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.

RADIC C.P.; ABELLEYRO M.M.; TEZANOS PINTO M; TETZLAFF T.; DE BRASI C.D.; LARRIPA I.B.; MARCHIONE V.D.; NEME D.; ROSSETTI L.C.

Berlín

Congreso; 26th Biennial Congress and 63rd Annual Scientific and Standardization Committee (SSC) Meeting of the International Society on Thrombosis and Haemostasis (ISTH).; 2017

International Society on Thrombosis and Haemostasis (ISTH)

Two Large F8 Deletions possiblyrelated to the mechanism of microhomology-mediated breakinduced replication(MMBIR) as a cause of severe haemophilia A and inhibitorsM.M.Abelleyro1, V.D. Marchione1, C.P. Radic1, T. Tetzlaff2, D. Neme3, M. TezanosPinto3,4, I.B. Larripa1, L.C. Rossetti1, C.D. De Brasi1,41IMEX-CONICET-AcademiaNacional de Medicina, Ciudad Autonoma de Buenos Aires, Argentina, 2Universidadde General Sarmiento, Ciudad Autonoma de Buenos Aires, Argentina, 3Fundación dela Hemofilia AlfredoPavlovsky, Ciudad Autonoma de Buenos Aires, Argentina, 4Instituto deInvestigaciones Hematológicas Mariano R. Castex (IIHEMA), ANM. Buenos Aires,Argentina, Ciudad Autonoma de Buenos Aires, ArgentinaBackground: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 inhibitorsagainst FVIII replacement therapy. Aims: Characterisation of the molecularevent in two F8 LDs, detected by consistent absence of exons 7-12 and 5-7 PCRanalysis in patients with severe HA (FVIII:C< 1IU/dL) and inhibitor tittersof 260 and 13,6BU/mL from family 1 (F#1) and 2 (F#2), respectively. Methods: Tochase LD breakpoints, PCR tagging schemes were designed by specific bipartitionanalysis on DNA samples from hemizygous probands. Long range-PCR (lrPCR)amplifications were performed with primers of the nearest 5´- and 3´-positivePCR tags. F#1´s primary lrPCR yielded a specific signal of 7.8kb and F#2´s, of5.1kb. Restriction analysis of lrPCR products allowed designing new LD-specificamplifications. 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 breakpointspermitted full characterisation of both events. Results: F#1 showed a LD of23kb (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 showedan insertion of 9bp at the recombination site (TGTATCCCA) which is also foundat inverted orientation 5bp upstream; and F#2 showed a microhomology of 2bp atthe breakpoint junction (Fig. 1). Bioinformatic scanning of sequencessurrounding the LD junctions revealed motifs associated with DNA instability(i.e., topoisomerase I consensus cleavage sites, DNA polymerase alpha pausesite core sequence, LINE L1 and LTR).