Warfarin Resistance Due to a Val29Leu Mutation in the VKORC1 Protein.

Warfarin resistance is a common clinical problem encountered by physicians of all subspecialities. Medical noncompliance and dietary indiscretions are felt to be the usual causes of a subject’s “resistance” to anticoagulation with coumarin derivatives. Warfarin’s target is the vitamin K epoxide reductase complex (VKOR). This complex functions within the vitamin K dependent γ-carboxylase system to recycle oxidized vitamin K. Until recently, the genetic sequence of components of this complex remained a mystery (Li et al., Nature 2004). Mutations in a subunit of this complex named vitamin K epoxide reductase complex subunit 1 (VKORC1) were isolated in human subjects with warfarin resistance (Rost et al., Nature 2004). Knowledge of this coding sequence has enabled us to study individual subjects suspected to have a genetic source for their warfarin resistance. Our study subject is a 24-year-old Caucasian female who received warfarin after developing bilateral pulmonary emboli. She required 30 mg of warfarin daily to achieve an international normalized ratio greater than 2.0. After institutional review board approval and with the patient’s informed consent, genomic DNA was extracted from the subject’s peripheral blood. The three exons comprising the VKORC1 gene were amplified by polymerase chain reaction (PCR). PCR primers were designed using the published sequence and online Primer3 software. Prolonged extension times were required to obtain good amplification of all three exons. After purification, the PCR products were sequenced and analyzed. The sequence analysis demonstrated a heterozygous G→T mutation in exon 1 at base pair 85. This results in a Val29Leu substitution, which has been described in a warfarin resistant patient by Rost et al. The second and third exons matched completely with the published sequence. The gene frequency of the mutation was then determined by examining 400 random, discarded DNA samples. The HypCH4 IV restriction enzyme cleaves at 2 positions in exon 1 in the wild-type genotype, but at just one position if our known mutation is present. The mutation was not identified in any sample.

In conclusion, we have identified a second patient with a Val29Leu substitution in vitamin K epoxide reductase resulting in warfarin resistance. Our analysis of 400 random samples has not identified any other example of this mutation, indicating that this is a rare occurrence. Genetic analysis of patients requiring more than 20 mg of warfarin daily would likely reveal more mutations in the VKORC1 protein. The identification of such mutations would provide valuable insight into the structure-function relationships of this protein complex.