PB0532 - Potential Structural and functional impact of the F8-variants influencing assay differences in patients with Haemophilia A and discrepancy in FVIII:C assays

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

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
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
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: In approximately one-third of patients with non-severe Hemophilia A (nsHA), 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: Analyze the potential structural and functional impact associated with different F8 variants over the FVIII protein in the OSA and CSA assays in patients with HA and FVIII:C discrepancy.

Methods: 139 patients having bleeding disorders with a diagnostic of HA between 2013 and 2023 were evaluated. We performed laboratory hemostasis studies in all samples (Prothrombin time, Activated Partial thromboplastin time (aPTT), Thrombin Time (TT), aPTT 1:1 mix, OSA, CSA, vWF:Ag, vWF:CoR, IL/ACL Top-300 and Siemens Healthineers/Sysmex CS-2500). The Initial classification of the disease was performed with OSA (Severe Hemophilia A: sHA < 0.01 IU/mL, Moderate Hemophilia A: modHA 0.01-0.05 IU/mL, Mild Hemophilia A: miHA 0.05-0.40 IU/mL). FVIII discrepancy was defined as OSA/CSA ≤ 0.5 or CSA/OSA ≥ 2.0, correlating it with the clinical phenotype. Genotyping: PCR-amplification method followed by conformation sensitive gel electrophoresis (CSGE) and Sanger sequencing.

Results: 109 patients were initially classified as sHA, and 30 patients were nsHA. 10 patients with nsHA showed discrepancies between the methods, F8-genotype was analyzed in the 10 cases. 30% of patients with nsHA showed discrepancies. A comprehensive analysis of the correlation of genotype and phenotype is showed in Table 1.

Conclusion(s): Potentially FVIIIa heterotrimer destabilisation and premature loss of cofactor activity could be induced by the F8-missense variants found involved in OSA/CSA and in the CSA/OSA case may possibly have a direct effect on binding of FVIII to vWF. We conclude that non-severe haemophilia patients could benefit if chromogenic assay is included in diagnosing and classification of severity haemophilia, and it also could help to individualize group of patients that could receive early replacement treatment.