Identification of Functional Genetic Variants Associated With Alcohol Dependence and Related Phenotypes Using a High-Throughput Assay.
paper
Primary
Public
- Authors
- Thapa, Kriti S; Chen, Andy B; Lai, Dongbing; Xuei, Xiaoling; Wetherill, Leah; Tischfield, Jay A; Liu, Yunlong; Edenberg, Howard J
- Year
- 2020
- Journal
- Alcoholism, clinical and experimental research
- PMID
- 33119910
- DOI
- 10.1111/acer.14492
- PMCID
- PMC7725989
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' untranslated regions (3'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 meta-analyses 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 meta-analysis p valuesΒ β€Β 0.001) that were located in 3'UTRs. RESULTS: We identified 60 SNPs that affected gene expression (false discovery rate [FDR]Β <Β 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.
PASSPORT-seq results in SH-SY5Y and SK-N-BE(2) cell lines. (A) Plot of the alternative allele effect (derived from the mixed effects generalized linear model) of the SNPs in SH-SY5Y [SH] and SK-N-BE(2) [SK] cell lines. SNPs with FDR < 0.05 in both cell lines are denoted in red. (B) Alternative allele frequency and total read depth (sum of reference and alternative alleles) for a few significant SNPs of interest. SH: SH-SY5Y; SK: SK-N-BE(2); ALT: alternate; FDR: false discovery rate; alt freq: alternative allele frequency.
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