THREE LARGE F8 DELETIONS POSSIBLY CAUSED BY THE MECHANISM OF MICRO-HOMOLOGY MEDIATED BREAK-INDUCED REPLICATION (MMBIR) AS A CAUSE OF SEVERE HEMOPHILIA A WITH THERAPEUTIC FVIII INHIBITOR DEVELOPMENT

ABELLEYRO, M.M.; ZIEGLER, BM; NEME, D.; ROSSETTI CL; RADIC CP; DE BRASI CD

Congreso; ISTH; 2023

Background: Large F8 deletions (LD) cause 8-15% of severe cases of hemophilia A (HA). LD genotyping are crucial because they typically associate with inhibitors against the FVIII replacement therapy.Aims: To characterize the F8 LD molecular events in three families with severe HA (FVIII:C< 1IU/dL), family 1 (F#1), 2 (F#2) and 3 (F#3), which showed absence of exon 7, exons 6-22 and 23-25; and inhibitor titters of 0.6, 0.6 and 1.3BU/mL , respectively.Methods: To chase the breakpoints of F#1 and F#3, case-specific bipartition PCR-tagging schemes were designed and applied in hemizygous probands. Long-range-PCR (lrPCR) amplifications were performed with primers in the nearest 5 ́- and 3 ́-positive PCR tags. F#1 lrPCR yielded a specific signal of 3.1kb and F#3, of 3.0kb. The F#2 proband’s aunt was diagnosed as HA carrier using MLPA analysis and a LD allele specific signal of 5.8kb was obtained by inverse lrPCR from the proband. In all three cases Sanger sequencing of the PCR products spanning the breakpoints permitted full characterization of the molecular events.Results: F#1 showed a LD of 5.6kb NM_000132.4:c.788-4718_4718_1009+679delinsAA, F#2 a LD of 103.2kb NM_000132.4:c.[670+394_6429+12440del; 6429+12447T>A] and F#3 a LD of 6.6kb NM_000132.4:c.6430-15877_6900+4699. F#1 event showed an insertion of 2bp at the recombination site (AA), F#2 and F#3 showed micro-homologies of 5 and 4bp, respectively (Figure 1). Bioinformaticzs of LD junctions revealed sequence motifs associated with DNA instability (e.g., Jurka, topoisomerase I consensus cleavage sites, LINE L1, LTR, secondary DNA and non-B DNA structures) (Table 1). Conclusion: The molecular evidence is compatible with micro-homology mediated break-induced replication (MMBIR) model in all LD events, which associate with the highest clinical severity in HA.