CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection

Naoya Hosono, Mamoru Kato, Kazuma Kiyotani, Taisei Mushiroda, Sadaaki Takata, Hiroko Sato, Hanae Amitani, Yumiko Tsuchiya, Keiko Yamazaki, Tatsuhiko Tsunoda, Hitoshi Zembutsu, Yusuke Nakamura, Michiaki Kubo

Research output: Contribution to journalArticle

53 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1546-1554
Number of pages9
JournalClinical Chemistry
Volume55
Issue number8
DOIs
Publication statusPublished - 01-08-2009
Externally publishedYes

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Cytochrome P-450 CYP2D6
Gene Dosage
Genes
Alleles
Assays
Genotyping Techniques
HapMap Project
Multiplex Polymerase Chain Reaction
Essential Genes
Gene Frequency
Cytochrome P-450 Enzyme System
Population
Real-Time Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Hosono, Naoya ; Kato, Mamoru ; Kiyotani, Kazuma ; Mushiroda, Taisei ; Takata, Sadaaki ; Sato, Hiroko ; Amitani, Hanae ; Tsuchiya, Yumiko ; Yamazaki, Keiko ; Tsunoda, Tatsuhiko ; Zembutsu, Hitoshi ; Nakamura, Yusuke ; Kubo, Michiaki. / CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection. In: Clinical Chemistry. 2009 ; Vol. 55, No. 8. pp. 1546-1554.
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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.",
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Hosono, N, Kato, M, Kiyotani, K, Mushiroda, T, Takata, S, Sato, H, Amitani, H, Tsuchiya, Y, Yamazaki, K, Tsunoda, T, Zembutsu, H, Nakamura, Y & Kubo, M 2009, 'CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection', Clinical Chemistry, vol. 55, no. 8, pp. 1546-1554. https://doi.org/10.1373/clinchem.2009.123620

CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection. / Hosono, Naoya; Kato, Mamoru; Kiyotani, Kazuma; Mushiroda, Taisei; Takata, Sadaaki; Sato, Hiroko; Amitani, Hanae; Tsuchiya, Yumiko; Yamazaki, Keiko; Tsunoda, Tatsuhiko; Zembutsu, Hitoshi; Nakamura, Yusuke; Kubo, Michiaki.

In: Clinical Chemistry, Vol. 55, No. 8, 01.08.2009, p. 1546-1554.

Research output: Contribution to journalArticle

TY - JOUR

T1 - CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection

AU - Hosono, Naoya

AU - Kato, Mamoru

AU - Kiyotani, Kazuma

AU - Mushiroda, Taisei

AU - Takata, Sadaaki

AU - Sato, Hiroko

AU - Amitani, Hanae

AU - Tsuchiya, Yumiko

AU - Yamazaki, Keiko

AU - Tsunoda, Tatsuhiko

AU - Zembutsu, Hitoshi

AU - Nakamura, Yusuke

AU - Kubo, Michiaki

PY - 2009/8/1

Y1 - 2009/8/1

N2 - 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.

AB - 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.

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Hosono N, Kato M, Kiyotani K, Mushiroda T, Takata S, Sato H et al. CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection. Clinical Chemistry. 2009 Aug 1;55(8):1546-1554. https://doi.org/10.1373/clinchem.2009.123620