LARGE DELETIONS OF THE FACTOR VIII (FVIII) GENE RESULT FROM BOTH HOMOLOGOUS AND NON-HOMOLOGOUS RECOMBINATION.

CARLOS DE BRASI, LILIANA ROSSETTI, ANNE GOODEVE, IRENE LARRIPA.

Bangkok, Tailandia

Congreso; XXVI International Congress of the Word Federation of Hemophilia.; 2004

Word Federation of Hemophilia.

Although large deletions from the FVIII gene are responsible for about 5% of cases of severe haemophilia A (seHA), few have been fully characterised. This abstract details characterization of two large partial deletions of the FVIII gene. Case 1: A seHA patient with a high titre inhibitor (5700BU/ml) demonstrated a consistent absence of PCR-amplification of exons 4 to 10 (DE4-10) of FVIII. Sequence alignment of FVIII introns 3 and 10 indicated Alu repeats to be present. Long-distance PCR (LD-PCR) amplification from exon 3 to exon 11 (also permitting carrier detection in the family) indicated unequal cross over between 86% homologous full-length Alu-sequences leading to a deletion of 30.9kb. A short stretch of intron 10 was embedded into intron 3 sequence, a typical hallmark of homologous recombination by the double-strand break repair model. Case 2: A seHA patient with high titre inhibitor (223BU/ml) showed a consistent failure of PCR-amplifications of exons 10 to 18 (DE10-18). LD-PCR was performed using primers in exons 9 and 19. DNA sequencing through the breakpoints defined a 62.5kb deletion, marked by a sequence shift from intron 9 nt 1012 (part of a LINE/L1 sequence) to intron 18 nt 1280. The absence of significant homology at the deletion breakpoints (only TG as “sticky ends”) indicated a mechanism of non-homologous recombination. The use of LD-PCR to obtain deletion-specific products permits isolation of the deletion breakpoints for further studies and enables detection of the defect in heterozygous females (carriers). Additionally, the examples studied here confirm that the FVIII gene suffers large deletions, which originate as a result of heterogeneous mechanisms, frequently involving interspersed repeats.