Cost-effective algorithm for molecular diagnosis of families with severe Haemophilia A in Argentina.

VANINA MARCHIONE; MARTÍN M ABELLEYRO; PRIMIANI L; CLAUDIA P. RADIC; DANIELA NEME; MIGUEL TEZANOS PINTO; CARLOS DE BRASI; LILIANA C. ROSSETTI

Berlin

Congreso; XXVI Bienal Congress and 63rd Annual Scientific and Standardization Committee (SSC) Meeting of the International Society on Thrombosis and Haemostasis (ISTH); 2017

Sociedad Internacional de Hemostasia y Trombosis

Haemophilia A (HA), the commonest X-linked coagulopathy, is caused by defects in the factor VIII gene (F8). Due to F8 size and complexity and mutational heterogeneity, gene testing in HA still represents a technical challenge. A half of the severe HA (sHA) is caused by recurrent invertions disrupting F8 at IVS33 (Inv22) and IVS1 (Inv1). The rest of sHAs are mostly caused by families-specific large or small del/ins and point mutations. Aims: Present a cost-effective gene testing algorithm for families with sHA suitable for developing countries.Methods: Leukocite-extracted genomic DNA from patients are subjected to sequential F8 genotyping protocols (fig 1): Inv22/Inv1 diagnosis by inverse shifting PCR, ver.2016; PCR amplicication targeting all relevant F8 sequences in 38 products to detect large deletions in inversion negative cases (designing specific gap-PCR or qPCR approaches for carrier diagnosis); and conformation sensitive gel electrophoresis (CSGE) screening for small mutations on the F8 amplimers in multiplex and characterization of the anomalous CSGE product by Sanger sequencing. Genotype/Phenotype assignment of the observed variant is achived by applying internationally accepted criteria.