PB0522 - Comprehensive Genotype and Phenotype characterization in siblings with Non-severe Hemophilia A with FVIII:C assay discrepancies

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Co-Author(s)

LR

Liliana C. Rossetti

Independent Researcher
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina (ANM)
Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina

Presenting Author(s)

Mirta Arias, MSc

Biochemist
Hospital Dr. Cesar Milstein
CABA, Ciudad Autonoma de Buenos Aires, Argentina

Co-Author(s)

ES

Emanuel Sueldo

Biochemistry
Hospital Dr. Cesar Milstein
CABA, Ciudad Autonoma de Buenos Aires, Argentina
BZ

Betiana Ziegler

MSc
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina (ANM)
CABA, Ciudad Autonoma de Buenos Aires, Argentina
PR

Pamela Radic

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

Martín Abelleyro

MSc
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina (ANM)
CABA, Ciudad Autonoma de Buenos Aires, Argentina
CD

Carlos De Brasi

MSc
Instituto de Medicina Experimental (IMEX), CONICET-Academia Nacional de Medicina (ANM)
CABA, Ciudad Autonoma de Buenos Aires, Argentina
AB

Alejandra Baques

Medical Doctor
Hospital Dr. Cesar Milstein
CABA, 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 two pair of sibling patients with HA-causative missense variants by genotype–phenotype correlation analysis.

Methods: Four patients with non-severe HA from two families associated with discrepant results in samples (OSA, ranging 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. F8-Genotyping: leukocyte-extracted genomic-DNA was mutational screened by PCR-amplification of all coding and regulatory regions followed by CSGE and selected-amplimers were characterized by Sanger sequencing. A detailed follow up of their clinical reports was done (Table1). 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: Two HA-causative missense variants were identified in the 4 patients: -both located in FVIII-A3-Domain. None of them was previously associated with assay discrepancy in EAHAD. In silico structural analysis shows several types of protein structural damages in the missense variants (Figure), which support their differential impact in the FVIII miss-function for the observed OSA>CSA level discrepancy.

Conclusion(s): In the two pairs of brothers, very similar FVIII:C values were observed for each assay (OSA/CSA), and the discrepancy between assays was evident and reproducible, indicating that the effect of the specific F8-variant is associated with both: FVIII:C level measured and discrepancy, even though there are some differences in the clinical phenotype, pointing the utility to complement with F8-genotyping studies in this patients.