Genetic Basis and Carrier Detection of Hemophilia B of Chinese Origin

Summary

We have characterized the genetic defects of 17 hemophilia B patients of Chinese origin by means of the polymerase chain reaction (PCR) and direct sequencing. The single-strand conformation polymorphism (SSCP) was used as an initial screening method to analyze the entire coding region and the flanking introns of each individual’s factor IX gene. The abnormal exons were subsequently amplified and the nucleotide sequence determined. Of the 17 patients studied, 16 had single point mutations and one had a gross gene deletion of exons VII and VIII of factor IX. Among these 16 factor IX variants with point mutations 13 were missense and two were nonsense mutations. The remaining one had a nucleotide deleted, resulting in frame shifting at amino acid residue 97. A total of ten novel mutations, including the one with gross gene deletion, are reported in this study which have not been described previously. Five of the remaining seven variants were missense mutations with novel amino acids substituted for residues 127, 132, 180, 207, and 215, respectively. Mutations containing different amino acid residues at those positions have been reported. The last two are variants that have already been described to contain mutations at amino acid residues 333 and 365, respectively. To evaluate the efficiency of SSCP analysis in assessing the mutated exons and to further confirm our results we sequenced the entire exons of all 17 factor IX genes. The mutations detected by SSCP method were indeed the only mutation identified in each factor IX variant. The SSCP analysis and direct sequencing have also allowed us to circumvent the difficulties of carrier determination for Chinese by direct detection of the abnormal factor IX alleles inherited by the females.

