Thiopurine methyltransferase pharmacogenetics: Human gene cloning and characterization of a common polymorphism
Thiopurine methyltransferase pharmacogenetics: Human gene cloning and characterization of a common polymorphism
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DOI：
10.1089/dna.1996.15.17
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发表时间：
1996-01-01
影响因子：
3.1
通讯作者：
Weinshilboum, R
中科院分区：
文献类型：
--
作者：
Szumlanski, C;Otterness, D;Weinshilboum, R
巯基嘌呤甲基转移酶（TPMT）催化巯基嘌呤药物的S-甲基化，这些药物的毒性和疗效的个体差异与控制人体组织中TPMT活性和免疫反应蛋白水平的共同遗传多态性有关，由于该酶的“药物遗传学”调节的临床意义，克隆人类TPMT基因并研究遗传多态性的分子基础将是重要的。作为克隆TPMT基因的第一步，我们使用基因组DNA末端的快速扩增来获得TPMT特异性内含子序列，该DNA序列被用于设计聚合酶链反应（PCR）的引物，这使得有可能确定TPMT的活性基因位于人类6号染色体上。从人6号染色体特异性基因组DNA文库中分离到一个TPMT阳性粘粒克隆，用荧光原位杂交法将该基因定位于染色体6p22.3带。根据cDNA末端5 ′-快速扩增的结果，转录起始发生在先前报道的TPMT cDNA的翻译起始密码子上游89个核苷酸处或附近。一旦确定了TPMT基因的结构，可以用与编码酶蛋白的每个外显子的内含子序列互补的引物进行PCR，所述外显子具有从具有已知TPMT遗传多态性表型的受试者。该DNA分离自来自4名具有遗传低TPMT活性的无关受试者和4名具有高TPMT活性的无关受试者的血液样品。所有TPMT活性低的受试者都是两个点突变的纯合子-外显子7中核苷酸460处的G->A转换和外显子10中核苷酸719处的A->G转换。两种突变均导致氨基酸序列的改变，分别为Ala-154 → Thr和Tyr-240 → Cys。从具有高TPMT活性的受试者血液中分离的所有DNA样品均含有“野生型”序列，当发现来自具有高TPMT活性的四个人肝样品的DNA具有野生型TPMT活性时，证实了用这些血液样品获得的结果。在核苷酸460和719处的型序列，而三个具有中等酶活性的肝脏样品（即，假定为多态性杂合的样品）对于纯合低受试者的血液样品中存在的外显子7和外显子10突变是杂合的。在COS-1细胞中瞬时表达TPMT表达构建体，其包含外显子7和10中的突变，以及各自独立的突变，证明了每个突变，以及两者一起，导致TPMT酶活性和免疫反应蛋白表达降低。TPMT基因的分子克隆和结构表征以及常见TPMT遗传多态性的分子基础的阐明将有助于开发基于DNA的多态性诊断测试，并确定其导致这种重要药物代谢酶表达降低的机制。
Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs, Individual variation in the toxicity and therapeutic efficacy of these drugs is associated with a common genetic polymorphism that controls levels of TPMT activity and immunoreactive protein in human tissues, Because of the clinical significance of the ''pharmacogenetic'' regulation of this enzyme, it would be important to clone the gene for TPMT in humans and to study the molecular basis for the genetic polymorphism, As a first step toward cloning the gene for TPMT, we used the rapid amplification of genomic DNA ends to obtain a TPMT-specific intron sequence, That DNA sequence was used to design primers for the polymerase chain reaction (PCR), which made it possible to determine that the active gene for TPMT is located on human chromosome 6. A TPMT-positive cosmid clone was then isolated from a human chromosome 6-specific genomic DNA library, and the gene was sublocalized to chromosome band 6p22.3 by fluorescence in situ hybridization, The gene for TPMT was found to be approximately 34 kb in length and consisted of 10 exons and 9 introns. On the basis of the results of 5'-rapid amplification of cDNA ends, transcription initiation occurred at or near a point 89 nucleotides upstream from the translation initiation codon of previously reported TPMT cDNAs, Once the structure of the TPMT gene had been determined, it was possible to perform the PCR with primers complementary to the sequences of introns nanking each exon that encodes enzyme protein with template DNA obtained from subjects with known phenotypes for the TPMT genetic polymorphism. This DNA was isolated from blood samples from 4 unrelated subjects with genetically low TPMT activity and 4 unrelated subjects with high TPMT activity. All subjects with low TPMT activity were homozygous for two point mutations-a G-->A transition at nucleotide 460 in exon 7 and an A-->G transition at nucleotide 719 in exon 10. Both mutations resulted in alterations in amino acid sequence, with Ala-154-->Thr and Tyr-240-->Cys, respectively, All DNA samples isolated from the blood of subjects with high TPMT activity contained ''wild-type'' sequence, Results obtained with these blood samples were confirmed when DNA from four human liver samples with high TPMT activity were found to have wild-type sequence at nucleotides 460 and 719, while three liver samples with intermediate enzyme activity (i.e., samples presumed to be heterozygous for the polymorphism) were heterozygous for the exon 7 and exon 10 mutations present in the blood samples of homozygous low subjects, Transient expression in COS-1 cells of TPMT expression constructs that contained both of the mutations in exons 7 and 10, as well as each independently, demonstrated that each mutation, as well as both together, resulted in decreased expression of TPMT enzymatic activity and immunoreactive protein. Molecular cloning and structural characterization of the TPMT gene as well as elucidation of the molecular basis for a common TPMT genetic polymorphism will help make it possible to develop DNA-based diagnostic tests for the polymorphism and to determine the mechanism by which it results in decreased expression of this important drug-metabolizing enzyme.