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Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA
GENOMICS
6,65-71
(1990)
Characterization of Mutations in the Factor VIII Gene by Direct Sequencing of Amplified Genomic DNA MIYOKO HIGUCHI,* CORINNE WONG,* LOTHAR KocHHAN,t KLAUS OLEK,j’ SOPHIA ARONIS,* CAROL K. KA~PER,§ HAIG H. KAZAZIAN, JR.,* AND S~VLIANOS E. ANTONARAKIS*,’ *Genetics Unit, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 2 1205; tlnstitute for Clinical Biochemistry, University of Bonn, Bonn, Germany; Koagulation Laboratory, “Agia Sophia” Children’s Hospital, Athens, Greece; and §Orthopedic Hospital, University of Southern California, Los Angeles, California 90007 Received
June 1, 1989;
revised
INTRODUCTION
Hemophilia A is a common disorder of blood coagulation caused by deficiency or abnormality of factor VIII (FVIII) and is inherited as an X-linked recessive trait. Plakma FVIII circulates as a metal-ion stabilized complex (Rotblat et al., 1985; Eaton et al., 1986; Fay, 1988) and participates as a cofactor for activation of factor X by factor IXa in the intrinsic pathway of the blood coagulation cascade (Mann, 1984; Sadler and Davie, 1987). Recent studies on plasma- and recombinant DNA-derived FVIII have demonstrated that the procoagulant activity of FVIII is regulated by several correspondence
should
7, 1989
proteolytic modifications including cleavages by thrombin at amino acid positions 372, 740, and 1689 which lead to maximal coagulant activity of FVIII (Eaton et al., 1986, 1987). The functional importance of proteolytic processing has been demonstrated following site-directed mutagenesis of codons for arginines (Arg) at residues 372 and 1689 (Pittman and Kaufman, 1988). The gene for human FVIII is 186 kb in length and is composed of 26 exons which encode an approximately 9-kb mRNA with a probable transcription start site at nucleotide -170 (initiation codon at $1) and an 1805nucleotide-long 3’ untranslated region (Toole et aZ., 1984; Gitschier et al., 1984). The sequence GATAAA, a presumptive “TATA”-box element located 30 bp upstream from nucleotide -170, is thought to be important for proper and efficient transcription of the gene. A number of mutations in the FVIII gene resulting in hemophilia A have been reported. These mutations [deletions (Gitschier et al., 1985; Antonarakis et al, 1985; Youssoufian et al., 1987; Higuchi et al., 1988), insertions (Kazazian et al., 1988), and point mutations (Gitschier et al., 1985, 1986; Antonarakis et at., 1985; Youssoufian et al., 1986, 1988a,b; Inaba et al., 1989)] were identified mainly by Southern analysis using several restriction enzymes including TcrqI and several FVIII cDNA fragments as molecular probes. In roughly 10% of patients with hemophilia A the molecular defect can be detected by this procedure (for review see Antonarakis and Kazazian, 1988); however, the majority of deleterious mutations remain undetected. Recently, discriminant oligonucleotide hybridization was used to detect two point mutations, one of which was located in a thrombin cleavage site at amino acid 1689 (Gitschier et al., 1988). In this report we describe the identification of mutations in selected regions of the FVIII gene using amplification of genomic DNA by polymerase chain re-
In order to search for mutations resulting in hemophilia A that are not detectable by restriction analysis, three regions of the factor VIII gene were chosen for direct sequence analysis. Short segments of genomic DNA of 127 unrelated patients with hemophilia A were amplified by polymerase chain reaction. A total of 136,017 nucleotides were sequenced, and four mutations leading to the disease were found: a frameshift at codon 360 due to deletion of two nucleotides (GA), a nonsense codon 1705 due to a C + T transition, and two missense codons at positions 1699 and 1708. The first missense mutation (A --* T) results in a Tyr + Phe substitution at a putative von Willebrand factor binding site. The second results in an Arg + Cys substitution at a thrombin cleavage site. In addition, we identified three rare sequence variants: a silent C + T transition at codon 34 which does not result in an amino acid change, a G + C change at codon 345 (Val + Leu), and an A + G change at the third nucleotide of intron 14. Direct sequence analysis of amplified DNA is a powerful but labor-intensive method of identifying mutations in large genes such as the human factor VIII gene. o ISBO Academic PRW. I~C.
1 To whom
August
be addressed.
65 All
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66
HIGUCHI
action (PCR) and direct sequencing regions of the FVIII gene. MATERIALS
AND
ET
6 min at 94’C, followed by 30 cycles of amplification using a step program (denaturation at 94’C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min) on a DNA thermal cycler (Perkin-Elmer-Cetus). Amplified DNA was purified by spin dialysis using Centricon 30 (Amicon, Danvers, MA) and subjected to direct sequencing.
of the amplified
METHODS
Subjects Our subjects were 127 unrelated hemophilia A patients who showed no detectable gross gene alterations or point mutations when their genomic DNA was screened by Southern analysis using several restriction enzymes including TaqI and 3 cDNA subfragments of the FVIII gene as hybridization probes (Toole et al., 1984; Antonarakis et aZ., 1985). These patients included IO7 patients from the series of Youssoufian et al. (for review see Antonarakis and Kazazian, 1988) and 20 German patients from Higuchi et al. (1989). According to clinical features and hematologic data, 86 of these patients were classified as having severe, 14 as having moderate, and 27 as mild hemophilia A. Fifteen patients with severe hemophilia A had developed antibodies against FVIII after multitransfusion therapy (inhibitor-positive patients). None of the patients with moderate or mild hemophilia A was inhibitor-positive. Amplification
Sequencing of Amplified Products Purified PCR products were directly sequencedwith T7 DNA polymerase (Sequenase, United State Biochemicals, Cleveland, OH) as previously described (Wong et al., 1987; Higuchi et al., 1988). Sequencing primers were end-labeled with [y-32P]ATP (3000 Ci/ mmol; New England Nuclear) using T4 DNA polynucleotide kinase (BRL, Gaithersburg, MD). Approximately 80 ng of PCR product was annealed with 10 ng of appropriate sequencing primer (Table 1) in a volume of 11 ~1 on ice for 10 min. After heat denaturing for 5 min at 95°C 2.5 ~1 of the annealed sample was added to four tubes with 3 ~1of the sequencing mixture containing nonradioactive dNTPs at a concentration of 62 PM, ddNTPs at a concentration of 6.2 PM, and 2 units of T7 DNA polymerase in buffer (25 mM TrisHCl, pH 7.5, 10 mM MgCls, 70 mM NaCl, and 7 mM dithiothreitol). The mixture was incubated for 15 min at 37”C, followed by the addition of 3 ~1of stop solution (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyan01 FF). Samples were boiled for 3 min before 3 ~1 was loaded onto a 6% polyacrylamide/B M urea gel. After electrophoresis at 58 W for 2-2.5 h, gels were dried and exposed to Kodak XAR5 film (Eastman Kodak Co., Rochester, NY) for 16 h. Sequencing reactions were repeated for areas in which the nucleotide composition was unclear in the first gel.
