CYP2D6 Genotyping for Functional-Gene Dosage Analysis by Allele Copy Number Detection
CYP2D6 Genotyping for Functional-Gene Dosage Analysis by Allele Copy Number Detection
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DOI：
10.1373/clinchem.2009.123620
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发表时间：
2009-08-01
影响因子：
9.3
通讯作者：
Kubo, Michiaki
中科院分区：
文献类型：
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作者：
Hosono, Naoya;Kato, Mamoru;Kubo, Michiaki
背景技术背景：细胞色素P450 2D 6（CYP 2D 6）是最重要的药物代谢酶之一，其编码基因（细胞色素P450，家族2，亚家族D，多肽6）的变异影响酶活性。对于CYP 2D 6的药物遗传学研究，准确测量功能基因的剂量是必不可少的;然而，目前的基因分型技术是不够的，因为它们无法提供确切的拷贝数的功能CYP 2D 6基因。我们开发了3种定量实时PCR（qPCR）检测方法，用于估计CYP 2D 6基因的总拷贝数，以及用于估计每个变异基因座处的等位基因比率的基于24-多重PCR的实时侵入测定（mPCR-RETINAs）。在确定每个位点的等位基因拷贝数后，我们估计了CYP 2D 6等位基因在人群中的频率，并通过CNVphaser（拷贝数变异相位器）估计了每个个体的二倍型。结果：42例（9.2%）有1个CYP 2D 6基因拷贝，207例（45.5%）有2个拷贝，161例（35.4%）有3个拷贝，40例（8.8%）有4个拷贝，5例（1.1%）有5个拷贝。我们发现了16个不同的CYP 2D 6等位基因，频率与以前的报告中描述的相似。在二倍型分析中，我们观察到CYP 2D 6 *1/*1和 *1/*10-*36是我们人群中最常见的二倍型（约20%）。我们相信我们的方法将促进和加速CYP 2D 6的详细药物遗传学分析。(C)2009年美国临床化学协会
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. (C) 2009 American Association for Clinical Chemistry