MOLECULAR INSIGHTS INTO THE MECHANISM OF LARGE F8-DELETIONS CAUSING SEVERE HAEMOPHILIA A IN TWO FAMILIES: FULL BREAKPOINT CHARACTERIZATION BY NON-CONVENTIONAL APPROACHES AND BIOINFORMATIC ANALYSIS OF IMPLICATED DNA ELEMENTS.

ABELLEYRO MM; WAISMAN K; ZIEGLER, BM; MARCHIONE VD; RADIC CP; ROSSETTI CL; DE BRASI C

Congreso; World Federation of Hemophilia Virtual Congress; 2020

IntroductionLarge F8-deletions cause 8-15% of cases with severe-haemophilia A (HA) and predispose to develop FVIII inhibitors, the major complication in HA treatment.An experimental objective was to develop approaches to diagnose and characterise large F8-deletions in hemizygous and heterozygous state. A theoretical objective was to estimate large F8-deletions mechanisms by looking at their breakpoints.MethodsInverse-PCR-based approaches, i.e., conventional inverse-PCR with unknown range (iPCR) and inverse shifting-PCR (IS-PCR), were adapted to resolve two large F8-deletions involved in families with severe-HA.ResultsFamily 1 includes a proband with a deletion of F8-exon 24-26, and family 2 includes a proband with a F8-exon 5-6 deletion and two female relatives (his mother and sister). The iPCR approach combined with long distance-PCR amplification conditions succeeded in amplifying the breakpoint junctions in family 1, and IS-PCR permitted a specific detection of the F8-e5_e6deletion involved in family 2 in hemizygous (proband) and heterozygous (females-at-risk) state. Sanger sequencing allowed full breakpoint characterization in both cases. F8-deletion-specific iPCR or IS-PCR amplification signals were present in probands and absent in normal control samples. Carrier diagnosis in family 2 indicated that proband?s mother and sister resulted carriers of the F8-e5_e6deletion. In addition, DNA-break stimulating elements (n=46, mined from the literature), interspersed repeats, non-B-DNA and secondary-structures were analysed in specific intervals around breakpoints vs null-hypotheses based on computer simulations and frequency based probabilities. Both F8-deletions showed neither micro-homologies nor non-B-DNA at junctions. Family 1 F8-e24_e26deletion showed no evidence of involving significantly unexpected recombinogenic motifs. However, it showed a LINE L2 element on the 3´ breakpoint and a stable secondary-structure at the 5ꞌ rupture site. Family 2 F8-e5_e6deletion showed recombinogenic sequences at both sides, including a Jurka motif on 3´ and a Murine Parvovirus recombination hotspot on the 5? breakpoint. It also showed a stable-secondary DNA-structure and an AluSx element on the 3´ breakpoint.ConclusionOur findings suggest the involvement of the retroposition machinery and secondary-structures playing significant roles in the origin of both F8-deletions. IS-PCR and iPCR approaches prove their value in characterization and diagnosis of severe-HA causative large F8-deletions in hemizygous/heterozygous state, showing potential to extend their application to other X-linked genes.