Am. J. Hum. Genet. 49:537-544, 1991

The Varying Frequencies of Five DNA Polymorphisms ofX-linked Coagulant Factor IX in Eight Ethnic GroupsJohn 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

SummaryFive RFLPS of X-linked coagulation factor IX were evaluated in more than 500 normal persons (723-804X 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', wereBamHI, XmnI, TaqI, MnI, and HhaI. A PCR procedure was developed for three previously describedRFLPs-XmnI, TaqI, and MnII; a PCRP procedure was developed for BamHI, and a PCRP which had beendescribed by others was used for HhaI. Europeans were the most polymorphic, African-Americans and EastAfricans were intermediate, and Orientals were the least polymorphic. Extragenic 3' HhaI was highly polymor-phic in most groups, and extragenic 5' BamHI was polymorphic only in persons with African ancestry. Twomajor haplotypes predominated among 247 men, and the expected and observed heterozygosities were concor-dant among women. Allelic association was very strong between the three intragenic PCRPs; it was presentbut weak between 5' extragenic BamHI and XmnI. No association was found between 3' extragenic HhaI andMnnI.

IntroductionThe 35-kb human coagulation factor IX (F.IX) genehas been completely sequenced (Yoshitake et al.1985). This makes very feasible the analysis of its poly-morphisms by rapid DNA (i.e., PCR) procedures. Wedescribe conditions sufficient for such analysis of fiveRFLPs of F.IX. Oligonucleotide primers developed forfour of the polymorphisms are described, as are theresults which were obtained by using both them andprimers developed by Winship et al. (1989) for thefifth polymorphism.

All five polymorphisms have been examined in thesame population, z500 normal persons from eightethnic groups (723-804 chromosomes). Three sets ofsubjects 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 in1986-87. They were evaluated for XmnI, TaqI, andMnlI RFLPs by Southern analysis, and the results havebeen published (Lubahn et al. 1987; Graham et al.1988). Anglo-Americans, African-Americans, andBasques were sampled in 1988, and East Africanswere sampled in 1990. Most of the data presented arenew and were obtained using PCR technology. Thepopulation genetics are collated by recording all dataon all subjects for all F.IX polymorphisms studied byall methods.Three polymorphisms are intragenic, and two are

extragenic. Their locations with respect to each otherand the eight exons are indicated in figure 1.The primers used for PCR analysis, as well as the

digestion patterns obtained, the gene frequencies, theeffective heterozygosities, the haplotype frequencies,and the allelic associations, are recorded and com-pared both with each other and with three other poly-morphisms-two F.VIII and one VWF-examined inthe same subjects.

537

Graham et al.

-590 7050 11082 20392B X T M H----mm.' .L .- * AW

1 2 3 4 5 6 7 8 8kb0 Kb 35

Figure I F.IX gene. The start codon and end of the 5' un-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. Inthe GenBank sequence, coding begins at 2995, and the BamHIdimorphic base site is at 2405. Other numbers are from the Gen-Bank sequence.

Material and MethodsSource of DNADNA was prepared from white blood cells obtained

under the Rules of Informed Consent as described else-where (Lubahn et al. 1987).Ethnic GroupsThe Anglo-Americans were University of North

Carolina medical students all of whose known ances-tors can be traced to the British Isles. The Swedes werepersons of Swedish descent presently living in Malmo,Sweden. The African-Americans were residents of Ev-ans County, Georgia.The Basques live in Boise, Idaho-a U.S. center of

Basque culture - and all four of the grandparents ofeach subject were of Basque origin. The East Indians,Chinese, and Malays were students or staff of the Uni-versity of Malaysia in Kuala Lumpur. The East Afri-cans were native students attending school in Harare,Zimbabwe. Care was taken at each site to test unre-lated persons. Essentially all subjects of each ethnicgroup were analyzed for the five polymorphisms.PCR ConditionsThe basic PCR procedure, adapted from the work

of Kogan et al. (1987), has been described by Grahamet al. (1989). Several recent modifications include (1)amplification DNA reduced to 200 ng, (2) initial dena-turation for 4 min at 93C, (3) Taq polymerase re-duced from 3 to 1 unit/amplification, (4) number ofcycles increased from 30 to 35, (5) amplified productnot always purified before restriction, (6) restrictionenzyme increased from 3 to 15 units / 10 gl of amplifiedproduct, (7) restricted fragments electrophoresed inagarose (Nusievem), and (8) external controls of diges-tion designed for and used in the BamHI and HhaIPCRPs.

