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

Abstract

Summary Background 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. Objectives 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. Methods Endogenous VKOR activity in HEK293 cells was knocked out by transcription activator-like effector nucleases (TALENs)-mediated genome editing. Results and Conclusions 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 ten naturally occurring VKOR mutants that were reported previously to have no activity in an in vitro assay. All ten 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.

Recent trends in the metabolism and cell biology of vitamin K with special reference to vitamin K cycling and MK-4 biosynthesis

“…In contrast to the in vitro assay data described above, these cell systems showed much better correlations of IC 50 values with warfarin dosage requirements in patients ( 86,98 ). However, 5 out of 10 variants that previously had been found to be associated with a resistant phenotype in humans ( 92 ) did not show resistance when expressed in cells with the double-gene knockout ( 86 ). This suggested that other factors apart from VKOR activity were responsible for warfarin resistance.…”

“…Both groups expressed putative resistant variants of VKOR in HEK293 cells in which either full-length factor IX ( 98 ) or factor IXgla-PC ( 86 ) had been coexpressed as the reporter for VKOR activity. One difference was that Stafford's group knocked out both VKOR and VKORL1 in HEK293 cells to eliminate endogenous K>O reductase activity ( 86 ). In contrast to the in vitro assay data described above, these cell systems showed much better correlations of IC 50 values with warfarin dosage requirements in patients ( 86,98 ).…”

“…Recently, more physiologically representative methods for assessing structure-function relationships of VKOR variants in humans or animals with a warfarin-resistant phenotype were independently introduced by the groups of Oldenburg ( 98 ) and Stafford ( 86 ). Both groups expressed putative resistant variants of VKOR in HEK293 cells in which either full-length factor IX ( 98 ) or factor IXgla-PC ( 86 ) had been coexpressed as the reporter for VKOR activity.…”

“…One difference was that Stafford's group knocked out both VKOR and VKORL1 in HEK293 cells to eliminate endogenous K>O reductase activity ( 86 ). In contrast to the in vitro assay data described above, these cell systems showed much better correlations of IC 50 values with warfarin dosage requirements in patients ( 86,98 ). However, 5 out of 10 variants that previously had been found to be associated with a resistant phenotype in humans ( 92 ) did not show resistance when expressed in cells with the double-gene knockout ( 86 ).…”

“…In a refi nement to their cell-based model, transcription activator-like effector nucleases were used to knockout the endogenous VKOR activity in HEK293 cells ( 86 ). This enabled the study of the reduction and possible recycling of vitamin K in VKOR -defi cient cells without using warfarin.…”