Evaluation of warfarin resistance using transcription activator-like effector nucleases-mediated vitamin K epoxide reductase knockout HEK293 cells.

ABSTRACT: Single nucleotide polymorphisms in the vitamin K epoxide reductase (VKOR) gene have been successfully used for warfarin dosage prediction. However, warfarin resistance studies of naturally occurring VKOR mutants do not correlate with their clinical phenotype. This discrepancy presumably arises because the in vitro VKOR activity assay is performed under artificial conditions using the non-physiological reductant dithiothreitol.The aim of this study is to establish an in vivo VKOR activity assay in mammalian cells (HEK293) where VKOR functions in its native milieu without interference from endogenous enzymes.Endogenous VKOR activity in HEK293 cells was knocked out by transcription activator-like effector nucleases (TALENs)-mediated genome editing.Knockout of VKOR in HEK293 cells significantly decreased vitamin K-dependent carboxylation with vitamin K epoxide (KO) as substrate. However, the paralog of VKOR, VKORC1L1, also exhibits substantial ability to convert KO to vitamin K for carboxylation. Using both VKOR and VKORC1L1 knockout cells, we examined the enzymatic activity and warfarin resistance of 10 naturally occurring VKOR mutants that were reported previously to have no activity in an in vitro assay. All 10 mutants are fully active; five have increased warfarin resistance, with the order being W59R>L128R?W59L>N77S?S52L. Except for the L128R mutant, this order is consistent with the clinical anticoagulant dosages. The other five VKOR mutants do not change VKOR's warfarin sensitivity, suggesting that factors other than VKOR play important roles. In addition, we confirmed that the conserved loop cysteines in VKOR are not required for active site regeneration after each cycle of oxidation.

SUBMITTER: Tie JK 

PROVIDER: S-EPMC3745541 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature