Comprehensive Genotype Characterization in Non-severe Hemophilia A. Mutational Profile and Assessment of the Bleeding Phenotype in Argentine Patients

BLANCO AN; RADIC C; WAISMAN K; SANCHEZ LUCEROS ANALIA; ROSSETTI, LILIANA CARMEN; PAIVA J; ABELLEYRO M; ROMERO ML; DE BRASI C; WOODS AI; MARCHIONE V; ZIEGLER B; CASINELLI MM

Philadelphia

Congreso; ISTH 2021 VIRTUAL CONGRESS; 2021

International Society on Thrombosis and Haemostasis

Background: Mild-moderate (non-severe, NS) Hemophilia A (HA) associates with minor reduction in factor VIII (FVIII:C) (1-40IU/dL) and involve about 60% of HA cases worldwide. Despite the importance of NS-HA, patients are rarely genotyped, particularly in developing countries. Clinical manifestations and laboratory phenotype are frequently mistaken with VWD2N.Aims: Characterize the F8-genotype and phenotype, exclude VWD2N, in a series of Argentine patients with FVIII:C 1-40IU/dL. Methods: Thirty-eight unrelated-families including 50 individuals (probands and relatives) were studied. The bleeding phenotype was obtained from clinical records.Laboratory: FVIII:C (one-stage method), VWF:Ag (ELISA), VWF:RCo (aggregometry). Mixing studies of the patient/control plasmas to evaluate the presence of inhibitors were undertaken by APTT and VWF:RCo. Genotyping: peripheral blood leukocyte-extracted genomic-DNA was mutationalscreened by PCR-amplification of all coding and regulatory regions of the F8 followed by conformation sensitive gel electrophoresis (CSGE) and selected amplimers were characterized by Sanger sequencing. Exons 17-27 of the VWF were PCR-amplified and directly Sanger-sequenced.Results: Table 1: F8-genotype and the associated phenotype in the Argentinian population withNS-HA.A HA-causative F8-genotype was identified in all 38 families including 28 pathogenic variants (PV) (Table 1). Eighteen (47%) of them showed 8 recurrent PV (range 2-3 families/PV), whereas the remnant 20 (53%) showed non-recurrent (private) F8-PV (Figure 1). No variants were found in VWF. Patients with major hemorrhages: 48.6%. Laboratory phenotype is shown in Table 1.Molecular distribution of F8 variants characterized in our series of NS-HA patients. Conclusions: Our practical approach is adequate to characterize the PV in NSHA patients and relatives. The confirmed diagnosis of NS-HA allows an appropriate treatment and genetic counseling for X-linked inheritance highlighting the role of genetic testing and phenotypic assays. Combined HA+VWD2N was excluded in all patients. The predominance of missense PV (30/38 cases, 79%) mostly targeting the FVIIIA2-domain and the high frequency of recurrent PVs found in NS-HA may reflect a reduced mutational loss due to the relatively conserved reproductive fitness of probands hence preserving the gene pool in the population.