of Genomic DNA
Three regions of the FVIII gene containing (i) the 5’ end with the putative promoter region and exon 1, (ii) a thrombin cleavage site in exon 8, and (iii) a thrombin cleavage site in the 3’ end of exon 14 were amplified by the method of Saiki et al. (1985) using Tuq DNA polymerase (Perkin-Elmer-Cetus, Norwalk, CT) (Saiki et al., 1988) and 3 pairs of PCR primers (Table 1). Amplification of genomic DNA (200-500 ng) isolated from leukocytes was performed in a reaction mixture of 100 ~1 of buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgClz, and 0.02% gelatin), 400 nM each PCR primer, 200 PM each dNTP, and 2 units of Taq DNA polymerase. The mixture was heated for
RESULTS
Three regions of the FVIII gene of patients were chosen for direct sequence analysis: the 5’ portion of
TABLE Sequence Amplified
Promoter,
Exon
region of factor VIII gene 5’ UT, and exon
6
3’ end of exon
14
1
of Oligonucleotide PCR
1
AL.
Primers
Used
primer
Sequencing
primer
PCR PCR
3 4
5’-GCT 5’-CGA
CCT TCA
GTT GAC
CAC TTT CCT ACA
GAC GGA
TT-3’ CA-3’
SP3 SP4 SP5
5’-TTC 5’-AAT 5’-CAC
TGA CCA CTG
TTA GTG CTT
AAG CAG ACT-3’ GGT AAA GTT-3’ CTT TCT GTG-3’
PCR PCR
1 2
5’-CTC 5’-AGA
TGG GAG
TAT TAC
AGA CAA
ACA TAG
GCC TCA
TA-3’ AA-3’
SPl SP2
5’-ATA 5’-GAA
TAG ATG
CAA GAT
GAC GTG
ACT CTG-3 GTC AGG-3’
PCR PCR
5 6
5’-CCT 5’-AGC
GGG AGA
CAA GCA
AGC AAG
AAG GAA
GTA TAA
GG-3’ CC-3’
SP6 SP7
5’-ACT 5’-GAT
GAA ‘ITT
AGG GAC
CTG ATT
TGC TAT
TCT-3’ GAT-3’
FACTOR
VIII
the FVIII gene including the putative promoter region and regions surrounding two thrombin cleavage sites encoded in exon 8 and the 3’ end of exon 14, respectively (Fig. 1). After 136,017 nucleotides were sequenced, four hemophilia A-producing mutations and three rare normal variants were identified in the 127 patients (Table 2). 5’ End of FVIII
Gene Including
Promoter
Region
The 529-bp segment of the 5’ portion of the FVIII gene was amplified using primers PCR3 and PCR4. This region comprises the probable “TATA”-box element, the 5’mRNA start site, the 5’untranslated region, and the entire exon 1 which codes for the 19-aminoacid “leader peptide” and the N-terminal portion of the mature FVIII (Gitschier et aZ., 1984; Toole et al., 1984). Among 58,420 nucleotides sequenced in 127 un-
GENE
67
MUTATIONS
related patients with hemophilia A in this region using three sequencing primers (SP3, SP4, and SP5), only one nucleotide substitution, a C + T transition at the third position of codon 34 in patient JH-43, was found (data not shown). Because this nucleotide substitution does not change the amino acid at position 15 of the mature FVIII molecule and was found in only one of 127 patients tested, it must represent a rare normal variant. Exon 8 Region Amplification of the exon 8 region using primers PCRl and PCR2 yields a 365-bp segment comprising the entire exon 8. This exon codes for the middle portion of the 90-kDa heavy chain of FVIII with the thrombin cleavage site at amino acid residue 372 (codon 391) (Vehar et al., 1984; Eaton et al., 1986). The PCR
PCR4
PCR3
A.
5
PCRI
B.
PCR2
5 345
360
PCR5
PCR6 SP6
-bSP7
FIG. 1. Schematic representations of the regions of the factor VIII gene that denotes the primers used for amplification, and SP denotes the sequencing primers. of the polymorphisms found, and filled circles denote the hemophilia A mutations
+1
were amplified using the polymerase chain reaction. PCR Numbers denote codons. Open circles denote the positions found.
HIGUCHI
68
ET
TABLE Hemophilia
A: Search PCR
Exon Exon Exon Total Total
VIII
sequenced:
2
Mutations
by Direct
Nucleotides sequenced
product MS)
460 325 286 1071
529 365 326 1220
1 8 14
nucleotides
for Factor
AL.
DNA
Sequencing
PCR
Exon
IVS
5’ UT
143 262 253 658
28 63 33 124
289
289
127 X 1071 = 136,017
Nucleotide
substitutions
or deletions Severity of hemophilia”
Mutation
Patient JH 43
Codon
34
GAC
+
GAT
aa 15 Asp -* Asp
JH 30
Codon
345
GTA
+ CTA
aa 326 Val --* Leu
JH 31
Codon
360”
GAA
+ de1 GA
Frameshift
Severe
JH 40
Codon
1699’
TAT
+ TTT
aa 1680 Tyr -* Phe
Mild
JH 36
Codon
1705’
CAG
+
Nonsense aa 1686 Gln -* STOP
Severe
JH 39
Codon
1708’
CGC
+ TGC
aa 1689 Arg --* Cys
Moderate
JH 42
after
IVS 14 nt3
TAG
de rwvob
? no ?
Yes Mother
19
A+G
Note. Data from 127 unrelated patients. ’ Severity of hemophilia is not reported for JH-43, JH-30, and JH-42 because the disease-producing mutation b The mothers of patients JH-31 and JH-36 are carriers of the abnormal gene, but the maternal grandparents ’ Mutations causing hemophilia A.
product includes the acceptor splice site of intron 7 and the donor splice site of intron 8. Sequence analysis of PCR products using two sequencing primers, SPl and SP2 (41,275 nucleotides sequenced), showed two alterations, a frameshift at codon 360 due to the deletion of two nucleotides (GA) in patient JH-31 (Fig. 2) and a G --f C substitution at codon 345 in patient JH30 which results in Val-Leu substitution at amino acid position 326 (data not shown). Although the frameshift mutation is clearly responsible for the severe FVIII deficiency in patient JH-31, it is highly unlikely that the nucleotide substitution in patient JH-30 causessevere hemophilia because the mother of patient JH-30 and his sister also have Val-Leu substitution but their FVIII activity and antigen levels are within the normal range (mother, FVIII:C 90%, FVIII:Ag 150%; sister, FVIII:C 80%, FVIII:Ag 110%). In addition, the substitution of Leu for Val alters neither the overall charge nor the conformation of FVIII.
is unknown. were unavailable
for study.
3’ Portion of Exon 14 The 326-bp fragment from the 3’ end of exon 14 and the 5’ end of intron 14 was amplified using primers PCR5 and PCRG. This region encodes the NHz-terminal portion of the 80-kDa light chain of FVIII and contains two codons at positions 1667 and 1708 for arginine residues (amino acid residues 1648 and 1689) which are involved in proteolysis of FVIII (Eaton et al., 1986, 1987; Kaufman et al., 1987, Pittman and Kaufman, 1988). Sequence analysis of a total of 36,322 nucleotides in this region revealed four single-nucleotide substitutions. A nonsense mutation due to a C + T transition at codon 1705 was found in patient JH36 with severe hemophilia A (Fig. 2). A C --* T transition at codon 1708 resulting in an Arg-Cys substitution was identified in patient JH-39. The substitution of Cys for Arg affects the thrombin cleavage site at amino acid position 1689. The second missense mutation, an A + T change at codon 1699 resulting in a
FACTOR Mutant A
C
VIII
Normal G
f
JH-31
JH-36
C A FIG. 2. in patients
Partial nucleotide sequences JH-31, JH-36, and JH-40.
of three
selected
mutations
Tyr-Phe substitution, was detected in a patient with mild hemophilia A (JH-40) (Fig. 2). This mutation is located within a short segment of 41 predominantly acidic amino acids that contains the putative von Willebrand factor (vWF) binding site (Foster et al., 1988; Lollar et al, 1988). In addition, an A + G substitution in the third position of the splice donor site in intron 14 was found in JH-42 (data not shown). This nucleotide alteration occurs within the consensus donor splice site (ZAGJGTPuAGT) (Shapiro and Senapathy, 1987). Among a large number of donor splice sites analyzed, A is the third nucleotide in the intron in 60% and G is the third nucleotide in 35%. Therefore, this mutation probably represents a rare sequence variation with no deleterious effect in the expression of the FVIII gene. DISCUSSION We have initiated a search for mutations in selected regions of the FVIII gene that are thought to be functionally important using direct sequencing of PCRamplified genomic DNA. In our survey of 127 unrelated hemophilia A patients, four disease-producing mutations were detected. A dinucleotide deletion resulting in a frameshift at codon 360 identified in patient JH31 was found within a short repeat sequence, GAA,
GENE
MUTATIONS
69
suggesting strand slippage as a possible mechanism for the GA deletion (Efstratiadis et aZ., 1980). A nonsense codon at position 1686 was identified in a severely affected individual without inhibitor production, which would result in a truncated protein lacking the functionally important 73-kDa light chain. Two missense mutations leading to hemophilia A found in this study are of particular interest. The first mutation, a Cys for the Arg at amino acid residue 1689 (patient JH-39), occurred at the thrombin cleavage site within the 80-kDa light chain (Gitschier et al., 1985), suggesting that this mutant FVIII is resistant to thrombin. Analysis of the variant FVIII revealed that the apparently normal 80-kDa light chain was not cleaved by thrombin to the normal 73-kDa fragment (Arai et al., submitted for publication). In addition, a missense mutation, a G + A substitution (Arg + His) at codon 391, preventing thrombin cleavage of the 90kDa fragment has previously been reported (Arai et al., 1989). The second missense mutation, an A + T alteration at codon 1699, occurred in patient JH-40 with mild hemophilia A. This single-nucleotide substitution converts Tyr to Phe at amino acid residue 1680 in the region that is thought to contain a binding domain to the von Willebrand factor (Foster et al., 1988; Lollar et al., 1988). The biosynthetic pathway of FVIII in mammalian cells has recently been clarified (Kaufman et al., 1988). After complex post-translational modifications including the sulfation of Tyr residues in the acidic regions, the mature single-chain FVIII is processed in the Golgi apparatus to generate the aminoterminus of the light chain. In the presence of vWF, the cleaved heavy and light chains assemble to a metalion stabilized complex. The importance of the sulfated Tyr residues at amino acid positions 1664 and/or 1680 in the acidic region of the light chain was demonstrated by analysis of mutants of FVIII obtained by in vitro site-directed mutagenesis (Pittman and Kaufman, 1987). The lack of vWF binding, however, does not completely prevent the appearance of biologically active FVIII molecule in the culture medium, suggesting that vWF binding site mutations may not be responsible for severe hemophilia A. This is consistent with our finding that patient JH-40 is affected with a mild hemophilia and has 10% of normal FVIII activity and 20% of normal FVIII antigen. Interestingly, no deleterious mutations were found in the promoter region of the FVIII gene. Promoter mutations identified in patients with /3+-thalassemia (Kazazian and Boehm, 1988) and analysis of singlebase substitutions in the mouse major @globin gene promoter generated by in vitro mutagenesis (Myers et al., 1986) suggest that mutations in this region of the FVIII gene may lead to a subhemophilic condition
70
HIGUCHI
(FVIII activity level of 15 to 40% of normal), which can only be diagnosed after severe injuries or major surgical operations. In addition to the four mutations that cause hemophilia A, three nucleotide sequence variations, two in coding regions of the factor VIII gene and one in an intervening sequence, were found. It was reported from restriction endonuclease analyses using a large number of probes that the frequency of genetic variation at restriction sites on the X chromosome is three times less than that of autosomes (Cooper et al., 1985; Hofker et al., 1986). For example, Hofker et al. estimated that the probability of variability per base pair in the X chromosome is about l:llOO, while that of the autosomes is on the order of 1:300. Furthermore, studies of the /I-globin gene cluster on chromosome llp (Chakravarti et al., 1984a), the human growth hormone gene cluster on chromosome 17p (Chakravarti et al, 1984b), and the APOAl-APOCS-APOA4 gene cluster on chromosome llq (Antonarakis et al., 1988) revealed that about 1:300-1:400 nucleotides differ between two randomly chosen chromosomes. In this limited study of 127 patients the number of neutral substitutions found was extremely small. The calculated nucleotide variability for the area sequenced is 4.4 X 10e5 f 1.2 X lop5 (3.8 X lop5 f 1.8 X lop5 for the 5’ untranslated region and introns and 4.8 X 10e5 + 1.6 X lop5 for exons), which is one to two orders of magnitude less than that calculated in the above-mentioned studies for sequences on the X chromosome and the autosomes. Direct nucleotide sequencing of amplified DNA fragments, which is extremely labor intensive, may not be the method of choice to screen for mutations in a large gene with small, scattered exons, such as the FVIII gene. Alternative methods of screening for mutations such as denaturing gradient gel electrophoresis (Sheffield et aZ., 1989) and chemical mismatch cleavage (Cotton et aZ., 1988) using amplified DNA fragments may be utilized first. Direct nucleotide sequencing will then identify mutations in the selected regions that show abnormalities identified by the screening methods.
ET
AL.
2.
3.
4.
ARAI, M., INABA, H., HIGUCHI, M., ANTONARAKIS, S. E., KAZAZIAN, H. H., JR., FUJIMAKI, M., AND HOYER, L. W. (1989). Direct characterization of factor VIII in plasma: Detection of a mutation detecting a thrombin cleavage site (arginine-372 + histidine). Proc. Natl. Acad. Sci. USA 86: 4277-4281.
5.
CHAKRAVARTI, A., BUETOW, K. H., ANTONARAKIS, S. E., WABER, P. G., BOEHM, C. D., AND KAZAZL~N, H. H., JR. (1984a). Nonuniform recombination within the human /3-globin gene cluster. Amer. J. Hum. Genet. 36: 1239-1258. CHAKRAVARTI, A., PHILLIPS, J. A., MELLITS, K. M., BUETOW, K. H., AND SEEBURG, P. H. (1984b). Patterns of polymorphisms and linkage disequilibrium suggest independent origins of the human growth hormone gene cluster. Proc. Natl. Acad. Sci. USA 81: 6085-6089. COOPER, D. N., SMITH, B. A., COOKE, H. J., NEIMANN, S., AND SCHMIDTKE, J. (1985). An estimate of unique DNA sequence heterozygosity in the human genome. Hum. Genet. 69: 210205. COTTON, R. G. H., RODRIGUEZ, N. R., AND CAMPBELL, R. D. (1988). Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc. Natl. Acad. Sci. USA 85: 4397-4401. EATON, D. L., RODRIGUEZ, H. R., AND VEHAR, G. A. (1986). Proteolytic processing of human factor VIII: Correlation of specific cleavages of thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 26: 505-512. EATON, D. L., HASS, P. E., RIDDLE, L., MATHER, J., WIEBE, M., GREGORY, R., AND VEHAR, G. A. (1987). Characterization of recombinant human factor VIII. J. Biol. Chem. 262: 32853290.
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ACKNOWLEDGMENTS 13. We thank Drs. L. Hoyer for determinations of FVIII antigen and activity levels, and A. Chakravarti for valuable suggestions. We also thank P. Stevens for expert secretarial assistance and J. Strayer for the artwork. The study was supported by an NIH grant to H.H.K. and S.E.A.
14.
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FACTOR
VIII GENE MUTATIONS
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20. HIGUCHI, M., KOCHHAN, L., SCHWAAB, R., EGLI, H., BRACKMANN, H. H., HORST, J., AND OLEK, K. (1989). Molecular defects in hemophilia A: Identification and characterization of mutations in the factor VIII gene and family analysis. Blood 74: 1045-1051. 21. HOFKER, M. H., SKRAASTAD, M. I., BERGER, A. A., WAPENAAR, M. C., BAKKER, E., MILLINGTON-WARD, A., VAN OMMEN, G. J., AND PEARSON, R. L. (1986). The X chromosome shows less genetic variation at restriction sites than the autosomes. Amer. J. Hum. Genet. 39: 438-451. 22. INABA, H., FUJIMAKI, M., KAZAZIAN, H. H., JR., AND ANTONARAKIS, S. E. (1989). Mild hemophilia A resulting from Arg to Leu substitution in exon 26 of the factor VIII gene. Hum. Genet. 81:335-338. 23. KAUFMAN, R. J., PI’ITMAN, D. D., WASLEY, L. C., FOSTER, B. W., AMPHLETT, G. W., AND GILES, A. R. (1987). Directed mutagenesis in the study of the requirements for factor VIII activity in vitro and in vivo. Thromb. Haemostasf 68: 537 (abstract). 24. KAUFMAN, R. J., WASLEY, L. C., AND DORNER, A. J. (1988). Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J. Biol. Chem. 263: 6352-6362. 25. KAZAZIAN, H. H., JR., WONG, C., YOUSSOUFIAN, H., SCOT-I’, A. F., PHILLIPS, D. G., AND ANTONARAKIS, S. E. (1988). Haemophilia A resulting from de novo insertion of Ll sequences represents a novel mechanism for mutation in man. Nature (London) 332: 164-166. 26. KAZAZIAN, H. H., JR., AND BOEHM, C. D. (1988). Molecular basis and prenatal diagnosis of @-thalassemia. Blood 72: 11071116. 27. LOLLIR, P., HILL-EUBANKS, D. C., AND PARKER, C. G. (1988). Association of the factor VIII light chain with von Willebrand factor. J. Biol. Chem. 263: 10451-10455. 28. MANN, K. G. (1984). Membrane-bound enzyme complexes in blood coagulation. In “Progress in Hemostasis and Thrombosis” (T. H. Spaet, Ed.), Vol. 7, pp. l-23, Grune & Stratton, Orlando. 29. MYERS, R. M., TILLY, K., AND MANIATIS, T. (1986). Fine structure genetics analysis of a /3-globin promoter. Science 232: 613618. 30. PITTMAN, D. D., AND KAUFMAN, R. J. (1987). Internal deletions of factor VIII identify potentially important peptide sequences for binding to von Willebrand factor. Blood 70: 392 (abstract). 31. PI~MAN, D. D., AND KAUFMAN, R. J. (1988). Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII). Proc. Natl. Acod. Sci. USA 85: 2429-2433.
