Haemophilia A Genotyping in Patients with Discrepancy in FVIIIActivity Levels Measured with One-stage and Chromogenic Assays

ROSSETTI LC; MARCHIONE VD; ARIAS, M; SUELDO, E; RADIC CP; ZIEGLER, BM; ABELLEYRO, M; WAISMAN K; BAQUES, A; DE BRASI C

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

Background: In approximately one-third of patients with mild-moderate haemophilia A (HA), there is a discrepancy between the results of FVIII activity (FVIII:C) measured by one-stage-assay (OSA) and chromogenic-substrate-assays (CSA), phenomenon which has been associated with particular missense variants.Aims: Characterize the F8-genotype in persons with HA (PwHA) from Argentina showing discrepancy between FVIII:C assays and compare them with those reported in the International F8-variant database EAHAD (European Association for Haemophilia and Allied Disorders, https://f8-db.eahad.org/).Methods: PwHA samples (N=5 with OSA ranging mild-moderate phenotypes, from 4 families) that showed discrepancy in the measurement of FVIII:C were included. OSA and CSA FVIII:C was determined using two reagent/coagulometer systems, IL/ACL Top 300 and Siemens/Sysmex CS-2500.Genotyping: peripheral blood leukocyte-extracted genomic-DNA was mutational screened by PCR-amplification of all coding and regulatory regions of F8 followed by conformation sensitive gel electrophoresis (CSGE) and selected-amplimers were characterized by Sanger sequencing. Pathogenicity ofF8-variants was classified according the ACMG criteria.Results: The HA-causative F8-genotype was identified in all 5 PwHA (Table 1) and classified as missense-type: one located in FVIII-C1-Domain and 4 in FVIII-A3-Domain. None of them was previously associated with assay discrepancy in EAHAD. Two of them were not reported (c.6246C>A/p.(Ser2082Arg); c.5508G>T/p.(Trp1836Cys)), whereas the reported ones were associated with different (mild-moderate-severe) phenotypes but only based on presumed OSA (Table 1).Conclusions: Thus far, in EAHAD database, only 28 missense HA-causative variants were associated with FVIII:C assay discrepancy, and none of them matched with our 5 cases from Argentina, which showed different phenotype severities in EAHAD when were reported. Our data may reveal some degree of misclassification of phenotype severity in HA, which may be associated with the rare application of CSA to measure FVIII:C complementing OSA levels. Consequently, our findings reinforce the convenience to base the initial diagnosis of non-severe-HA phenotype on the results of both, one-stage and chromogenic assays.