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

MARCHIONE VD; NEME D; ROSSETTI LC; RADIC CP; DE TEZANOS PINTO M; DE BRASI CD; ABELLEYRO MM; TETZLAFF T; LARRIPA IB

CIUDAD AUTONOMA DE BUENOS AIRES

Jornada; I Jornada de Divulgación de las Actividades del Departamento de Microbiología, Inmunología, Biotecnología y Genética. Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires; 2018

Text: 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). Conclusions: The microhomology between 5´ and 3´ ends, the insertion/synthesis of new material at the breakpoint from vicinal sequence templates and the insertion of nucleotides at the junctions observed in both LD events are consistent with the mechanism of MMBIR.