First Molecular Characterization of an Unequal Homologous Alu-mediated Recombination Event Responsible for Hemophilia

Summary

The large number of Alu repeats in the human genome provides abundant opportunities for unequal homologous recombination events that are responsible of several human diseases. We here describe a novel large FVIII gene deletion from a severe hemophilia A patient in which Alu-repetitive elements are directly involved in the origin of the mutation. Using a long-fragment PCR method, a ∼23 kb deletion was delimited between introns 24 and 25. The resulting FVIII gene had a hybrid 2317-bp intron and lacked exon 25. Absence of exon 25 was confirmed at the RNA level. Multiple sequence alignment of this hybrid intron and normal introns 24 and 25 provided evidence of an homologous recombination event between two Alu repeats and the exact breakpoints were delimited to a 16 bp region. To our knowledge, this is the first report of hemophilia caused by unequal homologous Alu/Alu recombination. This mechanism, commonly related to genetic human disorders, may be involved in a significant number of hemophilia cases considering that FVIII is coded by an Alu-rich gene.

Keywords

F8 - haemophilia - Alu - deletion

• References

• 1 Gitschier J, Wood WI, Goralka TM, Wion KL, Chen EY, Eaton DH, Vehar GA, Capon DJ, Lawn RM. Characterization of the human factor VIII gene. Nature 1984; 312: 326-30.
• 2 Kemball-Cook G, Tuddenham EGD, Wacey AI. The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4. Nucleic Acids Res 1998; 26: 216-9.
• 3 Antonarakis SE, Rossiter JP, Young M, Horst J, de Moerloose P, Sommer SS, Ketterling RP, Kazazian HHJ, Nègrier C, Vinciguerra C. et al. Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood 1995; 86: 2206-12.
• 4 Lakich D, Kazazian HHJ, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet 1993; 05: 236-41.
• 5 Van de Water N, Williams R, Ockelford P, Browett P. A 20.7 kb deletion within the factor VIII gene associated with LINE-1 element insertion. Thromb Haemost 1998; 79: 938-42.
• 6 Millar DS, Steinbrecher RA, Wieland K, Grundy CB, Martinowitz U, Krawczak M, Zoll B, Whitmore D, Stephenson J, Mibashan RS. et al. The molecular genetic analysis of haemophilia A; characterization of six partial deletions in the factor VIII gene. Hum Genet 1990; 86: 219-27.
• 7 Youssoufian H, Antonarakis SE, Aronis S, Tsiftis G, Phillips DG, Kazazian Jr. HH. Characterization of five partial deletions of the factor VIII gene. Proc Natl Acad Sci USA 1987; 84: 3772-6.
• 8 Krepelov A, Vorlov Z, Zavadil J, Brdicka R. Factor VIII gene deletions in haemophilia A patients in Czechoslovakia. Br J Haematol 1992; 81: 271-6.
• 9 Bardoni B, Sampietro M, Romano M, Crapanzano M, Mannucci PM, Camerino G. Characterization of a partial deletion of the factor VIII gene in a haemophiliac with inhibitor. Hum Genet 1988; 79: 86-8.
• 10 Figueiredo MS, Tavella MH, Simes BP. Large DNA inversions, deletions, and TaqI site mutations in severe haemophilia A. Hum Genet 1994; 94: 473-8.
• 11 Mikami S, Nishimura T, Naka H, Kuze K, Fukui H. A deletion involving intron 13 and exon 14 of factor VIII gene in a haemophiliac with anti-factor VIII antibody. Jinrui Idengaku Zasshi 1988; 33: 401-7.
• 12 Woods-Samuels P, Kazazian HHJ, Antonarakis SE. Nonhomologous recombination in the human genome: deletions in the human factor VIII gene. Genomics 1991; 10: 94-101.
