Variants in CHRNA5/CHRNA3/CHRNB4 and nicotine dependence

描述(由申请人提供)：吸烟会上瘾，被认为是由尼古丁依赖(ND)驱动的。几十年来，数据表明ND风险增加的遗传基础。最近，几项研究证实了rs16969968与尼古丁依赖之间的联系。Rs16969968是一个非同义SNP，将CHRNA5基因第398位氨基酸由天冬氨酸(D)变为天冬酰胺(N)(D398N)。我们推测，CHRNA5基因的这个非同义变异可能会改变含有5个亚基的nAChRs的功能特性，最终改变吸烟和新城疫的风险。为了验证这一假设，该项目有四个目标：1.建立稳定表达人a4b2*-或a3b4*-nAChRs的细胞系，并以这些细胞系为模型系统，体外研究主要亚型(a4b2*和a3b4*)的性质。2.用膜片钳技术研究野生型和D398Na5亚基对a4b2*和a3b4*-nAChR的功能和药理作用。3.检测rs16969968是否参与吸烟启动，以及基因与环境的相互作用--即确定环境暴露对吸烟行为的影响是否被rs16969968基因改变。4.利用a5基因敲除小鼠建立含a5基因的nAChR亚型在ND中的作用。我们的目标是定义天然nAChR中a5亚基的存在或不存在如何影响对尼古丁的急性反应(焦虑和运动活动)、尼古丁奖励(条件性位置偏爱，CPP)和主观影响(尼古丁辨别)。 公共卫生相关性：吸烟和尼古丁依赖是一个重大的公共卫生问题。许多流行病学研究提供了令人信服的证据，证明遗传因素在尼古丁依赖的发展中起着重要作用。最近，几项大规模的遗传学研究发现，尼古丁受体α5、α3和β4基因的几个变异与重度吸烟和尼古丁依赖显著相关，包括一个功能多态rs16969968或D398N。这项目前的研究旨在建立模型系统，以检查D398N在培养细胞、动物模型和人类行为水平上的影响。我们采取的方法是1)建立一个细胞模型系统来研究N398D的分子、药理和电生理特性；2)研究rs16969968的遗传变异如何与环境因素相互作用，最终导致吸烟成瘾和依赖；以及3)在动物模型中研究这些基因的行为效应。

DESCRIPTION (provided by applicant): Tobacco smoking is addictive and thought to be driven by nicotine dependence (ND). For decades, data have suggested genetic bases for an increased risk of ND. More recently, several studies validated the association of rs16969968 with nicotine dependence. rs16969968 is a non-synonymous SNP, changing the 398th amino acid from aspartic acid (D) to asparagine (N) (D398N) in the CHRNA5 gene. We hypothesize that this non-synonymous variant in the CHRNA5 gene may alter the functional properties of nAChRs containing 5 subunit, ultimately altering risks for tobacco smoking and ND. To test this hypothesis, the project has four aims: 1. To generate stably transfected cell lines expressing human a4b2*- or a3b4*-nAChRs containing wild- type or variant a5 subunits and use these cell lines as model system to study the properties of major subtypes (a4b2* and a3b4*) in vitro. 2. To characterize functional and pharmacological effects of the presence of wild-type or D398N a5 subunits on a4b2*- and a3b4*-nAChR using patch-clamp technique. 3. To test whether rs16969968 is involved in smoking initiation and to test for gene-environment interactions -that is, to determine if the impact of environmental exposures on smoking behaviors are modified by the rs16969968 genotypes. 4. To establish roles of a5-containing nAChR subtypes in ND using a5 knock-out mice. The goal is to define how the presence or absence of a5 subunits in native nAChR affects acute responses to nicotine (anxiety and locomotor activity), nicotine reward (conditioned place preference, CPP), and subjective effects (nicotine discrimination). PUBLIC HEALTH RELEVANCE: Tobacco smoking and nicotine dependence is a major public health issue. Many epidemiology studies have provided compelling evidence that genetic factors play a significant role in the development of nicotine dependence. More recently, several large scale genetic studies have found that several variants in the nicotinic receptor alpha 5, alpha 3 and beta 4 genes are significantly associated with heavy smoking and nicotine dependence, including one functional polymorphism rs16969968 or D398N. This current study is designed to create model systems to examine the effects of D398N at the levels of cultured cells, animal model and human behaviors. The approaches we take are 1) To develop a cellular model system to study molecular, pharmacological and electrophysiological properties of N398D; 2) To examine how genetic variants at rs16969968 interact with environmental factors and eventually lead to smoking addiction and dependence; and 3) To study the behavioral effects of these genes in animal models.