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Keywords:

  • haemophilia B;
  • factor IX;
  • mutation;
  • inhibitor;
  • mutation frequency

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The present series comprises all families (n = 77) with haemophilia B in Sweden and may be considered to be representative for the purposes of a population-based study of mutational heterogeneity. The 77 families (38 severe, 10 moderate, 29 mild) had 51 different mutations in total. Thirteen families had total, partial or small deletions, two had mutations in the promoter, eight families had splice site mutations, 14 had nonsense and the remaining 41 had missense mutations. Ten of the mutations, all C[RIGHTWARDS ARROW]T or G[RIGHTWARDS ARROW]A, recurred in 1–6 other families. Using haplotype analysis of seven polymorphisms in the factor IX (FIX) gene, we found that the 77 families carried 65 unique, independent mutations. Of the 48 families with severe or moderate haemophilia, 23 (48%) had a sporadic case of haemophilia compared with 31 families out of 78 (40%) in the whole series. Five of those 23 sporadic cases carried de novo mutations, 11 out of 23 of the mothers were proven carriers and, in the remaining seven families, it was not possible to determine carriership. Eleven of the 48 patients (23%) with severe haemophilia B developed inhibitors and all of them had deletions or nonsense mutations. Thus, 11 out of 37 (30%) patients with severe haemophilia B as a result of deletion/nonsense mutations developed inhibitors compared with 0 out of 11 patients with missense mutations. The ratio of male to female mutation rates was 5·3 and the overall mutation rate was 5·4 × 10−6 per gamete per generation.

Haemophilia B is an X-linked hereditary disorder affecting half the sons of carrier females. The disease is caused by deficient or defective coagulation factor IX (FIX) and, depending on the plasma concentration of FIX clotting activity (FIXC), haemophilia B is classified as severe (FIXC < 1 U/dl), moderate (FIXC 1–4 U/dl) or mild (FIXC 5–25 U/dl). The FIX gene is located in the distal part of the long arm of the X-chromosome (Xq27.1). Yoshitake et al (1985) elucidated the entire nucleotide sequence, which is 33·5 kb long, has eight exons (a–h) and manifests strong homology with other vitamin K-dependent coagulation factors. The Factor IX protein is a serine protease synthesized in the liver via a vitamin K-dependent process and occurs in plasma as a single glycoprotein of 415 amino acids with a molecular weight of 57 000 (Di Scipio et al, 1977).

Haemophilia B is caused by a wide variation of mutations distributed over the entire FIX gene. Since 1990, an annually updated database has been published of characterized point mutations, small deletions and insertions (Giannelli et al, 1998). However, patients with complete or partial deletions or more complex rearrangements are excluded (Thompson, 1991). In the latest update of the database, a total of 1713 mutations are listed, of which 652 are unique molecular events, while the remainder are repeats (Giannelli et al, 1998). It is not clear how many of the repeats are unrelated events and how many may be the result of a founder effect. Most patients with severe or moderate haemophilia and identical mutations may have unique mutations, whereas many patients with mild haemophilia may have mutations with a common derivation (Ketterling et al, 1991). CpG dinucleotides have been shown to be hot-spots for mutations and account for many of the identical mutations found in unrelated families (Cooper & Krawczak, 1990; Green et al, 1990).

The Swedish haemophilia B families have been carefully registered at the haemophilia centres for many decades, during which time pedigrees have been drawn up and maintained. The present series comprises all families with haemophilia B in Sweden and may be considered to be representative for the purposes of a population-based study of mutational heterogeneity. The aim of this study was to use this complete national database of mutations to study the type of mutations, to elucidate whether families with identical mutations share a common ancestor, to define the frequency of mutation and sporadic cases and, finally, to study the correlation between type of mutation and inhibitor development.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Subjects The series comprises all known families with haemophilia B in Sweden (n = 77) with a total of 148 affected members. A few immigrant families were excluded. Thirty-eight families (49·3%) comprising 48 haemophiliacs (32·6%) had severe haemophilia B (FIXC < 1 U/dl), 10 families (13·0%) comprising 15 haemophiliacs (10·2%) had moderate haemophilia B (1–4 U/dl) and 29 families (37·7%) comprising 85 haemophiliacs (57·8%) had mild haemophilia B (5–25 U/dl). Sweden has centralized the care of haemophilia and all known patients are registered in a national registry. Thirty-one (40%) of the families were sporadic, with only one known case of haemophilia, the remaining 46 had several affected members and/or known haemophilia in previous generations.

