Warfarin anticoagulation therapy is complicated by its narrow therapeutic index and by wide inter-individual differences in
dosing requirements arising, in part, from genetic factors. The present report describes the development, validation and feasibility
testing of a rapid genotyping assay that concurrently detects the
CYP2C9*2 and *3 variants along with the
VKORC1 C1173T polymorphism. The study employed melting curve analysis using labeled probes and compared two detection instruments
(the HR-1 and the R.A.P.I.D. LT) to two previously validated methods, 5′ nuclease allelic discrimination (Taqman
®) assay and cycle sequencing. The HR-1 detected 189 true negatives and 113 true positives; 1 wild-type sample was mistyped
as a heterozygote by both instruments. Sequencing of that sample confirmed it to be a CC homozygote; however, a rare C > T
polymorphism was discovered 1 base 5′ from the *2 polymorphic site, presumably causing the mistaken genotype by melting curve.
Both methods had sensitivity = 1.00 and specificity > 0.99. Combined with a method for rapid buccal swab DNA extraction, genotyping
results were obtained in a median of 59 min. These methods should facilitate genotype-driven warfarin dosing in “real-time”
clinical practice.
Keywords Genotype - Warfarin - Melting-curve analysis