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Am. J. Hum. Genet.

49:537-544, 1991

The Varying Frequencies of Five DNA Polymorphisms of


X-linked Coagulant Factor IX in Eight Ethnic Groups
John B. Graham,* Glenna R. Kunkel,* Nejat K. Egilmez,T Anders Wallmark,t Dana M. Fowikes,*
and Susan T. Lord *
*Department of Pathology and the Curriculum in Genetics, University of North Carolina, Chapel Hill; tUniversity of Arkansas Medical Center,
Little Rock; and tMalmo General Hospital, Malmo, Sweden

Summary
Five RFLPS of X-linked coagulation factor IX were evaluated in more than 500 normal persons (723-804
X chromosomes) of both sexes who belonged to eight ethnic groups: Anglo-Americans, Basques, Swedes,
African-Americans, East Africans, East Indians, Chinese, and Malays. The polymorphisms, 5' to 3', were
BamHI, XmnI, TaqI, MnI, and HhaI. A PCR procedure was developed for three previously described
RFLPs-XmnI, TaqI, and MnII; a PCRP procedure was developed for BamHI, and a PCRP which had been
described by others was used for HhaI. Europeans were the most polymorphic, African-Americans and East
Africans were intermediate, and Orientals were the least polymorphic. Extragenic 3' HhaI was highly polymorphic in most groups, and extragenic 5' BamHI was polymorphic only in persons with African ancestry. Two
major haplotypes predominated among 247 men, and the expected and observed heterozygosities were concordant among women. Allelic association was very strong between the three intragenic PCRPs; it was present
but weak between 5' extragenic BamHI and XmnI. No association was found between 3' extragenic HhaI and
MnnI.

Introduction

The 35-kb human coagulation factor IX (F.IX) gene


has been completely sequenced (Yoshitake et al.
1985). This makes very feasible the analysis of its polymorphisms by rapid DNA (i.e., PCR) procedures. We
describe conditions sufficient for such analysis of five
RFLPs of F.IX. Oligonucleotide primers developed for
four of the polymorphisms are described, as are the
results which were obtained by using both them and
primers developed by Winship et al. (1989) for the
fifth polymorphism.
All five polymorphisms have been examined in the
same population, z500 normal persons from eight
ethnic groups (723-804 chromosomes). Three sets of
subjects were investigated successively over a period
Received January 15, 1991; revision received April 12, 1991.
Address for correspondence and reprints: John B. Graham,
M.D., Department of Pathology, University of North Carolina,

Chapel Hill, NC 27599-7525.


i 1991 by The American Society of Human Genetics. All rights reserved.
0002-9297/91 /4903-0006$02.00

of 5 years by an evolving set of procedures. Swedes,


East Indians, Chinese, and Malays were sampled in
1986-87. They were evaluated for XmnI, TaqI, and
MnlI RFLPs by Southern analysis, and the results have
been published (Lubahn et al. 1987; Graham et al.
1988). Anglo-Americans, African-Americans, and
Basques were sampled in 1988, and East Africans
were sampled in 1990. Most of the data presented are
new and were obtained using PCR technology. The
population genetics are collated by recording all data
on all subjects for all F.IX polymorphisms studied by
all methods.
Three polymorphisms are intragenic, and two are
extragenic. Their locations with respect to each other
and the eight exons are indicated in figure 1.
The primers used for PCR analysis, as well as the
digestion patterns obtained, the gene frequencies, the
effective heterozygosities, the haplotype frequencies,
and the allelic associations, are recorded and compared both with each other and with three other polymorphisms-two F.VIII and one VWF-examined in
the same subjects.
537

Graham et al.

538
-590

7050

11082

20392

.L6

----mm.'
4
23
1

.7

AW
8kb

35

Kb

F.IX gene. The start codon and end of the 5' unFigure I
translated region are marked with solid circles (@). Solid bars
(-U-) along the

gene locate the

exons.

