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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A single point mutation leading to loss of catalytic activity in human thiopurine S-methyltransferase.
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A single point mutation leading to loss of catalytic activity in human thiopurine S-methyltransferase.

机译：单点突变导致人硫嘌呤S-甲基转移酶催化活性的丧失。

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摘要

Thiopurine S-methyltransferase (TPMT; S-adenosyl-L-methionine:thiopurine S-methyltransferase, EC 2.1.1.67) activity exhibits genetic polymorphism, with approximately 0.33% of Caucasians and African-Americans inheriting TPMT deficiency as an autosomal recessive trait. To determine the molecular genetic basis for this polymorphism, we cloned the TPMT cDNA from a TPMT-deficient patient who had developed severe hematopoietic toxicity during mercaptopurine therapy. Northern blot analysis of RNA isolated from leukocytes of the deficient patient demonstrated the presence of TPMT mRNAs of comparable size to that in subjects with high TPMT activity. Sequencing of the mutant TPMT cDNA revealed a single point mutation (G238-->C), leading to an amino acid substitution at codon 80 (Ala80-->Pro). When assessed in a yeast heterologous expression system, this mutation led to a 100-fold reduction in TPMT catalytic activity relative to the wild-type cDNA, despite a comparable level of mRNA expression. A mutation-specific PCR amplification method was developed and used to detect the G238-->C mutation in genomic DNA of the propositus and her mother. This inactivating mutation in the human TPMT gene provides insights into the genetic basis for this inherited polymorphism in drug metabolism.
机译：硫嘌呤S-甲基转移酶（TPMT; S-腺苷-L-蛋氨酸：硫嘌呤S-甲基转移酶，EC 2.1.1.67）活性表现出遗传多态性，约0.33％的白种人和非裔美国人遗传TPMT缺乏症是常染色体的隐性特征。为了确定这种多态性的分子遗传基础，我们从一名在患有嘌呤嘌呤治疗期间发生了严重造血毒性的TPMT缺陷患者中克隆了TPMT cDNA。从缺陷患者白细胞中分离的RNA的RNA印迹分析表明，TPMT mRNA的大小与具有高TPMT活性的受试者相当。突变TPMT cDNA的测序揭示了单点突变（G238-> C），导致第80位密码子的氨基酸取代（Ala80-> Pro）。当在酵母异源表达系统中评估时，尽管mRNA表达水平相当，但该突变导致TPMT催化活性相对于野生型cDNA降低了100倍。开发了一种突变特异性PCR扩增方法，该方法可用于检测性腺及其母亲的基因组DNA中的G238-> C突变。人TPMT基因中的这种失活突变为药物代谢中这种遗传多态性的遗传基础提供了见识。

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