Genetic Determinants of COMT Expression

描述（由申请人提供）：儿茶酚- o -甲基转移酶，COMT，代谢内源性儿茶酚底物，因此在包括神经传递在内的各种过程中很重要。儿茶酚水平失调与神经行为障碍有关。大量的遗传关联研究表明，COMT与精神分裂症和恐慌症等脑部疾病有关。许多这些等位基因关联涉及改变酶活性的功能性COMT DNA。这些关联结果往往是相互矛盾的，暗示在COMT位点或附近有另一个或多个变体。此外，已经观察到使用这种功能性COMT变体的等位基因特异性mRNA表达。本提案概述了一项可行性研究，以表征个体的DNA序列和表达变化，以了解序列变化对疾病人群中COMT表达的潜在影响。我们假设儿茶酚- o -甲基转移酶基因区域的DNA变异有助于COMT mRNA表达的变异。为了验证这一假设，我们将使用来自犹他州的30个高加索CEPH三胞胎的淋巴母细胞样细胞系，该人群已由HapMap Consortium进行基因分型，以表征人类群体的遗传多样性。在这个群体中，我们将使用定量逆转录-聚合酶链反应来表征细胞系COMT mRNA的表达。对于转录本中存在的DNA变异杂合的受试者，我们将使用单碱基扩展方法测量等位基因特异性mRNA表达。然后，我们将测试基因型与COMT mRNA表达变化之间的相关性。我们将在COMT位点和COMT上游约340kb的区域内对样本进行常见单核苷酸多态性（snp）的基因型分析，并使用在HapMap项目中同一区域的同一样本中已经进行基因型分析的约100个snp的基因型。单标记和多标记单倍型将用于检测mRNA表达变异的相关性。最后，我们将利用雌激素对COMT活性的调节作用，通过评估用雌激素处理细胞系后COMT的表达，然后测量与SNP基因型的关联，包括预测雌激素反应元件的变异。与COMT表达差异相关的遗传多态性可用于鉴定顺式调控元件，以预测COMT功能差异。这项体外工作可能有助于了解COMT对疾病易感性的遗传贡献，特别是关于COMT位点的顺式调控。

DESCRIPTION (provided by applicant): Catechol-O-methyltransferase, COMT, metabolizes endogenous catechol substrates, and thus is important in a variety of processes, including neurotransmission. Dysregulation of catechol levels is implicated in neurobehavioral disorders. Numerous genetic association studies suggest the involvement of COMT in brain diseases like schizophrenia and panic disorder. Many of these allelic associations involve a functional COMT DNA that alters enzymatic activity. These association results are often contradictory and implicate another variant or variants at or near the COMT locus. Additionally, allele-specific mRNA expression using this functional COMT variant has been observed. This proposal outlines a feasibility study to characterize the DNA sequence and expression variation in individuals in an effort to understand the potential impact of sequence variation on COMT expression in a disease population. We hypothesize that DNA variation in the catechol-O-methlytransferase gene region contributes to variability in COMT mRNA expression. In order to test this hypothesis, we will use lymphoblastoid cell lines from 30 Caucasian CEPH trios from Utah, a population that has been genotyped by the HapMap Consortium in order to characterize genetic diversity in the human population. With this population, we will characterize cell line COMT mRNA expression using quantitative reverse transcription-polymerase chain reaction. For subjects heterozygous for DNA variants present in transcripts, we will measure allele-specific mRNA expression using a single base extension method. We will then test the correlation between genotype and mRNA expression variation in COMT. We will genotype our sample for common single nucleotide polymorphisms (SNPs) within the COMT locus and across a region approximately 340kb upstream of COMT, as well as use genotypes from ~100 SNPs already genotyped in this same sample in this same region during the HapMap project. Single markers and multiple-marker haplotypes will be used to test for correlation with variation in mRNA expression. Finally, we will exploit observations of the regulatory role that estrogen plays in COMT activity by assessing COMT expression after treating cell lines with estrogen, and then measuring association to SNP genotypes, including variants in predicted estrogen response elements. Genetic polymorphisms correlated with differences in COMT expression can be used to identify cis- acting regulatory elements for predicting COMT functional differences. This in vitro work may be useful for the understanding of the genetic contribution of COMT to disease susceptibility, particularly with regard to the cis regulation of the COMT locus.