title: Detection and identification of cytochrome P-450 2C9 alleles *1, *2, and *3 by high-resolution melting curve analysis of PCR amplicons.
abstract: High-resolution melting curve analysis using a fluorescent DNA binding dye can detect sequence variations in a closed-tube system without labeled primers or probes. We developed and verified a melting analysis assay for common single nucleotide polymorphisms of cytochrome P-450 (CYP) 2C9 that affect warfarin metabolism. We used this method to genotype 84 patients receiving warfarin. For wild-type, *1/*1, 50% fluorescence corresponded to a mean+/-SD of 87.17+/-0.05 degrees C, whereas *2/*2 was 0.4 degrees C lower. The *1/*2 melting curve was easily distinguished from *1/*1 and *2/*2 based on transition temperature and shape. Exon 7 showed a more complex melting curve; however, genotypes *1/*1, *1/*3, and *3/*3 were easily distinguishable. Melting curves were highly reproducible (SD of temperature for multiple fluorescence values 0.04 degrees C-0.11 degrees C; mean, 0.06 degrees C). Heterozygotes (*1/*2 or *1/*3) required significantly lower mean maintenance warfarin doses compared with wild-type (30.67 and 29.56 vs 42.81 mg/wk; P<.05). High-resolution melting analysis provides a simple and accurate method for genotyping of CYP2C9.
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Hill
authors.forename: Alexander
authors.initials: A
authors.lastname: Duncan
authors.forename: Diane
authors.initials: D
authors.lastname: Wirth
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Nolte
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D000925:Anticoagulants / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044366:Transition Temperature; D014859:Warfarin / Q000378:metabolism
chemical_list: D000925:Anticoagulants; D005456:Fluorescent Dyes; D014859:Warfarin; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases