J Med Genet: first published as 10.1136/jmg.22.5.377 on 1 October 1985. Downloaded from http://jmg.bmj.com/ on 22 January 2019 by guest.

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Journal of Medical Genetics, 1985, 22, 377-381

The first observation of Hb D Punj ab thalassaemia

in an English family with 22 cases of unsuspected
thalassaemia minor among its members
SHEILA WORTHINGTON* AND HERMANN LEHMANNt
From *the Pathology Laboratory, George Eliot Hospital, Nuneaton; and tthe Department of Biochemistry,
University of Cambridge, Cambridge.

SUMMARY A 36 year old local Englishman from Nuneaton was referred to hospital with
suspected glandular fever. Relevant tests were negative and the symptoms subsided in due
course. The finding of a hypochromic microcytic blood picture without iron deficiency led to the
discovery that he was heterozygous for Hb D and ,1 thalassaemia. Hb D trait was established in
the father of the proband and Pi thalassaemia in his mother and a brother. The father's ancestors
were miners who came to Nuneaton from Monmouthshire in the 19th century. The mother's
ancestors have belonged to the indigenous population of Nuneaton and neighbouring
Leicestershire since the 18th century. Twenty local members of her wider family also had
thalassaemia. All thalassaemias had a low MCH and raised level of Hb A2. The Hb F level,
however, was normal in five, demonstrating the independent segregation of genetic factors

copyright.
influencing the Hb F level in ,1 thalassaemia trait.

A generation ago thalassaemia was considered rare
or absent in Britons. Early descriptions were based
on the finding of red cell abnormalities and a raised
level of Hb F. In one such case necropsy showed
leukaemia.1 A description in 1955 of 'target cell
anaemia' in two English women2 is well compatible
with thalassaemia. Once it had been shown that
raised Hb A2 levels could be a diagnostic feature, it
was possible to report an indisputable case from
East Anglia, and this was followed by many more
observations.
Hb D was originally described as a variant of Hb
A which on electrophoresis behaved like Hb S but
did not cause the sickling phenomenon.6 It was soon
found that this description covered a number of
variants differing by other investigations,7 and
denotes a haemoglobin differing from Hb A by an
additional positive charge or loss of a negative
charge on either both the two a or the two fi chains
of the Hb a212 tetramer. The most widely distri-
buted Hb D which has a frequency of 2 to 3% in the
Punjab8 was found to have a glutamine instead of a
glutamic acid at position 121 of the 146 residues PI
chain.9 When it was found that the original Hb D
tSince this paper was submitted, Professor Lehmann has died.
Received for publication 25 August 1984.
Accepted for publication 19 December 1984.
377
described in Los Angeles was identical to Hb D
Punjab, the term Hb D Los Angeles was intro-
duced. The association between Hb D and j3
thalassaemia was first described in a Persian girl'0
and most cases of thalassaemia associated with Hb
D Punjab have been I3 thalassaemias.5 The con-
dition is fairly mild and no more severe than I8
thalassaemia minor on its own. It is more severe,
however, than the homozygous state for the Hb D
gene. "
Materials and methods
Blood counts were evaluated on a Coulter Counter
Model S and other haematological methods, includ-
ing measurement of serum iron, iron binding
capacity,12 and G6PD, followed established
routine.13 Preparation of haemolysates, estimation
of Hb F, electrophoresis and quantification of Hb
fractions, preparation of globin and its tryptic
digestion, preparation of two dimensional chro-
matograms of tryptic peptides (fingerprints), and
amino acid analysis followed established routine.14
Globin chain separation was performed according to
Clegg et al.'5 Blood groups were determined with
monoclonal antisera for ABO groups and immune
antisera for Rhesus and other groups.
 _
oo
I_~ J Med Genet: first published as 10.1136/jmg.22.5.377 on 1 October 1985. Downloaded from http://jmg.bmj.com/ on 22 January 2019 by guest. Protected by
378 Sheila Worthington and Hermann Lehmann

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 E,o J Med Genet: first published as 10.1136/jmg.22.5.377 on 1 October 1985. Downloaded from http://jmg.bmj.com/ on 22 January 2019 by guest. Protected by
The first observation of Hb D Punjab P3 thalassaemia in an English family 379

