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.
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