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

描述（由申请人提供）：人类遗传学研究已确定15号染色体上的烟碱受体基因簇（CHRNA 5/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）相关的强有力候选基因。这些基因也与肺癌有关，但目前尚不清楚这种关联的哪些方面可能通过吸烟行为介导。此外，我们发现证据表明，CHRNA 3和CHRNB 4基因之间的基因间区域与更一般的去抑制行为措施有关，包括行为障碍，这是早期开始使用药物和后期问题的已知风险因素。有证据表明，从分子，药理学和动物研究，这些基因的转录调控可能是共同调节和复杂的，13和24亚基可能是良好的目标，为戒烟药物的发展。我们已经启动了研究，以评估在我们的人类遗传学研究中涉及的SNP等位基因的推定功能差异。我们的研究结果提供的证据表明，至少有两个SNPs在这一地区可能会导致基因表达的差异，在永生化细胞培养系和原代小鼠神经元胚胎细胞培养物中使用β-淀粉酶基因检测。本应用程序的目标是以几种方式扩展这些发现。首先，我们将使用这些检测来评估这些SNP周围的DNA序列如何影响它们差异驱动基因表达的能力（“启动子冲击”）。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，我们将用尼古丁挑战细胞，以评估这种药物是否改变基因表达模式（基因x环境相互作用）。第四，我们将确定是否等位基因特异性的基因表达的变化，使用荧光素酶检测导致相应的差异蛋白质亚基和受体表达。最后，我们将评估烟碱受体的功能，其亚基成分是使用我们的体外系统产生的，该系统含有单个SNP和SNP单倍型的不同等位基因。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到其他尼古丁受体基因的未来研究，也可能与药物滥用相关的行为。 公共卫生相关性：该项目的结果将有助于更好地了解CHRNA 3和CHRNB 4基因中自然发生的变异如何有助于导致与药物使用和滥用相关的行为个体差异的潜在分子机制。这些知识应导致发展更好的预防和治疗患有这些疾病的个人。

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.