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

We really appreciated the work presented in the comment by Lee et al., which adds an experimental contribution to our published manuscript. The last decade has brought a new era in hemophilia treatment, with several important improvements in terms of biological properties of therapeutics based on factor and nonfactor products, which overall have the potential to meet the different needs of patients under the guidance of clinicians [3]. Particularly for hemophilia B (HB), replacement therapy with either standard or enhanced half-life products remains the gold standard. The classification of hemophilia as severe, moderate, and mild is important to design and optimize therapy regimens to achieve the highest efficacy, with possibly the widest therapeutic window as a need to minimize the burden for patients. However, the severity classification, crucial as a therapeutic guideline, is paradoxically tied to functional evaluations, placing minor emphasis on the presence of circulating dysfunctional factor (F)IX, which primarily arises from missense variants. These HB-associated endogenous FIX protein forms, expressed at variable levels as a function of the underlying F9 genotype and characterizing a relevant proportion of severe/moderate patients (30%-35% cross-reacting material-positive patients with missense variants), may have implications on the therapeutic outcome, even in terms of balance between plasma and extravascular FIX distribution, the latter contributing to hemostatic function [4,5]. Thus, understanding the genotype–phenotype relationship of HB-associated missense variants would help interpret, characterize, and classify them as well as evaluate their impact on FIX structure/function, with HEK293 cells representing good tools for expressing wild type as well as FIX variants [6], even in our experience .