The Oligonucleotide PrimersBamRl.-Primer 1 was 5' GAA GTT TGA CCT

AAA CAT CAT AC, and primer 2 was 5' TTG AGTCTG AAA CAG GAA GTG A.

Xmnl. - Primer 1 was 5' CAGAGA CTG CTG ATTGAC TT, and primer 2 was 5' ACA GCC AGA TAAAGC CTC CA.

Taqi. -Primer 1 was 5' TAT AAT GGG AAT TCTCCA CAT, and primer 2 was 5'AGTAGAAAG TGAATT CCT CA.

Mnfi. - Primer 1 was 5' GAT TTG AAA ACT GCTCAT GAA AAT AAC, and primer 2 was 5' AAA GTACCT GCC AAG GGA ATT GAC CTG.

Hhal. -Primer 1 was 5' ACA GGC ACC TGC CATCAC TT, and primer 2 was 5' AGA TTT CAA GCTACC AAC AT.

ResultsThe Individual ProceduresThe essential characteristics of the PCRPs are

shown in table 1. The location of the dimorphic orenzyme-recognition site, the region amplified, the am-plification parameters, the sizes of the digestion frag-ments, and the type of control of digestion are noted.The Mn1I product contains an internal control of di-gestion, an invariant Mn1I site, and external controlswere engineered for the BamHI and HhaI polymor-phisms. Digestion controls were not engineered forXmnI and TaqI because (1) all groups except the EastAfricans had been studied by Southern analysis and(2) 27 Basque and Anglo-American samples had beenanalyzed by both PCR and Southern techniques, with-out a discrepancy.The BamHI polymorphism results from dimor-

phism of a basepair (bp 2405 in GenBank) 590 bp 5'to the F.IX start codon. All samples were examinedby the PCR procedure.XmnI and TaqI polymorphisms, which are intronic,

were examined in seven groups by Southern analysisusing the genomic probe pAla36 (Lubahn et al. 1987).Only the East Africans were examined for the XmnIand TaqI polymorphisms solely by the PCR proce-dure.The MnlI polymorphism had been examined earlier

in Swedish females by Southern analysis of dried gelsby using labeled oligonucleotides (Graham et al.1988), and some of the results had been ambiguous.All were retested by the PCR procedure, which dis-criminates precisely, and several putative Swedish ge-

538

DNA Polymorphisms of Factor IX

Table IComparison of PCR Polymorphisms of F.IX

Polymorphism Name, Size of Fragments Control of RestrictionGenBank Locationa Region Amplified Amplification Parameters (bp) Enzyme Digestion

BamHI, bp 2405 ........... 2258-2782 1.5 mM Mg"+, denatur- 525, 379, and 146 2692 fragment ofation for 30s at 91 IC and pBR322 with oneannealing/extension for BamHI site (larger4 min at 600C fragment when

pBR322 is cut by PvuIand PvuII)

XmnI, bp 10041-10050. 9979-10199 4.5 mM Mg++, denatur- 220, 153, and 67 Noneation for 30 s at 910C andannealing/extension for2 min at 600C

TaqI, bp 14076-14079... 13980-14279 6.0 mM Mg" +, denatur- 299, 203, and 96 Noneation for 30 s at 91'C andannealing/extension for2 min at 600C

MnlI (Malm6), bp 23387 23131-23537 6.0 mM Mg++, denatur- 278, 120, and 159 Invariant MnO site atation for 30 s at 91 C and 23247-23250; 126-annealing/extension for bp fragment4 min at 600C

HhaI, 8 kb 3' to F.IX gene ? 6.0 mM Mg+ +, denatur- 230, 150, and 80 1,059-bp fragment ofation for 30 s at 91C and (pX 174 with two HhaIannealing/extension for (smaller fragment when4 min at 600C (pX is cut with StuI

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

notypes were changed. All other females and all males,except the Swedish, were studied for the Mnln poly-morphism by the PCR procedure. Swedish males weregenotyped immunologically (Graham et al. 1988).The HhaI PCRP has been described by Winship et

al. (1989). We followed their procedure except forusing a different control of digestion, a fragment fromDX 174 (table 1). Our primer 1 is their FIXH1, andour primer 2 is their FIXH2.Gel Patterns of the Individual PolymorphismsThe MnlI digestion patterns have been described by

