deletions account for the majority o f CPEO, KSS and
Pearson M arrow /Pancreas Syndrom e cases seen clinically. Patients harboring
spontaneous deletions are observed sporadically as such deletions are somatic m u
tational events and are therefore no t transm itted to successive generations.
Over 120 d ifferent spontaneous, pathogenic mtDNA deletions have been
m apped and some com m on genetic features are evident (Wallace et al., 1991).
First, although such deletions vary widely in position and size, ranging from
roughly 2 kb to 9 kb, the two origins o f DNA replication are usually m aintained,
thus confining spontaneous deletions to the two m ajor arcs o f the mtDNA defined
by the position o f the two origins (M oreas et al., 1989; Goto et al., 1990b; Wallace
et al., 1991) (Figure 4). Second, b o th tRNA an d protein-coding genes are com
m only deleted (Wallace et al., 1991). T hird, sequence analysis o f the breakpoints
of over 50 spontaneous deletions revealed th at these deletions m ost often occur
within directly re p eated mtDNA sequences (Shoffner et al., 1989; Schon et al.,
1989; Mita et al., 1990; Wallace et al., 1991). D irect repeats are com m on in mtD
NA, in part because o f the disproportionate n u m b er o f H -strand g uanine residues.
Some direct repeats, which range from th ree to 13 base pairs in length, are
hotspots for deletion form ation (Wallace et al., 1991). T he best exam ple of such
a hotspot is a 13 base pair perfect re p eat ( 5 - ACCTCCCTCACCA 3'), found in the
ATPase 8 gene (from n p 8470 to np 8482) an d the ND5 gene (from np 13447
to np 13459) (Wallace et al, 1991; Wallace, 1992a). This re p eat is found at the
b reakpoint o f the m ost com m only-occurring pathogenic deletion of 4977 base
pairs, which has o ccu rred independently over 100 times (Wallace et al., 1991)
(Figure 4).
T he association of spontaneous deletions with direct repeats suggests a com m on
m utagenic m echanism . H om ologous recom bination has no t been proven to exist
in m itochondria and therefore probably does not account for somatic deletion
events. However, a "slipped replication" m odel as proposed by Shoffner et al.
(1989) does n o t rely on strand cross-over, yet is d e p e n d e n t on direct repeats for
deletion form ation. U n d e r the "slipped replication" m odel, the upstream , dis
placed, H -strand direct rep eat base-pairs with the com plem entary dow nstream L-
strand re p eat exposed by the DNA replication fork. A strand breakage event ju st
dow nstream from this pairing generates a 3'-OH for subsequent H -strand synthesis
an d results in the degradation o f the single-stranded H -strand dow nstream o f the
breakage event back to double-strand DNA. Thus, slipped replication resolves one
wild-type an d one deleted mtDNA m olecule. Since a m inority o f deletion events
occur in the absence of directly re p eated sequences, slipped replication cannot ac
count for all large spontaneous deletions. O th er m echanism s, such as illegitim ate
recom bination and topoisom erase II-m ediated deletion form ation, have been
proposed to account for those deletions n o t associated with direct repeats (Mita
e ta l., 1990).
484 MICHAEL D. BROWN ET AL.

Patients usually h arb o r a single deletion which is found in varying heteroplas-

mic ratios in different tissues of the body. Typically, a high pro p o rtio n o f deleted
m olecules are p resen t in the highly-oxidative, post-mitotic tissues which are the
m ost affected tissues in these patients. Thus, it is n o t uncom m on to detect 80%
deleted mtDNAs in the skeletal muscle of ocular myopathy patients. Lower
am ounts o f deletion are detected in the rapidh-dividing tissues such as the blood
and fibroblasts, presum ably due to selection against functionally com prom ised mi
tochondria. T he widespread tissue distribution and lack o f genetic transm ission
indicate that the deletion likely occurred in the ovum or at an early stage o f em
bryonic developm ent. T he tim ing of the deletion event d u rin g developm ent and
subsequent replicative segregation may account for patient-to-patient differences
ill deletion tissue distribution and heteroplasm ic ratios. This, in tu rn , explains the
variability in clinical expression seen in patients harboring mtDNA deletions as
KSS patients are likely to have a higher p ro p o rtio n of deleted mtDNAs in m ore tis
sues than CPEO patients.
Disease states resulting from mtDNA deletions progress with age. This is likely
due to an increase in the deleted mtDNA to norm al mtDNA ratio over tim e in pa
tient tissues. In a longitudinal study involving KSS patients, Larsson et al. (1990)
d em onstrated that the p roportion of deleted m olecules in skeletal m uscle in
creased with age an d that this increase in the percentage o f deleted m olecules was
associated with the progression o f clinical symptoms. T h e en rich m en t o f m utant
mtDNAs with time can be explained if the sm aller deleted m olecules h ad a repli
cative advantage over larger und eleted m olecules, a likely p h en o m en o n given the
rate-determ ining step in mtDNA replication is probably the size o f the tem plate
m olecule (Shoffner e ta l., 1989).
D eterm ination o f the precise pathogenic m echanism of such deletions has been
im paired by the lack o f correlation betw een the size and position of the various
deletions and the observed OXPHOS defects. In general, patients h arb o r high lev
els o f d eleted mtDNA an d low levels o f norm al mtDNA in affected tissue. N orth
ern blot analysis o f mtRNA isolated from cybrid cells containing d eleted mtDNAs,
in situ hybridization studies an d in intro transcription data indicate th at both wild-
type and d eleted mtDNA m olecules are transcribed (Mita et al., 1989; Nakase et
al., 1990; Hayashi e ta l., 1991; M oreas et al., 1992). However, im m unochem ical
studies and experim ents labeling m itochondrial translation products with '^S-Met
show th at translation is severely im paired in m itochondria containing a high pro
p ortion o f deleted mtDNAs (Hayashi et al., 1991; Ballinger et al, 1992; M oreas et
al., 1992). Since alm ost all deletions elim inate at least one of the m itochondrial-
encoded tRNAs, it is probable that overall m itochondrial translation would be af
fected by the loss of tRNAs. If so, intraorganellar tRNA com plem entation m ight
be expected to occur in those m itochondria that h arb o r a sufficient n u m b er of
wild-type m olecules, as tRNAs transcribed from the norm al mtDNAs com pensate
for those lost in the deleted mtDNAs. This appears to be the case in cybrid cells,
as Hayashi et al. (1991) dem onstrated that in cybrids containing below 60% delet
ed mtDNAs, translation occurs in p ro p o rtio n to mRNA levels and that a fusion
 MITOCHONDRIAL DNA MUTATIONS AND THE EYE 485

protein (mRNA coding region spans the deletion breakpoint) can be detected
u pon SDS-PAGE o f S-Met labeled m itochondrial proteins. However, in cybrids
containing greater than 60% deleted mtDNA, overall m itochondrial translation
decreased dramatically, as did synthesis o f the fusion protein. Thus, a generalized
translation defect due to tRNA deficit is likely in those cells harboring m itochon
dria with high prop o rtio n s o f deleted mtDNA. Such a pathogenic m echanism
would explain why ocular m yopathy is a com m on phenotypic result am ong pa
tients harboring m arkedly d ifferent deletions.
Pathogenic mtDNA deletions can also be inherited. These deletions are less
com m on than spontaneous deletions an d can be fu rth e r subdivided on the basis
o f m aternal versus M endelian inheritance. O ne well-characterized m aternally in
herited deletion o f 10.4 kb was d etected in a three generation pedigree exhibiting
adult-onset diabetes m ellitus and hearin g loss (Ballinger et al., 1992). All mater-
nally-related family m em bers studied h arb o red the heteroplasm ic deletion in m ul
tiple tissues. Unlike pathology associated with spontaneously-occurring deletions,
m em bers of this pedigree did n o t p resen t with m ild or severe form s o f ocular my
opathy. Also, unlike spontaneously-occurring deletions, this large deletion elim i
nates the light-strand origin of DNA replication (Figure 4). It is possible th at the
absence of the light-strand origin decreases DNA replication efficacy an d thereby
does no t allow the sm aller deleted m olecule a replicative advantage. Thus, p ro
gressive en rich m en t may n o t occur o r be greatly reduced in p atien t tissues, ac
counting for the unique pathology an d inheritance associated with this deletion.
Several pedigrees exhibiting the autosom al dom inantly transm itted propensity
for harboring mtDNA deletions have b een identified (Zeviani et al., 1989; Zeviani
et al., 1990; C orm ier et al., 1991). KSS an d bilateral cataract form ation are com
m on clinical m anifestations in many o f these pedigrees. In contrast to both spon
taneous and m aternally-inherited deletions, affected individuals h arb o r m ultiple
mtDNA deletions. D eletion breakpoints can vary am ong family m em bers, strongly
suggesting th at a predisposition to de novo deletion form ation is inherited, n o t the
deletions themselves. These deletions always spared both origins o f DNA replica
tion and occurred within d irect repeats. Zeviani et al. (1989, 1990) have identified
a m utational hotspot for deletion form ation near the tRNAPr/D -loop ju n ctio n
(nps 16070-16080). T he non-coding D-loop region contains im p o rtan t sequence
elem ents (including PL, PH, an d O h ,) that interact in trans with nuclear gene
products to allow mtDNA replication. Thus, it is quite possible th at a m utation in
a nuclear-encoded, trans-acting factor involved in mtDNA replication prom otes
deletion form ation, perhaps by facilitating slipped replication or intram olecular
recom bination. Such deletions have been found in both leukocyte and skeletal
m uscle mtDNA and the n u m b er o f deletions can increase with tim e in an individ
ual (C orm ier et al., 1991; Servidei et al., 1991). This would explain the progressive
clinical course seen in affected family m em bers an d is consistent with the consti
tutive presence o f a nuclear-encoded m u tan t gene product.
486 MICHAEL D. BROWN ET AL.

MtDNA Duplications

MtDNA duplications have also been associated with progressive ocular m yopathy
an d Pearson M arrow /Pancreas Syndrom e (P oulton et al., 1989a,b; Rotig et al.,
1990). All patients h arb o r a single, partially-duplicated mtDNA m olecule present
in heteroplasm ic ratios in m ultiple tissues. Rotig et al. (1991) have described the
m aternal transm ission o f a 26 kb mtDNA m olecule from a m other to two daugh
ters. In this pedigree, the m o th er had ocular m yopathy an d extrem ely low levels
o f rearra n g ed mtDNA while the daughters had m itochondrial myopathy, diabetes
m ellitus, proxim al tubulopathy and ataxia an d h arb o red higher am ounts o f the
partial duplication. As is the case with mtDNA deletions, disease courses are p ro
gressive in these patients, possibly due to two sets o f DNA origins found on these
rearra n g ed m olecules. Both the genetic m echanism producing the partial dupli
cations and the pathogenic m echanism p roducing the phenotype are unknow n.

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 GLOSSARY

*m ethods A term s # abbreviations

A3 end: T he e n d o f a nucleic acid strand th at contains a free 3 hydroxyl group

on the sugar portio n o f the nucleotide. DNA synthesis proceeds from this en d of
the strand when there is a DNA tem plate (DNA polym erase), an RNA tem plate
(reverse transcription), o r nonspecific addition (e.g. term inal deoxynucleotide
transferase)

A5end: T he en d o f a nucleic acid strand th at contains a free 5 hydroxyl group

on the sugar po rtio n o f the nucleotide. In DNA cleaved with enzymes, this
hydroxyl group is usually phosphorylated bu t can be rem oved and replaced by
P using a com bination o f phosphatase an d kinase. Like DNA replication, both
transcription and translation o f nucleic acid m olecules proceed in a 5 to 3 direc
tion along the DNA o r RNA strand.

Aacceptor splice site: T he ju n c tio n betw een the 3' e n d o f an in tro n an d the 5'
e n d o f an adjacent exon.

*agarose gel electrophoresis: T he separation o f charged m olecules (e.g. nega

tively charged nucleic acids) using an electric field across an agarose m atrix (see
acrylam ide gel electrophoresis). Agarose is a polysaccharide p rep ared from sea
weed an d can be used for the separation o f small as well as very large nucleic
acids. Because the gel is fo rm ed by hydrogen-bonding of the sugar chains, agar
ose gels are easily d isru p ted by heat a n d /o r d en atu rin g agents.

Aallele: An allele is an alternative form o f a single gene o r o th er locus. A single

allele is in h erited separately from each parent. In situations w here a genetic
m arker locus is defined by a nucleic acid sequence or probe, an allele may be
defined as a single nucleotide difference that causes a change in a restriction
endonuclease site (see RFLP), a conform ation alteration (seeSSCP, DGGE), a mis
m atch betw een the sequences from the two chrom osom es {see heteroduplex anal
ysis), a deletion or insertion o f DNA, o r variations in the length o f a m icrosatellite
re p eat (see CA repeats).

Aallele-specific oligonucleotide: An appropriately selected oligonucleotide can

be labelled an d used for hybridisations with sufficient stringency (specificity) that
single nucleotide differences as well as small rearrangem ents can be d etected in
genom ic DNA. W hen used in conjunction with PCR, allele-specific oligonucle
otides can be attached to a m em brane an d used to type a large set o f alleles rap
idly for a given DNA sequence, such as HLA antigen genes.
492 GLOSSARY

*Alu PCR: T he use o f sequences from Alu repeats (see below) as prim ers for PCR
(see polym erase chain reaction). Generally the sequences are selected from Alu
segm ents th at have evolved after speciation so that hum an sequences can be
detected in d ependently of DNA from o th er organism s. Because there are so
m any Alu repeats within the genom e, short DNA sequences betw een adjacent Alu
repeats can be am plified from many regions o f the genom e. T he technique has
b een used to identify an d characterize radiation hybrids, somatic cell hybrids and
YACs containing hum an genom ic DNA.

AAlu repeats: T here are an estim ated 3-5 x 10 repetitive elem ents in m am m alian
DNA th at are p a rt of a family re ferred to as Alu repeats. These repetitive ele
m ents are 300 bp in length and were first recognized when genom ic DNA was cut
with the restriction endonuclease, Alul. They are scattered th ro u g h o u t the
genom e an d because subfamilies o f the Alu repeats have arisen since m am m alian
speciation, specific Alu prim ers can be used to selectively amplify hum an geno
mic DNA from a m ixture o f yeast or ro d e n t DNA (see Alu PCR).

Aantisense strand: see sense strand

#ASO: see allele-specific oligonucleotide

*autoradiography: T he detection o f radioactively labeled m aterial using X-ray

film o r a com parable photographic em ulsion. Can be used for in situ hybridisa
tion o f chrom osom es, cells, an d tissue slices as well as for detection o f radioac
tively labeled bands or spots from do t blots, N ortherns, and Southerns.

Aavidin: A tetram eric protein derived from egg whites that tightly binds biotin
(see strep tav id in /b io tin ).

#b: see base

Abacteriophage: A virus th at specifically infects bacteria. T he m ost com m only

used bacteriophages in m olecular biology are derived from lam bda phage. Typi
cally, these virus-derived vectors can hold 5-20 kb o f inserted, foreign DNA.
A lthough less ideal for m anufacturing large am ounts of an inserted DNA than
plasm ids o r cosmids, bacteriophage are an im p o rtan t host for cloning because
the DNA can be packaged in vitro into phage particles th at e n te r bacteria with
very high efficiency. T he genetic inform ation from the bacteriophage also cause
infected cells to lyse, allowing for sim ple and sensitive screening o f large nu m b er
o f plaques (see plaque).

