Most variants found in clinical DNA sequencing are classified as variants of uncertain significance
(VUS), meaning these variants do not have enough information to be acted on clinically. Furthermore, of the
variants that are classified as likely pathogenic or pathogenic, the clinical outcomes for patients with these
variants are not often apparent from the classification alone. In particular, in hemophilia B, caused by variation
in the F9 gene, it is known that the precise genetic variant is directly tied to clinical outcomes like disease
severity, spontaneous bleeding risk, and risk of developing neutralizing antibodies (inhibitors) to replacement
therapy. However, because of the high rate of de novo variation in this gene, many patients have novel
variants for which clinical outcomes are not easily predictable. To combat this problem, this project aims to
combine and utilize a set of experimental tools to characterize each of the 8,759 possible variants in F9 in high
throughput with multiple functional assays, yielding a powerful dataset for reinterpreting VUS and predicting
clinical outcomes in F9.
As Factor IX is normally secreted from the cell, the first goal will be to generate a membrane-tethered
version of Factor IX that can be displayed on the surface of mammalian cells, so that it is amenable to deep
mutational scanning. This will allow me to assay key functions of Factor IX, including secretion and -
carboxylation for all possible missense variants of Factor IX. Second, I will permeabilize and assess
abundance of intracellular Factor IX as a proxy for detecting null alleles which are correlated with inhibitor risk.
Each of these high-throughput assays will annotate each missense variant with a quantitative score
documenting the effect of that variant on the selected phenotype. Once the data are collected, the relative
contribution of each Factor IX function to disease will be examined by comparing which functions are lost in
variants to sequencing-based data from MyLifeOurFuture, a cohort of 1,632 patients with hemophilia B.
Through this study, interpretable functional scores for thousands of Factor IX variants will be generated,
potentially informing clinical decision-making. Collectively, these experiments will generate valuable data for
the hemostasis community, while also establishing a new approach for scalable and accurate functional testing
of variants in secreted proteins.