CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection.
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.
Hosono
Naoya
N
Laboratory for Genotyping Development, Laboratory for Medical Informatics, Laboratory for Pharmacogenetics, Center for Genomic Medicine, RIKEN, Yokohama, Japan. nhosono@src.riken.jp
Kato
Mamoru
M
Kiyotani
Kazuma
K
Mushiroda
Taisei
T
Takata
Sadaaki
S
Sato
Hiroko
H
Amitani
Hanae
H
Tsuchiya
Yumiko
Y
Yamazaki
Keiko
K
Tsunoda
Tatsuhiko
T
Zembutsu
Hitoshi
H
Nakamura
Yusuke
Y
Kubo
Michiaki
M
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*
D004247:DNA; D019389:Cytochrome P-450 CYP2D6