Developing Systemic in vivo Massively Parallel Reporter Assay to Elucidate the Genetic Etiology of Problematic Alcohol Use

Abstract Problematic alcohol use (PAU), a complex phenotype that combines alcohol use disorder (AUD) and alcohol- related problems identified by a survey (AUDIT-P), is a major public health issue that arises from intricate interactions between genetic and environmental mechanisms. Genome-wide association studies (GWAS) have identified genomic regions associated with PAU, but it is difficult to determine which genomic variants functionally contribute to the development of PAU, largely because many of these variants are located in the noncoding genome. Recent advances in high-throughput sequencing technologies now permit the functional characterization of thousands of noncoding, disease-associated variants simultaneously with massively parallel reporter assays (MPRA). However, MPRA studies have traditionally been conducted in vitro which limits our ability to identify tissue- and sex-specific variant effects. Because the regulatory genome is sensitive to factors like cell state, tissue type, and environmental perturbation, it is crucial to study the regulatory function of noncoding variants within the intact physiological context. The goal of this project is to identify which PAU risk variants alter transcriptional regulatory capacity in the intact brain and liver under natural physiological conditions and to determine whether this modulation will differ in response to ethanol exposure. To determine the regulatory activity of variants in a tissue- and sex-specific manner, I will perform in vivo MPRA via intracerebroventricular and systemic tail vein injections (Aim 1). I will then perturb the animals with acute or chronic ethanol administration via intraperitoneal injection which may induce variant regulatory activity that is not present in the homeostatic state (Aim 2). The results of this work will pinpoint the putatively causal PAU-associated variants in a tissue- and sex-specific manner as well as which variants exhibit functional regulatory activity only in the environmentally perturbed state. Successful completion of this project will positively contribute to the neurogenetics field by determining which variants should be pursued for further study within their natural genomic context, thus elucidating the underlying molecular mechanisms of genetic predisposition for PAU.