Abase: A nitrogenous base used in nucleic acids consisting eith er o f a purine

(adenine, guanine) o r pyrim idine (thym ine, cytosine (DNA) or uracil (RNA))
residue. A nucleotide base is one o f the m onom eric units within nucleic acid and
 GLOSSARY 493

consists o f a phosphoric acid group attached to a deoxyribose (DNA) or ribose

(RNA) sugar and a p u rin e o r pyrim idine base. A 500 base RNA refers to a nucleic
acid strand com prised o f 500 nucleotides.

Abase pair: A pair o f hydrogen-bonded nucleotide bases (i.e a purine and pyrim
idine each attached to a ribose an d phosphate groups). T he sizes o f double
stranded nucleotide chains are often indicated by the # o f base pairs that are
shared. A 500 base pair (bp) DNA fragm ent consists o f two strands o f DNA, each
containing 500 nucleotides an d a total m olecular weight o f approxim ately 330
kilodaltons.

Abeta-galactosidase: A bacterial enzyme that cleaves-galactose. T he activity o f this

enzyme can be m o nitored by using indicator substrate m olecules such as X-gal or
Blue-gal an d the appearance o f a blue color. T he expression of beta-galactosidase
in troduced into bacteria as p art o f a recom binant plasm id is used to distinguish
ing non-recom binant an d recom binant plasm ids or cosmids. T he enzyme is often
coupled to d ifferent prom oters an d used as an indicator gene in transfections
and transgenic animals.

Abiotin: see S treptavidin/b iotin

#bp: see base pair

Abreakpoint: T he location at which th ere is a break in the continuity of a ch ro

m osom e allowing two previously noncontiguous pieces to becom e jo in e d (see
crossing over, recom bination, translocation).

*bubble PCR: A m plification of specific fragm ents of DNA w hen only one prim er
o f known sequence can be constructed. T he genom ic DNA is cleaved into pieces
(either specifically with restriction endonucleases or nonspecifically) and then
special DNA adaptors are ligated on to the exposed ends. T he special adaptors,
known as bubble adaptors, have m ism atching internal sequences but m atching
ends th at allow for stable ligation. T he bubble prim er is m ade to be identical (not
com plem entary) to one of the strands o f DNA within the bubble. W hen PCR is
perform ed, the sequence-specific PCR prim er allows the Taq polymerase to syn
thesize a com plem entary strand o f DNA. T he polym erase synthesizes the com ple
m en t to one o f the strands within the bubble. In the next ro u n d of prim er
annealing an d am plification, the bubble p rim er is able to anneal to this newly
synthesized DNA and allow a new com plem entary strand to be m ade. W ithout
the first synthesis step directed by the sequence-specific prim er, the bubble
p rim er is unable to anneal and prim e DNA synthesis. Using this technique, n o n
specific am plification o f DNA fragm ents th at contain identical ligated adaptor
m olecules is avoided.
494 GLOSSARY

ACA repeats: T here are h u n dreds of thousands o f m icrosatellite repeats within

the m am m alian genom e. These dinucleotide, trinucleotide and tetranucleotide
repeats include (CA)n, (CAA)n, (CAAT)n, (AT)n, and others. T he m ost com
monly used rep eat for PCR-based genotyping and linkage m apping is the CA
repeat. T housands of CA-repeat based m arkers have been isolated and m apped
to the hum an genom e. For many o f these repeats, there is considerable variations
(alleles) am ong individuals which allows them to be used for linkage studies.

#cDNA: see com plem entary DNA

*cDNA synthesis and cloning: cDNA synthesis begins with eith er total or
polyA+RNA and an oligonucleotide p rim er that will anneal to the 3 polyA tract
of the RNA (oligodT) o r a set of random prim ers that will an n eal/hybridise
along the length of the RNA. Reverse transcriptase is used to synthesize a com ple
m entary DNA (see above). By a variety of m ethods, the RNA of the RNA/DNA
hybrid is replaced by DNA so that a double-stranded DNA contains the original
genetic inform ation that was encoded in the RNA. T here are num erous varia
tions in the m ethod, including the use o f prim ers th at are already attached to a
vector, using PCR to amplify the double-stranded DNA, different techniques to
prim e the second strand synthesis, and different strategies for inserting this syn
thesized DNA into a vector. O nce the process o f building an d incorporating the
cDNA into the vector is com pleted, then the recom binant m aterial is in troduced
into E. coli eith er by transfection (for bacteriophage vectors), transform ation
(introducing un packaged DNA into cells) or electroporation (creating transient
holes in the cellular m em brane with an electric shock so that the DNA can en ter
the cells). Cells are spread an d grown so that individual colonies can be estab
lished. A ntibiotic selection is used to ensure that only cells that have taken up the
vector will grow. T he individual colonies can then be screened by a variety of
m ethods to identify those that contain a specific cDNA of interest.

Acell cycle: T he sequence o f stages th at occur between successive cell divisions.

T he period o f mitosis is referred to as the M phase, followed by a growth period
(G j), a DNA synthesis period (S) an d a second growth period, (G2) before en ter
ing the M phase again. Cells th at have stopped dividing an d have en tered a sta
tionary phase are said to be in G q.

AcentiM organ: A unit of m easure of recom bination frequency betw een two m ark
ers such that one centim organ equals a 1% chance th at a recom bination event
would occur in a single generation. While th ere is a rough correlation o f physical
distances (Mb) and genetic distances (cM), with 1 Mb equivalent to about lcM
in m an, there are regions o f red u ced o r increased recom bination that lim it direct
com parisons.
 GLOSSARY 495

Acentiray: A unit of m easure sim ilar to cendm organ bu t the centiray is based on
the probability o r frequency of DNA breaks induced by radiation. This m easure
o f genetic distance between m arkers is used in radiation hybrid m apping and
m ore closely correlates with physical distance than recom bination frequencies.
In general, one centiray equals approxim ately 50 kb o f DNA.

Achem ilum inescence: T he pro d u ctio n of light by a chem ical reaction. An enzyme
(e.g. horseradish peroxidase) th at can catalyze a chem ilum inescent reaction is
covalently linked to a m olecule (avidin o r an antibody) that also binds a DNA
"probe" (such as a specific DNA labelled with biotin or digoxygenin). Specific
d etection o f DNA fragm ents com plem entary to the probe can be achieved after
hybridisation by the addition of ap p ro p riate substrate m olecules an d the detec
tion o f light on photographic film. A lthough it is a m ore com plicated procedure
than autoradiography, the m ethod has the advantage o f creating stable reagents,
high sensitivity, rapid exposure times, an d avoidance of radioactivity.

Achrom osom e: A self-replicating genetic elem ent within the nucleus o f a eukary
otic cell that is com prised o f a single double-stranded m olecule o f DNA and a
large n u m b er o f proteins. Each chrom osom e contains a centrom ere w here spin
dle fibers attach during cell division an d telom eres at the ends o f each chrom o
some th at are req u ired for replication. All of the genetic inform ation in the
nucleus o f hum an cells is contained within a set of 23 pairs o f chrom osom es
which range in size from 50 to 250 Mb (average 150 M b), com prising a total of
three billion base pairs of haploid DNA. T he size o f the hum an m itochondrial
chrom osom e is 16,569 base pairs.

^chrom osom e banding: W hen cells in m etaphase are lysed an d spread on glass
slides, the chrom osom es are easily visible by light microscopy. Because the struc
ture an d com position o f the chrom osom es are n o t hom ogeneous, d ifferent dyes
(such as Giemsa and quinacrine) can be used that will create a standardised
b anding pattern along the length of each chrom osom e. These patterns can be
used to identify specific chrom osom es, establish a physical ru le r along the chro
m osom e by which genes can be m apped, and aid in the detection o f chrom o
somal rearrangem ents such as deletions and translocations.

chrom osom e painting: Alu-PCR (see Alu-PCR) can be used to amplify hum an-
specific genom ic DNA from flow-sorted chrom osom es or som atic cell hybrids (see
flow cytom etry and som atic cell hybrids), so that a hybridisation reagent can be
produced that will specifically label all or part o f specific chrom osom es. FISH
analysis can be carried o u t (jw fluorescence in situ hybridisation) by using fluo
rescence detection of the labeled PCR m aterial that will p ain t all or p a rt o f a
chrom osom e. These reagents are now com m ercially available an d are widely used
to d etect subtle chrom osom al rearrangem ents that were previously undetectable
by standard cytogenetic m ethods including chrom osom e banding.
496 GLOSSARY

#cM: see centi M organ

Acoding region: T he coding region is that po rtio n of the mRNA transcript (the
processed RNA synthesized from the gene) that contains the genetic inform ation
for the am ino acid sequence o f the p rotein product, see exon, intron, splicing,
m essenger RNA, open reading fram e.

Acodon: T he nucleotide triplet in mRNA that encodes for 1) the beginning of

p ep tid e synthesis (initiation codon), 2) a specific am ino acid to be added to the
p eptide chain o r 3) the term ination o f peptide synthesis (term ination codon).

Acolony: A population o f cells that have arisen from a single p ro g en ito r cell. In
plasm id, cosm id o r yeast libraries, individual colonies can be identified by spread
ing the library on agar plates. Libraries gen erated in bacteriophage vectors can
also be spread on plates to identify hom ogeneous, clonal populations o f viruses
which cause discrete spots o f bacterial lysis, known as plaques.

Acom plem entary DNA (cDNA): T he strand o f DNA th at is synthesised using a

specific o r non-specific p rim er to an RNA tem plate (R N A -dependent DNA poly
m erase such as reverse transcriptase) o r to a DNA tem plate (D N A -dependent
DNA polym erase). T he sequence o f this synthesized DNA is d eterm in e d by the
rules o f base pairing. T he term , cDNA, is frequently used to specifically identify
DNA th at has been m ade to be com plem entary to m essenger RNA or
polyA+RNA.

Acontigs: A group of clones th at provide overlapping coverage of one or m ore

regions of a genom e.

*contig m apping: T he process o f ordering the clones from a library to establish a

genom ic m ap based upon the relative o rd e r and overlap o f these sm aller genetic
elem ents.

Acosmids: A type of cloning vector that is derived from plasm ids bu t contains
the COS site of lam bda bacteriophage so that the recom binant vector can be
packaged an d transfected into E. coli. Cosmids are generally used for cloning
genom ic DNA in the size range of 25-40 kb because o f their greater efficiency at
en terin g bacteria by transfection com pared to the transform ation o f bacteria with
plasm ids containing large inserts.

AC ot-l DNA: M uch of the DNA within the m am m alian genom e is repetitive (see
repetitive DNA) and this DNA can be pari tally purified by d en atu rin g total
genom ic DNA and allowing it to reanneal to itself. Because repetitive sequences
are present in m any copies, the majority o f the DNA that initially reanneals will
contain these repetitive elem ents. DNA th at has rean n ealed u n d e r certain
 GLOSSARY 497

defined conditions is called Cot-1 DNA and contains the m ost highly repetitive
DNA within the genom e. This m aterial can be used in hybridisations to com pete
o u t repetitive DNAs on target m olecules, allowing unique sequences that are re p
resented in the hybridisation probes to be detected.

ACpG island: T he distribution o f nucleotides is n o t random th ro u g h o u t the

genom e and regions that are rich in C followed by G dinucleotides (CpG) are rel
atively rare. Studies have shown th at these CpG islands are often n ear the pro
m oter regions of genes. T he identification o f CpG islands is m ade with rare
cutting restriction endonucleases th at recognize these rare sequences. T he pres
ence o f CpG islands is one o f the indicators of potential genes in a large segm ent
o f genom ic DNA.

#cR: w c e n tiR a y

Acrossing over: Crossing over occurs w hen two paired chrom osom es exchange
m atching regions o f their DNA. see recom bination

90 etc*
AdATP, y- P o r a- P labelled: D eoxyadenosine triphosphate with the y- posi-
09
don phosphate containing P is used for 5 end-labeling o f DNA or oligonucle-
otides using T4 polynucleotide kinase, a - P-dATP is used for labelling DNA
m olecules by incorporation o f the nucleotide into the polynucleotide chain using
a DNA polymerase.

^denaturing gradient gel electrophoresis (DGGE): This type o f polyacrylam ide

gel electrophoresis m eth o d distinguishes DNA fragm ents (100-500 bp) o f similar
sizes but o f different nucleotide sequences an d is used in m utation detection.
T he d enaturation o r m elting properties of d ifferent regions o f a double-stranded
DNA m olecule are very sensitive to the nucleotide com position and sequence.
T he partial a n d /o r com plete d en a tu ratio n of the DNA strands dram atically alters
the mobility of the m olecules in a polyacrylam ide gel. A d en atu rin g gradient is
established by a gradient of a d e n a tu ra n t such as form am ide within the gel
(DGGE) o r less com m only by a therm al gradient (TGGE). PCR-generated frag
m ents are the source o f DNA for DGGE. A dditional sequences (GC clamps) are
often in tro d u ced on to the ends o f the fragm ent o f interest in o rd e r to improve
the sensitivity and effectiveness o f the detection m ethod.

Adeoxyribonucleic acid: A covalent polym er of deoxynucleotide subunits (see

nucleotide).

Adigoxygenin: A steroid derived from the digitalis p lan t that can be covalently
linked to DNA by incorporation o f derivatized nucleotides. T he labelled DNA
can then be detected by a variety o f m ethods (chem ilum inescence, enzymatic
498 GLOSSARY

stain, fluorescence) using a specific antibody conjugated to an appropriate indi

cator m olecule o r enzyme.

#DGGE: w d en atu rin g gradient gel electrophoresis

#DNA: see deoxyribonucleic acid

Adonor splice site: T he ju n ctio n between the 3' end o f an exon an d the 5' en d of
an adjacent intron.

*dot blotting: A m ethod o f detecting a n d /o r quantitating the presence o f a spe

cific nucleic acid fragm ent by hybridising a labeled probe to DNA or RNA that
has been spotted and fixed to a m em brane surface.

#dsDNA: double-stranded DNA

*electroporation: A m ethod for perm eabilizing cell m em branes to allow for the
passage o f large m olecules such as DNA into the cell. T he cells are suspended in
a special cuvette and exposed to a b rief electrical pulse that causes transient holes
in the m em brane. T he electrical conditions vary considerably am ong different
cell types. T he technique can be used for eukaryotic cells or bacteria.

#EST: see expressed sequence tag

Aexon: A region o f a gene th at is transcribed an d is p resen t in the final m essen

ger RNA (mRNA) transcript. T he exons contain the 5 an d 3 u ntranslated por
tions o f the mRNA as well as the sequences that code for the am ino acid
sequence o f the protein pro d u ct (see. intron, splicing, coding region, m essenger
RNA).

Aexpressed sequence tag: Equivalent to the sequence tagged site (STS) except
th at the un iq u e sequence that is used for construction o f the prim ers is within an
exon o f a gene.

Aexpression vector: A special vector (see Vector) that allows for the expression of
a foreign gene in an appropriate host. Expression vectors are used when a gene
can only be recognized by detection of its protein p roduct (im m unologically or
functionally), o r as a m eans o f m anufacturing the protein for biochem ical and
cell biology studies.

Aexpressivity: T he range of altered features (phenotypes) associated with a spe

cific genetic constitution o f an individual (genotype).
 GLOSSARY 499

*field inversion gel electrophoresis: A special type o f agarose gel electrophoresis

that uses an asym m etric altern atin g electric field to move the DNA through the
gel m atrix. T he in term itten t reversal o f the electric field allows for the separation
o f m uch larger DNA fragm ents then can norm ally be achieved by standard agar
ose gel electrophoresis. T he m eth o d is effective up to about 1 Mb of DNA. To
separate larger DNA fragm ents, pulsed-field gel electrophoresis m ethod is m ore
often used, see agarose gel electrophoresis, pulsed-field gel electrophoresis.

