Nicotinic receptor genes & substance abuse: Functional studies of associated SNPs

描述（由申请人提供）：人类遗传学研究已经确定了15号染色体上的尼古丁受体基因簇（CHRNA5/A3/B4）是与药物滥用相关行为（包括吸烟、酒精表型和可卡因依赖）相关的强有力候选者。这些基因也与肺癌有关，但目前尚不清楚这种联系的哪些方面可能通过吸烟行为介导。此外，我们已经发现证据表明，CHRNA3和CHRNB4基因之间的基因间区域与更一般的去抑制行为有关，包括品行障碍，这是早期开始使用药物和后来出现问题的已知危险因素。来自分子、药理学和动物研究的证据表明，这些基因的转录调控可能是共同调控的，并且是复杂的，13和24亚基可能是开发戒烟药物的良好靶点。我们已经启动了研究，以评估在我们的人类遗传研究中涉及的snp等位基因的假定功能差异。我们的研究结果提供了证据，在永生化细胞培养系和原代小鼠神经胚胎细胞培养中使用荧光素酶基因检测，该区域至少有两个snp可能导致基因表达差异。这个应用程序的目标是以几种方式扩展这些发现。首先，我们将使用这些分析来评估这些snp周围的DNA序列如何影响它们差异驱动基因表达的能力（“启动子撞击”）。其次，我们将研究这些snp在不同细胞类型中的作用，包括各种永生化神经元细胞、肺癌细胞和原代小鼠神经元胚胎细胞（基因x细胞类型相互作用）。第三，我们将用尼古丁刺激细胞来评估这种药物是否会改变基因表达模式（基因与环境的相互作用）。第四，我们将利用荧光素酶测定确定基因表达的等位基因特异性变化是否会导致蛋白质亚基和受体表达的相应差异。最后，我们将评估烟碱受体的功能，这些受体的亚基成分是用我们的体外系统产生的，该系统含有不同的单个snp和snp单倍型的等位基因。我们希望开发和应用于这些基因的方法可以很容易地应用于其他可能与药物滥用相关行为相关的尼古丁受体基因的未来研究。公共卫生相关性：本项目的结果将有助于更好地了解CHRNA3和CHRNB4基因的自然变异如何有助于导致与药物使用和滥用有关的行为的个体差异的潜在分子机制。这些知识应该有助于改进对这些疾病患者的预防和治疗。

DESCRIPTION (provided by applicant): Human genetic studies have identified the cluster of nicotinic receptor genes (CHRNA5/A3/B4) on chromosome 15 as strong candidates for association with drug abuse-related behaviors, including smoking, alcohol phenotypes, and cocaine dependence. These genes have also been associated with lung cancer, but it is remains unclear what aspects of this association may be mediated through smoking behavior. In addition, we have found evidence that the intergenic region between the CHRNA3 and CHRNB4 genes is associated with more general measures of disinhibitory behavior, including conduct disorder, which is a known risk factor for early initiation of drugs and later problems. There is evidence from molecular, pharmacological, and animal studies that transcriptional regulation of these genes is likely to be co-regulated and complex, and that the 13 and 24 subunits might be good targets for development of smoking cessation drugs. We have initiated studies to assess the putative functional differences of alleles for SNPs that have been implicated in our human genetic studies. Our results provide evidence that at least two SNPs in this region may lead to differences in gene expression, using luciferase-gene assays in immortalized cell culture lines and in primary mouse neuronal embryonic cell cultures. The goals of this application are to extend these findings in several ways. First, we will use these assays to evaluate how the DNA sequence surrounding these SNPs may affect their ability to differentially drive gene expression ("promoter bashing"). Second, we will examine the effects of these SNPs in different cell types, including a variety of immortalized neuronal cells, lung cancer cells, and primary mouse neuronal embryonic cells (gene x cell type interaction). Third, we will challenge the cells with nicotine to assess whether this drug modifies gene expression patterns (gene x environment interaction). Fourth, we will determine whether allele-specific changes in gene expression using the luciferase assays lead to corresponding differences in protein subunit and receptor expression. Finally, we will assess function of nicotinic receptors whose subunit constituents were generated using our in vitro system containing different alleles for individual SNPs and haplotypes of SNPs. We expect the methods developed and applied to these genes to be easily applied to future studies of other nicotinic receptor genes that may also be associated with drug abuse-related behaviors. PUBLIC HEALTH RELEVANCE: Results from this project will facilitate a better understanding of how naturally occurring variations in the CHRNA3 and CHRNB4 genes might contribute to the underlying molecular mechanisms responsible for individual differences in behaviors relevant to drug use and abuse. Such knowledge should lead to the development of improved prevention and treatment of individuals who suffer from these disorders.