基于聚合-连接探针的TPMT SNP新型高通量检测技术的研究

The thiopurine methyltransferase (TPMT) is one of the critical metabolic enzymes that involved in thiopurine drug metabolisms. Single nucleotide polymorphisms (SNP) of its genetic coding regions could affect TPMT activities, and therefore could be directly associated with the clinical responses and toxicity levels of thiopurine drugs. Although there have been various methods that could be used to analyze SNP, no one could satisfy the special requirements of TPMT SNP analysis because of its diversities and complexities. At present, there were still no any systematic studies on the effects of 37 kinds of TMPT SNP on the TPMT activity levels. In the present project, a novel method based on ligation-during- polymerization (LDP) probes was developed to highly-throughput analyze all the 37 TPMT SNP in a single closed tube. And then, the effects of single or multiplex SNP on the TPMT activity levels would be systematic studied. The SNP analysis method based on LDP probes developed in the present project has completely independent intellectual property rights. It could be used to simultaneously analyze 120 to 160 various SNP in a single closed tube. Therefore, this novel method has broad application prospects in clinical genetic polymorphisms studies. Moreover, the SNP effects on TPMT activities, which would be analyzed and certified in the present project, could provide highly useful laboratory data for the personalized therapy of thiopurine drugs.

硫嘌呤甲基转移酶(TPMT)是硫嘌呤类药物在体内代谢的关键酶，其编码基因的单核苷酸多态性(SNP)可以决定TPMT活性，进而直接影响硫嘌呤类药物的临床疗效和毒性程度。尽管现在已有多种SNP分析技术，但尚不能满足TPMTSNP多样性和复杂性对检测方法的特殊需求，TPMT 37种SNP对TPMT活性的影响程度尚缺乏系统性研究。本项目拟在前期工作基础之上，研发一种基于聚合-连接(LDP)探针原理、能够一次性单管并行检测TPMT 37种SNP的新型高通量检测技术，在此基础上，系统地分析和确认TPMT单个或多个并存SNP对TPMT活性的影响程度。本项目研发的SNP检测技术具有完全自主知识产权，可在理论上一次完成120-160种SNP的单管并行分析，具有广阔的应用前景；本项目所获得的单个或多个SNP对TPMT活性的影响程度可以为硫嘌呤类药物个体化治的合理用药及药物组合提供有效的实验室依据。

本研究系统地分析了TPMT基因型，以及TPMT基因型与硫嘌呤类药物疗效的相关性，为TPMT基因型检测的临床应用提供了详实的理论与数据支撑。成功构建了本项目所需的TPMT的37种SNP基因型对应的野生型和突变型质粒，为本项目的TPMT SNP检测技术的研究及相关技术参数的优化提供了参考品和标准品。成功构建了基于LDP探针原理的TPMT SNP检测体系，实现了多种SNP的单管平行检测，并且成功地对240例临床标本的基因型进行了4个因素的最优水平评价。外周血红细胞TPMT活性的鉴定及其酶活性的表型分析已经完成，基因型分析正在进行中。并在此基础之上成功构建了能够识别单碱基变异的CRAS-PCR反应体系，有望成为SNP检测新技术；同时开发了新型AS-PCR反应体系DCAS-PCR，消除了AS-PCR因嗜热性DNA聚合酶的热力驱动力而普遍存在的非特异性扩增。目前已用开发的DCAS-PCR成功地分析了中国重庆地区汉族人群200个临床血液样本中TPMT*3B和*3C的基因型。结果表明：TPMT*3C杂合子只有7个，占总样本数的3.5%，TPMT*3B均为野生型基因型。本项目开发的一系列检测方法可以满足TPMT SNP多样性和复杂性对检测方法的特殊需求。相关研究成果主要获得中华医学科技奖三等奖，国家发明专利授权1件，国家实用新型专利授权1件，SCI论文3篇，国内期刊论文2篇，培养硕士研究生2名。