Wang Liewei, Thomae Bianca, Eckloff Bruce, Wieben Eric, Weinshilboum Richard
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Graduate School-Mayo Clinic-Mayo Foundation, Rochester, MN 55905, USA.
Biochem Pharmacol. 2002 Aug 15;64(4):699-710. doi: 10.1016/s0006-2952(02)01223-6.
Histamine N-methyltransferase (HNMT) catalyzes one of two major metabolic pathways for histamine. The levels of HNMT activity and immunoreactive protein in human tissues are regulated primarily by inheritance. Previous studies of HNMT identified two common single nucleotide polymorphisms (SNPs), including a functionally significant nonsynonymous coding SNP (cSNP), (C314T, Thr105Ile), but that polymorphism did not explain all of the phenotypic variation. In the present study, a genotype-to-phenotype strategy was used to search for additional genetic factors that might contribute to the regulation of human HNMT activity. Specifically, we began by resequencing the human HNMT gene using 90 ethnically anonymous DNA samples from the Coriell Cell Repository and identified a total of eight SNPs, including the two that had been reported previously. No new nonsynonymous cSNPs were observed, but three of the six novel SNPs were located in the 5'-flanking region (5'-FR) of the gene-including a third common polymorphism with a frequency of 0.367 (36.7%). That observation directed our attention to possible genetic effects on HNMT transcription. As a first step in testing that possibility, we created and studied a series of reporter gene constructs for the initial 1kb of the HNMT 5'-FR. The core promoter and possible regulatory regions were identified and verified by electrophoresis mobility shift assays. We then studied the possible functional implications of the new common HNMT 5'-FR SNP. However, on the basis of reporter gene studies, that SNP appeared to have little effect on transcription. Phenotype-genotype correlation analysis performed with 112 human kidney biopsy samples that had been phenotyped for their level of HNMT activity confirmed that the common 5'-FR SNP was not associated with the level of HNMT activity in vivo. In summary, this series of experiments resulted in the identification of several novel HNMT polymorphisms, identification of the HNMT core promoter, and a comprehensive functional genomic study of a common HNMT 5'-FR SNP. These results represent an additional step in the definition of molecular genetic mechanisms involved in the regulation of this important autacoid-metabolizing enzyme in humans.
组胺N-甲基转移酶（HNMT）催化组胺两条主要代谢途径之一。人体组织中HNMT活性和免疫反应性蛋白水平主要受遗传调控。以往对HNMT的研究鉴定出两种常见的单核苷酸多态性（SNP），包括一个具有功能意义的非同义编码SNP（cSNP），即（C314T，Thr105Ile），但该多态性并不能解释所有表型变异。在本研究中，采用基因型-表型策略来寻找可能有助于调控人类HNMT活性的其他遗传因素。具体而言，我们首先使用来自科里尔细胞库的90份无种族信息的DNA样本对人类HNMT基因进行重测序，共鉴定出8个SNP，包括之前报道的2个。未观察到新的非同义cSNP，但6个新SNP中的3个位于该基因的5'侧翼区（5'-FR），包括一个频率为0.367（36.7%）的第三个常见多态性。这一发现将我们的注意力引向对HNMT转录可能的遗传效应。作为检验这一可能性的第一步，我们构建并研究了一系列针对HNMT 5'-FR最初1kb的报告基因构建体。通过电泳迁移率变动分析鉴定并验证了核心启动子和可能的调控区域。然后我们研究了新的常见HNMT 5'-FR SNP可能的功能影响。然而，基于报告基因研究，该SNP似乎对转录影响很小。对112份已根据HNMT活性水平进行表型分析的人类肾活检样本进行的表型-基因型相关性分析证实，常见的5'-FR SNP与体内HNMT活性水平无关。总之，这一系列实验鉴定出了几个新的HNMT多态性，确定了HNMT核心启动子，并对一个常见的HNMT 5'-FR SNP进行了全面的功能基因组研究。这些结果代表了在定义参与人类中这种重要自分泌代谢酶调控的分子遗传机制方面又迈出的一步。
