Summary
The molecular characterization of two haemophilia B defects, Calgary 1 and Calgary 2, was carried out using polymerase chain reaction (PCR) amplification and direct dideoxy sequencing. It had been previously shown that the Calgary 1 mutation affects the 5′ TaqI restriction site of exon VIII, whereas Calgary 2 involves the loss of the 3′ TaqI site of exon VIII of the factor IX gene. Sequencing data has now revealed that each of these alterations involves a C-to-T transition within a CpG dinucleotide. In each instance an arginine residue is replaced by a stop codon. These cases represent the recurrence of each particular alteration, both of which are predicted to result in the production of a truncated protein lacking a significant part of the catalytic region. A recently developed technique that reveals base substitutions as single-strand conformation polymorphisms (SSCP) was adapted for modelling in the detection of point mutations. Referred to here as single-strand conformation (SSC) analysis, this procedure, used in association with PCR, provided a reliable and sensitive system for molecular diagnosis in each of the cases presented. Computer-generated secondary structure predictions demonstrated a strong correlation with experimental results and the technique was used to screen 11 additional patients in the same region. A change detected by SSC analysis in one patient was localized to 55 base pairs, sequenced, and identified as a conservative amino acid substitution. This patient is now referred to as Calgary 3.
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References
Bird AP (1980) DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res 8:1499–1504
Brownlee GG (1987) The molecular pathology of haemophilia B, Biochem Soc Trans 15:1–8
Chen S-H, Scott CR, Lovrien EW (1989) Factor IX inPortland: a nonsense mutation (CGA to TGA) resulting in hemophilia B. Am J Hum Genet 44:567–569
Dimnik LS, Fraser BM, Poon M-C, Hoar DI (1990) Molecular diagnosis of single strand DNA conformational variation (abstract). Am J Hum Genet 47:A214
Driscoll MC, Bouhassira E, Aledort LM (1989) A codon 338 nonsense mutation in the factor IX gene in unrelated hemophilia B patients: factor IX338 New York. Blood 74:737–742
Fraser BM (1990) Molecular characterization of exon VIII mutation in the factor IX gene of two haemophilia B patients. Masters thesis, University of Calgary, Calgary, Alberta
Freedenberg DL, Chen S-H, Scott R (1989) A C to T mutation in the second TaqI site of exon VIII in the factor IX gene: detection by PCR amplification and direct sequencing (abstract). Am J Hum Genet 45 [Suppl]:A186
Gianelli F, Green PM, High KA, Lozier JN, Lillicrap DP, Ludwig M, Olek K, Reitsma PH, Goossens M, Yoshioka A, Sommers S, Brownlee GG (1990) Haemophilia B: database of point mutations and short additions and deletions. Nucleic Acids Res 18:4053–4059
Green PM, Bentley DR, Mibashan RS, Nilsson IM, Gianelli F (1990) The incidence and distribution of CpG and TpG transitions in the coagulation factor IX gene: a fresh look at CpG mutational hotspots. Nucleic Acids Res 18:3227–3231
Innis MA, Myambo KB, Gelfand DH, Brow MAD (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct dideoxy sequencing of polymerase chain reaction-amplified material DNA. Proc Natl Acad Sci USA 85:9436–9440
Ludwig M, Schwaab R, Eigel A, Horst J, Egli H, Brackman H-H, Olek K (1989) Identification of a single nucleotide C-to-T transition and five different deletions in patients with severe hemophilia B. Am J Hum Genet 45:115–122
Madisen L, Hoar DI, Holroyd CD, Crisp M, Hodes ME (1987) DNA banking: the effects of storage of blood and isolated DNA on the integrity of DNA. Am J Med Genet 27:379–390
Maniatis T, Fritsch EF, Sambrock J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989a) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766–2770
Orita M, Suzuki Y, Sikiya T, Hayashi K, (1989b) A rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874–879
Osterberg G, Sommer R (1981) Computer support of DNA sequence analysis. Comput Prog Biomed 13:101–109
Poon M-C, Chui DHK, Patterson M, Starozik DM, Dimnik LS, Hoar DI (1987) Haemophilia B (Christmas disease) variants and carrier detection analyzed by DNA probes. J Clin Invest 79:1204–1209
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
Siguret V, Amselem S, Vidaud M, Assouline Z, Kerbiriou-Nabias D, Pietu G, Goossens M, Larrieu MJ, Bahnak B, Meyer D, Lavergne JM (1988) Identification of a CpG mutation in the coagulation factor IX gene by analysis of amplified DNA sequences. Br J Haematol 70:411–416
Taylor SAM (1990) Molecular analysis of mutations within the human factor IX gene. PhD thesis, Queen's University, Kingston, Ontario
Yoshitake S, Schach BG, Foster DC, Davie EW, Kurachi K (1985) Nucleotide sequence of the gene for human factor IX. Biochemistry 24:3736–3750
Zuker M, Stiegler P (1981) Optimal computer folding of large RNA sequences using thermodynamics. Nucleic Acids Res 9:133–148
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Fraser, B.M., Poon, MC. & Hoar, D.I. Identification of factor IX mutations in haemophilia B: application of polymerase chain reaction and single strand conformation analysis. Hum Genet 88, 426–430 (1992). https://doi.org/10.1007/BF00215677
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DOI: https://doi.org/10.1007/BF00215677