Abstract
In an attempt to characterize disease producing mutations in the factor VIII gene we screened exons 4, 7, 8, 11, 12 and 16 by PCR-SSCP (polymerase chain reactionsingle strand conformation polymorphism), in 12 randomly selected haemophilia A patients. These exons were chosen because they have been reported to harbour a disproportionately high number of mutations relative to their size. Using this strategy we detected a frame-shifting 5-bp deletion (TACCT, involving nucleotides 519–523), which is predicted to result in a severely truncated factor VIII polypeptide, terminating approximately midway through the conserved A1 domain and resulting in the observed severe phenotype. We also showed that the sequence in the vicinity of the observed deletion is concordant with the modified “slipped-mispairing at DNA replication” model of Krawczak and Cooper.
References
Antonarakis SE, Kazazian HH (1988) The molecular basis of hemophilia A in man. Trends Genet 4:233–237
Gitschier J, Wood WI, Goralka TM, Wion KL, Chen EY, Eaton DE, Vehar GA, Capon DJ, Lawn RM (1984) Characterization of the human factor VIII gene. Nature 312:326–330
Grompe M (1993) The rapid detection of unknown mutations in nucleic acids. Nature Genet 5:111–117
Gyllensten UB, Erlich HA (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 85:7652–7656
Higuchi M, Antonarakis SE, Kasch L, Oldenburg J, Economou-Peterson E, Olek K, Arai M, Inaba H, Kazazian HH (1991) Molecular characterization of mild-to-moderate haemophilia A: Detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 88:8307–8311
Krawczak M, Cooper DN (1991) Gene deletions causing human disease: mechanisms of mutagenesis and the role of the local DNA sequence environment. Hum Genet 86:425–441
Lakich D, Kazazian HH, Antonarakis SE, Gitshier J (1993) Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nature Genet 5:236–241
Mgone CS, Lanyon WG, Moore MR, Connor JM (1992) Detection of seven point mutations in the porphobilinogen deaminase gene in patients with acute intermittent porphyria by direct sequencing of in vitro amplified cDNA. Hum Genet 90:12–16
Michaud J, Brody LC, Steel G, Fontaine G, Martin LS, Valle D, Mitchell G (1992) Strand-separating conformational polymorphism analysis: Efficacy of detection of point mutations in the ornithine delta-aminotransferase gene. Genomics 13:389–394
Naylor JA, Green PM, Montandon AJ, Rizza CR, Giannelli F (1991) Detection of three novel mutations in two haemophilia A patients by rapid screening of whole essential region of factor VIII gene. Lancet 337:635–639
Naylor JA, Green PM, Rizza CR, Giannelli F (1993a) Analysis of factor VIII mRNA reveals defects in everyone of 28 haemophilia A patients. Hum Mol Genet 2:11–17
Naylor J, Brinke A, Hassock S, Green PM, Giannelli F (1993b) Characteristic mRNA abnormality found in half the patients with severe haemophilia A is due to large DNA inversions. Hum Mol Genet 2:1773–1778
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphism of human DNA by gel electrophoresis as single strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766–2770
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sheffield VC, Beck JS, Kwitek AE (1992) Analysis of the efficiency of single base substitution detection by SSCP. Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York, pp 1–149
Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL, Pittman DD, Kaufman RJ, Brown E, Shoemaker C, Off EC, Amphlett GW, Foster WB, Coe ML, Knutson GJ, Fass DN, Hewick RM (1984) Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 312:342–347
Tuddenham EGD, Cooper DN, Gitschier J, Higuchi M, Hoyer LW, Yoshioka A, Peake IR, Schwabb R, Olek K, Kazazian HH, Lavergne JM, Giannelli F, Antonarakis SE (1991) Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene. Nucleic Acids Res 19:4821–4833
Vehar GA, Keyt B, Eaton D, Rodriguez H, O'Brien D, Roblat F, Oppermann H, Keck R, Wood WI, Harkins RN, Tuddenham EGD, Lawn RM, Capon DJ (1984) Structure of human factor III. Nature 312:337–342
Wood WI, Capon DJ, Simonsen CC, Eaton DL, Gitschier J, Keyt B, Seeburg PH, Smith DH, Hollingshead P, Wion KL, Delwart E, Tuddenham EGD, Vehar GA, Lawn RM (1984) Expression of active human factor VIII from recombinant DNA clones. Nature 312:330–337
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bidichandani, S.I., Lanyon, W.G. & Connor, J.M. Characterisation of a 5-bp deletion in exon 4 of the factor VIII gene: concordance with slipped-mispairing at DNA replication. Hum Genet 94, 447–449 (1994). https://doi.org/10.1007/BF00201612
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00201612