An integrated multitool analysis contributes elements to interpreting unclassified factor IX missense variants associated with hemophilia B

J Thromb Haemost. 2024 Oct;22(10):2724-2738. doi: 10.1016/j.jtha.2024.07.008. Epub 2024 Jul 15.

Abstract

Background: Dissection of genotype-phenotype relationships in hemophilia B (HB) is particularly relevant for challenging (mild HB) or for HB-associated but unclassified factor (F)IX missense variants.

Objective: To contribute elements to interpret unclassified HB-associated FIX missense variants by a multiple-level approach upon identification of a reported, but uncharacterized, FIX missense variant associated with mild HB.

Methods: Molecular modeling of wild-type and V92A FIX variants, expression studies in HEK293 cells with evaluation of protein (ELISA, western blotting) and activity (activated partial thromboplastin time-based/chromogenic assays) levels after recombinant expression, and multiple prediction tools.

Results: The F9(NM_000133.4):c.275T>C (p.V92A) variant was found in a mild HB patient (antigen, 45.4 U/dL; coagulant activity, 23.6 IU/dL; specific activity, 0.52). Newly generated molecular models showed alterations in Gla/EGF1-EGF2 domain conformation impacting Ca++ affinity and protein-protein interactions with activated factor XI (FXIa). Multitool analysis indicated a moderate impact on protein structure/function of the valine-to-alanine substitution, in accordance with patient and modeling data. Expression studies on the V92A variant showed a specific activity (0.49 ± 0.07; wild-type, 1.0 ± 0.1) recapitulating that of the natural variant, and pointed toward a moderate activation impairment as the main determinant underlying the p.V92A defect. The validated multitool approach, integrated with evidence-based data, was challenged on a panel (n = 9) of unclassified FIX missense variants, which resulted in inferred protein (secretion/function) outputs and HB severity.

Conclusion: The rational integration of multitool and multiparameter analyses contributed elements to interpret genotype/phenotype relationships of unclassified FIX missense variants, with implications for diagnosis, management, and treatment of HB patients, and potentially translatable into other human disorders.

Keywords: genotype–phenotype association; hemophilia B; missense mutation; molecular models; prediction tools.

Publication types

  • Case Reports

MeSH terms

  • Blood Coagulation / genetics
  • Factor IX* / genetics
  • Factor IX* / metabolism
  • Genetic Association Studies
  • Genetic Predisposition to Disease
  • HEK293 Cells
  • Hemophilia B* / blood
  • Hemophilia B* / diagnosis
  • Hemophilia B* / genetics
  • Humans
  • Male
  • Models, Molecular
  • Mutation, Missense*
  • Partial Thromboplastin Time
  • Phenotype*
  • Protein Conformation
  • Structure-Activity Relationship

Substances

  • Factor IX