title: An allele-specific PCR system for rapid detection and discrimination of the CYP2C19∗4A, ∗4B, and ∗17 alleles: implications for clopidogrel response testing.
abstract: CYP2C19 is involved in the metabolism of clinically relevant drugs, including the antiplatelet prodrug clopidogrel, which has prompted interest in clinical CYP2C19 genotyping. The CYP2C19∗4B allele is defined by both gain-of-function [c.-806C>T (∗17)] and loss-of-function [c.1A>G (∗4)] variants on the same haplotype; however, current genotyping and sequencing assays are unable to determine the phase of these variants. Thus, the aim of this study was to develop an assay that could rapidly detect and discriminate the related ∗4A, ∗4B, and ∗17 alleles. An allele-specific PCR assay, composed of four unique primer mixes that specifically interrogate the defining ∗17 and ∗4 variants, was developed by using samples (n = 20) with known genotypes, including the ∗4A, ∗4B, and/or ∗17 alleles. The assay was validated by testing 135 blinded samples, and the results were correlated with CYP2C19 genotyping and allele-specific cloning/sequencing. Importantly, among the six ∗4 carriers in the validation cohort, after allele-specific PCR testing both samples with a ∗1/∗4 genotype were reclassified to ∗1/∗4A, all three samples with a ∗4/∗17 genotype were reclassified to ∗1/∗4B, and a sample with a ∗4/∗17/∗17 genotype was reclassified to ∗4B/∗17. In conclusion, this rapid and robust allele-specific PCR assay can refine CYP2C19 genotyping and metabolizer phenotype classification by determining the phase of the defining ∗17 and ∗4 variants, which may have utility when testing CYP2C19 for clopidogrel response.
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address: stuart.scott@mssm.edu.
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Scott
authors.forename: Qian
authors.initials: Q
authors.lastname: Tan
authors.forename: Usman
authors.initials: U
authors.lastname: Baber
authors.forename: Yao
authors.initials: Y
authors.lastname: Yang
authors.forename: Suparna
authors.initials: S
authors.lastname: Martis
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Bander
authors.forename: Ruth
authors.initials: R
authors.lastname: Kornreich
authors.forename: Jean-Sébastien
authors.initials: JS
authors.lastname: Hulot
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Desnick
mesh_terms: D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001483:Base Sequence; D016022:Case-Control Studies; D000077144:Clopidogrel; D003324:Coronary Artery Disease / Q000628:therapy; D065731:Cytochrome P-450 CYP2C19; D056726:Genetic Association Studies; D006801:Humans; D062645:Percutaneous Coronary Intervention; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D016133:Polymerase Chain Reaction / Q000592:standards*; D011355:Prodrugs / Q000494:pharmacology* / Q000627:therapeutic use; D012015:Reference Standards; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D010975:Platelet Aggregation Inhibitors; D011355:Prodrugs; D000077144:Clopidogrel; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D013988:Ticlopidine