Prenatal nicotine and lung development: role of CHRNA5, addiction and epigenetics

描述（由申请人提供）：母亲在怀孕期间吸烟仍然是围产期发病率的主要原因，并导致肺功能终身下降和后代哮喘风险增加。尽管通过公共卫生运动和戒烟干预措施为减少孕产妇吸烟做出了重大努力，但约有13%的妇女自述在怀孕期间吸烟，这影响了每年40多万婴儿的终身呼吸健康。尼古丁成瘾显然是一些妇女在怀孕期间无法戒烟的驱动力，GWAS和基因分型研究都发现了一种常见的多态性（rs 16969968），在5烟碱乙酰胆碱受体（nAChR）中，与大量吸烟和减少戒烟有关。值得注意的是，我们的初步数据表明，这种增加母亲在怀孕期间吸烟可能性的相同多态性也增加了母亲在怀孕期间吸烟对后代肺功能产生不利影响的程度。因此，本研究的主要目的是确定nAChR亚基的改变介导母亲在妊娠期间吸烟对肺发育的影响的机制，并评估尼古丁成瘾与尼古丁对肺的直接影响的相对贡献。尼古丁成瘾的作用与尼古丁对细胞过程的直接后果这一基本问题适用于所有吸烟相关疾病，最终必须得到解决，以预防和治疗吸烟相关疾病。为了解释成瘾介导的效应和围产期尼古丁的直接终末器官介导的效应，我们将采用结合成瘾和肺发育范式的转基因小鼠模型。具体来说，我们将首先使用<$5基因敲除小鼠来描述<$5在肺发育中的作用。接下来，我们将使用表达野生型<$5亚基的小鼠和具有多态性<$5亚基敲入的小鼠来比较设定剂量的尼古丁与具有多态性<$5亚基的小鼠自我施用的更高剂量的尼古丁的效果。最后，我们将研究DNA甲基化在靶基因调控中的作用，以了解子宫内尼古丁诱导肺持续变化的机制。这一目标将通过在小鼠和人组织中参与肺结构和功能以及nAChR基因的选择基因的启动子区域内通过焦磷酸测序进行定量CpG甲基化分析来实现。通过学习研究小鼠肺发育和功能的基本方法以及临床研究的基础知识，将达到本建议的目标和培训内容。拟议的研究项目还将引入成瘾的概念和模型，以成功地揭示nAChR多态性在高度成瘾的情况下影响肺部发育的机制。这项研究具有重大的公共卫生意义，可能确定特定基因型增加妊娠期间母亲吸烟的后果和可能性的分子机制。

DESCRIPTION (provided by applicant): Maternal smoking during pregnancy remains a major cause of perinatal morbidity and causes lifelong decreases in pulmonary function and increased risk of asthma in offspring. Despite significant efforts to reduce maternal smoking through public health campaigns and smoking cessation interventions, approximately 13% of all women self-report smoking during pregnancy and this effects the lifelong respiratory health of over 400,000 infants each year. Nicotine addiction is clearly a driving force in the inability of some women to quit smoking during pregnancy and both GWAS and genotyping studies have identified a common polymorphism (rs16969968) in the ¿5 nicotinic acetylcholine receptor (nAChR) that is associated with heavier cigarette use and reduced smoking cessation. Remarkably, our preliminary data suggests that this same polymorphism that increases the likelihood of maternal smoking during pregnancy also increases the degree to which maternal smoking during pregnancy adversely affects offspring pulmonary function. Thus, the primary objective of this proposal is to determine the mechanism by which alterations in the ¿5 nAChR subunit mediate the effects of maternal smoking during pregnancy on lung development and to assess the relative contribution of nicotine addiction versus the direct effects of nicotine on lung. This fundamental question of the role of nicotine addiction versus direct consequences of nicotine on cellular processes is applicable to all smoking related diseases and ultimately must be addressed to prevent and treat smoking related diseases. To decipher between addiction mediated effects and direct end organ mediated effects of perinatal nicotine, we will employ transgenic mouse models combined with both addiction and lung development paradigms. Specifically, we will first use ¿5 knockout mice to delineate the role of ¿5 in lung development. Next we will use mice expressing the wild-type ¿5 subunit and mice with a knockin for the polymorphic ¿5 subunit to compare the effects of a set dose of nicotine to that of the higher doses of nicotine that are self-administered by mice with the polymorphic ¿5 subunit. Lastly, we will examine the role of DNA methylation in the regulation of target genes so as to understand the mechanisms by which in utero nicotine induces persistent changes in lung. This goal will be met by performing quantitative CpG methylation analysis by pyrosequencing within the promoter regions of select genes involved in pulmonary structure and function and in nAChR genes in both mouse and human tissues. The objectives and training components of this proposal will be met by learning basic approaches to studying lung development and function in mice and basics of clinical research. The proposed research project will additionally introduce concepts and models of addiction to successfully unravel the mechanism by which nAChR polymorphisms influences lung development within the setting of heightened addiction. This research has great public health significance to potentially identify the molecular mechanisms by which specific genotypes increase the consequences and likelihood of maternal smoking during pregnancy.