Analysis of large structural changes of the factor VIII gene, involving intron 1 and 22, in severe hemophilia A

Abstract

Background and objectives: Hemophilia A (HA), the deficiency of coagulation factor VIII (FVIII), is the most common, sex-linked inherited bleeding disorder. The disease is caused by FVIII gene intron 22 inversion in approximately 50% of the patients, and by intron 1 inversion in 5% of the patients with severe HA. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22, and their extragenic copy located telomeric to the FVIII gene. The goal of the present study was to analyze the presence of large structural changes in the FVIII gene in patients with severe hemophilia A.

Design and methods: We studied 104 unrelated, severe HA-patients or obligate carriers for the presence of intron 22 and intron 1 inversions by Southern blotting, long-distance polymerase chain reaction (PCR), and simple PCR.

Results: We found altered intron 22 restriction profiles by Southern analyses in 58 cases: 43 type 1, 11 type 2 inversions and 4 unusual patterns. Upon further examination of the last 4 cases, large deletions involving intron 22 were demonstrated in two cases. In the remaining two patients extra homologous regions were detected by Southern analysis, and long-distance PCR showed the presence of unaltered intra- and extragenic copies together with one inversion-affected copy, suggesting that an additional intronic fragment participated in the inversion process and was inserted in the genome. During screening for intron 1 inversion among 43 patients, who were intron 22 inversion negative, we identified only wild type individuals.

Interpretation and conclusions: The relatively large proportion of unusual patterns further supports the observation that the structure of FVIII intron 22 represents a hot spot for large gene rearrangements with various mechanisms, while intron 1 inversion seems to be not common in Hungary.