Arrows

(f) point

to

RFLPs,

B = BamHI; X = XmnI; T = TaqI; M = MnlI; H = HhaI. In


the GenBank sequence, coding begins at 2995, and the BamHI
dimorphic base site is at 2405. Other numbers are from the GenBank sequence.

Material and Methods


Source of DNA

DNA was prepared from white blood cells obtained


under the Rules of Informed Consent as described elsewhere (Lubahn et al. 1987).

The Oligonucleotide Primers

BamRl.-Primer 1 was 5' GAA GTT TGA CCT


AAA CAT CAT AC, and primer 2 was 5' TTG AGT
CTG AAA CAG GAA GTG A.
Xmnl. - Primer 1 was 5' CAG AGA CTG CTG ATT
GAC TT, and primer 2 was 5' ACA GCC AGA TAA
AGC CTC CA.
Taqi. -Primer 1 was 5' TAT AAT GGG AAT TCT
CCA CAT, and primer 2 was 5' AGT AGA AAG TGA
ATT CCT CA.
Mnfi. - Primer 1 was 5' GAT TTG AAA ACT GCT
CAT GAA AAT AAC, and primer 2 was 5' AAA GTA
CCT GCC AAG GGA ATT GAC CTG.
Hhal. -Primer 1 was 5' ACA GGC ACC TGC CAT
CAC TT, and primer 2 was 5' AGA TTT CAA GCT
ACC AAC AT.
Results

Ethnic Groups

The Individual Procedures

The Anglo-Americans were University of North


Carolina medical students all of whose known ancestors can be traced to the British Isles. The Swedes were
persons of Swedish descent presently living in Malmo,
Sweden. The African-Americans were residents of Evans County, Georgia.
The Basques live in Boise, Idaho-a U.S. center of
Basque culture and all four of the grandparents of
each subject were of Basque origin. The East Indians,
Chinese, and Malays were students or staff of the University of Malaysia in Kuala Lumpur. The East Africans were native students attending school in Harare,
Zimbabwe. Care was taken at each site to test unrelated persons. Essentially all subjects of each ethnic
group were analyzed for the five polymorphisms.

The essential characteristics of the PCRPs are


shown in table 1. The location of the dimorphic or
enzyme-recognition site, the region amplified, the amplification parameters, the sizes of the digestion fragments, and the type of control of digestion are noted.
The Mn1I product contains an internal control of digestion, an invariant Mn1I site, and external controls
were engineered for the BamHI and HhaI polymorphisms. Digestion controls were not engineered for
XmnI and TaqI because (1) all groups except the East
Africans had been studied by Southern analysis and
(2) 27 Basque and Anglo-American samples had been
analyzed by both PCR and Southern techniques, without a discrepancy.
The BamHI polymorphism results from dimorphism of a basepair (bp 2405 in GenBank) 590 bp 5'
to the F.IX start codon. All samples were examined
by the PCR procedure.
XmnI and TaqI polymorphisms, which are intronic,
were examined in seven groups by Southern analysis
using the genomic probe pAla36 (Lubahn et al. 1987).
Only the East Africans were examined for the XmnI
and TaqI polymorphisms solely by the PCR procedure.
The MnlI polymorphism had been examined earlier
in Swedish females by Southern analysis of dried gels
by using labeled oligonucleotides (Graham et al.
1988), and some of the results had been ambiguous.
All were retested by the PCR procedure, which discriminates precisely, and several putative Swedish ge-

PCR Conditions

The basic PCR procedure, adapted from the work


of Kogan et al. (1987), has been described by Graham
et al. (1989). Several recent modifications include (1)
amplification DNA reduced to 200 ng, (2) initial denaturation for 4 min at 93C, (3) Taq polymerase reduced from 3 to 1 unit/amplification, (4) number of
cycles increased from 30 to 35, (5) amplified product
not always purified before restriction, (6) restriction
enzyme increased from 3 to 15 units / 10 gl of amplified
product, (7) restricted fragments electrophoresed in
agarose (Nusievem), and (8) external controls of digestion designed for and used in the BamHI and HhaI
PCRPs.

