F9 missense variant hot spots associated with qualitative protein defects causing hemophilia B

Abstract

Hemophilia B, a rare bleeding disorder, is caused by genetic variations in F9. Although quantitative factor IX (FIX) deficiencies are successfully treated by protein replacement therapy, qualitative defects may result in dysfunctional proteoforms in the circulation, potentially interfering with prophylactically or therapeutically administered recombinant FIX (rFIX) concentrates. To delineate the F9 missense variants associated with qualitative defects, we integrated genotype and phenotype from the European Association for Haemophilia and Allied Disorders F9 Coagulation Factor Variant Database. Of the 663 patients for whom activity (FIX:Act) and antigen (FIX:Ag) data were available, 40% of patients with severe hemophilia (n = 248), 50% of patients with moderate hemophilia (n = 244), and 47% of patients with mild hemophilia (n = 171) had cross-reactive material defined as FIX:Ag ≥40%. Variants associated with qualitative defects were predominantly localized (1) at proteolytic sites for FIX processing and activation, (2) within exosite II of the serine protease domain, and (3) at calcium ion coordinating residues within the Gla/EGF-1 domain of the FIX light chain. To study the effect of dysfunctional FIX proteoforms on thrombin generation in the presence of rFIX, we investigated 2 individuals with hemophilia B harboring a variant of unknown significance with an unexplained bleeding phenotype despite prophylaxis. Ex vivo therapeutic monitoring using patient plasma supplemented with rFIX concentrates, bypassing agent or emicizumab, enabled head-to-head comparison and revealed limited normalization of the prolonged initiation phase in coagulation. Alignment of information on genotype with functional proteotype may clarify the heterogeneity in bleeding phenotype and treatment response and provide a stepping stone for personalized care.