• 13 Deininger PL, Batzer MA. Alu repeats and human disease. Mol Genet Metab 1999; 67: 183-93.
• 14 Schmid CW. Alu: structure, origin, evolution, significance and function of one-tenth of human DNA. Prog Nucleic Acid Res Mol Biol 1996; 53: 283-319.
• 15 Rowold DJ, Herrera RJ. Alu elements and the human genome. Genetica 2000; 108: 57-72.
• 16 Chandley AC. Asymmetry in chromosome pairing: a major factor in de novo mutation and the production of genetic disease in man. J Med Genet 1989; 26: 546-52.
• 17 Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation [see comments]. Tissue Antigens 1992; 39: 225-35.
• 18 Vidal F, Farssac E, Altisent C, Puig L, Gallardo D. Rapid Hemophilia A Molecular Diagnosis by a Simple DNA Sequencing Procedure: Identification of 14 Novel Mutations. Thromb Haemost 2001; 85: 580-3.
• 19 Liu Q, Nozari G, Sommer SS. Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in hemophilia A. Blood 1998; 92: 1458-9.
• 20 Bowen DJ, Hampton KK. Analysis of the BglI restriction fragment length polymorphism in the human factor VIII gene using virtual PCR – a novel approach employing the polymerase chain reaction in the absence of sequence information for the locus. Hum Genet 1996; 98: 219-22.
• 21 Batzer MA, Deininger PL, Hellmann-Blumberg U, Jurka J, Labuda D, Rubin CM, Schmid CW, Zietkiewicz E, Zuckerkandl E. Standardized nomenclature for Alu repeats. J Mol Evol 1996; 42: 3-6.
• 22 Rudiger NS, Gregersen N, Kielland-Brandt MC. One short well conserved region of Alu-sequences is involved in human gene rearrangements and has homology with prokaryotic chi. Nucleic Acids Res 1995; 23: 256-60.
• 23 Stahl FW. Special sites in generalized recombination. Annu Rev Genet 1979; 13: 7-24.
• 24 Myers RS, Stahl FW. Clinical analysis of the RecBC D enzyme of Escherichia coli. Annu Rev Genet 1994; 28: 49-70.
• 25 Harteveld KL, Losekoot M, Fodde R, Giordano PC, Bernini LF. The involvement of Alu repeats in recombination events at the alpha-globin gene cluster: characterization of two alphazero-thalassaemia deletion breakpoints. Hum Genet 1997; 99: 528-34.
• 26 Youssoufian H, Kasper CK, Phillips DG, Kazazian HHJ, Antonarakis SE. Restriction endonuclease mapping of six novel deletions of the factor VIII gene in hemophilia A. Hum Genet 1988; 80: 143-8.
• 27 Higuchi M, Kochhan L, Schwaab R, Egli H, Brackmann HH, Horst J, Olek K. Molecular defects in hemophilia A: identification and characterization of mutations in the factor VIII gene and family analysis. Blood 1989; 74: 1045-51.
• 28 Shibata M, Shima M, Morichika S, McVey J, Tuddenham EG, Tanaka I, Suzuki H, Nogami K, Minamoto Y, Hato T, Saenko EL, Scandella D, Yoshioka A. An alloantibody recognizing the FVIII A1 domain in a patient with CRM reduced haemophilia A due to deletion of a large portion of the A1 domain DNA sequence. Thromb Haemost 2000; 84: 442-8.
• 29 Wulff K, Gazda H, Schroder W, Robicka-Milewska R, Herrmann FH. Identification of a novel large F9 gene mutation-an insertion of an Alu repeated DNA element in exon e of the factor 9 gene. Hum Mutat 2000; 15: 299.
• 30 Vidaud D, Vidaud M, Bahnak BR, Siguret V, Gispert SSanchez, Laurian Y, Meyer D, Goossens M, Lavergne JM. Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene. Eur J Hum Genet 1993; 01: 30-6.
• 31 Sukarova E, Dimovski AJ, Tchacarova P, Petkov GH, Efremov GD. An Alu insert as the cause of a severe form of hemophilia A. Acta Haematol 2001; 106: 126-9.