DNA Polymorphisms of Factor IX

S39

Table I
Comparison of PCR Polymorphisms of F.IX
Region Amplified

BamHI, bp 2405 ...........

2258-2782

XmnI, bp 10041-10050.

9979-10199

TaqI, bp 14076-14079...

13980-14279

MnlI (Malm6), bp 23387

23131-23537

HhaI, 8 kb 3' to F.IX gene

Control of Restriction
Enzyme Digestion

Size of Fragments

Polymorphism Name,
GenBank Locationa

Amplification Parameters
1.5 mM Mg"+, denaturation for 30s at 91 IC and
annealing/extension for
4 min at 600C

(bp)
525, 379, and 146

4.5 mM Mg+ +, denaturation for 30 s at 91 0C and

220, 153, and 67

fragment when
pBR322 is cut by PvuI
and PvuII)
None

299, 203, and 96

None

278, 120, and 159

Invariant MnO site at


23247-23250; 126-

annealing/extension for
2 min at 600C
6.0 mM Mg" +, denaturation for 30 s at 91'C and
annealing/extension for
2 min at 600C
6.0 mM Mg++, denaturation for 30 s at 91 C and
annealing/extension for
4 min at 600C
6.0 mM Mg+ +, denaturation for 30 s at 91C and
annealing/extension for
4 min at 600C

bp fragment

Gel Patterns of the Individual Polymorphisms

The MnlI digestion patterns have been described by


Graham et al. (1989), and the patterns specific for the
other four polymorphisms are shown in figure 2. The
BamHI and HhaI patterns are complex, because external controls of enzymic digestion are also present, the
control fragments in each being indicated by an antecedent (c). The BamHI control produces two fragments, both larger than the amplified product. The
HhaI external control contains two HhaI restriction
sites, which produce three control fragments. The
smallest (1 90-bp) fragment lies between the undi-

1,059-bp fragment of
(pX 174 with two HhaI
(smaller fragment when
(pX is cut with StuI
and XhoI)

230, 150, and 80

a In GenBank this is known as HUMFIXG-PART1. The dimorphic base is known for BamHI and

notypes were changed. All other females and all males,


except the Swedish, were studied for the Mnln polymorphism by the PCR procedure. Swedish males were
genotyped immunologically (Graham et al. 1988).
The HhaI PCRP has been described by Winship et
al. (1989). We followed their procedure except for
using a different control of digestion, a fragment from
DX 174 (table 1). Our primer 1 is their FIXH1, and
our primer 2 is their FIXH2.

2692 fragment of
pBR322 with one
BamHI site (larger

MnlI.

BamHI

(A)

(B)

Males

Femoles
(und.) (- 1 +) (-,-) (+-,) (+.,-

bp
(c)2692

c)1692-

XmnI
Females

Males

bp

1+1

1-)

l+tl) (-,-)

(a,-)

220153-

(cl 1600 -

67-

525-

379-

(D)

HhaI
Males

146-

1
..51

"

Fer
,"

"

bp
(c)1025-

(C)
Males

Taq I

(c0800-

Females

-(-) (+.+) (_A (,_)


299203-_
96-

23015080-

(c)l90(c) (35)1

Figure 2 Ethidium bromide-stained agarose gels showing


fragments produced by digestion of amplified products by restriction enzymes. Primers used for amplification, as well the amplification conditions, are described in the text.

Graham et al.

540

gested (230-bp) and digested (150-bp) fragments of


the F.IX PCR product.
The XmnI and TaqI patterns are simple. In the absence of an external control the ( - ) and ( -, - ) genotypes may sometimes be difficult to distinguish from
nondigested amplified product. However, when the
East African samples were analyzed, every gel contained (+), ( + + ), or ( + ) individuals digested at
the same time under the same conditions which served
as controls.
,

-j
-J04

Cr

0.3

0
>.

