1. IN SILICO STRUCTURAL ANALYSIS OF FVIII MOLECULAR CHANGES FROM MISSENSE PATHOGENIC VARIANTS IN NON-SEVERE HEMOPHILIA A PATIENTS DISPLAYING FVIII:C DISCREPANCY BETWEEN ONE-STAGE VS CHROMOGENIC ASSAYS

LILIANA C. ROSSETTI; ZIEGLER, BETIANA; ARIAS M; SUELDO E; MARCHIONE VD; CLAUDIA P. RADIC; MIGUEL M. ABELLEYRO; BAQUES A; DE BRASI CD

Londres

Congreso; XXX Congress of the International Society on Thrombosis and Haemostasis (ISTH),; 2022

Background: Hemophilia A (HA) associates with a qualitative orquantitative deficiency in the coagulation factor VIII (FVIII). Aboutone-third of patients with non-severe HA carry particular missensevariants, which show discrepant results between FVIII activity lev-els (FVIII:C) measured by one-stage-assay (OSA) or chromogenic-substrate-assays (CSA).Aims: Analyze possible mechanisms underlying OSA vs. CSA FVIII:Cdiscrepancy in five patients with HA-causative missense variants byin silico structural analysis.Methods: Five patients with non-severe HA from four families as-sociated with discrepant results in samples (OSA, ranging mild-moderate, higher than CSA levels) were included. OSA and CSAFVIII:C were determined using two reagent/coagulometer sys-tems, IL/ACL Top 300 and Siemens/Sysmex CS-2500. Genotyping:leukocyte-extracted genomic-DNA was mutational screened byPCR amplification of all coding and regulatory regions of F8 followedby CSGE and selected-amplimers were characterized by Sanger se-quencing. Pathogenicity of F8-variants was classified according theACMG criteria using the International F8-variant database EAHAD(European Association for Hemophilia and Allied Disorders). 3D-structural analysis of missense variants were performed in silicousing publicly available resources (Figure).Results: Four HA-causative missense variants were identified in all5 patients from Argentina: -one located in FVIII-C1-Domain and 4in FVIII-A3-Domain. None of them was previously associated withassay discrepancy in EAHAD. In silico analysis revealed several typesof protein structural damages in the missense variants (Figure), whichsupport their differential impact in the FVIII miss function and regionchanges accounting for the observed OSA>CSA level discrepancy.Conclusion(s): Our results points the utility to complement the ge-netic studies of missense pathogenic variants in HA affected pa-tients showing FVIII:C assay discrepancy (OSA vs. CSA) with in silicostructural analysis of the molecular impact in the FVIII protein toimprove diagnosis, interpretation of FVIII:C assay discrepancy andthe correct assessment of the HA severity.