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

描述（由申请人提供）：孕妇在怀孕期间吸烟仍然是围产期发病率的主要原因，并导致终生肺功能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产妇吸烟做出了重大努力，但约有13%的妇女在怀孕期间自我报告吸烟，这每年影响到40多万婴儿的终生呼吸健康。尼古丁成瘾显然是一些妇女在怀孕期间无法戒烟的一个驱动因素，GWAS和基因分型研究都发现了5尼古丁乙酰胆碱受体（nAChR）的一种常见多态性（rs16969968），这种多态性与大量吸烟和戒烟减少有关。值得注意的是，我们的初步数据表明，这种多态性增加了母亲在怀孕期间吸烟的可能性，也增加了母亲在怀孕期间吸烟对后代肺功能不利影响的程度。因此，本研究的主要目的是确定怀孕期间母亲吸烟对肺发育影响的机制，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与吸烟有关的疾病，最终必须加以解决，以预防和治疗与吸烟有关的疾病。为了解释尼古丁成瘾介导效应和直接终器官介导效应之间的关系，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在肺部发育中的作用。接下来，我们将使用表达野生型¿5亚基的小鼠和敲入多态性¿5亚基的小鼠来比较固定剂量尼古丁与具有多态性¿5亚基的小鼠自我施用的高剂量尼古丁的效果。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部持续变化的机制。这一目标将通过在小鼠和人类组织中参与肺结构和功能的特定基因的启动子区域以及nAChR基因中进行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，本课程的目标和训练内容将得到满足。拟议的研究项目还将引入成瘾的概念和模型，以成功揭示nAChR多态性在成瘾加剧的情况下影响肺发育的机制。这项研究具有重大的公共卫生意义，可以潜在地确定特定基因型增加孕妇在怀孕期间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.