0.2

D0.1
0

Malays

Chins

Eat
Indians

Population Genetics of the Polymorphisms

ORIENTAL

Table 2 records the gene frequencies of the five polymorphisms in the eight ethnic groups. The frequencies
are weighted averages between males and females,
since there was not a statistically significant sex difference for any RFLP in any ethnic group. The frequencies of the F.IX polymorphisms are strongly affected
by ethnicity. Occidentals (Anglo-Americans, Swedes,
and Basques) are much more polymorphic than Orientals (East Indians, Chinese, and Malays). AfricanAmericans and East Africans tend to be intermediate
for four polymorphisms, but they are highly polymorphic for BamHI whereas other groups are essentially
monomorphic for it. The frequencies of the rarer alleles of each polymorphism are shown in the bar
graphs of figure 3.
The expected heterozygosities in table 3 are calculated from the gene frequencies and compared with

Africans

Amerans

Aans

AFRICAN

OCCIDENTAL

Frequencies of rarer alleles of each F.IX RFLP, plotFigure 3


ted as bar graphs. Both the high degree of polymorphism in Europeans and the slight degree in Orientals are very striking. BamHI
polymorphism is obviously an African trait. 0 = TaqI; E = MnlI;
0 = HhaI; a = XmnI; l = BamHI.

those observed. Observed and expected values did not


differ significantly, except for BamHI in East Africans.
This resulted from the large proportion of homozygous ( + + ) women in the East African sample, which
skewed the genotype frequencies.
,

Haplotypes

The haplotypes for the five polymorphisms of 247


shown in table 4. (Female haplotypes could
not be analyzed in the absence of family studies.) They
men are

Table 2
Gene Frequencies of F.IX Polymorphisms
No. OF CHROMOSOMES, p( + ):q(-) OF POLYMORPHISM

Xmnlb

Taqlb

MnlI (Malmo)a

Hhala

ETHNIC GROUP

BamHIa

Anglo-Americans

72, .07:.93

73, .15:.85

73,.31:.69

73, .33:.67

Basques ...............
Swedes......................

96,.01:.99

104, .23:.77

104,.31:.69

104, .32:.68

104,

.48:.52

.30:.70
.05:.95
.04:.96
.06:.94

115, .26:.74
91, .10:.90
148, .04:.96
80, .09:.91
68, .01:.99d
69, .01:.99

173, .33:.67
91, .11:.89
148, .07:.93
80, .04:.96
68, .03:.97
67, .03:.97

105,
91,
148,
80,
68,
66,

.55:.45
.57:.43
.68:32
.18:.82
.18:.82
.06:.94

748

804

African-Americans
East Africans .............
East Indians ...............
Chinese ...............
.......

Malays.....................
Total...............

106, .02:.98
91, .21:.79
148, .36:.64
80, 0:1.0
67, 0:1.0
67, 0:1.0
723

115,
91,
148,
80,
68,
69,

.01:.99c
0:1.0
748

72,.51:.49

734

Frequencies were determined by a PCR procedure-except for those of the MnlI polymorphism in Swedish males, which were determined
immunologically.
bFrequencies were determined by using the Southern technique-except for those of East Africans, which were determined by PCR. Data
on Swedes and Orientals have been published elsewhere (Lubahn et al. 1987). For Basques, Anglo-Americans, and African-Americans,
neither PCR data nor Southern data have been published elsewhere.
c Erroneously reported, by Lubahn et al. (1987), as .03:.97.
dErroneously reported, by Lubahn et al. (1987), as .04:.96.
a

541

DNA Polymorphisms of Factor IX


Table 3
Heterozygosity (2pq) of F.IX Polymorphisms
EXPECTED/OBSERVED FREQUENCY OF POLYMORPHISM

ETHNIC GROUP (NO.

OF

WOMEN)

Anglo-Americans (22) ......................


Basques (32) ............................
Swedes (33) ............................
African-Americans (30) ....................
East Africans (56) ...........................
East Indians (26) ............................
Chinese (18) ............................
Malays (22) ............................