71
32. ROTBLAT, F., O’BRIEN, D. P., O’BRIEN, F. J., GOODALL, A. H., AND TUDDENHAM, E. G. D. (1985). Purification of human factor VIII32 and its characterization by Western blotting using monoclonal antibodies. Biochemistry 24: 4294-4300. 33. SADLER, J. E., AND DAVIE, E. W. (1987). Haemophilia A, haemophilia B, and von Willebrand’s disease. In “The Molecular Basis of Blood Disease” (Stamatoyannopoulos, Nienhuis, Leder, and Majerus, Eds.), pp. 575-630, Saunders, Philadelphia. 34. SAIKI, R. K., SCHARF, S., FALOONA, F., MULLIS, K. B., HORN, G. T., ERLICH, H. A., AND ARNHEIM, N. (1985). Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 120: 1350-1354. 35. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, S. J., HIGUCHI, R., HORN, G. T., MULLIS, K. B., AND ERLICH, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. 36. SHAPIRO, M. B., AND SENAPATHY, P. (1987). RNA splice functions of different classes of eukaryotes: Sequence statistics and functional implications in gene expression. Nucleic Acids Res. 15: 7155-7174. 37. SHEFFIELD, V. C., Cox, D. R., LERMAN, L. S., AND MYERS, R. M. (1989). Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc. Natl. Acad. Sci. USA 86; 232-236. 38. TOOLE, J. J., KNOPF, J. L., WOZNEY, J. M., SULTZMAN, L. A., BEUCKER, J. L., PI~MAN, D. D., KAUFMAN, R. J., BROWN, E., SHOEMAKER, C., ORR, E. C., AMPHLETT, G. W., FOSTER, B., COE, M. L., KNUTSON, G. J., FASS, D. N., AND\HEWICK, R. M. (1984). Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature (London) 312: 342-347. 39. VEHAR, G. A., KEYT, B., EATON, D., RODRIGUEZ,H., O’BRIEN, D. P., ROTBLAT, F., OPPERMAN, H., KECK, R., WOOD, W. I., KARKINS, R. N., TUDDENHAM, E. G. D., LAWN, R. M., AND CAPON, D. J. (1984). Structure of human factor VIII. Nature (London) 312:337-342. 40. WONG, C., DOWLING, C. E., SAIKI, R. K., HIGUCHI, R. G., ERLICH, H. A., AND KA~AZIAN, H. H., JR. (1987). Characterization of /3-thalassemia mutations using direct genomic sequencing of amplified single copy DNA. Nature (London) 330: 384-386. 41. YOUSSOWIAN, H., KAZAZIAN, H. H., JR., PHILLIPS, D. G., ARONIS, S., TSIFTIS, G., BROWN, V. A., AND ANTONARAKIS, S. E. (1996). Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots. Nature (London) 324: 380382. 42. YOUSSOUFIAN, H., ANTONARAKIS, S. E., ARONIS, S., TSIFTIS, G., PHILLIPS, D. G., AND KAZAZIAN, H. H., JR. (1987). Characterization of five partial deletions of the factor VIII gene. Proc. Natl.
Acad.
Sci. USA
84: 3772-3776.
43. YOUSSOUFIAN, H., ANTONARAKIS, S. E., BELL, W., GRIFFIN, A. M., AND KAZAZIAN, H. H., JR. (1988a). Nonsense and missense mutations in hemophilia A: Estimate of the relative mutation rate at CG dinucleotides. Amer. J. Hum. Genet. 42: 718725. 44. YOUSSOUFIAN, H., WONG, C., ARONIS, S., PLATOKOUKIS, H., KAZAZIAN, H. H., JR., AND ANTONARAKIS, S. E. (1988b). Moderately severe hemophilia A resulting from Glu-Gly substitution in exon 7 of the factor VIII gene. Amer. J. Hum. Genet. 42: 867-871.
6,65-71
(1990)
Characterization of Mutations in the Factor VIII Gene by Direct Sequencing of Amplified Genomic DNA MIYOKO HIGUCHI,* CORINNE WONG,* LOTHAR KocHHAN,t KLAUS OLEK,j’ SOPHIA ARONIS,* CAROL K. KA~PER,§ HAIG H. KAZAZIAN, JR.,* AND S~VLIANOS E. ANTONARAKIS*,’ *Genetics Unit, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 2 1205; tlnstitute for Clinical Biochemistry, University of Bonn, Bonn, Germany; Koagulation Laboratory, “Agia Sophia” Children’s Hospital, Athens, Greece; and §Orthopedic Hospital, University of Southern California, Los Angeles, California 90007 Received
June 1, 1989;
revised
INTRODUCTION
Hemophilia A is a common disorder of blood coagulation caused by deficiency or abnormality of factor VIII (FVIII) and is inherited as an X-linked recessive trait. Plakma FVIII circulates as a metal-ion stabilized complex (Rotblat et al., 1985; Eaton et al., 1986; Fay, 1988) and participates as a cofactor for activation of factor X by factor IXa in the intrinsic pathway of the blood coagulation cascade (Mann, 1984; Sadler and Davie, 1987). Recent studies on plasma- and recombinant DNA-derived FVIII have demonstrated that the procoagulant activity of FVIII is regulated by several correspondence
should
7, 1989
proteolytic modifications including cleavages by thrombin at amino acid positions 372, 740, and 1689 which lead to maximal coagulant activity of FVIII (Eaton et al., 1986, 1987). The functional importance of proteolytic processing has been demonstrated following site-directed mutagenesis of codons for arginines (Arg) at residues 372 and 1689 (Pittman and Kaufman, 1988). The gene for human FVIII is 186 kb in length and is composed of 26 exons which encode an approximately 9-kb mRNA with a probable transcription start site at nucleotide -170 (initiation codon at $1) and an 1805nucleotide-long 3’ untranslated region (Toole et aZ., 1984; Gitschier et al., 1984). The sequence GATAAA, a presumptive “TATA”-box element located 30 bp upstream from nucleotide -170, is thought to be important for proper and efficient transcription of the gene. A number of mutations in the FVIII gene resulting in hemophilia A have been reported. These mutations [deletions (Gitschier et al., 1985; Antonarakis et al, 1985; Youssoufian et al., 1987; Higuchi et al., 1988), insertions (Kazazian et al., 1988), and point mutations (Gitschier et al., 1985, 1986; Antonarakis et at., 1985; Youssoufian et al., 1986, 1988a,b; Inaba et al., 1989)] were identified mainly by Southern analysis using several restriction enzymes including TcrqI and several FVIII cDNA fragments as molecular probes. In roughly 10% of patients with hemophilia A the molecular defect can be detected by this procedure (for review see Antonarakis and Kazazian, 1988); however, the majority of deleterious mutations remain undetected. Recently, discriminant oligonucleotide hybridization was used to detect two point mutations, one of which was located in a thrombin cleavage site at amino acid 1689 (Gitschier et al., 1988). In this report we describe the identification of mutations in selected regions of the FVIII gene using amplification of genomic DNA by polymerase chain re-
In order to search for mutations resulting in hemophilia A that are not detectable by restriction analysis, three regions of the factor VIII gene were chosen for direct sequence analysis. Short segments of genomic DNA of 127 unrelated patients with hemophilia A were amplified by polymerase chain reaction. A total of 136,017 nucleotides were sequenced, and four mutations leading to the disease were found: a frameshift at codon 360 due to deletion of two nucleotides (GA), a nonsense codon 1705 due to a C + T transition, and two missense codons at positions 1699 and 1708. The first missense mutation (A --* T) results in a Tyr + Phe substitution at a putative von Willebrand factor binding site. The second results in an Arg + Cys substitution at a thrombin cleavage site. In addition, we identified three rare sequence variants: a silent C + T transition at codon 34 which does not result in an amino acid change, a G + C change at codon 345 (Val + Leu), and an A + G change at the third nucleotide of intron 14. Direct sequence analysis of amplified DNA is a powerful but labor-intensive method of identifying mutations in large genes such as the human factor VIII gene. o ISBO Academic PRW. I~C.
