An integrated multi-tool analysis contributes elements to interpreting unclassified factor IX missense variants associated with haemophilia B

Background: Dissection of genotype-phenotype relationships in haemophilia B (HB) is particularly relevant for challenging (mild HB) or for HB-associated but unclassified factor IX (FIX) missense variants. Here, the identification of a FIX missense variant associated with mild HB, reported but unclassified, prompted a multiple-level approach to contribute elements to interpret unclassified HB-associated FIX missense variants. Methods: Molecular modelling of wild-type (WT) and V92A FIX variants, expression studies in HEK293 cells with evaluation of protein (ELISA, Western blotting) and activity (aPTT-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 on Ca++ affinity and protein-protein interactions with FXIa. Multi-tool analysis indicated a moderate impact on protein structure/function of the valine-to-alanine substitution, in accordance with patient's and modelling data. Expression studies on the V92A variant showed a specific activity (0.49±0.07; WT, 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 multi-tool 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. Conclusions: The rational integration of multi-tool and multi-parameter 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.