PB0196 - 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

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Liliana C. Rossetti, PhD

Investigator
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina

Co-Author(s)

Betiana M. Ziegler

Doctoral Fellow
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
Mirta del valle Arias

Biochemist
Laboratorio de Hematología y Hemostasia. Hospital César Milstein
CABA Bs As, Ciudad Autonoma de Buenos Aires, Argentina
Rodolfo E. Sueldo

Biochemist
Laboratorio de Hematología y Hemostasia. Hospital César Milstein
Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
VM

Vanina D. Marchione

Research
Instituto de Medina Experimental (IMEX)-CONICET- Academia Nacional de Medicina (ANM)
Ciudad Autónoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
CR

Claudia P. Radic

Investigator
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
MA

Miguel M. Abelleyro

Investigator
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autónoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
AB

Alejandra Baqués

Hematologist, MD
Servicio de Hemofilia. Hospital Dr. César Milstein.
Ciudad Autónoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
CD

Carlos D. De Brasi

Investigator
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina
Ciudad Autonoma de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina

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/coagulometer systems, 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 silico using 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 and the correct assessment of the HA severity.