#FIGE: see field inversion gel electrophoresis

#FISH: see fluorescence in situ hybridisation

*flow cytom etry: Cells or subcellular fractions such as chrom osom es can be anal
ysed an d sorted by dispersing individual elem ents into m inute, electrostatically-
charged w ater droplets, d eterm in in g the optical or fluorescent pro p erties o f the
elem ent w ithin the droplet, an d then sorting the d roplet using a controlled mag
netic field. By this approach, individual chrom osom es can be isolated to 80-90%
purity a n d then used for the construction o f chrom osom e-specific genom ic
libraries.

Afluorescence: T he detection o f specific m olecules based upon th eir ability to

em it light at a hig h er wavelength than the light absorbed by the m olecule.
Because o f the separation between the excitation and em ission wavelengths, one
can use selective filters to d etect m olecules with high sensitivity an d low back
ground. Exam ples o f fluorescent m olecules used in m olecular studies include
ethidium brom ide, an d derivatives o f fluorescein, phthalocyanines, an d rhoda-
m ine.

*fluorescence in situ hybridisation: C om m only re ferred to as FISH, fluorescence

in situ hybridisation is the localization o f one o r m ore m arkers to specific chro
m osom es using hybridisation of a probe to m etaphase or interphase chrom o
somes arrayed on a glass slide and detection with one or m ore fluorescent dyes.
In addition to providing physical m apping data, FISH can also be used to detect
m icrodeletions in selected regions as well as aneuploidy and chrom osom al rear
rangem ents.

#G q. T he stationary growth phase of the cell cycle (see cell cycle).

# G 1: T he growth phase after mitosis during the cell cycle (see cell cycle).

*gel electrophoresis: see agarose gel electrophoresis, polyacrylam ide gel electro
phoresis
500 GLOSSARY

*gel re tard atio n assays: A m eth o d to identify proteins that bind to specific nucle
otide sequences within the pro m o ter regions of genes. D etection of specific bind
ing is achieved by dem onstrating th at the mobility o f a labeled DNA fragm ent in
a gel is specifically decreased by the binding o f one o r m ore proteins to an inter
nal sequence. U nlabelled oligonucleotides that contain the target sequ-ence can
be used to com petitively block this binding and allow the labeled DNA to m igrate
as if the binding protein was n o t present.

Agene: A fundam ental u n it o f genetic stru ctu re residing in a single location

within the genom e that encodes for inform ation that is capable o f being exp
ressed by one or m ore cells during the lifetim e o f the organism .

Agenetic m ap: T he linear ord erin g and spacing o f m arkers and genes along the
chrom osom es based upon the frequency o f recom bination betw een the linked
elem ents.

Agenom e: T he en tire set o f genetic inform ation contained within a single (hap-
loid) set o f chrom osom es.

Ah eterogeneous nuclear RNA: RNA that is found within the nucleus an d consists
o f unprocessed and partially processed transcripts, as well as small nuclear RNAs.
H eterogeneous nuclear RNA is rapidly processed by polyadenylation an d splicing
o u t o r removal o f the introns so th at the final transcript, the m essenger RNA,
only contains the exon regions o f the gene a n d can be exported to the cytoplasm.

Aheterozygosity: 1) T he condition of having dissim ilar alleles at a given locus. 2)

A m easure o f the nu m b er an d distribution o f alleles in the population associated
with a m arker see polym orphism inform ation content

#hnRNA: see heterogeneous nuclear RNA

Ah orseradish peroxidase: An enzyme that, w hen com bined with the appropriate
substrate, creates a chem ilum inescent or insoluble product th at can be visually
detected. By coupling the enzyme with an ap p ro p riate m olecule, e.g. an antibody
or avidin, it can be used for the detection o f specific DNA fragm ents o r proteins
(see chem ilum inescence, in situ hybridisation).

#HRP: see horseradish peroxidase

*in situ hybridisation: In situ hybridisation (ISH) is a m ethod for directly visual
izing the cellular o r subcellular location o f o ne or m ore DNA or RNA sequences
using hybridisation with a labeled probe a n d a suitable detection m ethod. In situ
hybridisation is used for the localisation o f one or m ore m arkers to specific chro
m osom es arrayed on a glass slide (see Fluorescence in situ hybridisation). ISH is
 GLOSSARY 501

also used to identify the location o f cellular or viral transcripts o r viral DNA on
histologic preparations o f cells and tissue and to evaluate the developm ental
a n d /o r tissue-specific expression o f specific genes.

Aintron: T h at po rtio n o f a gene th at in transcribed an d p resen t in the initial

transcript (hnRNA) bu t is then rem oved by splicing to produce the final tran
script (mRNA). T he sequences within the intron are generally no t used for the
coding o f the p rotein, b u t som e genes have alternative splicing in which some
introns are rem oved only p art of the tim e, thus creating m ore com plex final tran
scripts and heterogeneous protein products (seeexon, splicing, coding region).

jum ping libraries: A genom ic library that has been constructed by cutting
genom ic DNA with a rare-cutting restriction endonuclease. T he large fragm ents
(100-200 kb) are allowed to circularize an d ligated. T he ligated circles are then
cut with a second enzyme an d the fragm ents are cloned. Clones are selected for
the presence o f the first, rare-cutting restriction endonuclease site. These clones
contain small fragm ents th at were originally separated by the length of the origi
nal fragm ent. They provide the m eans for ju m p in g across a distance th at is
g reater than can be norm ally contained within a single cosm id o r phage clone.

Akaryotype: T he display o f a com plete set of chrom osom es from a given individ
ual or cell (see B anding).

#kb: A thousand bases o r base pairs

library: A collection o f reco m b in an t m olecules o r clones (foreign DNA inserted

within a vector and propagated in cells) that represents a heterogeneous source
o f DNA or RNA. Libraries can contain the population of transcripts (mRNA)
within a cell o r tissue (see cDNA cloning), the genom ic DNA from an entire
organism , DNA from a specific chrom osom e (i.e. chrom osom es sorted by flow
cytom etry or somatic cell hybrids), o r DNA from a lim ited region of a chrom o
some (e.g. a m icrodissected portion o f a chrom osom e or from a YAC containing
a large genom ic fragm ent). Subtracted libraries are those in which the popula
tion o f clones has been constrained by the elim ination o f those clones that are in
com m on betw een two populations o f DNA or RNA. Libraries are said to be n o rm
alised w hen steps have been taken to m inim ise the difference in copy num bers of
unique elem ents. For exam ple, cDNA libraries may contain clones representing
transcripts that have less than 0.001% abundance within a cell an d o th er tran
scripts that constitute m ore than 10% of the mRNA population.

library screening: T he identification o f specific clones (eith er genom ic or

cDNA) from a library (see above) using eith er a hybridisation probe (such as a
previously cloned DNA, an oligonucleotide or a PCR product) o r a detection
m ethod for a specific gene p ro d u c t (such as an antibody to a protein product, a
specific enzymatic o r biological assay).
 502 GLOSSARY

Alinkage: T he greater than chance co-inheritance o f two or m ore m ark e rs/

genes. W hen two genes are linked, they are sufficiently close to each o th er that
the probability o f being in h erited to g eth er is greater than 50% (the probability
o f in d e p e n d e n t assortm ent).

linkage mapping: see genetic m apping

Alocus: T he position that a gene or m arker occupies in a chrom osom e.

Alod score: see discussion of Linkage in C hapter 1 (II)

#LOH: see loss of heterozygosity

Aloss o f heterozygosity: Heterozygosity refers to the state o f having pairs of dis

sim ilar alleles. Loss o f heterozygosity can occur as a result o f structural deletions,
m itotic recom bination o r nondisjunction. In some instances the cell is left with a
single copy o f the gene and in o th er circum stances the cell contains two copies of
the same parental chrom osom e, leading eith er to a hemizygous (1 copy) o r a
hom ozygous state (2 copies). An exam ple o f loss o f heterozygosity is when a reti
nal cell containing a m utation in the Rb (retinoblastom a) locus loses its norm al
Rb allele by a nondisjunction event. T he retinal cell becom es hemizygous fo r the
Rb m utation an d is vulnerable to clonal expansion an d the developm ent of a
m alignant state.

Alymphoblastoid cells: see transform ation

#M: T he mitosis phase of the cell cycle (see cell cycle).

Amarker: A position on a chrom osom e which can be identified an d m o nitored

by the inheritance of a trait or gene p ro d u c t o r by a unique attribute o f the DNA
sequence.

#Mb: A m illion nucleotides or nucleotide base pairs.

*microdissection o f chromosomes: A m eth o d for isolating lim ited regions o f a

specific chrom osom e by cutting the chrom osom e with a m icropipette u n d e r
d irect vision with a light m icroscope. T he selected DNA region is eith er cloned
directly or am plified as small fragm ents using PCR and cloned to create a region-
specific genom ic library.

Amicrosatellite repeats: see CA repeats

Amessenger RNA: T he RNA in the cell that encodes the processed transcript
from a gene and is the tem plate for p ro tein synthesis. Most, but no t all, of the
m essenger RNA in eukaryotic cells is polyadenylated (see polyA+RNA).
 GLOSSARY 503

#mRNA: see m essenger RNA

*nick translation: A m ethod of labeling specific, pure fragm ents o f double

stranded DNA by introducing nicks into the m olecules with DNasel followed by
tem plate-specific synthesis of DNA incorporating radioactive o r derivatized
nucleotides. T he resulting m ixture o f small fragm ents re p resen t the entire
sequence o f the original tem plate a n d can then be used as a hybridisation probe
(see random p rim er labeling).

Anondisjunction: T he failure of two hom ologous chrom osom es to separate p ro p

erly during cell division an d miosis.

N orthern blot: A m ethod for the analysis of RNA. An RNA m ixture is fraction
ated by size using d en atu rin g agarose gel electrophoresis. D enaturing the RNA is
necessary to elim inate secondary stru ctu re and to allow the RNA m olecules to
m igrate in accordance with their size. These gels generally em ploy form aldehyde
or glyoxal, an d rarely, methyl m ercuric hydroxide, as the denaturants. T he RNA
is then transferred to a filter or support so as to preserve the size fractionation
achieved by gel electrophoresis. Specific RNA species can then be detected and
assessed for size and am ou nt using a hybridisation probe.

Anucleoside: A nitrogenous base (p u rin e or pyiim idine) attached to a ribose or

deoxyribose sugar.

Anucleotide: T he basic subunit of DNA (a deoxynucleotide) or RNA (a rib o n u

cleotide). Each nucleotide is com prised o f a nitrogenous base (adenine, guanine,
or cytosine in DNA an d RNA; thym ine in DNA and uracil in RNA), a sugar (deox
yribose for DNA and ribose for RNA), and a phosphoric acid m olecule that serves
as the backbone linkage between nucleotides.

nucleotide sequencing: T here are a n u m b er of m ethods for establishing the

nucleotide sequence o f DNA. T he m ost com m only used techniques em ploy enzy
matic synthesis o f DNA using a specific prim er, a DNA polym erase, and a m ixture
of deoxynucleotides (dATP, dCTP, dGTP, dTTP) that contain a small am ount of a
m odified deoxynucleotide (dideoxynucleotide) th at can be incorporated into
the growing DNA strand bu t then blocks fu rth e r elongation o f the chain. Four
sets o f reactions are norm ally carried o u t for each tem plate (the DNA to be
sequenced) an d prim er, with each reaction containing one o f the four possible
dideoxynucleotides (ddATP, ddCTP, ddGTP, ddTTP). Each reaction mix contains
a collection o f DNA strands th at were all synthesized from a com m on starting
point but are term in ated at varying points along the length o f the tem plate DNA
w herever a specific dideoxynucleotide has been incorporated. T he m ixtures are
then separated on adjacent lanes o f a high resolution, d en atu rin g polyacrylam ide
gel that can distinguish DNA m olecules differing by a single base in length. T he
504 GLOSSARY

p attern on the gel can be detected by autoradiography o r by fluorescence detec

tion if the prim ers are suitably labelled. T he com bined p attern o f bands provides
a direct co rrespondence to the nucleotide sequence of the tem plate DNA.

Aopen reading fram e: A region of genom ic DNA that potentially encodes for an
am ino acid sequence o f a protein . T he region is characterized by a stretch of
nucleotide sequence th at begins with an initiation codon, followed by a series of
triplets th at w ould encode for different am ino acids, an d ends with a term ination
codon that would en d peptide synthesis.

#ORF: see open reading fram e

#PCR: see polym erase chain reaction

Apedigree: A description o r diagram o f the ancestral history of a family. Females

are depicted as circles, males as squares, individuals whose sex is unknow n are
shown as diam onds. Solid o r blackened objects indicate individuals expressing
the trait o r disease. A diagonal line through the object indicates th at the person is
deceased. H eterozygous fem ale carriers of X-linked conditions are d en o ted by a
do t in the cen ter o f the circle. C onsanguinous m atings are d en o ted by a horizon
tal double line connecting the two individuals. T he birth o rd e r (oldest to young
est) is generally presented from left to right beneath the parents and twins are
shown by a com m on branch point. An arrow is used to indicate the p ro b an d (or
propositus) of the family.

Ap enetrance: T he pro p o rtio n of individuals sharing a com m on genotype who

show a particular phenotype. If all gene carriers display at least some evidence of
the phenotype, the p en etran ce is said to be com plete. A genotype may show com
plete p en etran ce b u t highly variable expressivity such that it is difficult to recog
nize individuals with subtle m anifestations o f the genotype.

#PFGE: see pulsed-field gel electrophoresis

Aphage: see bacteriophage

Aphysical m ap: A linear array indicating the o rd e r an d physical distance (i.e.

base pairs) betwreen two or m ore m arkers o r genes on a chrom osom e. Distances
can be established by a variety o f m ethods including nucleotide sequence, contig
m apping, overlapping hybrids, and m easurem ents from in situ hybridisation.

#PIC: ^ p o ly m o rp h is m inform ation content

 GLOSSARY 505

Aplaque: A plaque is an individual patch o f lysed bacteria on a culture plate that

is the result o f clonal expansion o f a single bacteriophage particle (see bacte
riophage) .

Aplasm ids: An extrachrom osom al genetic elem ent found in bacteria th at is capa
ble of self-replication. N aturally occurring plasm ids were first identified by their
ability to confer antibiotic resistance to bacteria. Plasmids are double-stranded
circular DNA's th at range in size from 1 to 200 kb. Most plasmids that are used as
cloning vectors range in size from 2.5 to 7 kb an d contain a nu m b er of unique
restriction sites for insertion of foreign DNA, drug-selection gene(s), and o th er
DNA sequences that allow for replication in the bacterial host, gene expression
or in vitro transcription.

*polyacrylam ide gel electrophoresis: T h e separation of charged m olecules (e.g.

negatively charged nucleic acids) using an electric field across a polyacrylam ide
m atrix or gel. For m olecules o f a given charge density (the same n u m b er of
charges p er u n it size), the separation is based upon size a n d /o r conform ation of
the m olecules. Polyacrylamide is a covalently crosslinked polym er that is resistant
to most d en atu rin g agents such as heat, urea, form am ide. Polyacrylamide gels
which contain the d en atu rin g agent, urea, are typically used for high resolution
analysis o f single-stranded DNA fragm ents such as for nucleotide sequencing.

ApolyA+ RNA: RNA that contains a 3 tail of adenine nucleotides (polyA). The
enzymatic addition of ad enine nucleotides occurs shortly after transcripation as
part o f the processing o f the prim ary RNA transcript in the cell nucleus. This
RNA represents the majority o f the m essenger RNA within a cell. Poly A+ RNA is
purified from total cellular RNA by selection with oligo dT (an oligonucleotide
consisting of 15-30 thym idine residues) th at can be b o u n d to cellulose, m em
branes, o r beads.

