文档介绍：ASPCR方法检测中国人群CYP2C19基因多态性
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作者:薛丽,叶玉兴,易国辉,白玉杰
【摘要】目的:建立一种快速、灵敏的细胞色素氧化酶P450 2C19基因多态性检测方法。方法:分离外周血淋巴细胞基因组DNA,分别用一对等位基因特异性(allelespecific, AS)的反向引物与共用正向引物进行PCR扩增,通过电泳分析对应的PCR产物来判断CYP2C19基因型。在AS引物3′端倒数第三位或第二位引入错配碱基改进特异性。采用优化后方法对278例健康人进行CYP2C19基因型分析。结果:ASPCR可检测CYP2C19基因多态性,引入第二个错配碱基可提高检测特异性。经直接测序验证结果完全一致。结论:建立和优化的ASPCR方法适用于CYP2C19基因多态性快速检测。
【关键词】细胞色素氧化酶2C19;遗传多态性;基因型;等位基因特异性PCR

[ABSTRACT] Objective: To develop a rapid and sensitive met
hod for genotype analysis of the CYP2C19 gene in Chinese population. Methods: The genomic DNA was extracted from lymphocytes. mon forward primer and one of the two allelespecific (AS) reverse primers were used in parallel PCR reactions. The PCR products were analyzed by electrophoresis and specific amplification indicated the presence of the allele. In order to enhance the PCR specificity, the extra mismatch based at the third or second nucleotide position from the 3′end of AS primers were introduced. Results: Two hundred and seventyeight DNA samples were analyzed by this new method. The CYP2C19 genotyping results derived from this ASAPCR were perfectly consistent with sequencing. Conclusion: The new method developed and optimized in this study is a suitable strategy for CYP2C19 genotyping. It also enables accurate and rapid detection of CYP2C19 polymorphisms.

[KEY WORDS] CYP2C19; ic polymorphism; Genotype; Allelespecific PCR
细胞色素氧化酶P450 2C19 (CYP2C19)是人体中重要的代谢酶。最初因催化S美芬妥英羟化代谢功能而被称为S美芬妥英羟化酶
[1], CYP2C19催化羟化、氧化及环化等反应,参与机体多种内源性及外源性化学物质的代谢。奥美拉唑、美芬妥英、安定、普萘洛尔等临床常用药物经CYP2C19代谢[2-4],CYP2C19的功能与疾病风险及药物疗效都密切相关。

CYP2C19活性存在个体间差异,以S美芬妥英为探针药物,依据其代谢速度分为强代谢表型(Extensive Metabolism,EM)和弱代谢表型(Poor Metabolism,PM)。研究发现个体间代谢表型差异的重要原因是其基因多态性,外显子5上的第681位密码子GA变异(c681GA,称为“m1突变”)和外显子4上的第636位密码子GA变异(c636GA,称为“m2突变”)是引起PM表型的最主要基因变异。亚洲人群95%以上PM表型由m1和m2变异决定[5,6]。

分析CYP2C19基因多态性可推断代谢表型,从而预测解毒能力和患病风险,还可预测药物代谢能力、药物疗效及毒副作用,指导临床合理用药。本研究基于等位基因特异性PCR(allelespecific PCR,ASPCR)原理,建立了快速CYP