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

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

Berlin

Congreso; XXVI International Society on Thrombosis and Hemostasis Congress 2017; 2017

International Society on Thrombosis and Haemostasis

Background: 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 severe HA (sHA) is caused by recurrent inversions disrupting F8 at IVS22 (Inv22) and IVS1 (Inv1). The rest of sHAs are mostly caused by family-specific large or small del/ins and point mutations.Aim: Present a cost-effective gene testing algorithm for families with sHA suitable for developing countries.Methods: Leukocyte-extracted genomic DNA from patients are subjected to sequential F8 genotyping protocols (Fig. 1): Inv22/Inv1 diagnosis by inverse shifting PCR, ver.2016; PCR amplification 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 characterisation of the anomalous CSGE product by Sanger sequencing. Genotype/Phenotype assignment of the observed variant is achieved by applying internationally accepted criteria.Results: We characterised the causative mutation in 325 families with sHA, whereas 13 families remain uncharacterised (3.8%). We found 153 cases with the Inv22 (47.1%) (82% Inv22 type 1 and 18%, type 2), 4 Inv1 (1.2%), 18 large deletions (5.5%), 58 small ins/del (17.8%), 48 missense (14.8%), 32 nonsense (9.8%), and 12 splicing defects (3.7%).Conclusion: The presented algorithm allowed characterisation of the sHA causative mutation in 96.2% of families in a relatively rapid and cost-effective way. The remnant 4% may be due to the intrinsic limitation of the CSGE screening (≈95%), the theoretical extent of the F8 PCR amplification scheme to detect deep intronic splicing defects and other rare mutations, and the failure of gene dosage analyses to detect duplications.