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

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

Berlin

Congreso; XXVI Bienal Congress and 63rd Annual Scientific and Standardization Committee (SSC) Meeting of the International Society on Thrombosis and Haemostasis (ISTH); 2017

Sociedad Internacional de Hemostasia y Trombosis

Background: Large F8 deletion (LD) genotyping in haemophilia A (HA) (8-15% of severe cases) is important because it associates with the highest risk to develop inhibitors against FVIII replacement therapy.Aims: Characterisation of the molecular event in two F8 LDs, detected by consistent absence of exons 7-12 and 5-7 PCR analysis in patients with severe HA (FVIII:C< 1IU/dL) and inhibitor titters of 260 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 bipartition analysis on DNA samples from hemizygous probands. Long range-PCR (lrPCR) amplifications were performed with primers of the nearest 5´- and 3´ positive PCR tags. F#1´s primary lrPCR yielded a specific signal of 7.8kb and F#2´s, of 5.1kb. Restriction analysis of lrPCR products allowed designing new 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 full characterisation 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 9bp at 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 of sequences surrounding the LD junctions revealed motifs associated with DNA instability (i.e., topoisomerase I consensus cleavage sites, DNA polymerase alpha pause site core sequence, LINE L1 and LTR).