PB0164 - Full characterization of three F8 deletions causing severe hemophilia A with high responding FVIII inhibitors may be associated with the mechanism of microhomology-mediated break-induced replication.

Sunday, June 25, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Miguel M. Abelleyro, PhD

Investigator
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autónoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina

Co-Author(s)

Betiana M. Ziegler

Doctoral Fellow
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
DN

Daniela Neme

Hematologists
Fundación de la Hemofilia Alfredo Pavlovsky
Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
Liliana C. Rossetti, PhD

Investigator
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
CR

Claudia P. Radic

Investigator
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
CD

Carlos D. De Brasi

Investigator
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina

Background: Large F8 deletions (LD) cause 8-15% of severe cases of hemophilia A (HA). LD genotyping is 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), whose hemizygous probands showed high responding inhibitors of 8, 614 and 134BU/mL, respectively and associated with molecular absence of exon 7, exons 6-22 and 23-25.

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 an LD allele specific signal of 5.8kb was obtained from her DNA sample in heterozygous status by inverse lrPCR. In all three cases Sanger sequencing of 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_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). Bioinformatics 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(s): 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.