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

PRIMIANI L.; TEZANOS PINTO M; ABELLEYRO M.M.; NEME D.; ROSSETTI L.C.; MARCHIONE V.D.; RADIC C.P.; DE BRASI C.D.

Berlín

Congreso; 26th Biennial Congress and 63rd Annual Scientific and Standardization Committee (SSC) Meeting of the International Society on Thrombosis and Haemostasis (ISTH).; 2017

International Society on Thrombosis and Haemostasis (ISTH).

PB 1986 | Cost-Effective Algorithmfor Molecular Diagnosis of Families with Severe Haemophilia A in ArgentinaV.D.Marchione1, M.M. Abelleyro1, L. Primiani2, C.P. Radic1, D. Neme2, M. de TezanosPinto2,3, C.D. De Brasi1,3, L.C. Rossetti11Institutode Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina (ANM),Ciudad Autonoma De Buenos Aires (CABA), Argentina, 2Fundación de la Hemofilia AlfredoPavlovsky, Ciudad Autónoma de Buenos Aires, Argentina, 3Instituto deInvestigaciones Hematológicas Mariano R. Castex (IIHEMA), ANM., Ciudad Autónomade Buenos Aires, ArgentinaBackground:Haemophilia A (HA), the commonest X-linked coagulopathy, is caused by defectsin the factor VIII gene (F8). Due to F8 size and complexity and mutationalheterogeneity, gene testing in HA still represents a technical challenge. Ahalf of severe HA (sHA) is caused by recurrent inversions disrupting F8 atIVS22 (Inv22) and IVS1 (Inv1). The rest of sHAs are mostly caused byfamily-specific large or small del/ins and point mutations. Aims: Present acost-effective gene testing algorithm for families with sHA suitable fordeveloping countries. Methods: Leukocyte-extracted genomic DNA from patientsare subjected to sequential F8 genotyping protocols (Fig 1):Inv22/Inv1diagnosis by inverse shifting PCR, ver.2016; PCR amplificationtargeting all relevant F8 sequences in 38 products to detect large deletions ininversion negative cases (designing specific gap-PCR or qPCR approaches forcarrier diagnosis); and conformation sensitive gel electrophoresis (CSGE)screening for small mutations on the F8 amplimers in multiplex andcharacterisation of the anomalous CSGE product by Sanger sequencing.Genotype/Phenotype assignment of the observed variant is achieved by applyinginternationally accepted criteria. Results: We characterised the causativemutation 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%, type2), 4 Inv1 (1.2%), 18 large deletions (5.5%), 58 small ins/del (17.8%), 48missense (14.8%), 32 nonsense (9.8%), and 12 splicing defects (3.7%). Conclusions:The presented algorithm allowed characterisation of the sHA causative mutationin 96.2% of families in a relatively rapid and cost-effective way. The remnant4% may be due to the intrínsico limitation of the CSGE screening (≈95%), thetheoretical extent of the F8 PCR amplification scheme to detect deep intronicsplicing defects and other rare mutations, and the failure of gene dosageanalyses to detect duplications.