Abstract
Large DNA inversions caused by an intrachromosomal recombination between homologous regions located in intron 22 and 5′ of the factor VIII gene have recently been identified in patients with severe haemophilia A. To evaluate better the prevalence of this large inversion and to estimate the overall sensitivity of the Southern blot/hybridization method we analysed the factor VIII gene of 49 unrelated patients with severe haemophilia A. All patients were screened for the inversion mutation, TaqI site mutations, and deletions. Mutations were identified in 31 (63%) patients, and comprised 24 large inversions, 4 partial deletions, and 3 point mutations. Three different haplotypes were characterised in the patients presenting the inversion mutation, confirming its independent origin. Two novel deletions are reported: a large one spanning from intron 14 to intron 22 and a deletion of 86 bp comprising the 3′ region of exon 1 and 39–41 bp of intron 1. DNA sequencing of the deletion junction showed no significant homology between normal 5′ and 3′ sequences around the breakpoints. A novel missense mutation is also reported: CGA→GGA, Arg-2209 to Gly. These results confirm that the inversion mutation is the most common cause of severe haemophilia A and indicate that the Southern blot/hybridization assay should be used as the first method for screening of mutations in severe haemophilia A.
Similar content being viewed by others
References
Antonarakis SE, Carpenter RJ Jr, Hoyer LW, Toole JJ, Coperland KL, Carta CA, Caskey CT, Kazazian HH Jr (1985) Prenatal diagnosis of haemophilia A by factor VIII gene analysis. Lancet 1:1407–1409
Casula L, Murru S, Pecorara M, Ristaldi MS, Restagno G, Mancuso G, Morfini M, DeBiasi R, Baudo F, Carbonara A, Mori PG, Cao A, Pirastu M (1990) Recurrent mutations and three novel rearrangements in the Factor VIII gene of hemophilia A patients of Italian descent. Blood 75:662–670
Economou EP, Kazazian HH Jr, Antonarakis SE (1992) Detection of mutations in the FVIII gene using single-stranded conformational polymorphism (SSCP). Genomics 13:909–911
Figueiredo MS, Bernardi F, Zago MA (1992) A novel deletion of FVIII gene associated with variable levels of FVIII inhibitor. Eur J Haematol 48:152–154
Gitschier J, Wood WI (1992) Sequence of exon-containing regions of the human factor VIII gene. Hum Mol Genet 1:199–200
Higuchi M, Antonarakis SE, Kasch L, Oldenburg J, Economou-Petersen E, Olek K, Arai M, Inaba H, Kazazian HH Jr (1991 a) Molecular characterization of mild-to-moderate hemophilia A: detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 88:8307–8311
Higuchi M, Kazazian HH Jr, Kasch L, Warren TC, McGinniss MJ, Phillips JA III, Kasper C, Janco R, Antonarakis SE (1991 b) Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene. Proc Natl Acad Sci USA 88:7405–7409
Kasper CK, Ewing NP (1982) Measurement of inhibitor to factor VIIIC (and IXC). In Bloom AL (ed) The hemophilias. Methods in hematology, vol 5. Churchill Livingstone, Edinburgh, pp 39–50
Lakich D, Kazazian HH Jr, Antonarakis SE, Gitschier J (1993) Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nature Genet 5:236–241
Levine PH (1987) Clinical manifestations and therapy of hemophilias A and B. In: Colman RW, Hirsh J, Marder VJ, Salzman EW (eds) Hemostasis and thrombosis, 2nd edn. J. B. Lippincott Company, Philadelphia, pp 97–111
Levinson B, Kenwrick S, Lakich D, Hammonds G Jr, Gitschier J (1990) A transcribed gene in an intron of the human factor VIII gene. Genomics 7:1–11
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 (1993) Analysis of factor VIII mRNA reveals defects in everyone of 28 haemophilia A patients. Hum Mol Genet 2:11–17
Rizza CR, Rhymes IL (1982) Coagulation assay of VIIIC and IXC. In: Bloom AL (ed) The hemophilias. Methods in hematology, vol 5. Churchill Livingstone, Edinburgh, pp 18–38
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Silva WA Jr, Figueiredo MS (1994) Analysis of factor VIII gene polymorphisms in Brazilian blacks reveals further differences in the black population. Hum Hered 44:252–260
Tuddenham EGD (1993) Flipping the tip of the X. Nature Genet 5:209
Tuddenham EGD, Cooper DN, Gitschier J, Higuchi M, Hoyer LW, Yoshioka A, Peake IR, Schwaab R, Olek K, Kazazian HH Jr, 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
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
Woods-Samuels, Kazazian HH Jr, Antonarakis SE (1991) Nonhomologous recombination in the human genome: deletions in the human factor VIII gene. Genomics 10:94–101
Youssoufian H, Phillips DG, Kazazian HH Jr, Antonarakis SE (1987) Mspl polymorphism in the 3′ flanking region of the human factor VIII gene. Nucleic Acids Res 15:6312
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Figueiredo, M.S., Tavella, M.H. & Simões, B.P. Large DNA inversions, deletions, and TaqI site mutations in severe haemophilia A. Hum Genet 94, 473–478 (1994). https://doi.org/10.1007/BF00211010
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00211010