Identification of Functional Genetic Variants Associated With Alcohol Dependence and Related Phenotypes Using a High‐Throughput Assay

Abstract

Background: Genome-wide association studies (GWAS) of alcohol dependence (AD) and related phenotypes have identified multiple loci, but the functional variants underlying the loci have in most cases not been identified. Noncoding variants can influence phenotype by affecting gene expression; for example, variants in the 3 0 untranslated regions (3 0 UTR) can affect gene expression posttranscriptionally. Methods: We adapted a high-throughput assay known as PASSPORT-seq (parallel assessment of polymorphisms in miRNA target sites by sequencing) to identify among variants associated with AD and related phenotypes those that cause differential expression in neuronal cell lines. Based upon metaanalyses of alcohol-related traits in African American and European Americans in the Collaborative Study on the Genetics of Alcoholism, we tested 296 single nucleotide polymorphisms (SNPs with metaanalysis p values ≤ 0.001) that were located in 3 0 UTRs. Results: We identified 60 SNPs that affected gene expression (false discovery rate [FDR] < 0.05) in SH-SY5Y cells and 92 that affected expression in SK-N-BE(2) cells. Among these, 30 SNPs altered RNA levels in the same direction in both cell lines. Many of these SNPs reside in the binding sites of miRNAs and RNA-binding proteins and are expression quantitative trait loci of genes including KIF6, FRMD4A,CADM2,ADD2,PLK2, and GAS7. Conclusion: The SNPs identified in the PASSPORT-seq assay are functional variants that might affect the risk for AD and related phenotypes. Our study provides insights into gene regulation in AD and demonstrates the value of PASSPORT-seq as a tool to screen genetic variants in GWAS loci for one potential mechanism of action.

Perspective on Beyond Statistical Significance: Finding Meaningful Effects

“…Studying populations of different ancestry, and thereby different linkage disequilibrium patterns can help narrow a locus. High throughput assays that identify functional SNPs are important methods for prioritizing SNPs for further study [21][22][23]. Identification of the functional SNP(s) and the gene or genes they affect provides a strong basis for studying the biology underlying the trait.…”

“…Open chromatin and potential binding sites for regulatory proteins can be identified by techniques such as ATAC-sequencing [22]. High throughput assays of SNP function (as promoters, enhancers, silencers, and modifiers of RNA stability) [21][22][23] can make important contributions. Many of these approaches have been carried out in bulk tissues, and those studies have shown the importance of tissue-specific effects.…”

Functional roles, regulatory mechanisms and theranostics applications of ncRNAs in alcohol use disorder

“…It is estimated that 40-60% of AUD risk can be attributed to hereditary factors, with the remainder being linked to individual physiopsychological features, social contextual variables and gene-environment interactions [6]. Long-lasting changes in the expression of brain protein-coding genes have been the most intensively studied in AUD patients and rodents [8,9]. However, these genes only accounted for less than 2% of the mammalian genome sequence, and the rest of genomic transcripts are noncoding RNAs (ncRNAs) that were previously assumed to be incapable of coding for proteins [10].…”

Gene-based polygenic risk scores analysis of alcohol use disorder in African Americans

“…Furthermore, for these disease-causing genes, it is highly likely that there is at least one disease-causing variant located within the gene boundaries, e.g. non-synonymous mutations that change gene products or 3' UTR variants that alter gene expression levels (23,24).…”