BamHI

XmnI

TaqI

MnlI

HhaI

.13/.09
.02/.03
.04/.03
.33/.23
.43/.14
0/0
0/0
0/0

.26/.36
.35/.30
.42/.44
.10/.13
.08/.05
.11/.12
.02/.06
0/0

.44/.45
.43/.44
.38/.28
.18/.20
.07/.05
.16/.19
.02/.06
.02/0

.48/.41
.44/.36
.44/.49
.20/.23
.13/.12
.08/.04
.06/.06
.06/.05

.501.59
.50/.58
.501.56
.49/.53
.44/.34
.30/.23
.30/.33
.11/.04

in Orientals, while the majority (76/87 = 87%) of


the ( + ) alleles are in Occidentals and Africans.
Class II haplotypes, predominantly European, are
a heterogeneous lot. All are ( - ) for BamHI and ( + )
for MnlI (Malmo), and the seven subtypes comprise
34/247 (14%) of all haplotypes.
The class III, or African, haplotypes constitute 9%
of the total. All are BamHI (+ ), and they are equally
divided between (+) and (-) HhaI alleles.

are arbitrarily separated into three classes by using


the criteria of frequency, heterogeneity, and African
ancestry.
In class I are the common haplotypes (192/247 =
78%) which are present in all ethnic groups. They are
( - ) for four polymorphisms (with a single exception)
but highly polymorphic (.54:.46) for the 3' extragenic
HhaI site. A significant ethnic difference exists. The
majority (67: /104 = 64%) of the HhaI ( - ) alleles are

Table 4
Haplotypes of 247 Men of Eight Ethnic Groups
ETHNIC GROUP

HAPLOTYPE

East
East
AfricanAngloBamHI XmnI TaqI MnlI HhaI Americans Basques Swedes Americans Africans Indians Chinese Malays Total

CLASS
I

................

.(-)

(-)(-)(-)(-)

(-)

(-) (-) (-) (+)

6
12

6
15

12
19

6
17

7
15

22
4

26
4

(-)(-)
(-) (-)(+)

Subtotal

(+) (+) (+) (-)

(-)

(+)

(+) (+) (+)

(-)

(-)

(+) (+)

(-)

(+) (-) (+) (+)

(-)

2
1
1
~~~314

12

12
~

2
~~ ~~~~~~~~~~~~~~~~~~~~
34

...

(+)

(-) (-) (-) (-)

(+)

(-)

(-) (-) (+)

Subtotal

Total ....

~~~~~~~~~~~~~~~~~~

(-)
(+)(-)
(-)(-)

III.

104
87

192

...

II............... (-)

Subtotal

19
1

21

28

31

39

31

36

28

32

22

247

542

Graham

Allelic Association (linkage disequilibrium)

Strong allelic association is known to exist between


the three intragenic sites (Graham et al. 1988). Association between adjacent BamHI and XmnI sites was
examined by combining the African-American and
East African subgroups. (Non-Africans were not included, because of their slight BamHI polymorphism.)
Association between Mn1l and HhaI was studied by
combining the Anglo-Americans, Swedes, Basques,
and African-Americans. (Orientals and East Africans
were not included, because of low polymorphism for
MnlI.) As shown in table 5, weak but significant allelic
association exists between BamHI and XmnI, but association was not present between MnlI and HhaI.
Discussion

We have developed PCR procedures for five widely


spaced F.IX polymorphisms. We have used them, together with Southern analysis, to determine RFLP frequencies in eight ethnic groups. The data provide a
fairly complete picture of the diversity among F.IX
RFLPs on three continents.
PCR procedures are now available for three previously reported and frequently used RFLPs: XmnI
(Winship et al. 1984), TaqI (Camerino et al. 1984),
and BamHI (Hay et al. 1986). The PCR procedures
have significant advantages over the earlier methods,
being much faster, being equally accurate, requiring
less target DNA, and avoiding isotopes.
The Mn1I polymorphism, studied elsewhere by us
(Graham et al. 1989), which relates to a dimorphic

et

al.

