Acute Prenatal Alcohol Exposure Potentiates Conotruncal Defects in the Setting of a Permissive Genetic Background

项目概要/摘要 近30%的胎儿酒精谱系障碍（FASD）患者患有先天性心脏缺陷 （CHD）。流出道（OFT）缺陷的代表性过高，虽然有不同的发病率和严重程度。的 大多数产前酒精暴露（PAE）研究集中在FASD的标志，慢性饮酒和 神经缺陷人群中最常见的PAE形式，即急性接触， 围概念期及其对心脏发育的影响尚未研究。我们将研究 通过多机构临床研究和我们的急性PAE小鼠研究， 模型，其中小鼠在心脏关键点接受两次腹膜内注射3g/kg的30%乙醇 器官发生我们假设PAE诱导的OFT缺陷的发生率和严重程度不同， 解释为PAE和其他无害突变的组合，导致遗传上的 宽容的背景我们相信Notch通路的突变建立了这样一种遗传上允许的 背景，并且PAE通过Notch的表观遗传调节与这些突变协同作用，导致 OFT畸形本研究的目的1将阐明PAE对OFT有害作用的特异性 对齐和定义分子通路在个人与PAE诱导的OFT缺陷中断。我们将 通过对PAE和CHD诊断和途径的相对风险分析以及随后的逻辑分析来实现这一目标。 全基因组测序数据的回归分析。目标2将定义酒精驱动的表观遗传调控 作为急性PAE和其他无害Notch途径突变协同作用的机制， 破坏OFT的发展。使用体外和体内分子测定和组织学分析的组合， 我们将测试这种联合致畸性损伤对第二心脏区域（SHF）生存能力的影响， 迁移到OFT。我们将建立PAE驱动的表观遗传调控，破坏Notch基因的可及性， 和转录，抑制Notch信号传导，并在体外使用泛组蛋白乙酰转移酶抑制剂， 证明预防PAE诱导的过度乙酰化足以拯救Notch信号传导和SHF 生存能力类似地，通过在体外过表达notch胞内结构域（NICD）来拯救SHF活力， 将急性PAE和Notch突变协同作用的点确定为Notch信号传导的丧失，从而抑制SHF活力。 作为检查临床相关急性PAE与心脏相互作用的首批研究之一， 发展，这项研究解决了NIAAA的优先事项，以确定非慢性暴露的影响 以及酒精对未被充分研究的器官系统的影响。这项研究确定的遗传途径，包括 Notch通路在PAE诱导的CHD中具有重要意义的新验证将为疾病治疗提供靶点。 预防和识别那些最有可能患上世界上最常见和最致命的出生缺陷的人。 该项目的完成得到了赞助团队的技术培训和指导， 培训中心的尖端设施，以及主要研究者的医学博士提供的教育-博士程序.

PROJECT SUMMARY/ABSTRACT Nearly 30% of patients with Fetal Alcohol Spectrum Disorder (FASD) have a congenital heart defect (CHD). Outflow tract (OFT) defects are over-represented, though with variable incidence and severity. The majority of prenatal alcohol exposure (PAE) research focuses on the hallmarks of FASD, chronic drinking and neurologic defects. The most common form of PAE in the population, acute exposure during the periconceptual period, and its effect on cardiac development have not been studied. We will study the effects of acute, periconceptual drinking both through a multi-institutional clinical study and with our acute PAE murine model, in which mice receive two intraperitoneal injections of 3g/kg of 30% ethanol at a point critical to cardiac organogenesis. We hypothesize the variable incidence and severity of PAE-induced OFT defects can be explained by a combination of PAE and otherwise non-deleterious mutations that result in a genetically permissive background. We believe mutations in the Notch pathway establish such a genetically permissive background, and that PAE acts synergistically with these mutations via epigenetic regulation of Notch to result in OFT malformation. Aim 1 of this study will address the specificity of the deleterious effects of PAE to OFT alignment and define the molecular pathways disrupted in individuals with PAE-induced OFT defects. We will achieve this through relative risk analysis of PAE and CHD diagnoses and pathway and subsequent logistical regression analyses of whole genome sequencing data. Aim 2 will define alcohol driven epigenetic regulation as the mechanism by which acute PAE and otherwise non-deleterious Notch pathway mutations synergistically disrupt OFT development. Using a combination of molecular assays and histologic analysis in vitro and in vivo, we will test the impact of this combined teratogenic insult on second heart field (SHF) viability and ability to migrate into the OFT. We will establish PAE driven epigenetic regulation, disrupting Notch gene accessibility and transcription, inhibits Notch signaling and use a pan-histone acetyltransferase inhibitor in vitro to demonstrate prevention of PAE induced hyperacetylation is sufficient to rescue Notch signaling and SHF viability. Similarly, rescue of SHF viability by overexpression of notch intracellular domain (NICD) in vitro will cement the point of acute PAE and Notch mutation synergy as loss of Notch signaling prohibiting SHF viability. As one of the first studies to examine the interaction of clinically relevant acute PAE with heart development, this study addresses the priorities of the NIAAA to define the impact of non-chronic exposure and alcohol’s effects on understudied organ systems. The genetic pathways identified by this study, including novel validation of the Notch pathway as significant in PAE-induced CHD, will provide targets for disease prevention and identification of those most at risk to develop the world’s most common and deadly birth defect. Completion of this project is ensured by the technical training and mentorship by the sponsorship team, the training site’s cutting-edge facilities, and education provided by the principal investigator’s M.D.-Ph.D. program.