Two Large F8 Deletions possibly related to the mechanism of microhomology-mediated break-induced replication (MMBIR) as a cause of severe haemophilia A and inhibitors

MIGUEL M. ABELLEYRO; VANINA D. MARCHIONE; CLAUDIA P. RADIC; TETZLAFF T; DANIELA NEME; MIGUEL DE TEZANOS PINTO; IRENE B. LARRIPA; LILIANA C. ROSSETTI; CARLOS D. DE BRASI

Berlin

Congreso; XXVI International Society on Thrombosis and Hemostasis Congress; 2017

International Society on Thrombosis and Hemostasis

Background: Large F8 deletion (LD) genotyping in haemophilia A (HA) (8-15% of severe cases) isimportant because it associates with the highest risk to develop inhibitors against FVIII replacementtherapy.Aims: Characterisation of the molecular event in two F8 LDs, detected by consistent absence ofexons 7-12 and 5-7 PCR analysis in patients with severe HA (FVIII:C< 1IU/dL) and inhibitor titters of260 and 13,6BU/mL from family 1 (F#1) and 2 (F#2), respectively.Methods: To chase LD breakpoints, PCR tagging schemes were designed by specific bipartitionanalysis on DNA samples from hemizygous probands. Long range-PCR (lrPCR) amplifications wereperformed with primers of the nearest 5 ́- and 3 ́-positive PCR tags. F#1 ́s primary lrPCR yielded aspecific signal of 7.8kb and F#2 ́s, of 5.1kb. Restriction analysis of lrPCR products allowed designingnew LD-specific amplifications. F#1 (IVS6newup+IVS12newlo) yielded a product of 1.45 kb and F#2(H617_Delup+H700_Dello), of 1.67kb. Sanger sequencing spanning the breakpoints permitted fullcharacterisation of both events.Results: F#1 showed a LD of 23kb (ChrX: 154,952,228-154,975,240) NM_000132.3: c.[787+9445_1903+1662del23013;787+9454insTGTATCCCA] and F#2, a LD of 21.4kb (ChrX:154,968,575-154,989,956) c.[601+2979_1009+745del21380]. F#1 event showed an insertion of 9bpat the recombination site (TGTATCCCA) which is also found at inverted orientation 5bp upstream;and F#2 showed a microhomology of 2bp at the breakpoint junction (Fig. 1). Bioinformatic scanning ofsequences surrounding the LD junctions revealed motifs associated with DNA instability (i.e.,topoisomerase I consensus cleavage sites, DNA polymerase alpha pause site core sequence, LINEL1 and LTR).Conclusions: The microhomology between 5 ́ and 3 ́ ends, the insertion/synthesis of new material atthe breakpoint from vicinal sequence templates and the insertion of nucleotides at the junctionsobserved in both LD events are consistent with the mechanism of MMBIR (Fig. 1).