basepair in the sixth exon of F.IX (and a Thr/Arg


dimorphism in the peptide sequence), was discovered
immunologically (Smith 1985; Wallmark et al. 1985).
Its genetics could not be satisfactorily evaluated immunologically in women, and Southern analysis was
difficult and ambiguous. The PCR procedure neatly
solved the dilemma and permitted us to use all the
female data in assessing the features of the (Malm6)
polymorphism.
The HhaI polymorphism, which was discovered by
Winship et al. (1989), should be very useful diagnostically, because it is highly polymorphic in all populations, including Orientals. Our data on allelic association agree with those of Winship et al. HhaI is not
in association with the other F.IX RFLPs.
Zhang et al. (1989) have developed a PCR procedure for the BamHI polymorphism discovered by Hay
et al. ( 1986). Our primers are different from theirs, but
the dimorphism is not. We found weak but significant
-

allelic association between BamHI and XmnI although we had expected it to be stronger, since the
sites are only 7.6 kb apart. The association between
XmnI and MnlI is sixfold greater even though they are
farther apart. Perhaps there is more recombination in
the 5' extragenic region of F.IX than within the gene
itself.
The frequencies of the five F.IX polymorphisms
vary greatly with ethnicity. BamHI and HhaI are the
only RFLPs sufficiently polymorphic to be useful diagnostically in Africans, and none would be very useful
in Malays. The ethnic variability of F.IX polymorphisms contrasts sharply with the BclI and HindIII
polymorphisms of F.VIII, the frequencies of the lat-

Table 5
Allelic Associations

Association between
BamHI and XmnI ....
XmnI and TaqId .......

Ethnic Group(s)

African-Americans and East Africans


Swedes
TaqI and MnhI.l....... Swedes
XmnI and Mnhld ...... Swedes
Anglo-Americans, Swedes, Basques,
MnlI and HhaI ........
and African-Americans

Disequilibrium coefficient.
b Correlation coefficient.
c
Probability of association.
d From Graham et al. (1988).
' .95 Significance at .1021.
a

No. of
Chromosomes

Distance (kb)

D-

233
115
113
113

7.6
4.0
9.3
13.3

.0104
.096
.112
.063

.1156
.468
.506
.318

>.95
>.999
>.999
>.999

373

z20

.0195

.0868e

<.95

rb

PC

DNA Polymorphisms of Factor IX

ters' rarer alleles being essentially uniform across seven


of the same populations (Graham et al. 1990). In a
subsample selected from the same subjects, the frequencies of an RsaI polymorphism of von Willebrand
factor varied like the F.IX polymorphisms, but less
markedly so (Kunkel et al. 1990).
The higher frequency of the ( - ) alleles for all F.IX
polymorphisms except HhaI-which may be special
and extragenic and which may have CpGs -suggests
that (-) may once have been the universal norm.
Change of a basepair in a ( + ) allele produces a ( - )
allele, while only a change in the specific basepair responsible for a ( - ) allele will yield a ( + ) allele. If it
is assumed that the primeval sequence was ( - ) at all
sites in all populations, it is odd that ( + ) is fairly
frequent in Europeans and rare in Orientals. Genetic
drift is a reasonable possibility, but there is very little
difference in the proportion of the ( + ) alleles among
three different, noninterbreeding Oriental populations. Another possibility is that the sequences of the
( - ) alleles of Orientals contain more mismatches than
do those of Europeans and are therefore more fixed.
We considered the possibility that CpG deamination might be the basis for the switching of alleles,
especially since the HhaI polymorphism had been discovered by workers acting on this premise. However,
among the five F.IX polymorphisms, only TaqI and
HhaI restriction sites contain the appropriate sequences. CpGs are not present in the BamHI, XmnI,
and MnlI restriction sites, and the dimorphism in
BamHI is known to have been T to G and that in Mnl
is known to have been A to G.
A final possibility is that natural selection may be
operating on the F.IX restriction-enzyme sites of normal persons. This would be a surprise, because
enzyme-recognition sites have been assumed to be selectively neutral. The possibility could be tested by
careful, multigenerational studies of suitable populations, but this is an effort beyond the scope of the
present investigation.

