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

项目摘要/摘要 近30%的胎儿酒精光谱障碍(FASD)患者有先天性心脏缺陷 (CHD)。流出道(OFT)缺陷被过度描述，尽管其发生率和严重性各不相同。这个 大多数产前酒精暴露(PAE)研究集中在FASD、长期饮酒和 神经缺陷。人群中最常见的PAE形式，在 围孕期，及其对心脏发育的影响尚未被研究。我们将研究这些影响 通过一项多机构的临床研究和我们的急性PAE小鼠对急性妊娠期饮酒的研究 模型小鼠在心脏关键部位两次注射3g/kg的30%乙醇 器官发生。我们假设PAE引起的OFT缺陷的不同发生率和严重程度可能是 由PAE和其他非有害突变的组合解释，这些突变导致基因上的 宽大的背景。我们认为，Notch途径中的突变建立了这样一种基因允许的 背景，PAE通过对Notch的表观遗传调节与这些突变协同作用，从而导致 在OFT畸形中。这项研究的目标1将解决PAE对OFT有害影响的特殊性 比对和确定PAE诱导的OFT缺陷个体中被破坏的分子通路。我们会 通过PAE和CHD诊断和路径的相对风险分析以及后续的后勤工作来实现这一点 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和其他无害的Notch通路突变协同作用的机制 扰乱OFT发展。采用体外和体内分子检测和组织分析相结合的方法， 我们将测试这种复合致畸侮辱对第二心区(SHF)活性和能力的影响。 迁移到OFT。我们将建立PAE驱动的表观遗传调控，扰乱Notch基因的可及性 和转录，抑制Notch信号转导，并在体外使用泛组蛋白乙酰转移酶抑制剂 证明预防PAE诱导的高乙酰化足以挽救Notch信号和SHF 生存能力。同样，在体外通过过表达缺口细胞内域(NICD)来挽救SHF的活性将 证实了急性PAE和Notch突变协同作用的观点是Notch信号的丢失，抑制了SHF的生存。 作为首批研究临床相关的急性PAE与心脏相互作用的研究之一 作为发展，这项研究阐述了NIAAA的优先事项，以确定非慢性暴露的影响 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 新近证实Notch通路在PAE诱导的CHD中具有重要意义，将为疾病提供靶点 预防和识别世界上最常见和最致命的出生缺陷高危人群。 这个项目的完成是由赞助团队的技术培训和指导确保的 培训现场的尖端设施，以及由首席调查员的医学博士课程提供的教育。

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.