焦磷酸測序檢測CYP2C19基因多態性方法的建立

楊楠楠+徐琴+羅振元+付雪++黃盛文

[摘要] 目的 建立一種基于焦磷酸測序技術的用于CYP2C19基因多態性快速檢測的方法。 方法 選擇2016年12月～2017年3月于貴州省人民醫院行健康體檢者50名，提取其基因組DNA，針對CYP2C19*2（681G>A）、CYP2C19*3（636G>A）、CYP2C19*17（-806C>T）3個單核苷酸多態性（SNP）位點分別設計一套PCR擴增引物和焦磷酸測序引物。將PCR擴增產物制備成單鏈測序模板，用Qiagen PyroMark Q24焦磷酸測序儀進行測序和基因型分析，采用Sanger一代測序技術對焦磷酸測序結果進行驗證。 結果 根據出峰的堿基和峰高可清楚判斷出3個SNP位點的不同基因型，檢測結果與Sanger測序結果完全一致。50名健康體檢者中檢出CYP2C19超快代謝型1例，快代謝型22例，中代謝性22例，慢代謝型5例。 結論 本研究建立的焦磷酸測序檢測CYP2C19基因多態性的方法具有快速、準確、成本低的特點，適用于臨床實驗室對CYP2C19基因多態性的分型。

[關鍵詞] CYP2C19；基因多態性；焦磷酸測序；氯吡格雷

[中圖分類號] R446.9 [文獻標識碼] A [文章編號] 1673-7210（2017）10（b）-0011-04

Establishment of genotyping methods for polymorphisms of CYP2C19 gene based on pyrosequencing technology

YANG Nannan1 XU Qin1 LUO Zhenyuan2 FU Xue2 HUANG Shengwen2▲

1.School of Clinical Laboratory Science， Guizhou Medical University， Guizhou Province， Guiyang 550004， China； 2.Department of Clinical Laboratory， Guizhou Provincial People′s Hospital， Guizhou Province， Guiyang 550002， China

[Abstract] Objective To establish a fast detection method for CYP2C19 genotyping based on pyrosequencing technology. Methods Genomic DNA was extracted from 50 blood samples of healthy subjects collected from December 2016 to March 2017 in Guizhou Provincial People′s Hospital， and a set of amplification primers and pyrophosphate sequencing primers were designed according to the single nucleotide polymorphism （SNP） sites of CYP2C19*2 （681G>A）， CYP2C19*3 （636G>A）， CYP2C19*17 （-806C>T）. Single-stranded templates were made from PCR products， then sequencing and genotyping analysis were performed on a Qiagen PyroMark Q24 Sequencer System. The sequencing results were verified via Sanger sequencing. Results Based on the type and peak height of bases of DNA sequence， the genotypes of three SNPs could be identified clearly， which were consistent with Sanger sequencing results. Among the 50 samples of healthy group， 1 case was ultra-rapidmetabolism， 22 cases were rapidmetabolism， 22 cases were medium metabolism and 5 cases were slow metabolism. Conclusion The detection method of CYP2C19 polymorphism based on pyrosequencing technology is quick， accurate and convenient， which can be used to analyze the genotyping of CYP2C19 gene polymorphism.

[Key words] CYP2C19； Gene polymorphism； Pyrosequencing； Clopidogrel

氯吡格雷是臨床上常用的抗血小板聚集的藥物之一，冠狀動脈介入術（percutaneous coronary intervention，PCI）后患者需長期服用氯吡格雷來進行抗血小板治療[1-2]，但臨床上部分患者會出現氯吡格雷抵抗[3]。氯吡格雷的主要代謝酶為細胞色素P4502C19[4-6]，其編碼基因CYP2C19的多態性是影響氯吡格雷療效的主要遺傳因素[7-8]。CYP2C19的等位基因以CYP2C19*1（野生型）、CYP2C19*2、CYP2C19*3和CYP2C19*17較常見[9]。超快代謝型的基因型為*1/*17、*17*17，快代謝型的基因型為*1/*1，中代謝型的基因型為*1/*2、*1/*3，慢代謝型的基因型為*2/*2、*3/*3和*2/*3[10-11]。本研究采用焦磷酸測序技術建立了一種CYP2C19基因多態性檢測方法，并用Sanger測序法對其準確性進行驗證。endpri……