Abstract
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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
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Validation Study
MeSH terms
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Anticoagulants / administration & dosage*
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Anticoagulants / adverse effects
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Anticoagulants / pharmacokinetics
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Aryl Hydrocarbon Hydroxylases / genetics*
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Aryl Hydrocarbon Hydroxylases / metabolism
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Blood Coagulation / drug effects*
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Blood Coagulation / genetics
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Cytochrome P-450 CYP2C9
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DNA / analysis*
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DNA / chemistry
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DNA Mutational Analysis
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Feasibility Studies
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Fluorescent Dyes
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Gene Frequency
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Genotype
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Humans
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Linkage Disequilibrium
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Mixed Function Oxygenases / genetics*
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Mixed Function Oxygenases / metabolism
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Molecular Diagnostic Techniques* / instrumentation
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Mouth Mucosa / chemistry
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Nucleic Acid Conformation
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Nucleic Acid Denaturation
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Pharmacogenetics / instrumentation
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Pharmacogenetics / methods*
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Phenotype
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Polymerase Chain Reaction
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Polymorphism, Genetic
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Reproducibility of Results
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Sensitivity and Specificity
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Taq Polymerase
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Temperature
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Time Factors
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Vitamin K Epoxide Reductases
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Warfarin / administration & dosage*
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Warfarin / adverse effects
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Warfarin / pharmacokinetics
Substances
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Anticoagulants
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Fluorescent Dyes
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Warfarin
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DNA
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Mixed Function Oxygenases
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CYP2C9 protein, human
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Cytochrome P-450 CYP2C9
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Aryl Hydrocarbon Hydroxylases
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VKORC1 protein, human
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Vitamin K Epoxide Reductases
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Taq Polymerase