Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, 3-3165 Higashi-tamagawa Gakuen, Machida, Tokyo 194-8543, Japan.
Clin Chim Acta. 2012 Oct 9;413(19-20):1675-7. doi: 10.1016/j.cca.2012.05.013. Epub 2012 May 23.
Genetic polymorphisms of the human CYP2D6 gene can affect the metabolism of many drugs in clinical use. As a first step toward identifying poor drug metabolizers in the clinical setting, we developed a new multiplex PCR-based genotyping method to detect CYP2D6 whole-gene deletion.
We validated the new method by analyzing 500 genomic DNA samples from a Japanese population with the conventional long-PCR method and the new multiplex PCR method. The long-PCR system used a forward primer for CYP2D7P (a pseudogene closely related to CYP2D6) and a common reverse primer for the untranslated region. The multiplex PCR system used the same two primers as the long PCR and an additional forward primer for CYP2D6.
With the long-PCR system, DNA samples identified as containing CYP2D65 (whole-gene deletion) formed 3.5-kb PCR products. With the multiplex PCR system, many samples yielded 4.7-kb PCR products (implying the existence of normal CYP2D6) and some DNA samples yielded 6.2-kb PCR products (probably indicating CYP2D610D). The long-PCR assay detected 64 CYP2D65 alleles among 1000 Japanese alleles; however, the new multiplex PCR system identified 5 of these 64 alleles as CYP2D610D.
The new multiplex PCR method is useful for detecting CYP2D65. This system could reliably discriminate CYP2D65 from homologous pseudogene CYP2D7P and functional CYP2D6*10D.
人类 CYP2D6 基因的遗传多态性会影响许多临床应用药物的代谢。为了在临床环境中识别不良药物代谢者，我们开发了一种新的基于多重 PCR 的基因分型方法来检测 CYP2D6 全基因缺失。
我们使用传统的长 PCR 方法和新的多重 PCR 方法对来自日本人群的 500 个基因组 DNA 样本进行了验证。长 PCR 系统使用 CYP2D7P（与 CYP2D6 密切相关的假基因）的正向引物和未翻译区的通用反向引物。多重 PCR 系统使用与长 PCR 相同的两个引物，外加一个 CYP2D6 的正向引物。
使用长 PCR 系统，被鉴定为含有 CYP2D65（全基因缺失）的 DNA 样本形成 3.5kb 的 PCR 产物。使用多重 PCR 系统，许多样本产生了 4.7kb 的 PCR 产物（暗示存在正常的 CYP2D6），而一些 DNA 样本产生了 6.2kb 的 PCR 产物（可能表示 CYP2D610D）。长 PCR 检测到 1000 个日本等位基因中的 64 个 CYP2D65 等位基因；然而，新的多重 PCR 系统将其中的 5 个鉴定为 CYP2D610D。
新的多重 PCR 方法可用于检测 CYP2D65。该系统可可靠地区分 CYP2D65 与同源假基因 CYP2D7P 和功能 CYP2D6*10D。
