Introduction: In patients with severe hemophilia A, large deletions in the clotting factor VIII gene (F8) are associated with the highest risk of developing FVIII-inactivating antibodies, a major complication of replacement therapy. Since only few DNA breakpoints in F8 have been characterized precisely so far, this study aimed at their exact localisation to identify molecular mechanisms of X-chromosomal mutagenesis and to facilitate genetic diagnosis and carrier detection.

Methods: Genomic DNA isolated from patients’ lymphocytes was used for standard or long-range PCR, primer walking techniques and F8 sequencing.

Results: The identification of novel breakpoints revealed that hemophiliacs with the diagnosis “large deletion” in F8, which is routinely based on the failure to amplify F8 exons by PCR, in fact represent a heterogeneous group with a variety of mutation types: mere deletions of distinct DNA regions, deletions with microinsertions, but also deletions combined with the recurrent int22h-related inversions and complex mutations involving a deletion, an inversion and an insertion in F8.

Conclusion: A detailed analysis of F8 deletions may allow detection of mutational hot spots and improvement of diagnostic techniques. Patients with unusual patterns in Southern blot or PCR for int22h-related inversions may carry additional deletions which increase dramatically the risk of an immune response to FVIII. Moreover, our data help to establish understanding of the mechanisms behind gross chromosomal rearrangements causing hemophilia A and other diseases.