Graham et al. (1989), and the patterns specific for theother four polymorphisms are shown in figure 2. TheBamHI and HhaI patterns are complex, because exter-nal controls of enzymic digestion are also present, thecontrol fragments in each being indicated by an ante-cedent (c). The BamHI control produces two frag-ments, both larger than the amplified product. TheHhaI external control contains two HhaI restrictionsites, which produce three control fragments. Thesmallest (1 90-bp) fragment lies between the undi-

(A) BamHIMales Femoles

bp (und.) (- 1 +) (-,-) (+-,) (+.,-(c)2692c)1692-(cl 1600-

525-379-

146-

(C) Taq IMales Females-(-) (+.+) (_A (,_)

299-203-_

96-

(B) XmnIMales Females

bp 1+1 1-) l+tl) (-,-) (a,-)220-153-

67-

(D) HhaIMales Fer

1.. "51 ,," "

bp(c)1025-(c0800-

230-(c)l90-150-80-

(c) (35)1

Figure 2 Ethidium bromide-stained agarose gels showingfragments produced by digestion of amplified products by restric-tion enzymes. Primers used for amplification, as well the amplifica-tion conditions, are described in the text.

S39

Graham et al.

gested (230-bp) and digested (150-bp) fragments ofthe F.IX PCR product.The XmnI and TaqI patterns are simple. In the ab-

sence of an external control the ( - ) and ( -, - ) geno-types may sometimes be difficult to distinguish fromnondigested amplified product. However, when theEast African samples were analyzed, every gel con-tained (+), ( + , + ), or ( + , - ) individuals digested atthe same time under the same conditions which servedas controls.

Population Genetics of the PolymorphismsTable 2 records the gene frequencies of the five poly-

morphisms in the eight ethnic groups. The frequenciesare weighted averages between males and females,since there was not a statistically significant sex differ-ence for any RFLP in any ethnic group. The frequen-cies of the F.IX polymorphisms are strongly affectedby ethnicity. Occidentals (Anglo-Americans, Swedes,and Basques) are much more polymorphic than Orien-tals (East Indians, Chinese, and Malays). African-Americans and East Africans tend to be intermediatefor four polymorphisms, but they are highly polymor-phic for BamHI whereas other groups are essentiallymonomorphic for it. The frequencies of the rarer al-leles of each polymorphism are shown in the bargraphs of figure 3.The expected heterozygosities in table 3 are calcu-

lated from the gene frequencies and compared with

w

-j-J04

Cr

0.3

0>. 0.2

D0.1

0 Malays Chins EatIndians Africans Aans Amerans

ORIENTAL AFRICAN - OCCIDENTAL

Figure 3 Frequencies of rarer alleles of each F.IX RFLP, plot-ted as bar graphs. Both the high degree of polymorphism in Europe-ans and the slight degree in Orientals are very striking. BamHIpolymorphism is obviously an African trait. 0 = TaqI; E = MnlI;0 = HhaI; a = XmnI; l = BamHI.

those observed. Observed and expected values did notdiffer significantly, except for BamHI in East Africans.This resulted from the large proportion of homozy-gous ( + , + ) women in the East African sample, whichskewed the genotype frequencies.HaplotypesThe haplotypes for the five polymorphisms of 247

men are shown in table 4. (Female haplotypes couldnot be analyzed in the absence of family studies.) They

Table 2

Gene Frequencies of F.IX Polymorphisms

No. OF CHROMOSOMES, p( + ):q(-) OF POLYMORPHISMETHNIC GROUP BamHIa Xmnlb Taqlb MnlI (Malmo)a HhalaAnglo-Americans 72, .07:.93 73, .15:.85 73,.31:.69 73, .33:.67 72,.51:.49Basques............... 96,.01:.99 104, .23:.77 104,.31:.69 104, .32:.68 104, .48:.52Swedes...................... 106, .02:.98 115, .30:.70 115, .26:.74 173, .33:.67 105, .55:.45African-Americans

....... 91, .21:.79 91, .05:.95 91, .10:.90 91, .11:.89 91, .57:.43East Africans ............. 148, .36:.64 148, .04:.96 148, .04:.96 148, .07:.93 148, .68:32East Indians ............... 80, 0:1.0 80, .06:.94 80, .09:.91 80, .04:.96 80, .18:.82Chinese ............... 67, 0:1.0 68, .01:.99c 68, .01:.99d 68, .03:.97 68, .18:.82Malays..................... 67, 0:1.0 69, 0:1.0 69, .01:.99 67, .03:.97 66, .06:.94

Total............... 723 748 748 804 734a 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.

