GENOTYPING PARTIAL F8 DELETIONS CAUSING SEVERE HAEMOPHILIA A IN HEMIZYGOUS AND HETEROZYGOUS STATE: NEW APPLICATIONS OF INVERSE-PCR.

ZIEGLER, BETIANA MICHELLE; DE BRASI, CARLOS DANIEL; WAISMAN, KAREN; RADIC, CLAUDIA PAMELA; MARCHIONE, VANINA DANIELA; ROSSETTI, LILIANA CARMEN

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias; 2019

Abstract/Resumen: Large F8 deletions are responsible for approximately 8 ? 15 % cases with severe haemophilia A (sHA) and predispose to the development of FVIII therapeutic inhibitors. This work presents two practical approaches for genotyping large deletions both based on inverse-PCR that permitted resolving the cases of two unrelated families with sHA. Family 1 includes an affected patient with a deletion of F8-exon 24?26 whilst family 2 is composed by a family proband with a F8-exon 5-6 deletion and two female relatives (his mother and sister). The objective was to develop cost-effective approaches to diagnose large F8 deletions in hemizygous patients and their potential heterozygous female carriers. We designed and developed a protocol of inverse-PCR (iPCR) combined with long distance-PCR (LD-PCR) to analyse and characterise the breakpoint junctions in family 1, and the approach of inverse shifting-PCR (IS-PCR) to detect the presence/absence of the specific F8-deletion of family 2 in the proband and their female relatives. Based on the BclI restriction map, a LD-iPCR amplification system was designed to discriminate the normal variant (3.5 kb) and the deletion variant associated with 4.2 kb using primers on F8-intron 22 (IVS22-lo) and on F8-intron 23 (24A). Standard-size IS-PCR discrimination system was designed to detect and recognize the 1 kb normal allele (primers Bup1B and IVS6M-up) and the deleted variant (product size of 1.3 kb) obtained from primers IVS65-lo and IVS6M-up. As it was expected, in both families, deletion-specific LD-iPCR or IS-PCR amplification products were obtained in familiar probands and not-observed in normal control samples. Carrier diagnosis in family 2 indicated that both the mother and the sister resulted heterozygous for the deletion. Our findings points the utility to apply cost-effective and reliable approaches such as LD-iPCR and IS-PCR to allow detection and diagnosis of large deletions on X-linked genes, like the F8, to provide valuable information for carrier detection and prenatal diagnosis in families with X-linked disorders like HA.