Polymorphism in the promoter and coding regions of human metallothionein IIA gene

人体组织中镉的浓度随着年龄的增长而增加，因为大部分积累的镉可能会留在体内而不排出体外。一般日本人通常吃大米，大米中镉的含量自然较高。另一方面，镉已经被认为可以诱导金属硫蛋白的合成，金属硫蛋白是一种降低重金属毒性的精神结合蛋白。在一般成年人中，镉在组织中的积累多以与金属硫蛋白的低毒复合物形式存在。我们发现一些日本成年人的肾脏金属硫蛋白浓度相对较低。“低金属硫蛋白”有可能是遗传疾病所致。为此，我们采用单链构象多态性方法（PCR-SSCP）对普通日本人金属硫蛋白基因及其启动子区核苷酸序列的异常进行了检测。从法医尸检标本（21例）的肾皮质提取基因组DNA样本。采用PCR-SSCP和直接测序方法，在部分样品中观察到金属硫蛋白基因及其启动子区核苷酸序列异常。在4个样品的启动子区和2个样品的外显子3中发现了单碱基取代。在转录起始点上游5个碱基处，所有4个样本的启动子区域都出现了替换（A->G）。另一方面，在两个样品的同一位点上观察到外显子3的突变（GCC->GTC），诱导了Ala->Val的氨基酸取代。在一个样品中，在启动子和外显子3的相同等位基因上观察到这两个替换。这些碱基取代对金属硫蛋白合成的影响将在未来进一步研究。

Cadmium concentration in the human tissues increase with aging because most of the accumulated cadmium may be remained in the bodies without excreting. General Japanese usually well eat rice which naturally containing relatively high concentration of cadmium. On the other hand, cadmium has been known to induce synthesis of metallothionein, a mental binding protein which reduces toxicity of heavy metals. In the general adults, most of cadmium accumulated in the tissues exist as low toxic complex with matallothionein. We found some Japanese adults whose kidneys have relatively low concentration of metallothionein. There is a possibility that the "low metallothionein" may be induced by genetic disorder. Therefore, we examined abnormality of nucleotide sequence of metallothionein gene and its promoter region of general Japanese by single-stranded conformation polymorphism method(PCR-SSCP). Genomic DNA samples were extracted from cortex of kidney of the forensic autopsy samples(21 examples). By PCR-SSCP and direct sequencing methods, abnormalities of nucleotide sequence of metallothionein gene and its promoter region were observed in some samples. Single base substitutions were found in the promoter region of four samples and in the exon 3 of two samples. The substitution(A->G) in the promoter region in all four samples was observed in 5 bases upstream from the transcription starting point. On the other hand, the mutation(GCC->GTC) of exon 3 was observed in the same site of the two samples, inducing the amino acid substitution of Ala->Val. In one sample, these two substitutions was observed in identical allele of promoter and exon 3. Effect of these base substitutions on the synthesis of metallothionein will be examined in future.