540

DNA Polymorphisms of Factor IX

Table 3Heterozygosity (2pq) of F.IX Polymorphisms

EXPECTED/OBSERVED FREQUENCY OF POLYMORPHISM

ETHNIC GROUP (NO. OF WOMEN) BamHI XmnI TaqI MnlI HhaIAnglo-Americans (22) ...................... .13/.09 .26/.36 .44/.45 .48/.41 .501.59Basques (32) ............................ .02/.03 .35/.30 .43/.44 .44/.36 .50/.58Swedes (33) ............................ .04/.03 .42/.44 .38/.28 .44/.49 .501.56African-Americans (30) .................... .33/.23 .10/.13 .18/.20 .20/.23 .49/.53East Africans (56) ........................... .43/.14 .08/.05 .07/.05 .13/.12 .44/.34East Indians (26) ............................ 0/0 .11/.12 .16/.19 .08/.04 .30/.23Chinese (18) ............................ 0/0 .02/.06 .02/.06 .06/.06 .30/.33Malays (22) ............................ 0/0 0/0 .02/0 .06/.05 .11/.04

are arbitrarily separated into three classes by using in Orientals, while the majority (76/87 = 87%) ofthe criteria of frequency, heterogeneity, and African the ( + ) alleles are in Occidentals and Africans.ancestry. Class II haplotypes, predominantly European, are

In class I are the common haplotypes (192/247 = a heterogeneous lot. All are ( - ) for BamHI and ( + )78%) which are present in all ethnic groups. They are for MnlI (Malmo), and the seven subtypes comprise( - ) for four polymorphisms (with a single exception) 34/247 (14%) of all haplotypes.but highly polymorphic (.54:.46) for the 3' extragenic The class III, or African, haplotypes constitute 9%HhaI site. A significant ethnic difference exists. The of the total. All are BamHI (+ ), and they are equallymajority (67:/104 = 64%) of the HhaI ( - ) alleles are divided between (+) and (-) HhaI alleles.

Table 4Haplotypes of 247 Men of Eight Ethnic Groups

HAPLOTYPE ETHNIC GROUP

Anglo- African- East EastCLASS BamHI XmnI TaqI MnlI HhaI Americans Basques Swedes Americans Africans Indians Chinese Malays Total

I................

.(-) (-)(-)(-)(-) 6 6 12 6 7 22 26 19 104(-) (-) (-) (-) (+) 12 15 19 17 15 4 4 1 87

(-) (-)(+)(-)(-) ~ ~~~~~~~~~~~~~~~~~~~~~~~~~1 1Subtotal ... 192

II............... (-) (+) (+) (+) (-) 1 7 3 1 12(-) (+) (+) (+) (+) 1 1 2 1 2 1 8(-) (-) (+) (+) (-) ~~~314

(-) (+) (-) (+) (+) 12 2(-)(-)(-)(+)(-) ~ ~~ ~~~~~~~~~~~~~~~~~~~~1 1 2

Subtotal ... 34III. (+) (-) (-) (-) (-) 5 4 9

(+) (-) (-) (-) (+) 1 1 2 5 9

Subtotal 21Total .... 28 31 39 31 36 28 32 22 247

541

Graham et al.

Allelic Association (linkage disequilibrium)Strong allelic association is known to exist between

the three intragenic sites (Graham et al. 1988). Associ-ation between adjacent BamHI and XmnI sites wasexamined by combining the African-American andEast African subgroups. (Non-Africans were not in-cluded, because of their slight BamHI polymorphism.)Association between Mn1l and HhaI was studied bycombining the Anglo-Americans, Swedes, Basques,and African-Americans. (Orientals and East Africanswere not included, because of low polymorphism forMnlI.) As shown in table 5, weak but significant allelicassociation exists between BamHI and XmnI, but as-sociation was not present between MnlI and HhaI.

