Spire-Vayron de la Moureyre C, Debuysère H, Sabbagh N, Marez D, Vinner E, Chevalier E D, Lo Guidice J M, Broly F
Laboratoire de Biochimie et Biologie Moléculaire, Hôpital Calmette, Centre Hospitalier Régional et Universitaire de Lille, France.
Hum Mutat. 1998;12(3):177-85. doi: 10.1002/(SICI)1098-1004(1998)12:3<177::AID-HUMU5>3.0.CO;2-E.
To detect mutations in the thiopurine S-methyltransferase gene (TPMT), we have developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The sensitivity of the method was first evaluated by analyzing DNA samples from five individuals, including two high methylators (HMs), two intermediate methylators (IMs), and one deficient methylator (DM). TPMT alleles and mutations in each of these individuals had previously been characterized by conventional PCR-based assays and direct sequencing analysis. All mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments, allowing their identification. We further tested the efficiency of the strategy to detect new TPMT mutations. For this purpose, additional DNAs from 15 IMs and 15 HMs were submitted to PCR-SSCP analysis. A total of 7 alleles were characterized, including two new alleles. The first one, termed TPMT1A, harbors a single mutation C-->T at nucleotide -178 in exon 1 and was detected in a HM subject. The second one, termed TPMT7, was characterized by a T-->G transversion at nucleotide 681 in exon 10. This allele should be a nonfunctional allele of the TPMT gene since it was observed in combination with a wild-type allele in an intermediate methylator. We conclude that the PCR-SSCP strategy we developed could be advantageously used to fully characterize the extent of allelic variation at the TPMT gene locus in populations and thus to improve our understanding of the genetic polymorphism of TPMT activity, which has considerable consequences for the toxicity and efficacy of therapeutically important and widely used drugs.
为了检测硫嘌呤S-甲基转移酶基因（TPMT）中的突变，我们开发了一种基于聚合酶链反应（PCR）扩增基因的单链构象多态性（SSCP）分析的策略。该方法的灵敏度首先通过分析来自五个人的DNA样本进行评估，这五个人包括两个高甲基化者（HMs）、两个中间甲基化者（IMs）和一个甲基化缺陷者（DM）。这些个体中每个个体的TPMT等位基因和突变先前已通过传统的基于PCR的检测方法和直接测序分析进行了表征。所有突变都与DNA片段电泳迁移率的特定变化相关，从而可以识别它们。我们进一步测试了该策略检测新的TPMT突变的效率。为此，将另外15名IMs和15名HMs的DNA进行PCR-SSCP分析。共鉴定出7个等位基因，包括两个新等位基因。第一个称为TPMT1A，在外显子1的核苷酸-178处有一个单一突变C→T，在一名HM受试者中检测到。第二个称为TPMT7，在外显子10的核苷酸681处有一个T→G颠换。由于在一名中间甲基化者中观察到该等位基因与野生型等位基因同时存在，因此该等位基因应该是TPMT基因的一个无功能等位基因。我们得出结论，我们开发的PCR-SSCP策略可有利地用于全面表征人群中TPMT基因座等位基因变异的程度，从而增进我们对TPMT活性基因多态性的理解，这对治疗上重要且广泛使用的药物的毒性和疗效具有相当大的影响。