Results
Findings in the proband were WBC 8-7x 109/1, RBC
z z z z z z z z z z z z z z z z z z
6 0x 10'2/1, Hb 12-7 g/dl, PCV 0-395 1/1, MCV 66 fl,
MCH 21-1 pg, MCHC 31-5 g/dl, and reticulocytes
c
1-8 per 100 RBC. The red cells were microcytic and
-
hypochromic with slight polychromasia, moderate
II c leptocytosis, and slight basophilic stippling. The
IC l IC differential white cell count was normal and glandu-
c
lar fever cells were not seen. Platelets were normal.
1l -6 cL
- The slide test for glandular fever was negative. The
- G6PD was normal. The serum iron level was 26
o
v)
[tmol/l (normal 13 to 32 ,molIl). Hb electrophoresis
c - b _ x _ IC
:%
on cellulose acetate at pH 9-2 showed no Hb A and
c
:e only a band migrating in the position of Hb D or S
o- 'Mcs s s 'tt es c)
amounting to 91%, Hb A2 7-2% (normal 2-0 to
-

0e
4-0%), and Hb F 1-4% (normal less than 0-9%). Hb
-
F was heterogeneously distributed among the red
v:
c)
-
cells. On agar gel electrophoresis at pH 6-2 the
o
abnormal haemoglobin did not migrate like Hb S
cJ
E
but like Hb A, which indicated that it was Hb D.
F:
!'! sN 0,r Z
.>
c
Fingerprinting and amino acid analysis showed that
o the Hb D was Hb D Punjab a2132 121 Glu-*Gln.
o The mother of the proband had , thalassaemia
trait and the father had Hb D trait. Their blood

copyright.
--et NN
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Ct
o
groups were compatible with the son's first degree
z z z+ + + z z z +
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relationship. On investigating the parents' families it
.t
was found that the mother's maternal aunt also had
s thalassaemia trait. This led us to examine the
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C)
mother's family. The aunt was the survivor of nine
-
-
:5
sibs. Two of them had no descendants. Of the other
E e
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six it was possible to demonstrate thalassaemia in
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descendants of four of them. The table shows the
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C) C
haematological findings and fig 1 shows the family
pedigree. It will be noted that in addition to the
_ _ proband, his mother, and brother, there were
; e.)
another 20 cases of unsuspected thalassaemia. Hb D
o
_
X
Q
was not demonstrated in any of the father's three
CV _
sibs but was found in a paternal cousin. It has not
-
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M
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V
It ee 1t
x 0
1t en
o a 4 4 N
.>
._
X
Q
been possible to examine more members of this
+ -
_
family.
E -c
o
=
C)
:3
Discussion
11 C
"0 N- 4
Z _
The proband is the first case of Hb D Punjab (Los
C
o
C Angeles) 130 thalassaemia found in a native English-
man. Schneider et al'6 described a similar case in a
rI -r 1.1I r.I :., I'? 1.1 C.l CIO .) CP C.l C? = 'IT I.-, It Cr C
-1, :t f-I 1- -- n :2 .! = c,:. m "t " :. r. CZ4 :. *-
V)
C.)
n
person of English, Irish, and Scottish ancestry in the
C -
USA. That thalassaemia was, however, of the r
C Q
o
type and the clinical picture was much more severe.
'lT
"
=
_
t-
e"
"
"
14
"
-
C,
X
"
m
-
b- C _ Although 13 thalassaemia trait is found occasionally
X C. among the indigenous population of Nuneaton (10
_
, n
Q
cases from different families have been diagnosed in
C -
the last five years), it was surprising to find 22
_t W _ " M W) c 1- O0 _ _
unsuspected cases among 57 relatives of the
i~ -e
^en m en e e _
_
o
C
proband. The proband, an engineer with the local

380
\
.\ 30
\K --V-I
VI
FIG 1 Family pedigree.
Water Board, had been well apart from the brief
illness mentioned and showed no abnormality on
physical examination later. The relatives with tha-
lassaemia trait were mostly well. Two of them,
however, had been treated previously for hypochro-
mic anaemia. The findings were consistent: Hb A2
was always raised and the MCH was near 20 pg. Hb
F level, however, was not raised in all thalassaemics.
Fig 2 shows the independent segregation of the
genetic factors which influence the Hb F levels in
,1 thalassaemia trait. It is noteworthy that in the two
J Med Genet: first published as 10.1136/jmg.22.5.377 on 1 October 1985. Downloaded from http://jmg.bmj.com/ on 22 January 2019 by guest. Protected by
Sheila Worthington and Hermann Lehmann
Io>
/ I
copyright.
/I
/
/
/
11
-1
Hb D trait carriers the Hb F is raised in one (III.20)
but normal in the other (III.24). Though thalas-
saemia is rare in Britons its significance is important
as there are genetic implications for those in whom it
is diagnosed. It is essential that in the differential
diagnosis of hypochromia the clinician is aware that
thalassaemia is a possibility and should be consi-
dered (among others with sideroblastic anaemia and
aleukaemic leukaemia) so that mistreatment with
iron is avoided. The Coulter Counter indices
produced by the majority of haematology depart-