Blood collection Citrated plasma was stored at −70°C until examined. DNA and RNA was extracted immediately from EDTA blood or the blood was stored at −40°C until required.

Factor IX coagulant activity (FIXC) FIXC was determined in a one-stage recalcification system with FIX-deficient plasma as the test base (Nilsson, 1974) (reference range 60–140 U/dl).

Factor IX coagulant antigen (FIXAg) FIXAg was determined using an immunoradiometric assay with monoclonal antibodies. The lowest concentration that could be measured was 0·025 U/dl. When no FIXAg could be detected, the result was expressed as < 0·1 U/dl (Wallmark et al, 1985).

Mutation analysis Characterization of mutations was performed by direct sequencing of genomic DNA using a modified dideoxy method. Briefly, the exon or part of the exon was amplified by polymerase chain reaction (PCR) using 50–100 ng of genomic DNA. The 50 μl samples contained 67 mmol/l Tris-HCl, pH 8·8, 16·6 mmol/l ammonium sulphate, 6·7 mmol/l magnesium chloride, 10 mmol/l β-mercaptoethanol, 1·7 mg/ml bovine serum albumin (BSA), 5 mmol/l dNTPs, 300 ng of each primer and 2·5 units of Amplitaq (Perkin-Elmer, USA). The conditions used in the amplification were: 5 min denaturation at 94°C; 60 s annealing at 58°C; 90 s polymerization at 72°C for 30 cycles and a final extension at 72°C for 5 min. Amplification was monitored by electrophoresis on a 4% Nusieve agarose gel and 1–2% agarose gel. The PCR product was purified using QIAquick PCR purification Kit (QIAGEN), according to the manufacturers' instructions. The exons were sequenced on an automated DNA sequencer (ABI-prism, model 373, Perkin-Elmer) or, during the initial phase of the study, using radiolabelled probes on a 6% polyacrylamide gel.

Intragenic polymorphisms To trace the origin of mutations and study haplotype differences, several polymorphisms were determined using various restriction enzymes:

HhaI, detecting a polymorphism located 8 kb 3′ to the factor IX gene with alleles 230/150 + 80 bp (Winship et al, 1989);

TaqI, detecting a polymorphism in intron 4 with alleles 1273/810 + 463 bp (Giannelli et al, 1984);

MnlI, detecting a polymorphism as a result of a single nucleotide variation in position 20422 in exon f (that causes a neutral amino acid polymorphism 148 thr/ala) with alleles 333/214 + 119 bp (Graham et al, 1989);

DdeI, in intron 1 (insertion 50 bp in position 5505–5554), with alleles 1·7/1·75 kb or 1419/1369 bp (Winship et al, 1984);

XmnI, detecting a polymorphism in intron 3, with alleles 11·5/6·5 kb (Winship et al, 1984);

MspI, in exon e with alleles 2·4/5·8 kb (Camerino et al, 1985);

BamHI, a polymorphism 5′of the transcription start with alleles 289/187 + 102 bp (Hay et al, 1986).