1 To whom
August
be addressed.
65 All
Copyright 0 1990 rights of reproduction
o&33-7543/90 $3.00 by Academic Press, Inc. in any form reserved.
66
HIGUCHI
action (PCR) and direct sequencing regions of the FVIII gene. MATERIALS
AND
ET
6 min at 94’C, followed by 30 cycles of amplification using a step program (denaturation at 94’C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min) on a DNA thermal cycler (Perkin-Elmer-Cetus). Amplified DNA was purified by spin dialysis using Centricon 30 (Amicon, Danvers, MA) and subjected to direct sequencing.
of the amplified
METHODS
Subjects Our subjects were 127 unrelated hemophilia A patients who showed no detectable gross gene alterations or point mutations when their genomic DNA was screened by Southern analysis using several restriction enzymes including TaqI and 3 cDNA subfragments of the FVIII gene as hybridization probes (Toole et al., 1984; Antonarakis et aZ., 1985). These patients included IO7 patients from the series of Youssoufian et al. (for review see Antonarakis and Kazazian, 1988) and 20 German patients from Higuchi et al. (1989). According to clinical features and hematologic data, 86 of these patients were classified as having severe, 14 as having moderate, and 27 as mild hemophilia A. Fifteen patients with severe hemophilia A had developed antibodies against FVIII after multitransfusion therapy (inhibitor-positive patients). None of the patients with moderate or mild hemophilia A was inhibitor-positive. Amplification
Sequencing of Amplified Products Purified PCR products were directly sequencedwith T7 DNA polymerase (Sequenase, United State Biochemicals, Cleveland, OH) as previously described (Wong et al., 1987; Higuchi et al., 1988). Sequencing primers were end-labeled with [y-32P]ATP (3000 Ci/ mmol; New England Nuclear) using T4 DNA polynucleotide kinase (BRL, Gaithersburg, MD). Approximately 80 ng of PCR product was annealed with 10 ng of appropriate sequencing primer (Table 1) in a volume of 11 ~1 on ice for 10 min. After heat denaturing for 5 min at 95°C 2.5 ~1 of the annealed sample was added to four tubes with 3 ~1of the sequencing mixture containing nonradioactive dNTPs at a concentration of 62 PM, ddNTPs at a concentration of 6.2 PM, and 2 units of T7 DNA polymerase in buffer (25 mM TrisHCl, pH 7.5, 10 mM MgCls, 70 mM NaCl, and 7 mM dithiothreitol). The mixture was incubated for 15 min at 37”C, followed by the addition of 3 ~1of stop solution (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyan01 FF). Samples were boiled for 3 min before 3 ~1 was loaded onto a 6% polyacrylamide/B M urea gel. After electrophoresis at 58 W for 2-2.5 h, gels were dried and exposed to Kodak XAR5 film (Eastman Kodak Co., Rochester, NY) for 16 h. Sequencing reactions were repeated for areas in which the nucleotide composition was unclear in the first gel.
of Genomic DNA
Three regions of the FVIII gene containing (i) the 5’ end with the putative promoter region and exon 1, (ii) a thrombin cleavage site in exon 8, and (iii) a thrombin cleavage site in the 3’ end of exon 14 were amplified by the method of Saiki et al. (1985) using Tuq DNA polymerase (Perkin-Elmer-Cetus, Norwalk, CT) (Saiki et al., 1988) and 3 pairs of PCR primers (Table 1). Amplification of genomic DNA (200-500 ng) isolated from leukocytes was performed in a reaction mixture of 100 ~1 of buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgClz, and 0.02% gelatin), 400 nM each PCR primer, 200 PM each dNTP, and 2 units of Taq DNA polymerase. The mixture was heated for
RESULTS
Three regions of the FVIII gene of patients were chosen for direct sequence analysis: the 5’ portion of
TABLE Sequence Amplified
Promoter,
Exon
region of factor VIII gene 5’ UT, and exon
6
3’ end of exon
14
1
of Oligonucleotide PCR
1
AL.
Primers
Used
primer
Sequencing
primer
PCR PCR
3 4
5’-GCT 5’-CGA
CCT TCA
GTT GAC
CAC TTT CCT ACA
GAC GGA
TT-3’ CA-3’
SP3 SP4 SP5
5’-TTC 5’-AAT 5’-CAC
TGA CCA CTG
TTA GTG CTT
AAG CAG ACT-3’ GGT AAA GTT-3’ CTT TCT GTG-3’
PCR PCR
1 2
5’-CTC 5’-AGA
TGG GAG
TAT TAC
AGA CAA
ACA TAG
GCC TCA
TA-3’ AA-3’
SPl SP2
5’-ATA 5’-GAA
TAG ATG
CAA GAT
GAC GTG
ACT CTG-3 GTC AGG-3’
PCR PCR
5 6
5’-CCT 5’-AGC
GGG AGA
CAA GCA
AGC AAG
AAG GAA
GTA TAA
GG-3’ CC-3’
SP6 SP7
5’-ACT 5’-GAT
GAA ‘ITT
AGG GAC
CTG ATT
TGC TAT
TCT-3’ GAT-3’
FACTOR
VIII
the FVIII gene including the putative promoter region and regions surrounding two thrombin cleavage sites encoded in exon 8 and the 3’ end of exon 14, respectively (Fig. 1). After 136,017 nucleotides were sequenced, four hemophilia A-producing mutations and three rare normal variants were identified in the 127 patients (Table 2). 5’ End of FVIII
Gene Including
Promoter
Region
The 529-bp segment of the 5’ portion of the FVIII gene was amplified using primers PCR3 and PCR4. This region comprises the probable “TATA”-box element, the 5’mRNA start site, the 5’untranslated region, and the entire exon 1 which codes for the 19-aminoacid “leader peptide” and the N-terminal portion of the mature FVIII (Gitschier et aZ., 1984; Toole et al., 1984). Among 58,420 nucleotides sequenced in 127 un-
GENE
67
MUTATIONS
related patients with hemophilia A in this region using three sequencing primers (SP3, SP4, and SP5), only one nucleotide substitution, a C + T transition at the third position of codon 34 in patient JH-43, was found (data not shown). Because this nucleotide substitution does not change the amino acid at position 15 of the mature FVIII molecule and was found in only one of 127 patients tested, it must represent a rare normal variant. Exon 8 Region Amplification of the exon 8 region using primers PCRl and PCR2 yields a 365-bp segment comprising the entire exon 8. This exon codes for the middle portion of the 90-kDa heavy chain of FVIII with the thrombin cleavage site at amino acid residue 372 (codon 391) (Vehar et al., 1984; Eaton et al., 1986). The PCR
PCR4
PCR3
A.