• References

• 1 Kurachi K, Davie EW. Isolation and characterization of a cDNA coding for human factor IX. Proc Natl Acad Sci USA 1982; 79: 6461-6464
• 2 Yoshitake S, Schach BG, Foster DC, Davie EW, Kurachi K. Nucleotide sequence of the gene for human factor IX (anti-haemophilic factor IX). Biochemistry 1985; 24: 3736-3750
• 3 Anson DS, Choo KH, Rees DJ, Gianelli F, Gould K, Huddleston JA, Brownlee GG. Gene structure of human anti-haemophilic factor. EMBO J 1984; 3: 1053-1064
• 4 Purello M, Alhadeff B, Esposito D, Szabo P, Rocchi M, Truett M, Masiarz F, Siniscalco M. The human genes for hemophilia A and hemophilia B flank the X chromosome fragile site at Xq27.3. EMBO J 1985; 4: 725-729
• 5 Jaje M, de la Salle H, Schamber F, Balland A, Kohli V, Findeli A, Toltoshev T, Lecocq JP. Isolation of a human antihaemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX. Nucleic Acids Res 1983; 11: 2325-2334
• 6 McGraw RA, Davis LM, Noyes CM, Lundblad RL, Roberts HR, Graham JB, Stafford DW. Evidence for a prevalent dimorphism in the activation peptide of human factor IX. Proc Natl Acad Sci USA 1985; b 82: 2847-2851
• 7 Brownlee GG. Haemophilia B: a review of patient defects, diagnosis with gene probes and prospects for gene therapy. In: Recent Advances in Haematology. Hoffbrand AV. (ed) Churchill Livinstone, Edinburgh: 1988. Vol 5 251-264
• 8 Giannelli F, Green PM, High KA, Sommer S, Lillicrap DP, Ludwig M, Olek K, Reitsma PH, Goosens M, Yoshioka A, Brownlee GG. Haemophilia B: database of point mutations and short additions and deletions. Nucleic Acid Res 1991; 19 Supplement 2193-2219
• 9 Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphism. Proc Natl Acad Sci USA 1989; 86: 2766-2770
• 10 Lin SW, Shen MC. Characterization of genetic defects of hemophilia B of Chinese origin. Thromb Haemostas 1991; 66: 459-463
• 11 Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual. 02.. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 1989
• 12 Saiki RK, Scharf S, Faloona F, Mullis KB, Hort GT, Erlich HA, Arnheim N. Enyzmatic amplification of β-globin genomic sequenced restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230: 1350-1354
• 13 Lozier JN, Monroe DM, Stanfield-Oakley S, Lin SW, Smith KJ, Roberts HR, High KA. Factor IX New London: Substitution of proline for glutamine a position 50 causes severe hemophilia B. Blood 1990; 75: 1097-1104
• 14 Mihovilovic M, Lee JE. An efficient method for sequencing PCR amplified DNA. Biotechniques 1989; 7: 14-16
• 15 Brow MAD. Sequencing with Taq DNA polymerase. In PCR Protocols: A Guide to Methods and Applications. Innis MA, Gelfand DH, Sninsky JJ, White TJ. (eds) Harcourt Brace Jovanovich Publishers. Academic Press, Inc; New York: 1989
• 16 Bentley AK, Ress DJG, Rizza C, Brownlee GG. Defective propeptide processing of blood clotting factor IX caused by mutation of arginine to glutamine at position -4. Cell 1986; 45: 305-307
• 17 Ware J, Diuguid DL, Liebman HA, Rabiet MJ, Kasper CK, Furie BC, Furie B, Stafford DW. Factor IX San Dimas. J Biol Chem 1987; 264: 11401-11406
• 18 Sugimoto M, Miyata T, Kawabata S, Yoshioka A, Fukui H, Iwanaga S. Factor IX Kawachinagano: impaired function of the Gla-domain caused by attached propeptide region due to substitution of arginine by glutamine at position -4. Br J Haematol 1989; 72: 216-221
• 19 Smith KJ, Thompson AR, McMullen BA, Frazier D, Lin SW, Stafford DW, Kiesel W, Thibideau SA, Chen SH, Smith L. Carrier testing in hemophilia B with an immunoassay with distinguishes a prevalent factor IX dimorphism. Blood 1987; 70: 1006-1013
• 20 Thompson AR, Chen SH, Smith KJ. Role of an immunoassay-detected polymorphism of factor IX in diagnosing hemophilia B carriers. Blood 1987; 70 (Suppl. 01) 382a
• 21 Sarkar G, Yoon HS, Sommer SS. Screening for mutations by rna single-strand conformation polymorphism (rSSCP): comparison with DNA-SSCP. Nucleic Acids Res 1992; 20: 871
• 22 Fraser BDM, Poon MC, Hoar DI. Identification of factor IX mutations in haemophilia B: application of polymerase chain reaction and single strand conformation analysis. Hum Genet 1992; 88: 426-430
• 23 Chen SH, Zhang M, Lovrien EW, Scott CR, Thompson AR. CG dinucleotide transitions in the factor IX gene account for about half of the point mutations in hemophilia B patients: a Seattle series. Hum Genet 1991; 87: 177-182
• 24 Green PM, Montandon AJ, Bentley DR, Ljung R, Nilsson IMJ, Giannelli F. The incidence and distribution of CpG → TpG transitions in the coagulation factor IX gene: a fresh look at CpG mutational hotspots. Nucleic Acids Res 1990; 18: 3227-3231
• 25 Sarkar G, Koeberl DD, Sommer SS. Direct sequencing of the activation peptide and the catalytic domain of the factor IX gene in six species. Genomics 1990; 6: 133-143
• 26 Noyes CM, Griffith MJ, Roberts HR, Lundblad RL. Identification of the molecular defect if factor IX Chapel Hill: Substitution of histidine for arginine at position 145. Proc Natl Acad Sci USA 1983; 80: 4200-4205
• 27 Griffith MJ, Breitkreutz. Trapp H, Briet E, Noyes CM, Lundblad RL, Roberts HR. Characterization of the clotting activities of structurally different forms of activated factors IX. J Clin Invest 1985; 75: 4-8
• 28 Giannelli F, Choo KG, Rees DJG, Boyd Y, Rizza CR, Brownlee GG. Gene deletions in patients with hemophilia B and anti-factor IX antibodies. Nature 1983; 303: 181-182
• 29 Matthews RJ, Anson DS, Peake IR, Bloom AL. Heterogeneity of the factor IX locus in nine hemophilia B inhibitor patients. J Clin Invest 1987; 79: 746-751
• 30 Green PM, Bentley DR, Mibashan RS, Nilsson IM, Gianelli F. Molecular pathology of hemophilia B. EMBO J 1989; 8: 1067-1071
• 31 Attree O, Vidaud D, Vidaud M, Amselem S, Lavergne JM, Goossens M. Rapid characterization of mutations in the catalytic domain of human coagulation factor IX. Blood 1988; 72 (Suppl. 01) 289a