*polym erase chain reaction: A m eth o d (p aten ted by Perkin Elm er Cetus C orpo
ration) for amplifying a defined segm ent of double-stranded DNA by using two
DNA prim ers, one m atching a unique sequence at the 5' en d o f the sense strand
an d the o th er corresponding to the sequence o f the 5' en d o f the antisense
strand (Figure 1). W hen the DNA tem plate is d en a tu red by heat and allowed to
cool, the prim ers can specifically anneal to the com plem entary DNA strands and
serve as starting points for DNA synthesis by a therm ostabile DNA polym erase
(e.g. Taq polym erase). As a result, two new strands o f DNA are synthesised that
are com plem entary to the original DNA tem plate and two double-stranded m ole
cules are g enerated where previously there was only one. T he process of d en a tu r
ation, prim er annealing and DNA synthesis is then repeated a n d both DNAs can
serve as tem plates. W ith each successive ro u n d o r cycle, there is a doubling o f the
synthesised m aterial. W ithin a small n u m b er o f cycles (30-35) the initial tem plate
can be am plified 10-106 fold. As the process continues, a nearly hom ogeneous
506 GLOSSARY

po pulation o f DNA fragm ents o f a defined length are synthesised (Figure 1).
T hese fragm ents can then be cloned o r directly analysed for m utations or by
nucleotide sequencing. For Alu-PCR, the prim ers are derived from hum an-spe-
cific regions o f the highly repetitive Alu sequences. For reverse transcription-
PCR o r R T/PC R , the initial step consists o f synthesising a com plem entary DNA
(cDNA) to an RNA tem plate using reverse transcriptase and then using the cDNA
as the tem plate for a standard polym erase chain am plification.

Apolym orphism : T he occurren ce o f two o r m ore alternative genotypes (or alle

les) within a population such th at the frequency of the least com m on allele is at
least 1 % in the population (i.e. at least 2% o f the population are heterozygotes).

Apolym orphism inform ation content: A m easure o f the inform ativeness o f a

m arker based u p o n the relative frequencies o f the different alleles in the popula
tion an d the p ro p o rtio n o f genotype com binations (m atings) that are inform a
tive (i.e. the parental alleles can be distinguished). A m arker that detects only two
alleles, o n e o f which is present in over 90% o f the population, would have a low
PIC value a n d would not be very useful for linkage m apping. A m arker that
detects m ultiple alleles, none o f which is p resen t in m ore than 50% o f the p o p u
lation, would have a high PIC value and would be m ore inform ative in a linkage
analysis.

Aprim er: An oligonucleotide o r pre-existing polynucleotide chain that can serve

as the starting p o in t for the addition o f new deoxyribonucleotides by DNA poly
m erase o r reverse transcriptase.

Ap roband: T he individual in a family who is the first to be identified o r to seek

the advice o f a genetic counsellor and after whom o th e r family m em bers are
ascertained. Also referred to as the propositus (female, proposita) (see pedigree).

Aprobe: Single-stranded DNA o r RNA m olecules representing a specific nucle

otide sequence th at are used to d etect com plem entary sequences in a com plex
nucleic acid m ixture. T he probe m olecules are m odified so that they can be
d etected eith er by autoradiography, fluorescence or by secondary binding o f
d etecto r m olecules (e.g. conjugates of antibodies or avidin with enzymatic indica
tors) (see m ark ers).

Ap rom oter: T h at portion o f the gene that is involved in the control o f gene
expression, including tissue an d developm ental specificity an d regulation. T he
p ro m o ter includes the site at which the RNA polym erase binds a n d initiates the
transcription o f RNA from the gene.

*pulsed-field gel electrophoresis: This m eth o d o f agarose gel electrophoresis

separates large DNA m olecules ranging in size from 100 kb to 10 Mb. T he tech
 GLOSSARY 507

nique is used for physical m apping o f genom ic DNA using long-range restriction
endonucleases and for the analysis o f YACs and yeast chrom osom es. T he cells to
be analysed are em b ed d ed in agarose plugs, lysed and de-proteinised with pro
teolytic enzymes because very large DNA m olecules are highly vulnerable to
shearing in aqueous solutions. T he extracted DNA m olecules are then separated
in an agarose gel using altern atin g directions o f electrical cu rren t that are at vary
ing angles to the final direction o f DNA m igration. This alternating application
of electric fields causes the DNA m olecules to shift their orientation and confor
m ation so th at they can gradually pass th ro u g h the gel m atrix and be separated
on the basis of size a n d shape (see FIG E).

^radiation hybrid mapping: This m eth o d of physical m apping o f m arkers relies

on the breaking up o f chrom osom al DNA within cells by application of X-irradia-
tion and then rescue of chrom osom al fragm ents within a som atic cell hybrid. A
radiation hybrid usually contains m ultiple fragm ents from different chrom o
somes. Statistical m ethods sim ilar to those used for genetic m apping (linkage
analysis) are used to establish w7h e th e r two or m ore m arkers are physically linked
within a retained fragm ent. Because the size of the retain ed fragm ents can be
regulated by the radiation dose, m arkers as close as 50 kb apart can be separated
and m apped. T he m ap unit used for radiation hybrid m apping is the centiRay,
cR.

*random primer labeling: T here are several m ethods of generating labeled DNA
that can be used as a probe for hybridisation. R andom p rim er labeling involves
the use o f small oligonucleotides (e.g. hexam ers) th at can anneal to the tem plate
DNA and serve as the starting points for DNA synthesis using a DNA polym erase
and nucleotides th at are radioactively labelled (35S, 32P,33P ). T he probe can then
be detected using autoradiography. If the nucleotides are m odified to contain
biotin, then a detection m ethod based upon the binding o f biotin an d an avidin-
conjugate can be used (horseradish peroxidase stain or chem ilum inescence)
O th e r DNA labeling m ethods include nick translation, incorporation o f ;~P on to
the 5' en d o f the probe or p rim er using T4 polynucleotide kinase, or the addition
of radioactive or derivatized nucleotides on to the 3' e n d o f a DNA probe using
term inal deoxynucleotide transferase.

Arecombination: T he generation o f new com binations o f genes o r m arkers in the

progeny th at differ from those in the parents as a result eith er o f in d ep en d e n t
assortm ent o f chrom osom es o r o f crossing-over between hom ologous chrom atids
in meiosis. Crossing-over occurs when two paired chrom osom es (chrom atids)
exchange m atching regions of th eir DNA.

Arepetitive DNA: DNA sequences th at are present in m ultiple copies in the

genom e. T h e m ost well-known repetitive elem ents in the m am m alian genom e
are Alu elem ents which are approxim ately 300 base pairs long and the LINE-1
508 GLOSSARY

elem ents (long interspersed nuclear elem ents, also known as LI repeats) that
range in size from 1 to 6 kb. T here are also h u n d red s o f thousands o f short rep et
itive elem ents known as m icrosatellite repeats (see m icrosatellite repeats, Cot-1
DNA).

Restriction endonuclease: A large n u m b er o f enzymes have been isolated from

bacteria a n d o th er prokaryotes that have the ability to recognize specific seq
uences of DNA an d direct the double-stranded cleavage o f the DNA in a re p ro
ducible fashion. T he length and sequence o f the recognized DNA plays an im por
tan t role in d eterm in in g how frequently the restriction endonuclease cuts the
genom e. Enzymes can cut the DNA so as to create blunt-ended m olecules, dou-
ble-stranded DNAs with 5' o r 3' single stran d ed overhangs, or even cut the DNA
at a location several bases away from the recognition site. Restriction e n d o n u
clease digestions are frequently used to m ap DNA, identify restriction fragm ent
length polym orphism s, characterise cloned segm ents of DNA, and to facilitate
the jo in in g or ligation of DNA fragm ents for the purpose o f cloning.

Restriction fragment length polymorphism: Variation in the length of a specific

DNA restriction fragm ent that is detectable in d ifferent individuals in a popula
tion. T he variation in DNA fragm ents are d u e to the gain, loss or displacem ent of
specific restriction endonuclease sites. DNAs from different individuals are cut
with a restriction endonuclease, fractionated by agarose gel electrophoresis and
analyzed by a S outhern blot. Polym orphism s are detected by the presence o f dif
feren t sized bands th at hybridise to a u n iq u e DNA probe (Figure 1).

*reverse transcription: T he synthesis o f a com plem entary DNA to an RNA tem

plate using a p rim er (such as oligo dT) an d an R N A -dependent DNA polym erase
such as reverse transcriptase.

#RFLP: see restriction fragm ent length polym orphism

Aribonucleic acid: A covalent polym er of ribonucleotides (see n u cleo tid e).

#RNA: see ribonucleic acid

*RNase protection: A m ethod to detect an d quantitate small am ounts o f a single

stranded RNA transcript using a labelled, hom ogeneous RNA probe that is com
p lem entary (antisense) to the target RNA transcript. T he target and probe are
hybridised together in solution and the unhybridised probe is degraded with
ribonuclease. T he probe th at hybridises to the target creates a double-stranded
RNA th at is resistant to certain ribonucleases. T he protected, labeled RNA can
th en be m easured directly o r purified by polyacrylam ide gel electrophoresis. It is
a m ore sensitive m ethod for RNA m easurem ents than a N o rth e rn blot because
the unhybridised probe is rem oved enzymatically (seeN orthern blot).
 GLOSSARY 509

AMPLIFICATION
[primer annealing (55C)
and extension (72C)]

M B
' [---------------------------------T -

2 2 0 bp ESS: = -< - 220bp

Figure 1 A m plification o f specific targ et se q u e n c e s u sin g th e p o ly m erase ch a in reactio n . A

co m p lex D NA m ix tu re su ch as to tal g e n o m ic DNA is d e n a tu re d by h e a t (94C) a n d a n n e a le d
to specific single s n a n d e d p rim e rs (ca. 20 bases lo n g ) a t a strin g e n t te m p e ra tu re (55C) th a t
re d u c e s non-specific a n n e a lin g . T h e two p rim e rs are sp aced 0 .0 5 -5 kb a p a rt a n d are co m p le
m e n ta ry to th e sense a n d a n tisen se stra n d s respectively th a t flank th e re g io n to b e am plified.
A heat-stable polym erase (e.g. Taq p o ly m erase) is a d d e d w hich rep licates th e DNA in a 5' to
3' d ire c tio n , c o m m e n c in g a t e a ch p rim e r o n o p p o site strand s. T h e m ix tu re is d e n a tu re d a n d
th e cycle re p e a te d u p to 40 tim es. T h e re s u lta n t a m p lificatio n is (1+ X )11 w h ere X is th e am
plificatio n efficiency o f each cycle (a b o u t 0.85) a n d n is th e n u m b e r o f cycles, a lth o u g h th e
re a c tio n usually re a c h e s a p la te a u a fter 2 0 -3 0 cycles (a b o u t lOf-fold a m p lific a tio n ). B, am p li
fied p ro d u c t; M, size m arkers.

#RT: see reverse transcription

*S1 nuclease m apping: A m ethod to identify the en d of an RNA transcript by

hybridising a labeled DNA fragm ent to a com plem entary RNA. T he single
stranded ends o f the m olecules th at do n o t m atch are digested with SI nuclease .
If the DNA contains m ore than the com plem ent to the entire RNA strand, then
the DNA fragm ent will be sh o rten ed by the enzyme, leaving only those regions
protected by the RNA. T he change in size can be d eterm in e d on a sequencing
(denaturing) polyacrylam ide gel an d the length of the original RNA m olecule
can be calculated.

#S phase: T he DNA synthesis phase o f the cell cycle (see cell cycle).

Asense strand: Also known as the coding strand, the sense strand is the strand of
duplex DNA whose sequence corresponds to the nucleotide sequence o f the
mRNA transcribed from that gene. T he com plem entary strand to the sense
strand is often referred to as the antisense strand o f DNA. T he antisense strand of
genom ic DNA is the tem plate for RNA transcription.
510 GLOSSARY

Asequence tagged site: A specific location in the genom e that is defined and
identified by m eans of flanking PCR p rim er sequences th at specifically amplify
that locus. T h e site can therefore be identified using PCR within genom ic clones,
som atic o r radiation cell hybrids. T he PCR products re p resen t the sequence
tagged sites, STSs, and are used for the construction o f genetic and physical
maps. T he presence of a given STS in two o r m ore genom ic clones is an indica
tion that the clones overlap and form part o f a contig (see PCR, physical maps,
contig m apping).

Ashuttle vectors: G enetic elem ents that allow for the propagation o f foreign
DNA in m ore than one cell type. For exam ple, a plasm id that allows for propaga
tion o f a foreign gene in bacteria and can also be stably transferred to a yeast host
o r to a eukaryotic cell for gene expression (see v ecto r).

^single strand conform ation polym orphism : Also called single strand conform a
tion analysis, this m ethod detects single base differences betw een two segm ents of
DNA (usually u n d e r 400 bp in length) by observing the subtle differences in
mobility o f the DNA when the single-stranded m olecules are allowed to form
intra-strand base pairing as they m igrate through a non-denaturing polyacryla
m ide gel. T he differences in mobility are due to the slightly d ifferent folding pat
terns shown by partially base-paired single-stranded DNA m olecules as a result of
nucleotide differences.

*som atic cell hybrid m apping: Somatic cell hybrids are cell lines th at contain the
m ajority o f their DNA from one species an d a sm aller am ount of DNA (a few
chrom osom es o r chrom osom al fragm ents) from an o th er species. Panels o f these
hybrid cells, containing a broad distribution of hum an chrom osom es or chrom o
somal fragm ents, can be readily used to localise new m arkers o r genes to specific
hum an chrom osom es o r chrom osom al regions.

*Southern blot: T he detection o f specific DNA fragm ents after im m obilisation

on a m em brane by hybridisation with a labelled probe. T he technique was nam ed
after its developer, E.M. S outhern. T he m ixture o f DNA fragm ents are first sepa
rated by agarose gel electrophoresis and then transferred or blotted to a m em
b rane so as n o t to disturb the physical array o f fragm ents. T he filter is then
hybridised to the probe and washed so th at only those probe m olecules that have
hybridised to precisely com plem entary DNA fragm ents are retained. A utoradiog
raphy or o th er detection m ethods are then used to visualise the retain ed probe
DNA (see agarose gel electrophoresis, probe, restriction fragm ent length poly
m orphism ) .

Asplicing, in tro n /ex o n : T he process by which the initial transcript o f the gene
(called h eterogeneous nuclear RNA o r hnRNA) is edited to remove the intron
regions and appropriately connect the exons that make up the final mRNA tran
script (see intron, exon).
 GLOSSARY 511

#SSCP: see single strand conform ation polym orphism

#ssDNA: single-stranded DNA

Astreptavidin/biotin: Streptavidin is a com m ercially available protein derived

from streptomyc.es avidini th at contains four binding sites for biotin m olecules. It
behaves similarly to avidin, a slightly larger protein derived from egg whites.
Streptavidin o r avidin are readily conjugated to o th e r m olecules that allow for the
im m obilisation o r detection o f biotinylated m olecules. DNA o r RNA can be bioti-
nylated by photochem ical linkage or by the incorporation o f biotinylated nucle
otides by enzymes such as DNA o r RNA polymerases.

#STS: see Sequence Tagged Site

ATATA Box: A nucleotide sequence, e.g. TATAAAA, within the pro m o ter region,
usually located 19-27 bp from the transcription start site of eukaryotic genes.

Atranscription: T he synthesis o f RNA th at is com plem entary to the antisense

strand o f a DNA tem plate by a D N A -dependent RNA polymerase.