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Table I shows the mutations found in families with severe haemophilia B (n = 38). Twelve out of 38 families had deletions [four total, two partial and six small deletions (< 10 bp), 14 nonsense mutations creating stop codons, nine missense mutations, two splicing defects and two mutations in the promoter]. The index case in family Malmö 7, with sporadic haemophilia, was found to have two mutations, one neutral and one nonsense (Montandon et al, 1990). Four mutations have not previously been reported from other centres to the International Haemophilia B database (Giannelli et al, 1998) (C 6693 T; Gln stop and T 31306 G; Tyr 395 stop and G 10391 C; acceptor splice site and G 17668 C; acceptor splice site). Twenty of the 27 point mutations affected CpG dinucleotides (C[RIGHTWARDS ARROW]T or G[RIGHTWARDS ARROW]A transitions). Two mutations, G −6 C and A 13 G, were in the promoter and caused the Leyden phenotype (Veltkamp et al, 1970). Two families with missense mutations had measurable but low FIXAg levels.

Table I.  Mutations in families with severe haemophilia B (n = 38).
Mö-IDNucleotide positionAmino acid changeFIXCFIXAg
  1. *Patient number Mö 7 had two mutations, this one being neutral (Montandon et al, 1990).

2total del00
34total del00
60total del00
68total del00
53exon a-f del00
82exon a–d, f–h del00
86398 del 2 bp (Fs)Glu 8 stop 1400
96466 del 1 bp (Fs)Val 31, stop 5700
106665 del 10 bpsplicing defect00·2
4420398 del 1 bp (Fs)Thr 140 stop 15600
1320510 del 1 bp (Fs)Asp 177 stop 19800
130950 del 8 bp (Fs)Asp 276, stop 28800
63G−6 Cpromoter00
79A 13 Gpromoter00
6C 6364 TArg −4 Trp026
4C 6460 TArg 29 stop00
69C 6460 TArg 29 stop00
52C 6693 TGln 44 stop00
83G 10391 Cacceptor splice00
11G 17668 Csplicing defect00
51C 17700 GCys 95 Trp00
7C 17761 TArg 116 stop00
71C 17761 TArg 116 stop00
12G 20375 TCys 132 Phe00
5G 20561 ATrp 194 stop00
61G 20561 ATrp 194 stop00
3C 30863 TArg 248 stop00
14C 30863 TArg 248 stop00
15C 30863 TArg 248 stop00
41C 30875 TArg 252 stop06
65C 30875 TArg 252 stop00
7C 30890 T*His 257 Tyr*00
50A 30927 TAsp 269 Val00
18G 31035 AGly 305 Asp00
58C 31118 TArg 333 stop00
24G 31128 ACys 336 Tyr00
47G 31170 ACys 350 Tyr00
49G 31276 ATrp 385 stop00
73T 31306 GTyr 395 Trp00

Table II shows the mutations found in the families with moderate haemophilia B. In one of the families, the only affected member was a woman with deletion of at least part of one of the FIX genes concomitant with extreme lyonization (Kling et al, 1991). The remaining nine patients had point mutations, five with the same mutation (C 6364 T; Arg −4 Trp) and eight mutations affecting CpG dinucleotides. One of the mutations (C 30114 C; His 221 Tyr) is in the proposed active site and had not been described before from other centres.

Table II.  Mutations in families with moderate haemophilia B (n = 10).
Mö-IDNucleotide changeProtein changeFIXCFIXAg
  • *

    Female.

45*exon del44
33G 122 Asplicing defect32
19C 6364 TArg −4 Trp336
20C 6364 TArg −4 Trp227
40C 6364 TArg −4 Trp130
48C 6364 TArg −4 Trp2 
75C 6364 TArg −4 Trp2 
66C 20413 TArg 145 Cys3 
76C 30114 THis 221 Tyr1 
26T 31041 GVal 307 Gly34
77T 31274 CTrp385 Arg3 

Table III shows the mutations found in families with mild haemophilia B. All patients had point mutations creating amino acid changes, except five families who had the same ‘silent’ mutation (G 17736 A; Val 107, silent) that had not been described before from other centres. Four other mutations had not been described by other centres before (G 10395 T; Gly 48 Val and C 10415 T; Pro 55 Ser and C 10416 T; Pro 55 Leu and C 31248 A; Thr 376 Asn). Twenty-two of the 29 mutations affected CpG dinucleotides.