5
PCRI
B.
PCR2
5 345
360
PCR5
PCR6 SP6
-bSP7
FIG. 1. Schematic representations of the regions of the factor VIII gene that denotes the primers used for amplification, and SP denotes the sequencing primers. of the polymorphisms found, and filled circles denote the hemophilia A mutations
+1
were amplified using the polymerase chain reaction. PCR Numbers denote codons. Open circles denote the positions found.
HIGUCHI
68
ET
TABLE Hemophilia
A: Search PCR
Exon Exon Exon Total Total
VIII
sequenced:
2
Mutations
by Direct
Nucleotides sequenced
product MS)
460 325 286 1071
529 365 326 1220
1 8 14
nucleotides
for Factor
AL.
DNA
Sequencing
PCR
Exon
IVS
5’ UT
143 262 253 658
28 63 33 124
289
289
127 X 1071 = 136,017
Nucleotide
substitutions
or deletions Severity of hemophilia”
Mutation
Patient JH 43
Codon
34
GAC
+
GAT
aa 15 Asp -* Asp
JH 30
Codon
345
GTA
+ CTA
aa 326 Val --* Leu
JH 31
Codon
360”
GAA
+ de1 GA
Frameshift
Severe
JH 40
Codon
1699’
TAT
+ TTT
aa 1680 Tyr -* Phe
Mild
JH 36
Codon
1705’
CAG
+
Nonsense aa 1686 Gln -* STOP
Severe
JH 39
Codon
1708’
CGC
+ TGC
aa 1689 Arg --* Cys
Moderate
JH 42
after
IVS 14 nt3
TAG
de rwvob
? no ?
Yes Mother
19
A+G
Note. Data from 127 unrelated patients. ’ Severity of hemophilia is not reported for JH-43, JH-30, and JH-42 because the disease-producing mutation b The mothers of patients JH-31 and JH-36 are carriers of the abnormal gene, but the maternal grandparents ’ Mutations causing hemophilia A.
product includes the acceptor splice site of intron 7 and the donor splice site of intron 8. Sequence analysis of PCR products using two sequencing primers, SPl and SP2 (41,275 nucleotides sequenced), showed two alterations, a frameshift at codon 360 due to the deletion of two nucleotides (GA) in patient JH-31 (Fig. 2) and a G --f C substitution at codon 345 in patient JH30 which results in Val-Leu substitution at amino acid position 326 (data not shown). Although the frameshift mutation is clearly responsible for the severe FVIII deficiency in patient JH-31, it is highly unlikely that the nucleotide substitution in patient JH-30 causessevere hemophilia because the mother of patient JH-30 and his sister also have Val-Leu substitution but their FVIII activity and antigen levels are within the normal range (mother, FVIII:C 90%, FVIII:Ag 150%; sister, FVIII:C 80%, FVIII:Ag 110%). In addition, the substitution of Leu for Val alters neither the overall charge nor the conformation of FVIII.
is unknown. were unavailable
for study.
3’ Portion of Exon 14 The 326-bp fragment from the 3’ end of exon 14 and the 5’ end of intron 14 was amplified using primers PCR5 and PCRG. This region encodes the NHz-terminal portion of the 80-kDa light chain of FVIII and contains two codons at positions 1667 and 1708 for arginine residues (amino acid residues 1648 and 1689) which are involved in proteolysis of FVIII (Eaton et al., 1986, 1987; Kaufman et al., 1987, Pittman and Kaufman, 1988). Sequence analysis of a total of 36,322 nucleotides in this region revealed four single-nucleotide substitutions. A nonsense mutation due to a C + T transition at codon 1705 was found in patient JH36 with severe hemophilia A (Fig. 2). A C --* T transition at codon 1708 resulting in an Arg-Cys substitution was identified in patient JH-39. The substitution of Cys for Arg affects the thrombin cleavage site at amino acid position 1689. The second missense mutation, an A + T change at codon 1699 resulting in a
FACTOR Mutant A
C
VIII
Normal G
f
JH-31
JH-36
C A FIG. 2. in patients
Partial nucleotide sequences JH-31, JH-36, and JH-40.