*transfection: 1) T he process o f intro d u cin g foreign DNA into cells by packag

ing the DNA into particles th at use viral m echanism s o f infecting cells, b u t are
n o t able to establish a continuing infection (i.e. propagation o f infectious parti
cles). 2) T he introduction o f DNA into cultured eukaryotic cells eith er to assess
transient gene expression or to obtain stable integration.

*transform ation: 1) T he in tro d u ctio n of foreign DNA into cells by transiently

altering the perm eability o f the cell m em brane to allow the m olecules to enter.
T ransform ation can be achieved by chem icals in the m edium , attached to the
DNA (e.g. calcium phosphate, lipofectin), or by an electric shock (see electropo
ration).
2) T ransform ation is also used to describe a change in the behavior of a cell th at
allows the cell to proliferate an d escape from growth control. An exam ple o f this
transform ation is observed when the retinoblastom a gene is inactivated an d the
cells begin to proliferate and form a malignancy. A nother exam ple o f cellular
transform ation is the transform ation o f blood lymphocytes by Epstein-Barr virus
to create lym phoblastoid lines th at can be continually grown in culture as a
replenishable source o f genom ic DNA.

*transgenic animal: A transgenic anim al is one that expresses foreign genetic

m aterial in addition to its own. It is a m eans o f studying the expression o f a syn
thetic or foreign gene in an intact anim al. Foreign genetic m aterial may be intro
duced by fusing m ouse em bryonic stem (ES) cells containing a m odified gene
with m ouse blastocysts and im plantation into a foster fem ale which results in the
512 GLOSSARY

producion of chim aeric offspring. Alternatively, a foreign gene is in troduced into

an anim al (e.g. m ouse, rat, pig, cow) by injecting the DNA directly into the pro
nucleus o f a fertilized egg. T he injected egg is im planted into a pseudo-pregnant
fem ale to allow for norm al developm ent. Single or m ultiple copies o f the foreign
gene (transgene) integrate in a random fashion within the host genom e. These
transgenes can be expressed with appropriate developm ental- and tissue-specific-
ity d ep e n d in g on 1) the pro m o ter elem ents that are included with the coding
region and 2) the effects o f the flanking sequences aro u n d the insertion site.
T ransgenic anim als are used for studies o f gene expression an d function and for
the creation o f anim al m odels o f genetic disease. Because the transgenes are in te
grated into the anim al's genom e, the genetic m aterial can be stably propagated
th rough the anim al's progeny, (see C hapter 1 (II) and Figure 5 of that C hapter)

translation: T he cellular process by which an mRNA sequence directs the

o rd e red assembly o f am ino acids into a peptide chain.

Atranslocation: A chrom osom al aberration in which regions of one o r m ore

chrom osom es are displaced to an o th er chrom osom al segm ent. T he p o in t at
which the two previously noncontiguous DNA segm ents are jo in e d is referred to
as the translocation breakpoint.

Avariable number tandem repeats: DNA segm ents that contain variable num bers
o f short tandem ly repeated segm ents with a particular sequence. G enom ic DNA
is cut with a restriction enzyme outside th e rep eated sequence, the specific frag
m en t is size fractionated using gel electrophoresis and then d etected by hybridis
ation. Because o f the variable nu m b er of repeats, the fragm ents d em onstrate dif
feren t lengths (polym orphism s) in d ifferent individuals which can be used as
alleles for that locus.

Avector: A genetic elem ent th at is used to carry a foreign piece o f genetic infor
m ation into a cell and will support the propagation of that foreign DNA within
the cell. Vectors can refer to plasmids, cosm ids an d phage which su p p o rt foreign
DNA in bacteria, as well as YACs in yeast a n d viral elem ents that in troduce DNA
into m am m alian cells.

#VNTR: see variable nu m b er tandem repeats

*Western blot: A m ethod th at is analogous to S outhern and N o rth e rn blotting,

in which proteins are fractionated by size using d en atu rin g polyacrylam ide gel
electrophoresis an d then transferred to a m em brane o r filter. T he detection of a
specific protein is achieved using an antibody that has been raised against all or
p art of that protein. Detection o f the b o u n d antibody can be achieved by radioac
tive an d nonradioactive m ethods.
 GLOSSARY 513

Ayeast artificial chrom osom e: An artificial chrom osom e that contains large frag
m ents o f foreign DNA (0.1-1.5 Mb) within a linear vector m olecule that can stably
propagate itself and segregate within the yeast Saccharomyces cerevisiae. A YAC con
tig (see contig) can define a large region of the genom e with a lim ited nu m b er of
clones. T he use o f these vectors is h am p ered by occasional instability o f the
inserted DNA, cloning artifacts such as rearrangem ents an d jo in in g o f unrelated
sm aller pieces o f DNA (chim aeric clones), an d difficulties in obtaining large
am ounts o f the purified YAC free of background yeast chrom osom es. YACs pro
vide a source of DNA for the identification of genes, Alu-PCR, fluorescence in situ
hybridisation and chrom osom e painting, lim ited genom ic libraries, an d contig
m apping o f large genom es (see Alu-PCR, chrom osom e painting, cloning, contig
m apping, fluorescence in situ hybridisation, and library).

#YAC: see yeast artificial chrom osom e

zinc linger proteins: Proteins th at contain one o r m ore segm ents that are capa
ble o f binding Zn to two closely-linked cysteine residues followed by two histidine
residues. T he binding of Zn creates loops or fingers th at are capable of binding
DNA and potentially regulating transcription.
 INDEX

3' e n d , 7, 491 A m in o acid seq u en c e , 309, 314, 389

5' en d , 7, 491 A m n io cen tesis, 211
11-W,v retin al. 161, 219 A n g elm an syn d ro m e, 105-106
A b e ta lip o p ro te in a em ia , 6 3 -6 4 A nim al m o d els albinism , 89 -1 1 3 , 104
A b so rp tio n sp e c tru m c o lo u r pig a n irid ia, 4 52-455
m en ts, 219, 222, 244 cata ract, 4 2 9 -4 3 4
A cce p to r splice site, 491 c h o ro id e re m ia , 316
A ch ro m ato p sia, 220 CSNB, 293
A garose gel ele c tro p h o re sis, 491 p la te le t sto rag e disease, 109-110
A g e-related m a c u la r d e g e n e ra tio n M arfan syndrom e, 392
(AM D), 173-191 re tin a l d e g e n e ra tio n , 123-139
d efin itio n o f AMD, 1 73-174 U sh e r sy n dro m e type II, 377
h eritab ility o f AMD, 179-181 A n irid ia, 4 45-462
h e te ro g e n e ity o f AMD, 174-179 AN1, AN2, 448
therapy, 190 clinical featu res, 44 5 -4 4 7
A geing, 179, 361, 413, 425, 472, 484 c lo n in g o f c a n d id a te a n irid ia genes,
A land eye disease, 28 8 -2 9 0 4 50-452
A lbinism , 89 -1 1 3 cytogenetic a b n o rm alities, 448-449
classification, 96 frequency, 447
d efin itio n o f h u m a n albinism , 9 3 -9 5 genetics, 447
d iagnosis, 112-113 hom o zy g o te (A N2), 453
o c u lar albinism , 89, 110-112 linkage studies, 44 7 -4 4 8
a u to so m al recessive o c u la r albi A n o p h th a lm ia , 4 5 3 -4 5 4
nism (O A 2 ), 111-112 A n te rio r se g m e n t defects, 321, 459
X -linked recessive O A (O A 1), A ntib o d ies, 204, 289, 334, 3 87-388,
110-111 392
o c u lo c u ta n e o u s alb in ism (O C A ), 89, A ntisense stran d , 492
9 5 -1 0 8 A p p aloo sa horses, 293
brow n OCA, 107 A rrestin , 128, 130-131, 163
m in im al p ig m e n t OCA, 9 7 -9 8 ASO, 158-160, 49 1 -4 9 2
m u ta tio n s in h u m a n TYR ge n e , A taxia, 359, 363, 447, 480 -4 8 1 , 486
9 8 -1 0 2 ATP, 469
p la tin u m OCA, 98 ATP synthase (C o m p lex V ), 469-470,
ru fo u s OCA, 107-108 480
te m p e ra tu re sensitive OCA, 98 A trophy, o f ch o ro id a n d RPE, 304
tyrosinase n egative OCA, 97 A ud iog ram s, 362
tyrosinase positive O CA (O C A 2), A u d ito ry evoked resp o n ses, 361-362
103-108 A utism , 323
tyrosinase re la te d O CA (O C A 1), A u to rad io g rap h y , 492
9 6 -1 0 3 A vidin, 492
yellow OCA, 9 7 -9 8 A xonem es, 371
tre a tm e n t, 113
A llele, 491
A llele-specific o lig o n u c leo tid e (A SO), 15 8 - B acterio p h ag e, 492
160, 491 B ardet-B iedl sy n d o m e, 368
A lu PCR, 492 Base, 1, 492
A lu re p e a ts, 5, 492 Base pair, 493
A m in o acid u ria, 403, 405 B eh av iou r d iso rd ers, 402
516
B ests disease see M acular dystrophy,
v itelliform
Beta-galactosidase, 493
B iettis cry stallin e dystrophy, 66 -6 7
B iotin, 493
B ip o lar cells, 74
B lindness, 259, 321, 367, 400, 477,
4 7 9 -4 8 0
B lood-retinal b arrier, 280
B lue-cone m o n o ch ro m acy , 71, 220,
242
bp, 493
B reak p o in t, 493
B ru c h 's m e m b ra n e , 175-176, 178
B ubble PCR, 493
B ull's eve dystrophies, 68-69
B utterfly d y stro p h y o f th e fovea, 6 6 ,
185-187
CA re p ea ts, 6 , 494
C alcium c h a n n e l, 40, 42
C alciu m signalling, 40, 408
C a n d id a te g e n e , i8 2 , 184, 189, 266,
315, 329, 372, 3 7 4 -3 7 6 , 407
C ap site, 7 -8
C a rrie rs in c h o ro id e re m ia , 62, 304
in N o rrie disease, 324
in O C R L syn d ro m e, 3 9 9 -4 0 1 , 408
in X -linked c o n g e n ital statio n ary
n ig h t b lin d n ess, 28 7 -2 8 8
in X -linked ju v e n ile retinoschisis,
347-348, 350-351
in X -linked retin itis p ig m en to sa, 57,
2 6 0 -2 6 1 ,2 6 9 "
C atalase g en es, 44 9 -4 5 0
C ataract. 260, 321, 367, 399, 401, 404,
408, 4 13 -4 3 5 , 445, 453, 459,
485
aging, 413, 425
a n te rio r p o la r cataract, 426
c o n g e n ita l, 399
C o p p o c k cataract, 427-428
d o m in a n t in h e rite d cata ract
in h e rite d fo rm s in m an , 420,
425
e m b ry o n a l cata ract, 427
h e re d ita ry c e n tra l cataract, 429
lam ellar cata ract, 425-426, 429
linkage stu d ies in h u m a n in h e rite d
cataracts, 427-429
M a rn e rs cata ract, 4 27-428
m orp h o lo g y , 426
IXDF.X
m u ta g e n -in d u ce d , 433
p o ste rio r c a p su lar cataract, 428
p o ste rio r p o la r cataract, 42 6 -4 2 8
p u l\ ui u h iit cataract, 426, 429
total cata ract, 426
z o n u la r cataract, 42 6 -4 2 7
C attle m o d el, M arfan syn d ro m e, 392
cDNA see C o m p le m e n ta ry DNA
C ell cycle, 206, 494
C en tiM o rg a n , 1 1 ,4 9 4
C entiray, 495
C e n tro m e re , 2
C EPH fam ilies, 14, 349, 375
C h a p e ro n e activity, 416
C h ed iak -H ig ash i sy n d ro m e, 109
C h e m ica l cleavage o f m ism atch , 189
C h e m ilu m in e sce n ce , 495
C h im aeric m ice, 16-17, 432, 460,
511 -5 1 2
C h o rio cap illaris, 62, 67, 69-7 0 , 175,
304
C h o rio n ic villus sam pling, 208
C h o ro id , 62, 66 -6 7 , 70, 30 3 -3 0 4 , 316
C h o ro id e re m ia , 61 -6 3
C h o ro id e re m ia (C H M ), 61 -6 3 ,
3 0 3 -3 1 6 , 341
c h ro m o so m al ab n o rm alities with
c h o ro id e re m ia , 30 5 -3 0 7
clinical aspects, 3 0 3 -3 0 4
clo n in g C H M g e n e , 307-311
co n tig u o u s g e n e d e le tio n
syndrom es, 304-305
critical reg io n , 305
fu n d u s ch a n g e s in hetero zy g o u s
fem ales, 62
g en o m ic stru c tu re C H M gene,
3 08-309
geran y lg eran y l transferase, 315316
m u rin e h o m o lo g u e o f C H M , 308,
314-315"
C U M I g en e, 308, 312, 314, 316, 375
C h ro m o so m a l ju m p in g , 306
C h ro m o so m a l localisation
d o m in a n t in h e rite d c a ta ra c t g en es
in m an, 420
hC H M L g e n e , 315
m ajo r h u m a n g en e s e x p ressed in
lens, 420
N D P g e n e , 327
p h o to tra n sd u c tio n genes, 131
RS g e n e , 350
RP2, RP3 g en es, 263
 INDEX
U sh e r sy n d ro m e genes, 360, 373
C h ro m o so m a l w alking, 306
C h ro m o so m e 1, h u m a n , 3 75-378
C h ro m o so m e 2, h u m a n , 417
C h ro m o so m e 3, h u m a n , 157-165, 169
C h ro m o so m e 4, h u m a n , 135
C h ro m o so m e 6 , h u m a n , 138-139,
165-169, 181, 184-188
C h ro m o so m e 7, h u m a n , 168
C h ro m o so m e 8 , h u m a n , 168-169
C h ro m o so m e 11, h u m a n , 9 8 -1 0 2 , 183,
3 72-374, 417, 4 4 7 -4 5 2
C h ro m o so m e 13, h u m a n , 197-211
C h ro m o so m e 14, h u m a n , 3 70-372
C h ro m o so m e 15, h u m a n , 106-107,
3 87-392
C h ro m o so m e 17, h u m a n , 417
C h ro m o so m e 21, h u m a n , 417
C h ro m o so m e 22, h u m a n , 417, 429
C h ro m o so m e X, h u m a n , 217-254,
25 9 -2 7 2 , 27 7 -2 9 5 , 303-316,
3 21-334, 3 39-353, 3 99-408
C h ro m o so m e b a n d in g , 495
C h ro m o so m e p a in tin g , 495
C h ro m o so m es, 2, 202, 495
C h ro n ic g ra n u lo m a to u s disease, 264
C h ro n ic progressive e x te rn a l
o p h th a lm o p le g ia (C P E O ),
48 1 -4 8 4
Cilia, 371
C iliary body hypoplasia, 445
C iliary processes, 322
C iliary zo n u les, 386
cM see ce n tiM o rg a n
C o ats disease, 367
C ochlea, 3 61-362
C o d in g re g io n , 496
C o d o n , 7, 496
C olo b o m a, 445-446, 459
Colony, 496
C o lo u r m atch in g , 218, 222, 224,
2 46-248
C o lo u r p ig m en ts long-w avelength,
219
m e d iu m w avelength, 219
n o rm a l variatio n , 223
short-w avelength, 219
C o lo u r p ig m e n t g e n e s 225, 228, 236
defects, 241
fu sio n g en es, 226-229, 233
sto ich io m etry , 23 4 -2 3 6 , 23 9 -2 4 0
stru c tu re , 2 3 5 -2 3 6
517
C o lo u r sen satio n , 219
C o lo u r vision, 21 7 -2 5 4 , 281, 288
variations in n o rm a l c o lo u r vision,
22 3 -22 5
defects, 70 -7 1 , 21 7 -2 5 4
C o m p le m e n ta ry DNA (cD N A ), 22,
1 3 6 ,4 9 4 ,4 9 6
C o m p le m e n ta tio n , g en etic, 484
C o m p lex I-V, re sp ira to ry ch ain , 469
C o m p o u n d eye, Drosophila, 30
C o m p o u n d hetero zy g o tes, 98
C o n e d efects co n g en ita l, 70-71
C o n e d ystrophies, 6 8 -6 9 , 181
C o n e p h o to re c e p to rs, 154, 217, 243
C o n e p ig m en ts see c o lo u r p ig m e n ts
C o n e-ro d dystrophies, 68 -6 9
C o n g e n ita l ach ro m a to p sia, 220
C o n g e n ital a d re n a l hypoplasia, 288
C o n g en ital cataract, see under c ata ract
C o n g e n ita l c o n tra c tu ra l
arachnodactyly, 390-391
C o n g e n ita l p h o to re c e p to r defects,
70-75
C o n g e n ita l re tin a l folds, 347
C o n g e n ita l ro d m o n o c h ro m a cy
(a c h ro m a to p sia ), 220
C o n g e n ita l statio n a ry n ig h t b lin d n ess
(CSNB), 71 -7 2 , 271-272,
2 77-295
au to so m a l d o m in a n t, 278-279, 294
carriers, X -linked, 28 7 -2 8 8
classification, 2 78-279
clinical m an ifestatio n s, 279-281,
2 91-292
c o m p le te tvpe, 283, 285
CSNB1, 289, 294
CSNB2, 289, 294
d a rk a d a p ta tio n , 291
electro p h y sio lo g ical ev aluation,
2 81-288
histopathology, 292
in c o m p le te type, 284286, 289, 294
in h e rita n c e , 279
m a p p in g g en e s fo r CSNB, 2 9 3 -2 9 4
N o u g a re t variety, 279
p ath o g e n e sis, 29 2 -2 9 3
p h o to p ic resp o n ses, 286
X -linked, 27 1 -2 7 2 , 27 9 -2 8 0
C o n n ectiv e tissue, 383, 387
C ontigs, 378, 496
C o n tig m a p p in g , 496
C o n tig u o u s g e n e d e le tio n syndrom e.
518
304, 36 8 -3 6 9 , 377, 447
G o p p er-b in d in g regions, 99
C o rn e a , 66, 321, 399, 445, 481
C osm ids, 329, 352, 496
Cot-1 DNA, 496
C pG island, 9, 264-265, 450, 452, 497
cR seecentiRay
C rossing over 10, 497
critical, 375
u n e q u a l, 220, 2 27-228
C rystallins, 413-435
a-crystallins, 41 5 -4 1 7
aA -crystallin g e n e in D ow ns
sy n d ro m e, 428
c h a p e ro n e activity, 416
c h ro m o so m a l localisation,
41 6 -41 7
d e v e lo p m en ta l e x p ressio n , 416
g e n e o rg a n isa tio n (aA a n d (3B),
41 5 -41 7
h e a t shock p ro te in hom ology, 416
P-crystallins, 417-421
ev o lu tio n , 419
g e n e o rg an isa tio n , 418
in te ra ctio n s, 419
p o st-tran slatio n al m o d ificatio n ,
418
p ro te in stru c tu re , 418
PB2-crystallin, 430-431
a lte re d p ro p e rtie s in Philly m o u se,
431
m u ta tio n s in Phillv m o u se, 430
Y-crystallins, 4 21-424, 428, 432, 434
ev o lu tio n . 423
g e n e o rg an isa tio n , 422
in cataract, 425
linkage stu d ies in m an , 428
p ro te in stru c tu re , 422
yE-crystallin g e n e m u ta tio n in Elo
m o u se, 432
5-crvstallin, 415, 434
^-crystallin, 432-433
enzym atic activity, 433
m u ta tio n in 1 3 /N g u in e a pig,
432-433
ag g reg a tio n in cataract, 425
enzym atic activity, 4 3 3 -4 3 4
po st-tran slatio n al m o d ificatio n ,
414, 418
taxon-specific, 414, 432, 434
C ybrids, 484
Cyclic CMP, 124, 129, 163
Cyclic G M P -p h o sp h o d iesterase, 38,
INDEX
123-136, 162-163, 170
stru c tu re, 128
a b n o rm a litie s in rd m o u se, 128-130
g e n e stru c tu re , ^-subunit, 134
Cyclic G M P-gated io n c h a n n e l,
162-163
Cysteine re p ea ts, 3 89-390
C v to ch ro m e c o xidase (C o m p lex IV ),
470, 475, 478
C ytogenetic analysis, 2 0 1 , 326, 4 47-448
C ytoskeletal p ro tein s, 43
D ark a d a p ta tio n , 60, 71, 73 -7 5 , 167,
259, 288, 29 0 -2 9 1 , 346
dATP, y-:,-P o r a-3-'P lab elled , 497
D eafness, 304, 32 3 -32 4, 359-361, 481,
485
D eletio n s 26 4 -2 6 5 , 305-306, 3 2 8 -
329, 324, 449, 458
c h ro m o so m al, 20 0 -2 0 1 , 288, 304,
447
in m t DNA, 481-485
sm allest re g io n o f o v erlap (SR O ),
449 -4 5 0
sub m icro sco p ic, 203, 2 64-265, 268,
307, 309-310, 329
D em en tia, 323-324, 481
D e n a tu rin g g ra d ie n t gel e le c tro p h o resis
(D G G E ), 186, 189, 497
D eo x y rib o n u cleic acid (DNA) 2, 497
b in d in g d o m ain s, 452
fra c tio n a tio n , 20
m e th o d s o f m odifying, 20-21
re p a ir g en es, 3 76-377
rep etitiv e, 5 -6
seq u en ce, 158-159, 190, 203, 210,
332, 329, 407, 457
D e ta c h e d re tin a , 321-322
D eu teran o m aly , see T rich ro m acy
D e u te ra n o p ia , i^ D ic h ro m a c y
D e v elo p m e n t o f th e eve, 452, 455,
459, 461
D ev elo p m e n tal switch, 391
D iabetes m ellitus 48 5 -4 8 6
cataracts in, 408
D iagnosis 112, 189, 324, 332, 334,
341, 350, 458
in in h e rite d retin a l d iso rd ers, 5 3 -7 6
d ifferen tial, 325, 347
Dichrom acy, 218, 220-221
d e u te ra n o p ia , 22 0 -2 2 1 , 225
m o le c u la r g en etics, 22 5 -2 2 6
p ro ta n o p ia , 220- 2 2 1 , 226
 INDEX 519