DiscussionWe have developed PCR procedures for five widely

spaced F.IX polymorphisms. We have used them, to-gether with Southern analysis, to determine RFLP fre-quencies in eight ethnic groups. The data provide afairly complete picture of the diversity among F.IXRFLPs on three continents.PCR procedures are now available for three pre-

viously reported and frequently used RFLPs: XmnI(Winship et al. 1984), TaqI (Camerino et al. 1984),and BamHI (Hay et al. 1986). The PCR procedureshave significant advantages over the earlier methods,being much faster, being equally accurate, requiringless target DNA, and avoiding isotopes.The Mn1I polymorphism, studied elsewhere by us

(Graham et al. 1989), which relates to a dimorphic

basepair in the sixth exon of F.IX (and a Thr/Argdimorphism in the peptide sequence), was discoveredimmunologically (Smith 1985; Wallmark et al. 1985).Its genetics could not be satisfactorily evaluated immu-nologically in women, and Southern analysis wasdifficult and ambiguous. The PCR procedure neatlysolved the dilemma and permitted us to use all thefemale data in assessing the features of the (Malm6)polymorphism.The HhaI polymorphism, which was discovered by

Winship et al. (1989), should be very useful diagnosti-cally, because it is highly polymorphic in all popula-tions, including Orientals. Our data on allelic associa-tion agree with those of Winship et al. - HhaI is notin association with the other F.IX RFLPs.Zhang et al. (1989) have developed a PCR proce-

dure for the BamHI polymorphism discovered by Hayet al. (1986). Our primers are different from theirs, butthe dimorphism is not. We found weak but significantallelic association between BamHI and XmnI- al-though we had expected it to be stronger, since thesites are only 7.6 kb apart. The association betweenXmnI and MnlI is sixfold greater even though they arefarther apart. Perhaps there is more recombination inthe 5' extragenic region of F.IX than within the geneitself.The frequencies of the five F.IX polymorphisms

vary greatly with ethnicity. BamHI and HhaI are theonly RFLPs sufficiently polymorphic to be useful diag-nostically in Africans, and none would be very usefulin Malays. The ethnic variability of F.IX polymor-phisms contrasts sharply with the BclI and HindIIIpolymorphisms of F.VIII, the frequencies of the lat-

Table 5Allelic Associations

No. ofAssociation between Ethnic Group(s) Chromosomes Distance (kb) D- rb PCBamHI and XmnI .... African-Americans and East Africans 233 7.6 .0104 .1156 >.95XmnI and TaqId ....... Swedes 115 4.0 .096 .468 >.999TaqI and MnhI.l....... Swedes 113 9.3 .112 .506 >.999XmnI and Mnhld ...... Swedes 113 13.3 .063 .318 >.999

Anglo-Americans, Swedes, Basques,MnlI and HhaI ........ and African-Americans 373 z20 .0195 .0868e

DNA Polymorphisms of Factor IX 543

ters' rarer alleles being essentially uniform across sevenof the same populations (Graham et al. 1990). In asubsample selected from the same subjects, the fre-quencies of an RsaI polymorphism of von Willebrandfactor varied like the F.IX polymorphisms, but lessmarkedly so (Kunkel et al. 1990).The higher frequency of the ( - ) alleles for all F.IX

polymorphisms except HhaI-which may be specialand extragenic and which may have CpGs -suggeststhat (-) 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 re-sponsible for a ( - ) allele will yield a ( + ) allele. If itis assumed that the primeval sequence was ( - ) at allsites in all populations, it is odd that ( + ) is fairlyfrequent in Europeans and rare in Orientals. Geneticdrift is a reasonable possibility, but there is very littledifference in the proportion of the ( + ) alleles amongthree different, noninterbreeding Oriental popula-tions. Another possibility is that the sequences of the( - ) alleles of Orientals contain more mismatches thando those of Europeans and are therefore more fixed.We considered the possibility that CpG deamina-

tion might be the basis for the switching of alleles,especially since the HhaI polymorphism had been dis-covered by workers acting on this premise. However,among the five F.IX polymorphisms, only TaqI andHhaI restriction sites contain the appropriate se-quences. CpGs are not present in the BamHI, XmnI,and MnlI restriction sites, and the dimorphism inBamHI is known to have been T to G and that in Mnlis 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 nor-mal persons. This would be a surprise, becauseenzyme-recognition sites have been assumed to be se-lectively neutral. The possibility could be tested bycareful, multigenerational studies of suitable popula-tions, but this is an effort beyond the scope of thepresent investigation.

