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

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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.