m o le cu la r genetics, 226 Elo m o u se see m o u se m u ta n ts

trita n o p ia, 220 E m b ry o n ic stem (ES) cells,
D ifferen tia tio n cellular, 205-206, 334 1 5 -1 7 ,5 1 1
D igoxygenin, 497 Emx-1, Emx-2 see h o m e o b o x g en es
DGGE see D e n a tu rin g g ra d ie n t gel E n h a n c e r seq u en ces, 10
e le c tro p h o resis E p id e rm a l grow th facto r m otif, 390,
D -loop m t DNA, 471 392
DNA see D eoxyribonucleic acid EST, see E x p ressed se q u e n ce tag
D og, Irish setter, 135 E sterase D, 200
D o m in a n t d ru se n , 179, 183 E thical issues, 211
D o m in a n t n egative effect, 392 E volution, o f crystallins, 419, 423
D o n o r splice site, 498 E volu tio n ary co n serv atio n , 308,
D o p a c h ro m e tau to m e ra se , 91, 93 4 51-452
D o t b lo ttin g , 498 E xons, 7, 498
D ow ns sy n d ro m e cata ract, 428 E x p ressed se q u e n c e tag (E S T ), 498
dsDNA, 498 E xpression vector, 498
D ow ns syn d ro m e, 428 Expressivity, 347, 44 5 -4 4 6 , 458, 475,
c a ta ra c t in , 428 4 80-481, 498
aA -crystallin g en e , 428 E x tracellu lar m atrix m o lecu les, 387
D ro so p h ila m elanogaster, 2 9 -4 6 Eye d ev elo p m e n t, 452, 455, 459, 461
a lle le -d e p e n d e n t p h o to re c e p to r
d e g e n e ra tio n , 45 Fam ilial ex udative v itreo -retin o p ath y
c o m p o u n d eye stru c tu re , 3 0 -3 2 (FEVR) sci- X -linked FEVR
m u ta n ts ninaE , 45 F an co n i sy n d ro m e, 403
ninaC , 34, 43, 46 Fatal in fan tile c ard io m y o p ath y
norpA, 34, 36, 41 (FICM ), 474
paired, 452 F ib re cell e lo n g a tio n , 431, 434
rdgA, 41, 45 F ibrillin genes, 3 87-392
rdgB, 34, 41 M arfan sy n d ro m e a n d m u ta tio n s in
rdgC, 34, 44 fibrillin g en e , 39 1 -3 9 2
re tin al d e g e n e ra tio n , 2 9 -4 6 Fib5 g e n e, 390-391
tip. 34, 3 9 -4 0 , 46 F ib l5 g e n e , 388
D ru sen , 174, 176, 179, 183, 344 F ib l7 g e n e , 390
D u p licatio n s m t DNA, 481, 48 5 -4 8 6 F ib ro b last grow th fa c to r 3 (FG F3), 372
D ystrophin, 289 Field inversion gel elec tro p h o resis,
307, 499
E l A p ro te in , 205 FIGE see Field inversion gel
E7 p ro te in , 205 ele c tro p h o re sis
E cto p ia lentis, 385 FISH see F lu o rescen ce in situ
Elastin, 393 hybrid isatio n
E lectro-nystagm ography, 363 Fleck re tin a o f K andori, 75, 278-279
E lectro -o cu lo g ram (E O G ), 65, 68-69, Flow cytom etry, 19, 499
345 F lu o rescein angiography, 56, 62, 6 6 ,
E lectro n tra n s p o rt im p a irm e n t, 480 6 8 , 74, 280, 346, 445
E lectro p ho resis, 18, 20, 499 F lu o rescen ce, 499
E le c tro re tin o g ra m (ER G ), 30 -3 1 , 42, F lu o rescen ce in situ
45, 56, 59, 6 5 -6 6 , 6 8 , 71-75, hybrid isatio n , 452, 458, 499
124, 167-168, 201, 259, F o u n d e r effect, 102, 339, 309
2 81-285, 287, 289-290, Fovea, 93 -9 4 , 186, 288, 342, 3 66-367,
3 46-347, 366 445
n egative ERG, 283, 287, 289 F re n c h A cadian p o p u la tio n , 372
S c h u b ert-B o rn sch ein , 2 8 3 -2 8 4 F u n d u s alb ip u n c ta tu s, 74, 278, 281
E le c tro p o ra tio n , 498 F u n d u s flavim aculatus, 6 7 -6 8
520 INDEX

F u n d u s reflecto m etry , 292 G yrate atrophy, 310

F u n d u s, retin al, 278, 280, 304, 3 64-366
H -stran d , m t DNA, 471
H a e m o rrh a g e s, in retinoschisis, 346
G, 499
H a llg re n sy n d ro m e, 368
G ;. 499
H aplo-insufficiency, 137, 457
G alacto k in ase, 426
H ap lo ty p e analysis, 26 7 -2 6 8
G alactosem ia, 408
H ap lo ty p e association, 351
G an g lio n cells, 72
H e a t sh o ck p ro te in s, 416
Gel elec tro p h o re sis, 499
H erm ansky-P udlak sy n d ro m e, 108-109
G el re ta rd a tio n assays, 500
H e te ro d u p le x analysis, 158, 189, 455
G en e, 5 -9 , 500
H e te ro g e n e ity clinical, 35 9 -3 7 9
G en e anatom y, 6 -1 0
gen etic, 14, 169, 262, 269, 350,
G en e co n v ersio n , 423
359 -3 7 9 , 388, 390,
G en e ex p ressio n - 424, 310, 333, 459
H e te ro g e n e o u s n u c le a r (h n ) RNA, 7,
c o lo r p ig m e n t g en es, 242-243,
500
24 8 -2 4 9 , 253
H eteroplasm y, 472, 480, 483, 4 85-486
y-crystallins, 424
Heterozygosity, 500
Pax-6, 452, 45 9 -4 6 0
H isto p ath o lo g y N o rrie disease, 323
G e n e stru c tu re c o lo u r opsins, 236
CSNB, 292
C H M , 30 7 -3 0 9
retin o b lasto m a, 197
crystallins, 41 5 -4 1 8 , 422
ju v e n ile retinoschisis, 341
cyclic GMP-PDE (3-subunit, 134
hnR N A see H e te ro g e n e o u s n u c le a r
PAX6 , 455-456
RNA
p e rip h e rin /R D S , 166
H o m e o b o x g en e s 452, 455, 461
m o u se rds, 184185
Emx-1, 461
N D P g e n e , 32 9 -3 3 2
Em x-2, 461
R B I, 2 0 3 -2 0 4
Hox-7, 461
TRP1, 92
H ox-8 , 461
tyrosinase, 92
XANF-1, 461
F ib l5 , 391
H o m ology h u m an -m o u se, 144
G e n e therapy, 39, 43 -4 4 . 395
H om oplasm y, 472, 474, 478
G en etic co d e, 1
H o rserad ish p e ro x id ase, 500
G e n e tic co u n sellin g , 351, 457, 462
H R P see H o rse ra d ish p ero x id a se
G en etic d istan ce, 5
H u m a n crystallin g en es c h ro m o
G en etic m ap , 12, 351, 500
som al localisation, 417
G en etic m a p p in g , 10-17
H u m a n m ito c h o n d ria l (m t) DNA, 470
g e n etic m ap len g th s, 12
H ybrid isatio n in situ , 500
g e n e tic m ark ers, 14
subtractive, 130, 136
G en etic silen cin g , 203
d ifferen tial, 130, 136
G e n ito u rin a ry an o m alies, 324, 447
H ybrids som atic cell, 137, 139, 200,
G en o m e, 500
266, 314, 327, 449
G e n o m e anatom y, 2 -6
H y p e rp h o sp h a tu ria , 403
G en o ty p e-p h en o ty p e c o rre la tio n s, 269,
H y p o p ig m e n ta tio n , 89 -1 1 3 , 288
333, 480
G illespie sy n d ro m e, 447, 459
G lau co m a, 321, 399, 404, 445 Im aging, CT a n d MRI, 393
G lutath io n e-S -tran sferase, 426 Im p rin tin g , 3, 106, 200
Glycerol kinase deficiency, 288 In b o rn e r r o r o f m etab o lism , 408
G lycosam inoglycans (GAGs), 406, 408 In n e r n u c le a r layer, 282
G oldm ann-F avre disease, 347 In n e r p lex ifo rm layer, 282
G uanylate cyclase, 162-163 In o sito l p h o sp h a te m etab o lism , 36 -37 ,
G u in e a pig, 1 3 /N , 43 2 -4 33 40, 4 07-408
 INDEX

In o sito l p o ly p h o sp h ate-5 -p h o sp h atase, Loss o f heterozygosity, 200, 206, 502