of three
selected
mutations
Tyr-Phe substitution, was detected in a patient with mild hemophilia A (JH-40) (Fig. 2). This mutation is located within a short segment of 41 predominantly acidic amino acids that contains the putative von Willebrand factor (vWF) binding site (Foster et al., 1988; Lollar et al, 1988). In addition, an A + G substitution in the third position of the splice donor site in intron 14 was found in JH-42 (data not shown). This nucleotide alteration occurs within the consensus donor splice site (ZAGJGTPuAGT) (Shapiro and Senapathy, 1987). Among a large number of donor splice sites analyzed, A is the third nucleotide in the intron in 60% and G is the third nucleotide in 35%. Therefore, this mutation probably represents a rare sequence variation with no deleterious effect in the expression of the FVIII gene. DISCUSSION We have initiated a search for mutations in selected regions of the FVIII gene that are thought to be functionally important using direct sequencing of PCRamplified genomic DNA. In our survey of 127 unrelated hemophilia A patients, four disease-producing mutations were detected. A dinucleotide deletion resulting in a frameshift at codon 360 identified in patient JH31 was found within a short repeat sequence, GAA,
GENE
MUTATIONS
69
suggesting strand slippage as a possible mechanism for the GA deletion (Efstratiadis et aZ., 1980). A nonsense codon at position 1686 was identified in a severely affected individual without inhibitor production, which would result in a truncated protein lacking the functionally important 73-kDa light chain. Two missense mutations leading to hemophilia A found in this study are of particular interest. The first mutation, a Cys for the Arg at amino acid residue 1689 (patient JH-39), occurred at the thrombin cleavage site within the 80-kDa light chain (Gitschier et al., 1985), suggesting that this mutant FVIII is resistant to thrombin. Analysis of the variant FVIII revealed that the apparently normal 80-kDa light chain was not cleaved by thrombin to the normal 73-kDa fragment (Arai et al., submitted for publication). In addition, a missense mutation, a G + A substitution (Arg + His) at codon 391, preventing thrombin cleavage of the 90kDa fragment has previously been reported (Arai et al., 1989). The second missense mutation, an A + T alteration at codon 1699, occurred in patient JH-40 with mild hemophilia A. This single-nucleotide substitution converts Tyr to Phe at amino acid residue 1680 in the region that is thought to contain a binding domain to the von Willebrand factor (Foster et al., 1988; Lollar et al., 1988). The biosynthetic pathway of FVIII in mammalian cells has recently been clarified (Kaufman et al., 1988). After complex post-translational modifications including the sulfation of Tyr residues in the acidic regions, the mature single-chain FVIII is processed in the Golgi apparatus to generate the aminoterminus of the light chain. In the presence of vWF, the cleaved heavy and light chains assemble to a metalion stabilized complex. The importance of the sulfated Tyr residues at amino acid positions 1664 and/or 1680 in the acidic region of the light chain was demonstrated by analysis of mutants of FVIII obtained by in vitro site-directed mutagenesis (Pittman and Kaufman, 1987). The lack of vWF binding, however, does not completely prevent the appearance of biologically active FVIII molecule in the culture medium, suggesting that vWF binding site mutations may not be responsible for severe hemophilia A. This is consistent with our finding that patient JH-40 is affected with a mild hemophilia and has 10% of normal FVIII activity and 20% of normal FVIII antigen. Interestingly, no deleterious mutations were found in the promoter region of the FVIII gene. Promoter mutations identified in patients with /3+-thalassemia (Kazazian and Boehm, 1988) and analysis of singlebase substitutions in the mouse major @globin gene promoter generated by in vitro mutagenesis (Myers et al., 1986) suggest that mutations in this region of the FVIII gene may lead to a subhemophilic condition
70
HIGUCHI
(FVIII activity level of 15 to 40% of normal), which can only be diagnosed after severe injuries or major surgical operations. In addition to the four mutations that cause hemophilia A, three nucleotide sequence variations, two in coding regions of the factor VIII gene and one in an intervening sequence, were found. It was reported from restriction endonuclease analyses using a large number of probes that the frequency of genetic variation at restriction sites on the X chromosome is three times less than that of autosomes (Cooper et al., 1985; Hofker et al., 1986). For example, Hofker et al. estimated that the probability of variability per base pair in the X chromosome is about l:llOO, while that of the autosomes is on the order of 1:300. Furthermore, studies of the /I-globin gene cluster on chromosome llp (Chakravarti et al., 1984a), the human growth hormone gene cluster on chromosome 17p (Chakravarti et al, 1984b), and the APOAl-APOCS-APOA4 gene cluster on chromosome llq (Antonarakis et al., 1988) revealed that about 1:300-1:400 nucleotides differ between two randomly chosen chromosomes. In this limited study of 127 patients the number of neutral substitutions found was extremely small. The calculated nucleotide variability for the area sequenced is 4.4 X 10e5 f 1.2 X lop5 (3.8 X lop5 f 1.8 X lop5 for the 5’ untranslated region and introns and 4.8 X 10e5 + 1.6 X lop5 for exons), which is one to two orders of magnitude less than that calculated in the above-mentioned studies for sequences on the X chromosome and the autosomes. Direct nucleotide sequencing of amplified DNA fragments, which is extremely labor intensive, may not be the method of choice to screen for mutations in a large gene with small, scattered exons, such as the FVIII gene. Alternative methods of screening for mutations such as denaturing gradient gel electrophoresis (Sheffield et aZ., 1989) and chemical mismatch cleavage (Cotton et aZ., 1988) using amplified DNA fragments may be utilized first. Direct nucleotide sequencing will then identify mutations in the selected regions that show abnormalities identified by the screening methods.
ET
AL.
2.
3.
4.
ARAI, M., INABA, H., HIGUCHI, M., ANTONARAKIS, S. E., KAZAZIAN, H. H., JR., FUJIMAKI, M., AND HOYER, L. W. (1989). Direct characterization of factor VIII in plasma: Detection of a mutation detecting a thrombin cleavage site (arginine-372 + histidine). Proc. Natl. Acad. Sci. USA 86: 4277-4281.
5.
CHAKRAVARTI, A., BUETOW, K. H., ANTONARAKIS, S. E., WABER, P. G., BOEHM, C. D., AND KAZAZL~N, H. H., JR. (1984a). Nonuniform recombination within the human /3-globin gene cluster. Amer. J. Hum. Genet. 36: 1239-1258. CHAKRAVARTI, A., PHILLIPS, J. A., MELLITS, K. M., BUETOW, K. H., AND SEEBURG, P. H. (1984b). Patterns of polymorphisms and linkage disequilibrium suggest independent origins of the human growth hormone gene cluster. Proc. Natl. Acad. Sci. USA 81: 6085-6089. COOPER, D. N., SMITH, B. A., COOKE, H. J., NEIMANN, S., AND SCHMIDTKE, J. (1985). An estimate of unique DNA sequence heterozygosity in the human genome. Hum. Genet. 69: 210205. COTTON, R. G. H., RODRIGUEZ, N. R., AND CAMPBELL, R. D. (1988). Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc. Natl. Acad. Sci. USA 85: 4397-4401. EATON, D. L., RODRIGUEZ, H. R., AND VEHAR, G. A. (1986). Proteolytic processing of human factor VIII: Correlation of specific cleavages of thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 26: 505-512. EATON, D. L., HASS, P. E., RIDDLE, L., MATHER, J., WIEBE, M., GREGORY, R., AND VEHAR, G. A. (1987). Characterization of recombinant human factor VIII. J. Biol. Chem. 262: 32853290.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENTS 13. We thank Drs. L. Hoyer for determinations of FVIII antigen and activity levels, and A. Chakravarti for valuable suggestions. We also thank P. Stevens for expert secretarial assistance and J. Strayer for the artwork. The study was supported by an NIH grant to H.H.K. and S.E.A.
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