Rapid melting curve analysis for genetic variants that underlie inter-individual variability in stable warfarin dosing.
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.
Department of Internal Medicine, Division of Cardiology, University of Utah School of Medicine, Salt Lake City, UT, USA. john.carlquist@intermountainmail.org
John F
JF
Carlquist
Jason T
JT
McKinney
Zachary P
ZP
Nicholas
Jessica L
JL
Clark
Samera F
SF
Kahn
Benjamin D
BD
Horne
Joseph B
JB
Muhlestein
Heidi T
HT
May
Jeffrey L
JL
Anderson
D000925:Anticoagulants / Q000008:administration & dosage* / Q000009:adverse effects / Q000493:pharmacokinetics; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001777:Blood Coagulation / Q000187:drug effects* / Q000235:genetics; D065729:Cytochrome P-450 CYP2C9; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004252:DNA Mutational Analysis; D005240:Feasibility Studies; D005456:Fluorescent Dyes; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D015810:Linkage Disequilibrium; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation; D009061:Mouth Mucosa / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010597:Pharmacogenetics / Q000295:instrumentation / Q000379:methods*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D013696:Temperature; D013997:Time Factors; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000008:administration & dosage* / Q000009:adverse effects / Q000493:pharmacokinetics
D000925:Anticoagulants; D005456:Fluorescent Dyes; D014859:Warfarin; D004247:DNA; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases; D019914:Taq Polymerase