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

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

CABA

Jornada; I Jornada de Divulgación de las Actividades del Departamento de Microbiología, Inmunología, Biotecnología y Genética; 2018

. Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires

Text: 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 at thebreakpoint from vicinal sequence templates and the insertion of nucleotides at the junctions observed in both LD events areconsistent with the mechanism of MMBIR (Fig.1).