Functional analysis of genetic variation in catechol-o-methyltransferase (COMT):: Effects on mRNA, protein, and enzyme activity in postmortem human brain
Functional analysis of genetic variation in catechol-o-methyltransferase (COMT):: Effects on mRNA, protein, and enzyme activity in postmortem human brain
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DOI：
10.1086/425589
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发表时间：
2004-11-01
影响因子：
9.8
通讯作者：
Weinberger, DR
中科院分区：
文献类型：
--
作者：
Chen, JS;Lipska, BK;Weinberger, DR
儿茶酚- o -甲基转移酶（COMT）是人类大脑前额叶皮层中消除多巴胺的关键酶。COMT基因（MIM 116790）的遗传变异与前额皮质功能改变和精神分裂症的高风险有关，但具体的等位基因及其功能意义一直存在争议。我们分析了COMT中几种单核苷酸多态性（snp）对mRNA表达水平（使用逆转录酶聚合酶链式反应分析）、蛋白质水平（使用Western blot分析）和酶活性（使用儿茶酚甲基化）的影响，这些影响主要来自于死后人类前额皮质组织的大样本（n = 108），该组织主要表达-膜结合亚型。一个共同的编码SNP， Val158Met (rs4680)，显著影响蛋白质丰度和酶活性，但不影响mRNA表达水平，这表明蛋白质完整性的差异解释了等位基因之间酶活性的差异。内含子1上的SNP （rs737865）和3'侧翼区域的SNP （rs165599）——两者都被报道导致等位基因表达差异，并作为Val单倍型的一部分与精神分裂症相关——对mRNA表达水平、蛋白质免疫反应性或酶活性没有影响。在来自47名受试者的淋巴细胞中，我们证实了Val/Met基因型样品对酶活性的类似影响，但对内含子1或3a snp的样品没有影响。单独的分析显示，受试者的性别以及P2启动子区域（rs2097603）中SNP的存在对COMT酶活性的影响很小。通过对小鼠COMT cDNA进行定点诱变，然后进行体外翻译，我们发现同源位置的Leu转化为Met或Val会逐渐显著降低酶活性。因此，尽管我们不能排除COMT功能影响的更复杂的遗传基础，但Val是决定前额叶皮层COMT活性较高的主要因素，这可能导致突触多巴胺水平降低和相对有害的前额叶功能。
Catechol-O-methyltransferase (COMT) is a key enzyme in the elimination of dopamine in the prefrontal cortex of the human brain. Genetic variation in the COMT gene (MIM 116790) has been associated with altered prefrontal cortex function and higher risk for schizophrenia, but the specific alleles and their functional implications have been controversial. We analyzed the effects of several single-nucleotide polymorphisms (SNPs) within COMT on mRNA expression levels (using reverse-transcriptase polymerase chain reaction analysis), protein levels (using Western blot analysis), and enzyme activity (using catechol methylation) in a large sample (n = 108) of postmortem human prefrontal cortex tissue, which predominantly expresses the -membrane-bound isoform. A common coding SNP, Val158Met (rs4680), significantly affected protein abundance and enzyme activity but not mRNA expression levels, suggesting that differences in protein integrity account for the difference in enzyme activity between alleles. A SNP in intron 1 (rs737865) and a SNP in the 3' flanking region (rs165599) - both of which have been reported to contribute to allelic expression differences and to be associated with schizophrenia as part of a haplotype with Val - had no effect on mRNA expression levels, protein immunoreactivity, or enzyme activity. In lymphocytes from 47 subjects, we confirmed a similar effect on enzyme activity in samples with the Val/Met genotype but no effect in samples with the intron 1 or 3 a SNPs. Separate analyses revealed that the subject's sex, as well as the presence of a SNP in the P2 promoter region (rs2097603), had small effects on COMT enzyme activity. Using site-directed mutagenesis of mouse COMT cDNA, followed by in vitro translation, we found that the conversion of Leu at the homologous position into Met or Val progressively and significantly diminished enzyme activity. Thus, although we cannot exclude a more complex genetic basis for functional effects of COMT, Val is a predominant factor that determines higher COMT activity in the prefrontal cortex, which presumably leads to lower synaptic dopamine levels and relatively deleterious prefrontal function.