建立單一螢光探針PCR法以檢測CYP2C19基因型來幫助臨床上clopidogrel的治療

Setup a Single Probe Hybridization PCR Method for Genotyping of CYP2C19 in Taiwanese Population to Support Clopidogrel Treatment

本研究中我們建立single probe hybridization PCR，設計3條專一性螢光探針分別標記ROX、Cy5與HEX，偵測CYP2C19*2、CYP2C19*3與CYP2C19*17 SNPs，利用融離曲線分析Tm值，區分正常型與變異型。並分析2011~2012年共150例彼此無親緣關係的一般族群台灣人DNA，發現有27%（82/300）為CYP2C19*2；7%（22/300）為CYP2C19*3，0.67%（2/300）為CYP2C19*17。我們也分析41例接受PCI（percutaneous coronary intervention,PCI）服用clopidogrel的病患CYP2C19基因型與預後情形，發現CYP2C19 SNPs的確影響心血管疾病患者服用clopidogrel的藥效與預後。此方法具有快速，原理簡易、成本低廉的優點，提供藥物基因CYP2C19篩檢與臨床用藥參考。

CYP2C19, one of the Cytochrome P450 enzymes, is involved in the metabolism of clopidogrel which is an antiplatelet agent and is used for treatment of cardiovascular disease. People with genotypes of CYP2C19*2 or CYP2C19*3 are poor metabolizer (PM) and easily develop second ischemic hit after treatment. In contrast, people with the genotype of CYP2C19*17 are ultra-rapid metabolizer which have higher bleeding risk after the same dosage of clopidogrel.In this study, we established real-time PCR methods with modified single hybridization probe principle for detection of CYP2C19*2, CYP2C19*3 and CYP2C19*17. Reactions were carried out on Roche LightCycler480, reporter probes were labeled with three different fluorophores. Melting temperature (Tm) of different fluorescent channel were used to distinguish normal type from variant type. Total 150 genomic DNA from general population of Taiwanese were analyzed. Results showed that 27% (82/300) were CYP2C19*2, 7% (22/300) were CYP2C19*3, and 0.67% (2/300) were CYP2C19*17. All PCR results were further confirmed by Sanger's sequencing. In addition, we investigated the curative effect of 41 patients who received percutaneous coronary intervention and prescribed 600 mg clopidogrel as loading dose. Thrombosis were found in 7% of patients with CYP2C19*1/*1, 12% with CYP2C19*1/*2, and 50% with CYP2C19*2/*2. The result indicated that CYP2C19 polymorphism correlates to clinical outcome. In conclusion, we have successfully developed an inexpensive and simple genotyping method for detecting CYP2C19 SNPs and it can support the therapeutic decision for physicians and prevent adverse events.