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.
While it is known that variation in the F9 gene causes hemophilia B, systematic mechanistic understanding of the effects of each of these variants is still lacking and prevents accurate clinical assessment of long term outcomes. This project proposes to develop genomic technologies to characterize all 8,759 possible single nucleotide variants in F9 in parallel for functional assessment of biochemical properties, including proper secretion, post-translational modification, and enzymatic activity. These data will then be integrated with existing clinical sequencing data from 1,632 patients with hemophilia B to interrogate the effects of each variant on clinical disease severity and risk of developing neutralizing antibodies (inhibitors) to treatment, thus providing a repository of detailed variant information for physicians and their patients.
