Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders

Xi Rao ,Andy B Chen ,Tatiana Foroud ,Alison Goate ,Hai Lin ,Yue Wang ,Robert O Messing ,Todd C Skaar ,Dongbing Lai ,Joseph Ipe ,Jay A Tischfield ,Manav Kapoor ,Jill L Reiter ,Howard J Edenberg ,Kriti Shrestha Thapa ,Katherine A Hargreaves ,Sean P Farris ,Hongmei Gu ,Hongyu Gao ,Xiaoling Xuei ,Yunlong Liu

doi:10.1038/s41380-019-0508-z

Abstract

Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3′ untranslated regions (3′UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3′UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.

Highlights

Alcohol use disorder (AUD) is a major public health problem [1,2,3]
We focused on the ~17,000–24,000 single nucleotide polymorphisms (SNPs) in each of the four brain regions that had more than 10 reads in at least five heterozygous samples in both the AUD and control groups
To examine whether there were allele-specific differences that varied between subjects with AUD and controls, a generalized linear mixed effect model (GLMM) was implemented; the coefficient of the interaction between allele and experimental group, β12, estimates the log2 ratio of the fraction of alternative allele reads between the control and AUD samples, and was used to evaluate whether the allele-specific expression (ASE) at a specific locus was significantly different between AUD and control groups

Summary

Introduction

Alcohol is a central nervous system depressant, and high levels of consumption over a long period may alter brain function to promote AUD and damage the brain, in part by altering gene expression levels [4, 5]. Understanding the molecular mechanisms by which alcohol affects the brain is important and might provide clues to the causes of AUD and ways to reverse the impact on the brain of heavy drinking. Variations in many genes influence the risk for AUD; aside from functional variants in two alcoholmetabolizing enzymes, alcohol dehydrogenase and aldehyde dehydrogenase, each individual variant has only a small effect [1, 3, 6, 7]. Genome-wide association studies (GWAS) identify regions in the genome that affect risk for complex diseases [8], but to date, only a few Allele-specific expression and high-throughput reporter assay reveal functional genetic variants

Methods

Results

Conclusion