Thefirst observation of Hb D Punjab /30 thalassaemia in an English family
O Normal (>O*9%)
* Slightly raised (0 9-1 0%)
Significantly raised (1 1-410%)
*
FIG2 Haemoglobin F levels associated with the f3
thalassaemia trait through four generations of the family.
ments these days give a clear indication that thalas-
saemia may be present, and when a low MCV, low
MCH, and normal MCHC are reported patients
should be investigated thoroughly to establish the
true diagnosis. Heterozygotes for thalassaemia can
be recognised easily, and family studies of thalas-
saemia adumbrate what will become possible, when
other heterozygous states which at present cannot
be diagnosed as easily as thalassaemia will become
recognised by restriction enzyme analysis of the
DNA.
We wish to thank for their help and advice: Dr E N
Trounson, Consultant Pathologist, and the technical
staff, Pathology Laboratory, George Eliot Hospital,
Nuneaton; Dr G W G Bird, Consultant Adviser,
Blood Group Reference Laboratory, Oxford; Miss
Laurene Robb, Abnormal Haemoglobin Reference
Laboratory, Addenbrooke's Hospital, Cambridge;
Mr A Camp, Director, Society of Genealogists
J Med Genet: first published as 10.1136/jmg.22.5.377 on 1 October 1985. Downloaded from http://jmg.bmj.com/ on 22 January 2019 by guest. Protected by
381
(London); Miss V D M Quant, Assistant Curator,
Nuneaton Museum; and all the family who so kindly
cooperated in this study.
References
Bywaters EGL. The Cooley syndrome in an English child. Arch
Dis Child 1938;13:173-87.
2
Israels MCG, Turner RL. A British target cell anaemia. Lancet
1955;ii: 1356-66.
3Havard CW, Lehmann H, Bodley-Scott R. Thalassaemia minor
in an English woman. Br Med J 1958;i:304-5.
Callender ST, Mallett BJ, Lehmann H. Thalassaemia in Britain.
Br J Haematol 1961;7:1-3.
5Weatherall DJ, Clegg JB. The thalassaemia syndromes. 3rd ed.
Oxford: Blackwell Scientific Publications, 1981.
6 Itano HA. Third abnormal hemoglobin associated with heredi-
tary hemolytic anemia. Proc Natl Acad Sci USA 1951;37:775-84.
7Benzer A, Ingram M, Lehmann H. Three varieties of human
haemoglobin D. Nature 1958;182:852-3.
8 Bird GWG, Lehmann H. Haemoglobin D in India. Br Med J
1956;i:514.
Baglioni C. Abnormal human haemoglobins. VIII. Chemical
studies on haemoglobin D. Biochim Biophys Acta 1962;59:437-
49.
1' Hynes M, Lehmann H. Haemoglobin D in a Persian girl:
presumably the first case of haemoglobin D thalassaemia. Br
Med J 1950;ii:923.
1 Politis-Tsegos C, Kynoch PAM, Lang A, et al. Homozygous
haemoglobin D Punjab. J Med Genet 1975;12:269-74.
12 Ramsey WMN. The determination of iron binding capacity of
copyright.
serum. Clin Chim Acta 1957;2:221.
13 Dacie JV, Lewis SM. Practical haematology. 5th ed. Edinburgh:
Churchill Livingstone, 1975.
14 Lehmann H, Huntsman RG. Man's haemoglobins. Revised ed.
Amsterdam: North-Holland, 1974.
5 Clegg JB, Naughton MA, Weatherall DJ. Abnormal human
haemoglobin. Separation and characterization of the a and fi
chains by chromatography. J Mol Biol 1966;19:91-108.
16 Schneider RG, Ueda S, Alperin JB, Levin WC, Jones RT,
Brimhall B. Hemoglobin D Los Angeles in two Caucasian
families: hemoglobin SD disease and hemoglobin D thalasse-
mia. Blood 1968;32:250-9.
Correspondence and requests for reprints to
Dr Sheila Worthington, Department of Haematology,
George Eliot Hospital, College Street, Nuneaton,
Warwickshire CV10 7DJ.