Identifying and protecting alcohol-sensitive epigenetic changes in congenital heart disease

该提案描述了一项为期五年的有指导的研究和培训计划，该计划将促进 斯蒂芬妮·福特博士作为先天性心脏病发病机制独立研究者的发展 疾病。以福特博士作为临床新生儿专家和基础科学家的背景为基础，她将获得 在小鼠研究、RNA-FISH和表观遗传机制设计方面的专业知识。她将通过以下途径获得技能 有组织的指导、动手的实验室体验、授课教学和凯斯的正式课堂作业 西部储备大学、美国国立卫生研究院FAES和杰克逊实验室。迈克尔·詹金斯博士，先驱 心脏光学成像，以及产前酒精暴露模型专家辛西娅·比勒博士将提供 他们对这个项目的专业知识和指导技能，促进了福特博士向研究独立性的过渡。 据估计，美国2.4-4.8%的新生儿患有胎儿酒精谱系障碍(FASD)，由 产前酒精暴露(PAE)。PAE引起的先天性心脏病(CHDS)尚未被研究为 尽管FASD的患病率很高(40%)，但与其他FASD结果一样严重。与以下疾病相关的先心病 FASD，主要是瓣膜间隔和流出道缺陷，危及生命，影响生长和健康。PAE 已知会影响甲基化和一碳代谢。正常的单碳代谢及其结果 DNA的甲基化对基因的正确表达至关重要。比较基因组学研究揭示了 在包括老鼠和鸟类在内的脊椎动物物种中有很强的表观遗传保守性，特别是 关键基因的高甲基化和低甲基化DNA序列。我们将在以下位置研究PAE引起的变化 小鼠和禽类心脏发育的关键时期(心内膜垫和四腔发育) 胚胎心脏。所有的心脏都将用光学相干断层扫描进行成像，以快速确定他们的心脏 表型。DNA甲基化变化将通过甲基-ATAC-Seq和亚硫酸氢盐的组合来确定 测序。DNA甲基化将在两个物种中进行比较，因为两个物种中保守的变化更多 可能与人类PAE诱导的缺陷有关。RNA-FISH将被用来确认基因表达的变化， 这将使我们能够精确定位3D心脏中的哪个部位，例如形成瓣膜，基因表达正在发生变化。 然后，我们将探索使用胆碱和谷胱甘肽来预防PAE的影响。胆碱和 众所周知，谷胱甘肽可以促进一碳代谢中的甲基化。胆碱已经在人类身上显示出来 预防PAE早期神经效应的研究。我们已经在一个鸟类模型中表明，谷胱甘肽可以防止 PAE后的CHDS和DNA甲基化异常。我们将同时使用鸟类和老鼠模型来 确定酒精+胆碱或谷胱甘肽对心脏结构、DNA甲基化和基因的影响 表情。我们假设，通过维持正常的甲基化，因此DNA 表达，我们选择的化合物将防止由PAE导致的CHD。一种化合物 可以预防PAE引起的先天性心脏病，每年可以帮助成千上万的儿童及其家庭。

This proposal describes a five-year mentored research and training plan that will facilitate the development of Dr. Stephanie Ford, MD as an independent investigator in the pathogenesis of congenital heart disease. Building upon Dr. Ford’s background as a clinical neonatologist and a basic scientist, she will attain expertise in design of mouse studies, RNA-FISH, and epigenetic mechanisms. She will gain skills through structured mentorship, hands-on laboratory experiences, didactic teaching, and formal classwork at Case Western Reserve University, FAES at the NIH, and Jackson Laboratories. Dr. Michael Jenkins, a pioneer in cardiac optical imaging, and Dr. Cynthia Bearer, an expert in prenatal alcohol exposure models, will provide their expertise and mentorship skills to this project, fostering Dr. Ford’s transition to research independence. An estimated 2.4-4.8% of newborns in the U.S. have fetal alcohol spectrum disorders (FASDs), caused by prenatal alcohol exposure (PAE). PAE induced Congenital Heart Diseases (CHDs) have not been studied as intensively as other FASD outcomes despite their high prevalence rate (40%). The CHDs associated with FASDs, mostly valvuloseptal and outflow tract defects, are life-threatening and impact growth and health. PAE is known to affect methylation and one-carbon metabolism. Normal one-carbon metabolism and its resulting methylation of DNA is crucial for the correct expression of genes. Comparative genomics studies have revealed that there is strong epigenetic conservation across vertebrate species including mice and avians, particularly the hyper-and hypo-methylated DNA sequences of critical genes. We will investigate the PAE-induced changes at times critical to heart development (endocardial cushion and 4 chamber development) in mouse and avian embryonic hearts. All hearts will be imaged with optical coherence tomography to rapidly determine their phenotype. DNA methylation changes will be determined with a combination of methyl-ATAC-seqand bisulfite sequencing. DNA methylation will be compared in both species, as conserved changes in two species are more likely to be relevant to human PAE-induced defects. RNA-FISH will be used to confirm gene expression changes, which will allow us to pinpoint where within the 3D heart, such as the forming valves, gene expression is changing. We will then explore the use of choline and glutathione to prevent the effects of PAE. Choline and glutathione are known to promote methylation in one-carbon metabolism. Choline has been shown in human studies to prevent early neurologic effects of PAE. We have shown in an avian model that glutathione prevents the CHDs and abnormal DNA methylation seen after PAE. We will use both avian and mouse models to determine the effects of alcohol + choline or glutathione on cardiac structure, DNA methylation, and gene expression. We hypothesize that by maintaining normal methylation, and therefore DNA expression, our chosen compounds will prevent the CHDs that result from PAE. Compounds that could prevent PAE-induced CHDs could help thousands of children and their families each year.