Vitamin K epoxide reductase (VKOR), encoded by the VKORC1 gene, is the target of warfarin, a commonly prescribed anticoagulant with an extremely narrow therapeutic window. Warfarin-related hemorrhages due to over-dosing results in greater than 21,000 hospitalizations yearly, while under-dosing with the drug may lead to therapeutic failures and an unknown number of thrombotic events. Coding-region variation in VKORC1 is well recognized to confer warfarin resistance. Thus, knowing a patient’s VKOR coding-region sequence before initiating treatment could allow for more precise dosing if we knew the associated warfarin resistance phenotype. To this end, we are measuring the warfarin resistance phenotype of all 3,260 VKOR missense variants using deep mutational scanning, a method that combines high throughput mutagenesis with deep sequencing. Libraries of VKOR variants will be binned according to functional activity using a flow cytometry assay across a range of warfarin doses, allowing us to calculate an IC50 value for each variant. These data will comprise a VKOR sequence-resistance map that reveals how each singly mutated variant we observe could potentially impact a patient’s warfarin resistance. Our ultimate goal is to develop a VKOR sequence-based dosing algorithm to help clinicians start patients on the appropriate dose of warfarin to minimize therapeutic failures. |