Rapid melting curve analysis for genetic variants that underlie inter-individual variability in stable warfarin...

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.