Characterisation of two novel large F8 deletions in patients with severe haemophilia A and factor VIII inhibitors

Abstract

SummaryLarge deletions are found in approximately 5% of patients with severe haemophilia A, but only a few deletion breakpoints have been char-acterised precisely so far. In this study we characterised the deletion breakpoints of two patients with severe haemophilia A, large deletions and factor VIII (FVIII) inhibitors, and subsequently established deletion-specific assays for the identification of carriers. Patient 1 had a deletion of 37,410 bp comprising exon 1 and the F8promoter region, and a 5 bp homology (GGGCC) is present at the chromosomal fusion site. In patient 2, a deletion of 22,230 bp including parts of intron 25, exon 26 and 3‘-UTR was identified. No homologous repetitive elements were found at the breakpoints. However, both breakpoints were located within long terminal repeats of endogenous retroviruses and the DNA motif TTTAAA – known to be able to bend DNA molecules – was identified at the centromeric breakpoint. By deletion-specific PCR experiments we were able to identify a heterozygous state in mother 2 (carrier) while mother 1 presented only with wild-type alleles (non-carrier). Both deletions are most likely created by DNA double strand breaks and subsequent DNA repair by the non-homologous end joining DNA repair pathway (NHEJ). The exact identification of the deletion breakpoints provides a reliable diagnostic tool for carrier identification in affected families by means of a deletion-specific PCR.