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

LILIANA C, ROSSETTI; WOODS, A I; BLANCO, A; PAIVA, J; MARCHIONE VD; RADIC CP; ABELLEYRO MM; ZIEGLER, BM; WAISMAN K; ROMERO ML; CASINELLI, MM; SANCHEZ LUCERO, ANALIA; DE BRASI C

Congreso; Virtual Meeting of the International Society on Thrombosis and Haemostasis (ISTH); 2021

Background: Mild- moderate (non- severe, NS) Hemophilia A (HA) as-sociates 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 mutational screened 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: A HA-causativeF8-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. Conclusions: Our practical approach is adequate to characterize the PV in NS-HA 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 FVIII-A2-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.