Acknowledgments
We would like to thank the following colleagues for help
in obtaining blood samples: Dr. John C. Day of Boise, Idaho
(Basques); Dr. Curtis Hames of Claxton, Georgia (AfricanAmericans); and Dr. John Bosco of Kuala Lumpur, Malaysia
(East Indians, Chinese, and Malays). We would also like to
thank Dr. Bruce Weir of NC State University for advice
concerning the population genetics. The research was supported in part by NIH grants HL 35152, HL 06350, and

543
HL 07225 and by Swedish Medical Research Council grant
19X00087.

References
Camerino G, Grzeschik KH, Jaye M, de la Salle H, Tolstoshev P, LecocqJP, MandelJL (1984) Regional localization
on the human X chromosome and polymorphism of the
coagulation factor IX gene (hemophilia B locus). Proc
Natl Acad Sci USA 81:498-502
Gianelli F, Choo KH, Rees DJG, Boyd Y, Rizza CR,
Brownlee GG (1983) Gene deletions in patients with haemophilia B and anti-factor IX antibodies. Nature 303:
181-182
Graham JB, Kunkel GR, Fowlkes DM, Lord ST (1990) The
utility of a HindIII polymorphism of F.VIII examined by
rapid DNA analysis. Br J Haematol 76:1-5
Graham JB, Kunkel GR, Tennyson GS, Lord ST, Fowlkes
DM (1989) The Malmo polymorphism of factor IX: establishing the genotypes by rapid analysis of DNA. Blood
73:2104-2107
Graham JB, Lubahn DB, Lord ST, Kirshtein J, Nilsson IM,
Wallmark A, Ljung R, et al (1988) The Malmo polymorphism of cogulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage
disequilibrium with two other F.IX polymorphisms. Am
J Hum Genet 42:573-580
Hay CW, Robertson KA, Yong S-L, Thompson AR, Growe
GH, McGillivary RTA (1986) Use of a BamHI polymorphism in the factor IX gene for the determination of hemophilia B carrier status. Blood 67:1508-1511
Kogan SC, Doherty M, Gitschier J (1987) An improved
method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences: application to hemophilia A. N Engl J Med 317:985-990
Kunkel GR, Graham JB, Fowlkes DM, Lord ST (1990) An
RsaI polymorphism in von Willebrand factor (VWF) detected by rapid DNA analysis. Nucleic Acids Res 18:4961
Litt M, Jorde LB (1986) Linkage disequilibria between pairs
of loci within a highly polymorphic region of chromosome
2Q. Am J Hum Genet 39:166-178
Lubahn DB, Lord ST, Bosco J, Kirshtein J, Jeffries OJ, Parker N, Levtzow C, et al (1987) Population genetics of
coagulant factor IX: frequencies of two DNA polymorphisms in five ethnic groups. Am J Hum Genet 40:527536
Smith KJ (1985) Monoclonal antibodies to coagulation factor IX define a high-frequency polymorphism by immunoassays. Am J Hum Genet 37:668-679
Wallmark A, Ljung R, Nilsson IM, Holmberg L, Hedner U,
Lindvall M, Sjogren H-O (1985) Polymorphism of normal
factor IX detected by mouse monoclonal antibodies. Proc
Natl Acad Sci USA 83:3839-3843
Winship PR, Anson DS, Rizza CR, Brownlee GG (1984)
Carrier detection in haemophilia B using two further re-

544
striction fragment length polymorphisms. Nucleic Acids
Res 12:8861-8872
Winship PR, Rees DJG, Alkan M (1989) Detection of polymorphisms at cytosine phosphoguanidine dinucleotides
and diagnosis of hemophilia B carriers. Lancet 1: 631-634
Yoshitake S, Schach BG, Foster DC, Davie EW, Kurachi K

Graham et al.
(1985) Nucleotide sequence of the gene for human factor
IX (antihemophilic factor B). Biochemistry 24:37363750
Zhang M, Chen S-H, Scott RC, Thompson AR (1989) The
factor IX Bam HI polymorphism: T to G transversion at
the nucleotide sequence - 561. Hum Genet 82:283-284