Table III.  Mutations in families with mild haemophilia B (n = 29).
Mö-IDNucleotide changeProtein changeFIXCFIXAg
27G 10395 TGly 48 Val19108
21C 10415 TPro 55 Ser1252
22C 10416 TPro 55 Leu20 
67T 10482 GPhe 77 Tyr1983
35G 17736 AVal 107, silent2114
37G 17736 AVal 107, silent1524
42G 17736 AVal 107, silent2024
55G 17736 AVal 107, silent18 
56G 17736 AVal 107, silent17 
32G 20414 AArg 145 His6115
36G 20414 AArg 145 His7110
38G 20414 AArg 145 His686
23G 20414 AArg 145 His7148
17G 20414 AArg 145 His1191
43G 20414 AArg 145 His6115
39T 30100 CIle 216 Thr44
28G 30150 AAla 233 Thr22 
29G 30150 AAla 233 Thr2212
30G 30150 AAla 233 Thr15 
31G 30150 AAla 233 Thr1113
46G 30150 AAla 233 Thr16 
78G 30864 AArg 248 Gln5 
25C 31008 TThr 296 Met415
59C 31008 TThr 296 Met7 
57C 31122 AAla 334 Asp27 
62C 31122 AAla 334 Asp23 
72G 30150 AAla 233 Thr9 
16C 31248 AThr 376 Asn1513

Haplotype analysis

Families with the same mutation were tested to see whether they were haploidentical, using seven different polymorphisms in the FIX gene. Different haplotypes for one or more polymorphisms were found in two out of two families with C 17761 T (one of the mothers was a non-carrier and only one of the families carried the neutral mutation C 30890 T), two out of two families with G 20561 A, three out of three families with C 30863 T (all three mothers were non-carriers), two out of two families with C 30875 T (the mothers were non-carriers), five out of five families with C 6364 T, one out of five families with G 17736 A, one out of six families with G 20414 A, and one out of five families with G 30150 A. Two families had C 6460 T and were unrelated, owing to different ethnic origin. Malmö 57 and 62 had the same mutation with identical alleles for TaqI, MnlI and HhaI. Malmö 25 and 59 had the same mutation with identical alleles for TaqI, MnlI and HhaI.

Estimation of sex-specific mutation rate

Of the 77 families, 31 had a sporadic case of haemophilia. If one considers only the 48 families with severe or moderate haemophilia (to avoid bias from possible undiagnosed mild haemophiliacs), 23 (48%) had a sporadic case of haemophilia. Five of these 23 sporadic cases carried de novo mutations as the mother did not carry the mutation (and no evidence of mosaicism could be found), 11 out of 23 of the mothers were proven carriers and in the remaining seven cases it was not possible to determine carriership [owing to deletion (three cases) and difficulties in obtaining samples (four cases)]. Five patients carried proven de novo mutations in the 16 families with sporadic mutations in which it was possible to perform a thorough investigation of all relevant family members. Thirty-one families had sporadic mutations and the number of de novo mutants in the whole Swedish male population (4·4 million) may be calculated as 5/16 × 31 = 9·7. The remaining mothers of the sporadic cases in the 16 investigated families carried de novo mutations, according to restriction fragment length polymorphism (RFLP) analysis. By extrapolation to the whole population, this implied that 11/16 × 31 = 21·3 women are new mutants carrying a FIX gene mutation. However, the probability of detecting a new heterozygous mutant through an affected offspring is equal to the risk that the carrier has given birth to a boy with haemophilia. This can be approximately calculated as P = S/4 × (1–C) [1·8/4 × (1–0·24) = 0·34], in which S is the average number of children born to Swedish women during the relevant period and C the fraction of Swedish women below childbearing age (< approximately 20 years) (figures for S and C from the National Bureau of Statistics). Thus, the total number of new female carriers of a FIX mutation in the population should be 21·3/0·34 = 62·6.

