F9 Missense mutations impairing factor ix activation are associated with pleiotropic plasma phenotypes

Background: Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in haemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels. Objectives: To investigate i) expression of a complete panel of missense mutations at FIX activation sites and ii) contribution of F9 genotypes on the FIX pharmacokinetics (PK). Methods: FIXag and activity assays in plasma and after recombinant expression of FIX variants. Analysis of infused FIX PK parameters in patients (n=30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42). Results: The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants result in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. PK analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 hrs, 95% CI 44.3-114.5) in patients (n=7) with the R191/R226 substitutions, which in regression analysis were independent predictors (β coefficient 0.699, p=0.004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX. Conclusions: FIXag levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients’ phenotypes and substitutive treatment.