title: CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection.
abstract: BACKGROUND
  Cytochrome P450 2D6 (CYP2D6), one of the most important drug-metabolizing enzymes, has been reported to possess variation in the encoding CYP2D6 gene (cytochrome P450, family 2, subfamily D, polypeptide 6) that affects enzymatic activity. For the pharmacogenetic study of CYP2D6, accurate measurement of the dosage of the functional gene is essential; however, current genotyping techniques are insufficient because of their inability to provide the exact copy number of functional CYP2D6 genes.
  
  
  METHODS
  We developed 3 quantitative real-time PCR (qPCR) assays for estimating the total copy number of the CYP2D6 gene, as well as 24-multiplex PCR-based real-time Invader assays (mPCR-RETINAs) for estimating the allele ratio at each variation locus. After determining the allele copy number at each locus, we estimated the frequencies of CYP2D6 alleles in a population and the diplotype in each individual by a CNVphaser (copy number variation phaser). The qPCR assays and RETINAs used for HapMap Japanese and Chinese samples were applied to 455 Japanese individuals.
  
  
  RESULTS
  Forty-two individuals (9.2%) had one CYP2D6 gene copy, 207 (45.5%) had 2 copies, 161 (35.4%) had 3 copies, 40 (8.8%) had 4 copies, and 5 (1.1%) had 5 copies of the CYP2D6 gene. We found 16 different CYP2D6 alleles, with frequencies similar to those described in previous reports. In the diplotype analysis, we observed that CYP2D6*1/*1 and *1/*10-*36 were the most common diplotypes (approximately 20%) in our population.
  
  
  CONCLUSIONS
  Our method is the first to determine the exact number of functional CYP2D6 gene copies. We believe our method will facilitate and accelerate the detailed pharmacogenetic analysis of CYP2D6.
authors.affiliation: Laboratory for Genotyping Development, Laboratory for Medical Informatics, Laboratory for Pharmacogenetics, Center for Genomic Medicine, RIKEN, Yokohama, Japan. nhosono@src.riken.jp
authors.forename: Naoya
authors.initials: N
authors.lastname: Hosono
authors.forename: Mamoru
authors.initials: M
authors.lastname: Kato
authors.forename: Kazuma
authors.initials: K
authors.lastname: Kiyotani
authors.forename: Taisei
authors.initials: T
authors.lastname: Mushiroda
authors.forename: Sadaaki
authors.initials: S
authors.lastname: Takata
authors.forename: Hiroko
authors.initials: H
authors.lastname: Sato
authors.forename: Hanae
authors.initials: H
authors.lastname: Amitani
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Tsuchiya
authors.forename: Keiko
authors.initials: K
authors.lastname: Yamazaki
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Tsunoda
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Zembutsu
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Michiaki
authors.initials: M
authors.lastname: Kubo
mesh_terms: D000483:Alleles; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D004247:DNA / Q000032:analysis / Q000235:genetics; D018628:Gene Dosage; D005787:Gene Frequency; D014644:Genetic Variation; D006801:Humans; D007564:Japan; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004247:DNA; D019389:Cytochrome P-450 CYP2D6