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

ROSSETTI LILIANA C.; ZIEGLER BETIANA M.; ARIAS M.; SUELDO E.; MARCHIONE VANINA D.; RADIC CLAUDIA PAMELA; ABELLEYRO MIGUEL MARTIN; BAQUES A.; DE BRASI CARLOS D

Londres

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

Background: Hemophilia A (HA) associates with a qualitative or quantitative deficiency in the coagulation factor VIII (FVIII). About one-­third of patients with non-­severe HA carry particular missense variants, which show discrepant results between FVIII activity levels (FVIII:C) measured by one-­stage-­assay (OSA) or chromogenic-­ substrate-­assays (CSA). Aims: Analyze possible mechanisms underlying OSA vs. CSA FVIII:C discrepancy in five patients with HA-­causative missense variants by in silico structural analysis. Methods: Five patients with non-­severe HA from four families associated with discrepant results in samples (OSA, ranging mild-­ moderate, higher than CSA levels) were included. OSA and CSA FVIII:C were determined using two reagent/coagulometersys-tems, IL/ACL Top 300 and Siemens/Sysmex CS-­2500. Genotyping: leukocyte-­extracted genomic-­DNA was mutational screened by PCR amplification of all coding and regulatory regions of F8 followed by CSGE and selected-­amplimers were characterized by Sanger sequencing. Pathogenicity of F8-­variants was classified according the ACMG 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 all 5 patients from Argentina: -­one located in FVIII-­C1-­Domain and 4 in FVIII-­A3-­Domain. None of them was previously associated with assay discrepancy in EAHAD. In silico analysis revealed several types of protein structural damages in the missense variants (Figure), which support their differential impact in the FVIII miss function and region changes accounting for the observed OSA>CSA level discrepancy. Conclusion(s): Our results points the utility to complement the genetic studies of missense pathogenic variants in HA affected patients showing FVIII:C assay discrepancy (OSA vs. CSA) with in silico structural analysis of the molecular impact in the FVIII protein to improve diagnosis, interpretation of FVIII:C assay discrepancy andthe correct assessment of the HA severity.