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与尼古丁依赖的关联。rs16969968是一个非同义SNP，将CHRNA5基因中的第398个氨基酸从天冬氨酸(D)变为天冬酰胺(N) （D398N）。我们假设，CHRNA5基因的这种非同义变体可能改变含有5个亚基的nAChRs的功能特性，最终改变吸烟和ND的风险。为了验证这一假设，该项目有四个目标：1。生成含有野生型或变异型a5亚基的表达人a4b2*-或a3b4*- nachrs的稳定转染细胞系，并以这些细胞系为模型系统，在体外研究主要亚型（a4b2*和a3b4*）的性质。2. 利用膜片钳技术表征野生型或d398na5亚基对a4b2*-和a3b4*- nachr的功能和药理作用。3. 为了测试rs16969968是否参与吸烟的开始以及测试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基因型所改变。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。目的是确定原生nAChR中a5亚基的存在或缺失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（条件位置偏好，CPP）和主观效应（尼古丁歧视）。

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.