407 Lowe o c u lo c e re b ro re n a l sy ndrom e
In te rfe re n c e , 11 (O C R L ), 39 9 -4 0 8
Inter-specific crosses, 15, 132, 144, 455 b io ch em ical findings, 4 06-407
In tro n s, 7, 501 clinical featu res, 3 99-405
Iris hypoplasia, 445, 453 fertility, 404
Iris p ig m e n t, 63, 94, 288 grow th, 402
Iso p ren y latio n site, 135 infectio n s, 404
o c u la r featu res, 40 0 -4 0 2
Ju n k DNA, 6 systemic featu re s, 40 2 -4 0 4
Ju m p in g libraries, 501 lin k ag e studies, 407
Ju v en ile retinoschisis, see X 4 ink ed p ath o lo g ic al featu res, 404405
ju v e n ile retin o sch isis p o sitio n al clo n in g , 4 0 7 -4 0 8
therapy, 408
K aryotype, 501 L -strand, m t DNA, 471
K earns-Sayre sy n d ro m e (KSS), L y m p h o b lasto id cells see tran sfo r
48 1 -4 8 4 m a tio n

L actate, 474, 481 M p h ase, cell cycle, 502

L arge T a n tig e n , 205
L au ren ce-M o o n syn d ro m e, 368
L e b e rs h e re d ita ry o p tic n e u ro p a th y
(L H O N ), 47 4 -4 8 0
clinical featu res, 474
high-risk m u tatio n s, 47 6 -4 77
m o d erate-risk m u ta tio n s, 47 6 -4 7 9
low-risk m u ta tio n s, 476, 4 7 9 -4 8 0
L e ig h s disease, 480
Lens a b n o rm a lities in an irid ia, 453
d ev elo p m en t, 399, 406, 413-414,
421
d e v elo p m e n ta l re g u la tio n , 421
dislocation, 445
in d u c tio n , 460
m ajo r h u m a n g en es ex p re sse d in,
420
stru c tu re , 415
L ibrary, 501
L ib rary screen in g , 501
L ight effects o n re tin a l d e g e n e ra
tio n , 38, 4 0 -4 1 , 4 3 -4 5 , 176,
190
L inkage analysis, 10-14, 106, 139, 145,
1 5 7 ,1 6 5 ,1 8 1 -1 8 2 ,1 8 9 , 200,
20 9 -2 1 0, 262, 266, 289,
2 93-294, 32 6 -3 2 7 , 348-349,
352, 372, 374, 38 7 -3 8 8 , 391,
407, 42 7 -4 2 9 , 447-448, 502M a tern a l in h e rita n c e , 472, 475
L ipofuscin, 65, 67, 183-184
Locus, 502
L ocus-control reg io n (L C R ), 220, 243
L od score, 12-13, 502
L O H see Loss o f heterozygosity
M acro m elan o so m es, 109, 111, 288
M acula, 6 4 -6 5 , 259, 341, 445
M acular d e g e n e ra tio n , 167, 242, 342,
314, 347
see A g e-related m ac u la r d e g e n e ra
tio n
M acular dystrophy, 59, 138, 169,
a d u lt vitelliform , 65, 168, 187
N o rth C aro lin a, 145, 170, 179, 181
p seu d o -in fla m m ato ry (Sorsbys),
69-70 , 179, 183
vitelliform , 64 -6 6 , 170, 179, 181, 183
MAO see m o n o a m in e oxidase
M ap p in g fu n ctio n s, 11
M arfan sy n d ro m e (M FS), 38 3 -39 5
an im al m o d el, 39 2 -3 9 3
clinical featu res, 38 3 -3 8 6
cardio v ascu lar featu res, 385, 393
m usculoskeletal featu res, 383-384,
394, 404
o c u la r featu res, 38 5 -3 8 6
in fan tile, 386
in h e rita n c e , 386
linkage studies, 3 87-388
m a n a g e m e n t, 39 3 -3 9 4
mortality, 393
m u ta tio n s in fibrillin g e n e , 391-392
M arker, 502
M axim um lik elih o o d , 13
Mb, 502
M cL eod sy ndrom e, 264
M elan in synthesis, 90-91
522 INDEX

M elan o m a, 202 m ic ro o p th a lm ia (m i), 139

M ental re ta rd a tio n , 109, 264, 304, 324, m ic ro p h th a lm ia defective iris
368, 402, 447 (m idl), 461
M eso d erm , 459 m o tc h , 461
M essen g er RNA, 502 m u sc u la r d y strophy (m dx), 17
M etastasis, 199 n e rv o u s (n r), 123
M icrocephaly, 324 p earl (pe) , 293
M icro d e le tio n s see D eletio n s Philly (Phil), 430-431
M icrodissection o f ch ro m o so m es, 502 pink-eyed d ilu tio n (p ), 104, 106
M icrofibrils, 3 87-389 p u rk in je cell d e g e n e ra tio n (ped),
M icro in jectio n , 17, 164 123
M icro p h th a lm ia , 321, 426, 431 re tin a l d e g e n e ra tio n (rd), 15, 39,
M icrosatellite m ark ers, 6 -1 4 , 165, 123-135
189, 351 re tin a l d eg e n e ra tio n -3 ( rd-3), 123,
re p e a ts see C A rep ea ts 377
M ito ch o n d ria l ab n o rm a litie s, 176, 399, R etin al d e g e n e ra tio n -4 (R d -4 ), 123
406, 4 8 2 -4 8 6 re tin a l d e g e n e ra tio n slow ( rds) , 15,
M ito ch o n d ria l DNA, 3, 46 9 -4 86 58, 123, 136-139, 160,
d ele tio n s, 48 2 -4 8 5 1 66-167
d u p lica tio n s, 48 5 -4 8 6 shaker-1 (sh-1), 374
m u ta tio n s a n d o p h th a lm ic disease, situs inversus visceru m (iv), 371
47 3 -4 8 6 slaty (sit), 91, 93
O x p h o s, 46 9 -4 7 2 Sm all eye (Sey) , 15, 45 2 -4 5 5
poly m o rp h ism s, 479 vitiligo (mi'"1), 123
re p lica tio n , 471, 48 3 -4 8 5 M ouse re tin a l d e g e n e ra tio n s, 123151
tra n sc rip tio n , 471 m RN A see M essenger RNA
M ito ch o n d ria l e n c e p h a lo p a th y M ucin, 333
(M ELAS), 474 M uller cells, 73 -7 4 , 341
M ito c h o n d ria l g e n o m e , 3 -5 , 470-471 M us sp retu s, 15, 132, 144, 455
M ito c h o n d rial m yopathy, 474, 4 8 0 -4 8 6 M uscular d y strophy D u c h e n n e type,
M izuo p h e n o m e n o n , 278 264, 288, 290
M o n o am in e oxidase (MAOA, M AOB), M u tag en -in d u ced cataract, 433
328 M utagenesis, 39, 4 3 -4 4
M o nochrom acy, 71, 21 8 -2 2 0 M u tatio n s 3, 35, 102, 203, 220, 235,
b lu e co n e m o n o ch ro m acy , 220 312, 325, 340, 3 5 1 ,4 3 2 ,4 5 3 ,
co n g e n ita l ro d m o n o ch ro m acy , 71, 45 5 -4 5 6 , 462
220 analysis, 332
M osaicism , 199 c lu sterin g , 99 -1 0 0 , 313
M ouse g e n etic m ap, 1417 crite ria fo r p athogenicity, 188
M ouse m o d els see A nim al m o d els d o m in a n t versus recessive, 36
M ouse m u ta n ts alb in o (c), 101-103 d e le tio n , 9, 138, 166-167, 186-187,
a p h a k ia ( ak), 461 2 0 4 ,3 1 2 , 3 9 2 ,4 0 7 , 473
b eig e ( bg), 109 d u p lica tio n , 473
b o u n cy (be), 139 e x o n skip p in g , 407
brow n (b), 92 e x p an sio n , 9
c a ta ra c t a n d sm all eye ( Cts), 461 fram esh ift, 9, 98, 186, 312, 457
co lo b o m a (Cm), 461 hot-spot, 485
c rib rifo rm d e g e n e ra tio n ( cri), 123 id en tificatio n , 210
dysgenic len s (d l), 461 in se rtio n , 137, 204, 312
e x tre m e d ilu tio n (c e), 112 m issense, 7 -9 , 98, 138, 168, 185, 187,
Eye len s o b so lescen ce (Elo) , 2 0 4 ,2 1 0 , 333, 3 9 2 ,4 0 7 ,
4 31-432, 461 47 3 -4 7 5 , 480
H im alyan ( ch), 101 in m t DNA, 4 6 9 -4 8 6
 INDEX
new, 197, 200, 386, 447
n o n se n se , 7, 98, 134-135, 168, 187,
204, 312, 392, 407, 455
pathw ays, 2 2 9 -2 3 0
nu ll, 137, 167-168
sp e c tru m a n d p h e n o ty p ic effects,
187
splice site, 9, 204, 312, 333, 455, 457
in stop c o d o n , 478
te m p e ra tu re sensitive, 98, 101
tJRNA, 4 7 3 -4 7 4
M yelin, 405
M yoclonic E pilepsy a n d R agged-R ed
Fiber D isease (M ERRF), 474
Myopia, 72, 261, 269, 271-272, 280,
2 8 3 ,2 8 8 ,2 9 3 ,3 0 4 ,3 8 6
M yopic d e g e n e ra tio n , 260
NADH d e h y d ro g e n a se (C o m p lex I ) ,
470, 475, 4 77-479
N D P g en e, see N o rrie disease
N eovascularization, 174
N e u ro e c to d e rm , 45 9 -4 6 0
N e u ro fib ro m ato sis type II (N F2),
4 28-429
c ata ra c t in , 428
c h ro m o so m e 22, 429
N e u ro g e n ic m uscle w eakness, atax ia a n d RP
(NARP), 474, 480-481
N eu ro lo g ical d is o rd e r in N o rrie disease,
32 3 -3 2 4
in NARP, 480-481
in L H O N , KSS, 474
N eu ro tra n sm issio n in CSNB, 293
N ick tran sla tio n , 503
N ig h t b lin d n ess, 154, 259-260, 271,
27 7 -2 9 5, 304, 366
N o n d isju n c tio n , 503
N o rrie disease (N D P ), 294, 321-334,
347
clinical featu res, 32 1 -3 2 4
cytogenetics, 3 2 6 -3 2 7
d iffe re n tia l diagnosis, 32 4 -3 2 5
g e n e isolation, 329
histology, 3 2 3 -3 2 4
linkage studies, 326
m u ta tio n analysis, 3 3 2 -3 3 3
p e ric e n tric inversion, 327
physical fin e m a p p in g o f N D P g en e ,
32 8 -3 2 9
d e le tio n studies, 32 8 -3 2 9
lo n g ra n g e re stric tio n m a p p in g ,
329
523
stru c tu re o f N D P g en e, 329-332
N o rth C aro lin a m a c u la r dystrophy
see u n d e r M acular dystrophy
N o rth e rn blot, 3 11-312, 329, 333, 503
N ucleic acids d e te c tio n , 2 5 -2 6
hy b rid isatio n , 2 6 -2 7
m a n ip u la tio n , 19
p ro d u c tio n o f p u re , 19, 2 2 -2 3
se p a ra tio n , 18-19
N u cleo sid e, 503
N u cle o tid e , 503
N u c le o tid e p y ro p h o sp h a ta se , 406
N u c le o tid e seq u e n c in g , 503
N u tritio n a l deficiency, 176
N ystagm us, 93, 220, 260, 280, 287-288,
399, 445
O c u la r alb in ism (O A ), 89, 110-112
au to so m a l recessive o c u la r albinism
(O A 2), 111-112, 288
X -linked recessive OA (O A 1),
109-111
O c u la r m yopathy, 48 5-4 8 6
O C R L g e n e , see Lowe o c u lo c e re b ro re n a l
syn d ro m e
O ct-1, Oct-2, 455
O c u lo c u ta n e o u s alb in ism (O C A ), 89,
9 5 -1 0 8
b ro w n OCA, 107
m in im al p ig m e n t OCA, 97 -98
m u ta tio n s in h u m a n TYR g en e,
98 -1 0 2
p la tin u m OCA, 98
ru fo u s OCA, 107-108
skin cancer, 93
te m p e ra tu re sensitive OCA, 98
tyrosinase n egative OCA, 97
tyrosinase positive O CA (O C A 2),
103-108
tyrosinase re la te d O CA (O C A 1),
96 -1 0 3
yellow OCA, 97, 100
O c u lo c e re b ro re n a l sy n d ro m e, see
Lowe o c u lo c e re b ro re n a l
syn d ro m e
O g u c h is disease, 7 3 -7 4 , 278, 280
O lfacto ry m a rk e r p ro te in (O M P1),
374
O m m a tid iu m , 32
O n c o g e n e s, 197
O p e n re a d in g fram e, 329, 332, 504
O p h th a lm o p le g ia , 473, 481
O p sin , 34, 219, 222
524 INDEX