AcknowledgmentsWe 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 (African-Americans); and Dr. John Bosco ofKuala Lumpur, Malaysia(East Indians, Chinese, and Malays). We would also like tothank Dr. Bruce Weir of NC State University for adviceconcerning the population genetics. The research was sup-ported in part by NIH grants HL 35152, HL 06350, and

HL 07225 and by Swedish Medical Research Council grant19X00087.

ReferencesCamerino G, Grzeschik KH, Jaye M, de la Salle H, Tolsto-

shev P, LecocqJP, MandelJL (1984) Regional localizationon the human X chromosome and polymorphism of thecoagulation factor IX gene (hemophilia B locus). ProcNatl Acad Sci USA 81:498-502

Gianelli F, Choo KH, Rees DJG, Boyd Y, Rizza CR,Brownlee GG (1983) Gene deletions in patients with hae-mophilia B and anti-factor IX antibodies. Nature 303:181-182

Graham JB, Kunkel GR, Fowlkes DM, Lord ST (1990) Theutility of a HindIII polymorphism of F.VIII examined byrapid DNA analysis. Br J Haematol 76:1-5

Graham JB, Kunkel GR, Tennyson GS, Lord ST, FowlkesDM (1989) The Malmo polymorphism of factor IX: es-tablishing the genotypes by rapid analysis ofDNA. Blood73:2104-2107

Graham JB, Lubahn DB, Lord ST, Kirshtein J, Nilsson IM,Wallmark A, Ljung R, et al (1988) The Malmo polymor-phism of cogulation factor IX, an immunologic polymor-phism due to dimorphism of residue 148 that is in linkagedisequilibrium with two other F.IX polymorphisms. AmJ Hum Genet 42:573-580

Hay CW, Robertson KA, Yong S-L, Thompson AR, GroweGH, McGillivary RTA (1986) Use of a BamHI polymor-phism in the factor IX gene for the determination ofhemo-philia B carrier status. Blood 67:1508-1511

Kogan SC, Doherty M, Gitschier J (1987) An improvedmethod for prenatal diagnosis ofgenetic diseases by analy-sis of amplified DNA sequences: application to hemo-philia A. N Engl J Med 317:985-990

Kunkel GR, Graham JB, Fowlkes DM, Lord ST (1990) AnRsaI polymorphism in von Willebrand factor (VWF) de-tected by rapid DNA analysis. Nucleic Acids Res 18:4961

Litt M, Jorde LB (1986) Linkage disequilibria between pairsof loci within a highly polymorphic region ofchromosome2Q. Am J Hum Genet 39:166-178

Lubahn DB, Lord ST, Bosco J, Kirshtein J, Jeffries OJ, Par-ker N, Levtzow C, et al (1987) Population genetics ofcoagulant factor IX: frequencies of two DNA polymor-phisms in five ethnic groups. Am J Hum Genet 40:527-536

Smith KJ (1985) Monoclonal antibodies to coagulation fac-tor IX define a high-frequency polymorphism by immuno-assays. Am J Hum Genet 37:668-679

Wallmark A, Ljung R, Nilsson IM, Holmberg L, Hedner U,Lindvall M, Sjogren H-O (1985) Polymorphism ofnormalfactor IX detected by mouse monoclonal antibodies. ProcNatl 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 Graham et al.

striction fragment length polymorphisms. Nucleic AcidsRes 12:8861-8872

Winship PR, Rees DJG, Alkan M (1989) Detection of poly-morphisms at cytosine phosphoguanidine dinucleotidesand diagnosis of hemophilia B carriers. Lancet 1: 631-634

Yoshitake S, Schach BG, Foster DC, Davie EW, Kurachi K

(1985) Nucleotide sequence of the gene for human factorIX (antihemophilic factor B). Biochemistry 24:3736-3750

Zhang M, Chen S-H, Scott RC, Thompson AR (1989) Thefactor IX Bam HI polymorphism: T to G transversion atthe nucleotide sequence - 561. Hum Genet 82:283-284