title: Single tube genotyping of CYP2A6 gene deletion based on copy number determination by quantitative real-time PCR.
abstract: The CYP2A6*4 allele, characterized as the whole deletion of this gene, is closely associated with nicotine dependence, cancer susceptibility, and drug responsiveness. It has long been a significant challenge for pharmacogenetics scientists to develop a reliable method to detect this molecular variant due to its high homology with its homologous genes CYP2A6 and CYP2A3 in the clinical setting. Here, we introduce a quantitative real-time PCR assay that specifically amplifies CYP2A6 by designing a specific set of primers and the probe, which effectively prevent the amplification of the CYP2A7 and CYP2A13 alleles. CYP2A6 gene copy numbers were normalized to albumin (ALB) which was co-amplified simultaneously in a single-tube duplex reaction and at a setting as the internal reference gene. The established assay was validated with a selection of previously genotyped DNA samples, which harbored none, one or two CYP2A6 gene copies. The results were in complete concordance with previously published data and no overlap between the three groups was observed. Further analysis of a cohort of 120 samples revealed high specificity and sensitivity of this assay as demonstrated by the agreement of determined gene copy numbers in all of the cases. In conclusion, this novel assay allows reliable and sensitive detection of the CYP2A6 gene deletion, which will be useful for pharmacogenetics studies and routine clinical settings.
authors.affiliation: National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University, Xi'an, PR China.
authors.forename: Jin-hui
authors.initials: JH
authors.lastname: Liu
authors.affiliation: National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University, Xi'an, PR China.
authors.forename: Xiao-jie
authors.initials: XJ
authors.lastname: Xun
authors.affiliation: National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University, Xi'an, PR China.
authors.forename: Cong
authors.initials: C
authors.lastname: Pang
authors.affiliation: National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University, Xi'an, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Ma
authors.affiliation: National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University, Xi'an, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zou
authors.affiliation: National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University, Xi'an, PR China.
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.affiliation: National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University, Xi'an, PR China. Electronic address: daipg@nuw.edu.cn.
authors.forename: Peng-gao
authors.initials: PG
authors.lastname: Dai
mesh_terms: D065637:Cytochrome P-450 CYP2A6 / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D017353:Gene Deletion; D018628:Gene Dosage; D005838:Genotype; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6
keywords: CYP2A6*4; Gene copy number; Gene deletion; Quantitative real-time PCR