Males who are de novo mutants provide a direct estimate of the female-specific mutation rate (u): 9·7/4·4 × 106 = 2·2 × 10−6 (the male population of Sweden is estimated to be 4·4 million). In carrier women who are new mutants, the mutation originates from the maternal grandmother or grandfather. The frequency of such women, 62·6/4·5 × 106 = 13·9 × 10−6, is an estimate of u + v, in which v is the mutation rate in males (the female population of Sweden estimated to be 4·5 million). Thus, v = (13·9–2·2) × 10−6 = 11·7 and the ratio of male to female mutation rates (v/u) = 11·7/2·2 = 5·3. The overall FIX gene mutation rate according to the formula µ = (v + 2 u)/3 is 5·4 × 10−6 per gamete per generation.

Inhibitor development and type of mutation

Eleven of the 48 patients (23%) with severe haemophilia B developed inhibitors and all of them had deletions or nonsense mutations (Table IV). Thus, 11 out of 37 (30%) patients with severe haemophilia B as a result of deletion/nonsense mutations developed inhibitors compared with 0 out of 11 with missense mutations.

Table IV.  Mutations in patients who developed inhibitors (n = 11).
Mö-IDMutationProtein change
2del total
68del total
82del exons A–D, F–H
4420398 del 1 bp (fs)fs Thr 140, stop 156
4420398 del 1 bp (fs)fs Thr 140, stop 156
130950 del 8 bp (fs)fs Asp 276, stop 288
4C 6460 TArg 29, stop
69C 6460 TArg 29 - stop
5G 20561 ATrp 194, stop
3C 30863 TArg 248, stop

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Owing to the careful registration for many decades of all cases of haemophilia in a national registry, the present series of haemophilia B families represents the entire population of Sweden. It was found that almost all families with haemophilia B had a unique mutation, as 84·4% (65 out of 77) of the mutations found were seen to be independent events when families with the same mutations were studied for haplotype identity in the FIX gene. This is a much higher figure than the one found in the latest international database of point mutations, deletions and insertions (Giannelli et al, 1998), in which only 38% were unique events (652 out of 1713), bearing in mind that large deletions were not included. If one considers only the severe and moderate families in our series, all families were unrelated and had unique mutations. Sharing the same haplotype does not prove that the families are unrelated. However, the results were repeated by testing several polymorphisms (avoiding linkage disequilibrium) in many families and, in some families that carried the same mutation, the results were confirmed because the origin of mutation or ethnic origin ruled out the fact that they were related.

Considering only the 66 unique mutations (in 65 families), 53% (35) were missense mutations, 21% (14) were nonsense/stop mutations, 20% (13) were deletions (total or partial), 3% (two) were splice site mutations and 3% (two) were mutations in the promoter (Leyden variants). Seventy-four percent of the mutations (40 of the 54 unique point mutations) were either C[RIGHTWARDS ARROW]T or G[RIGHTWARDS ARROW]A in our series, compared with 59% in the International database (Giannelli et al, 1998) and 50% in the Seattle series (Chen et al, 1991). For example, in the eighth edition of the database (Giannelli et al, 1998), 87 out of 1713 had the C 31008 T compared with only 2 out of 77 in our series. In the series reported by Attali et al (1999), G 20414 A (Arg 145 His) and G 30150 A (Ala 233 Thr) constituted 30% of the 70 unrelated cases. In our series, these mutations were found in 12 out of 29 families, but only 4 out of 12 could be proved as being unique by haplotype analysis. Certainly hot-spots for mutations do exist, but it could well be that certain types of mutations are more frequent in some ethnic groups.

An important issue in genetic counselling is calculating the risk that a mother of a sporadic case of haemophilia is a carrier. In an early report of the origin of mutations in sporadic cases of haemophilia B, we found that in 6 out of 10 carrier mothers of a sporadic case, the mutation had arisen in the healthy maternal grandfather (Kling et al, 1992), thus implying a higher frequency of mutations in the male than the female X-chromosome. In a subsample of the Swedish population, Montandon et al (1992) estimated the male to female mutation rate, v/u, to be 11·0. In a recent report, Green et al (1999) have studied a large subsample (n = 424 families) of the British population and estimated the male to female mutation rate to be 8·64, the female-specific rate 2·18 and the male-specific rate 18·82. Our figure, v/u = 5·3, is in good agreement with the British figure 8·64. Our figures for the female mutation rate are very similar, 2·18 versus 2·2, but our figure for the male mutation rate is slightly lower at 11·7 versus 18·82. Our estimate is also in good agreement with another estimate of overall male to female mutation ratio of 3·75, based on 59 families with known germline origin (Ketterling et al, 1999). In the latter study, it was found that the sex-specific mutation rate differed according to the mutation type, a finding not seen in the British series. Our results are in agreement with the British results: out of five female mutations, four were C[RIGHTWARDS ARROW]T and one was a total deletion, and, in seven proven male mutations, four were C[RIGHTWARDS ARROW]T, one was a 2 bp del, one was a splice and one a G[RIGHTWARDS ARROW]C. In our first report we found paternal age at birth (41·5 years) of a new female carrier to exceed the average paternal age in the general population, a finding that is still relevant in the present series and contradictory to the results of Ketterling et al (1999), who did not find this association between mutation and paternal age but rather a maternal age effect. A higher mutation rate in males has also been shown in haemophilia A (Becker et al, 1996).

The overall rate of FIX gene mutation per gamete per generation that we found in the Swedish population, 6·4 × 10−6, is in excellent agreement with the 7·7 × x 10−6 found by Green et al (1999) and with an old estimate of 3·1 × 10−6 made without the use of molecular biology by Ferrari & Rizza (1986) using a method by Haldane (1935).

In our series, 23% of patients with severe haemophilia B (11 out of 48) developed inhibitors, which is a high figure (Shapiro, 1979; Sultan, 1992). No patients with moderate or mild haemophilia B developed antibodies. The type of mutation is, as reported earlier (Giannelli et al, 1983; Ljung, 1995; Gill, 1999), a distinct predisposing factor for inhibitor development. In the present series, none of the 11 patients with missense mutations developed inhibitors while 11 out of 37 (30%) of the patients with deletions, nonsense or stop codon mutations developed inhibitors. However, in one family with total deletion, none of three affected males developed inhibitors (Wadelius et al, 1988). One may speculate on whether the high incidence of inhibitors in haemophilia B in Sweden is accidental, owing to the limited number of cases, or is real and as a result of, for example, the choice of concentrate or mode of delivery. One important factor is probably the fact that the frequency of large deletions (> 30 bp) seems to be higher in Sweden (8% of the families, but 13% of the total number of patients with severe haemophilia B) than elsewhere (2–5% of reported cases) (Thompson, 1991; Ketterling et al, 1994; Saad et al, 1994), bearing in mind that we do not have reliable data on the frequency of large deletions as these are not included in the International database. In the British series, large deletions were only found in 7 out of 412 families (1·7%) (Green et al, 1999). It is reasonable to assume population-specific differences in the frequency of various types of mutations that may have clinical implications for that population. A similar finding was recently reported in a small series of sporadic cases in Mexico (Jaloma-Cruz et al, 2000).

In summary, in this study of a whole population, a wide spectrum of mutations were found with unique mutations in all families with the severe and moderate forms, a very high proportion of mutations in CpG dinucleotides, and a higher than expected prevalence of deletions. Twenty-one percent of sporadic cases were de novo mutants, the overall frequency of mutations was 5·4 × 10−6 per gamete per generation, and the ratio of male to female mutation rate was 5·3. The type of mutation was a strong predictor of the risk for the development of inhibitors. This national database will be of great value for the genetic counselling of present and future generations in Sweden.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported in part by grants from the Swedish Medical Research Council (no. K20000–7110-13493–01 A and no.10409), research funds from the University of Lund (ALF), and regional funds from the county of Skåne and Malmö University Hospital, Sweden. We thank Professor F. Giannelli and P. Green, Division of Medical and Molecular Genetics, Guy's Hospital, London, UK, for co-operation and methodological support during the initial phase of this work.

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  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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