O p tic n eu ro p a th y , 473-481 Philly m o u se see m o u se m u ta n ts

O p tic n e rv e fib re m isro u tin g , 95, 105, P h o sd u c in , 130
1 1 0 ,2 8 8 P h o sp h atase, 44, 129
O p tic n e rv e hypoplasia, 445 P h o sp h o lip ase C g e n e m u ta n ts in
O re g o n eye disease, 289 D ro so p h ila ( norpA), 36 -3 9
ORE see O p e n re a d in g fram e P h o sp h au d y lin o sito l tra n sfe r p ro te in ,
O rn ith in e a m in o tra n sfe rase (O A T ), 42
310 P h o sp h o ry latio n , 42, 44, 205
O scillato ry p o ten tia ls, 2 82-284, 287 P h o to p h o b ia , 94, 220
O steo sarco m a, 206 P h o to re c e p to r 3 0 -3 2 , 153, 282, 304,
O u te r p lex ifo rm layer, 289 371
O xidative d am a g e to DNA, 472 d e g e n e ra tio n in D roso p h ila, 2 9 -4 6
O xidative p h o sp h o ry la tio n (O X P H O S ), in n o rm a l m o u se, 138
4 69-472 d y stro p h ies ce n tral, 6 4 -7 0
O xygen radicals, 472 p e rip h e ra l, 5 4 -6 4
defects, co n g e n ita l, 70 -7 5
in n e r seg m en t, 161
P locus, 106 o u te r seg m en t, 58, 60, 72, 123-124, 161,
P -elem en t, 39 185, 316
P aire d box, 452 R l-6 , 31 -3 2 , 45
P aralo g o u s reg io n s, 374 R 7 /8 , 31 -32 , 45
P a te rn a l age effects, 386 v e rte b ra te , 126
P a tte rn dystrophy, 6 6 , 168, 179, 183, P h o to tra n sd u c tio n - in v erteb rates,
185, 189 36 -3 7 , 42, 44
P a tte rn fo rm a tio n , 452 v erteb rates, 128, 155, 160
Pax g e n e family, 452 Physical m ap, 352, 451, 504
PAX6 g e n e in a n irid ia, 45 2 -4 6 2 P hytanic acid, 63
Pax-6 g e n e in m o u se, 452-455 PIC see P o ly m o rp h ism in fo rm a tio n
P C R ^ P o l y m e r a s e c h a in re a c tio n c o n te n t
P earso n m a rro w /p a n c re a s syn d ro m e, P ig m e n t yellow p. in m a c u la /le n s,
481, 483, 48 5 -4 8 6 , 473 222
P ed ig ree, 504 P ig m en t dystrophy, 168
P e n e tra n c e , 197, 199, 203-204, 325, P ig m e n ta tio n o f retin a , 89 -1 1 3 , 155,
347, 369, 458, 475, 480, 504 260, 304, 244, 366
P e r ip h e r in /r d s g e n e (m o u se ), P laq u e, 505
136-138 Plasm ids, 505
P e rip h e rin /R D S g e n e (h u m a n ), P la telet sto rag e p o o l d eficien cies see
5 8 -6 1 ,6 6 ,1 3 7 - 1 3 9 ,1 6 2 , A nim al m o d els
165-168, 183, 187 Pleiotropy, 368, 453
m u ta tio n in a d u lt vitelliform m ac u la r Polyacrylam ide gel elec tro p h o resis,
dystrophy, 169, 187 505
m u ta tio n s in au to so m al d o m in a n t PolyA* RNA, 505
m a c u la r dystrophy, 169, 187 Polym erase ch a in re a c tio n (PC R ), 21,
m u ta tio n s in a u to so m al d o m in a n t RP, 1 5 9 ,1 6 5 ,1 8 6 ,2 0 3 ,2 1 0 -2 1 1 ,
165-168, 187 246, 455, 505
m u ta tio n s in h u m a n , 58-61 P o ly m o rp h ism , 101, 166, 188, 242, 479,
m u ta tio n in retin itis p u n c ta ta 506
albescens, 169, 187 P o ly m o rp h ism in fo rm a tio n c o n te n t,
P e te rs anom aly, 459 506
PFGE see p ulsed-field gel e le c tro p h o re sis P osition effect, 265, 458
P h ag e, 504 P ositional clo n in g , 157, 329, 447
Phag o so m es, 185 Prader-W illi syn dro m e, 105-107
P h en o c o p ie s, 68 P re im p la n ta tio n screen in g , 211
 INDEX
P ren a ta l diagnosis, 269, 35 0 -3 5 1 , 395,
408, 457, 462
P re n a ta l scree n in g , 20 7 -2 0 8 , 211
P re su m ed o c u la r histoplasm osis sy ndrom e
(P O H S ), 174, 189
P rim ary colour, 218
Prim er, 506
P ro b a n d , 506
P ro b e, 506
P ro lin e u p ta k e , 406
P rom o ter, 9, 92, 204, 313, 332, 506
P rotanom aly, seeT richrom acv
P ro ta n o p ia , see D ichrom acy
P ro te in kinase C, 408
P ro te in kinase II, c a lc iu rn /c a lm o d u lin -
d e p e n d e n t (C am k2a), 139
P ro te in p h o sp h a ta se 2A, 129
P ro te in p ren y ltra n sfe ra se , 315
Proteolytic cleavage, 389
Provirus, 134
P seu d o g en es, 422
P se u d o in fla m m a to ry m acu lar
dy strophy see u n d e r
M acu lar dystrophy
Psychiatric diso rd er, 323, 368
P sychological co u n sellin g , 394
Ptosis, 473, 481
Pulsed-field gel e le c tro p h o re sis
(PFG E ), 19, 328,
4 5 0 -4 5 1 ,5 0 6
R ab 3A, 31 5 -3 1 6
Rab 3A G DP d issociation in h ib ito r
(G D I), 315
R ab g eran y lg eran y l tran sferase, 315
R ad iatio n h y b rid m a p p in g , 507
R an d o m p rim e r labelin g , 507
R ayleigh c o lo u r m a tch in g , 221
R e c e p to r p o te n tia l D ro so p h ila, 36,
39, 41
R e c o m b in a n t in b re d (RI) strains, 15
R eco m b in atio n , 10-12, 328, 507
re c o m b in a tio n fra c tio n , 11-12
R ecoverin, 162-163
R e d /g re e n c o lo u r b lin d n ess, see
d ic h ro m a c y /tric h ro m a c y
R efsum disease, 63
R eg u lato ry seq u en ces, 9, 92, 134, 137,
164, 204, 313, 332
R epetitive DNA, 5, 137, 165, 507
R eplicative seg reg atio n , 472
R ep licatio n slippage, 313, 483
R escue o f m u ta n t p h e n o ty p e , 134, 137
R esp irato ry c h a in , 469
R estrictio n en d o n u c le a se , 508
R estrictio n fra g m e n t le n g th
p o ly m orph ism s (RFLP), 3-4,
101, 140, 157, 326, 207-208,
348, 407, 508
R etin a lig h t m ic ro g ra p h o f n o rm a l
m o u se, 125
lig h t m ic ro g ra p h o f r d / rd m ouse,
127
R etin al d e g e n e ra tio n (rd) g e n e , 123
136
c h a ra cte risa tio n o f rd g e n e , 131-132
isolatio n o f rd g en e , 130-136
site o f rd m u ta tio n , 134-135
R etin al d e g e n e ra tio n in b lue-cone
m o n o ch ro m acy , 220
a n d c o n e p ig m e n t g en es, 242
R etinal d e g e n e ra tio n m u ta n ts D ro
sop h ila, 29-51
m u ta n ts o f n inaC g en e , 4 3 -4 4
m u ta n ts o f ninaE g e n e, 3 3 -3 6
m u ta n ts o f norpA g e n e , 3 6 -3 9
m u ta n ts o f rdgB g en e, 4 1 -4 2
m u ta n ts o f rdgC g e n e , 44
m u ta n ts o f trp g en e , 39 -40
R etin al d e g e n e ra tio n s in m o u se,
123-145
cyclic GMP levels, 123-127
rd m o u se, 124-139
rds m o u se, 136-139
R etin al d e g e n e ra tio n slow (rds) gene,
123
h u m a n c o u n te rp a rt o f m o u se rds
g e n e , 138-139
id e n tific atio n o f rds g e n e , 136-138
R etin al d e ta c h m e n t, 339, 346, 386
R etin al d ev elo p m e n t, 341
R etin al d iso rd e rs h u m a n in h e rite d ,
5 3 -8 8
R etin al fu n d u s B ests vitelliform
dystrophy, 184
in retin itis p ig m en to sa, 156
m ac u la r d e g e n e ra tio n , 177
n o rm a l, 155
R etin al p ig m e n t e p ith e liu m , 62, 6 5 -6 9 ,
74, 168, 174-175, 183, 185,
25 9 -2 6 0 , 282, 316
R etinitis p ig m e n to sa (RP) h u m a n , 35,
5 4 -6 1 , 138, 153-170, 184,
25 9 -2 7 2 , 292, 341, 359, 363,
480
a u to so m al d o m in a n t RP (ADRP),
526 INDEX

54 -5 6 , 58, 138, 145, 153-170 risk assessm ent, 20 8 -2 0 9

m u ta tio n o f g e n e o n c h ro m o so m e R etin o m a, 198, 203
7, 168 R etinoschisis au to so m a l d o m in a n t,
m u ta tio n o f g e n e o n c h ro m o so m e 347
8, 168 a u to so m al recessive, 347
m u ta tio n s o f p e r ip h e r in /r d s g en e, R etinoschisis, see X 4 in k ed ju v e n ile
165-168 retin o sch isis (RS)
m u ta tio n s o f rh o d o p sin g en e, R everse tra n sc rip ta se /p o ly m e ra se
158-165 ch a in re ac tio n (R T /P C R ),
au to so m a l recessive RP, 5 4 -5 5 , 57, 312, 333, 407, 455, 4 57-458
169-170, 371 R everse tra n sc rip tio n , 508
expressivity, 56 RFLP see R estrictio n fra g m en t
fu n c tio n a l studies, 5 5 -5 8 le n g th p o ly m o rp h ism
g e n e tic studies, 54 -5 5 R h a b d o m ere , 32, 40-41, 44
p rev alen ce, 262 R h o d o p sin , 33-37, 41, 44, 5 8 -6 0 , 72,
secto r RP, 5 5 -5 6 , 5 9 -6 0 74, 128-129, 138-139, 153,
sine p ig m e n to , 366, 377 157-165, 220, 29 3 -2 9 4
sp o rad ic, 5 4 -5 5 , 260 in m o u se, 139
tap e ta l reflex, 57, 261 m u ta n ts in D ro so p h ila ( ninaE ),
type I (diffuse) RP, 55, 59, 165 33 -3 6
type II (reg io n al) RP, 55, 60, 165 m u tatio n s in a u to so m al d o m in a n t
u n ila te ra l RP, 56 RP, 5 8 -6 0 , 158-165
X 4 in k ed RP, 5 4 -5 5 , 5 7 -5 8 , 145, 1 6 9 - tran sg e n ic m ice with ADRP m u ta
170, 25 9 -2 7 2 , 371 tio n , 164-165
clinical featu res, 260-261 R h o d o p sin kinase, 128-129, 163
g e n e tic m a p p in g , 26 2 -2 6 8 R ib o n u cleic acid, 508
RP2 g e n e , 262, 266-268, 294 a ltern ativ e splicing, 462
RP3 g e n e , 262, 264-265 h e te ro g e n e o u s nuclear, 7
g e n o ty p e -p h e n o ty p e c o rrelatio n s, p ro te c tio n , 508
269 splicing, 7
p rev alen ce, 262 Risk in ag e-related m ac u lar d e g e n
RP2 g en e, 58, 26 6 -2 68 e ra tio n , 190
RP3 g en e, 58, 26 4 -2 6 5 in L e b e rs h e re d ita ry o p tic n e u ro p a th y ,
R etinitis p u n c ta ta albescens, 74, 139, 475
166, 168, 187 in retin o b la sto m a , 20 8 -2 0 9
R e tin o b lasto m a (R b ), 197-211, 323 RNA see R ib o n u cleic acid
b ilateral, 198, 209 RNA p ro te c tio n , 508
cy to g en etic analysis o f Rb tu m o u rs, R od d efects c o n g en ital, 71-75
2 0 1 -2 0 4 ' R od m o n o c h ro m a tism , see m o n o c h ro m a c y
fam ily history, 197, 209 R od o u te r segm en t, 58, 60, 72, 123-124,
g e n e tic s o f re tin o b la sto m a , 197-199 161, 185, 316
p re n a ta l diagnosis, 2 07-208 ROM-1 g e n e , 61, 183, 185, 372
risk assessm ent, 20 8 -2 1 0 RT see R everse tra n sc rip tio n
trilate ra l, 207
u n ila te ra l, 198, 209, 211 SI n u clea se m ap p in g , 509
R etin o b lasto m a (R B I) g en e , 199-208 S p h ase, 509
fu n c tio n s o f RBI g e n e , 205-206 Sense stra n d , 509
id e n tific a tio n o f causative m u tatio n s, S eq u en ce tagged site, 510
210-211 S h u ttle vectors, 510
iso latio n o f RBI g e n e , 2 02-203 S ignal seq u e n c e , 333
pRB p ro te in , 20 5 -2 0 6 Single stra n d c o n fo rm a tio n p o ly m o rp h ism
RBI m u ta tio n s in o th e r tu m o u rs, (SSC P), 18, 138, 158-159, 166,187,
2 0 6 -2 0 7 189, 203, 246, 312-313, 334, 510
 INDEX
Sm all eye m o u se (Sey), 45 2 -4 5 5
S om atic cell h y b rid m ap p in g , 510
Sorsbys fu n d u s dystrophy, 6 9 -7 0
S o u th e rn blo t, 510
S pectral sensitivity o f opsins, 24 4 -2 4 5
Splicing, 7
Splicing, in tr o n /e x o n , 510
SSCP see Single stra n d c o n fo rm a
tio n p o ly m o rp h ism
ss DNA, 511
S ta rg a rd ts disease, 67, 70, 183
Stickler disease, 347
S tre p ta v id in /b io tin , 511
STS see S eq u e n c e ta g g e d site
Synaptic vesicle tra n sp o rt, 316
Synteny, 453
TATA box, 511
T elo m ere, 3
Trans-activation d o m ain , 455
T ra n sc rip tio n , 511
b re a k th ro u g h , 457
T ra n sc rip tio n factors, 204, 206, 455, 461
T ra n sd u c in , 128, 130, 139-140, 142, 161
T ran sfectio n , 511
T ra n sfo rm a tio n , 511
T ran sg en ic an im al, 15-17, 103, 134, 137,
1 6 4 ,1 8 8 ,1 9 0 ,3 9 5 ,5 1 1
T ran slatio n , 512
T ran slo catio n , 512
b alan ced , 202, 448, 450, 458
b re a k p o in t, 450-451
in c o n e dystrophy, 181
X /a u to s o m e , 304, 3 06-307, 309, 324,
326, 399, 407
T richrom acy, 218, 22 1 -2 2 3
a n o m alo u s, 221, 2 4 9 -2 5 2
d eu teran o m aly , 221, 226, 241,
m o le c u la r genetics, 226
p ro tan o m aly , 221, 226
m o le c u la r genetics, 226
tritanom aly, 221
T u m o u r analysis, 211
m ultifocal, 202
n on-ocular, 20 6 -2 0 7 , 211
p ro g ressio n , 207
su ppressor, 197
virus, 205
T yrosine tra n s p o rt p ro te in , 106
T yrosinase, 9 0 -9 3 , 112
m o u se tyrosinase (c-locus) m u tatio n s,
102-103
tyrosinase g e n e (TYR), 92
m u tatio n s in h u m a n TYR g en e,
9 8 -1 0 2
tyrosinase re la te d p ro te in -1 g en e
(T R P1), 9 2 -9 3
tyrosinase re la te d p ro tein -2 g en e
(T R P 2 ), 93
U b iq u in o k c y to c h ro m e c o x id o re d u c ta se
(C o m p e x III), 470,
475, 478-479
U sh er sy n d ro m e (U S), 3 59-379
b ra in ab n o rm alities, 369
classification, 35 9 -3 6 0
clinical featu res, 35 9 -3 6 9
h e a rin g loss, 3 60-362
in h e rita n c e , 36 9 -3 7 0
U sh e r type I, 170, 35 9 -3 7 5
linkage to c h ro m o so m e 11,
3 72-375
linkage to c h ro m o so m e 14q,
3 70-372
in P o itu -C h a re n te s reg io n , 370
U sh e r type II, 145, 170, 315,
359-370, 375-378
linkage to c h ro m o so m e lq ,
375-377
U sh er type III, 360-361
U sh e r type IV, 368
sp eech , 360-361
sp erm , 371
v estib u lar fu n ctio n , 363
V ariable n u m b e r ta n d e m rep eats, 512
Vector, 2 2 -2 3 , 512
V estibular h a ir cells, 371
V estibular responses, 360, 363
V isual evoked resp o n ses, 366
V isual fields, 154, 25 9 -2 6 0 , 281, 366,
V isual p ig m e n t see c o lo u r p ig m en ts,
o psin, rh o d o p sin
V itam in A, 41, 64
V itam in E, 64
V itellifo rm m a c u la r d y stro p h y (B ests
disease) see under M acular
d ystrophy
a d u lt vitelliform m a cu lar dystrophy,
6 5 -6 6 ,1 6 8 -1 6 9 ,1 8 7
atypical m a c u la r dystrophy, 181
Von W illeb ran d factor, 333
V N T R ^ V a r ia b l e n u m b e r ta n d e m
re p e a ts
528
W ag n er disease, 347
WAGR sy ndrom e, 44 7 -4 4 8 , 451
d e le tio n s le a d in g to m a p p in g o f
a n irid ia locus, 44 8 -4 5 0
W estern blo t, 512
W ilm s tu m o u r, 4 47-448, 450, 458
X 4 n activation, 3, 203, 260, 2 87-288,
304, 324, 326, 399
X 4 in k e d fam ilial exudative
v itreo re tin o p ath y , 325
X 4 in k ed ju v e n ile retin o sch isis (RS),
' 33 9 -3 5 3
INDEX
clinical featu res, 34 1 -3 4 8
e p id em io lo g y & prevalence,
339-341, 351
linkage studies, 348-352
p re n a ta l diagnosis, 350-351
therapy, 346
Yeast artificial c h ro m o so m e , 266, 329,
352, 407, 513
YAC see Yeast artificial c h ro m o so m e
Y oung41elm holtz theory, 2 27-228
Z inc fin g er p ro tein s, 513