From the, Department of Biochemistry and Molecular Biology, (KST, HJE), Indiana University School of Medicine, Indianapolis, Indiana.
Department of Medical and Molecular Genetics, (ABC, DL, XX, LW, YL, HJE), Indiana University School of Medicine, Indianapolis, Indiana.
Alcohol Clin Exp Res. 2020 Dec;44(12):2494-2518. doi: 10.1111/acer.14492. Epub 2020 Nov 17.
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.
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 values ≤ 0.001) that were located in 3'UTRs.
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.
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.
全基因组关联研究（GWAS）发现了多个与酒精依赖（AD）和相关表型相关的基因座，但大多数情况下尚未确定这些基因座下的功能变异。非编码变异可以通过影响基因表达来影响表型；例如，3'非翻译区（3'UTR）中的变异可以在后转录水平影响基因表达。
我们采用了一种称为 PASSPORT-seq（通过测序平行评估 miRNA 靶位点多态性）的高通量检测方法，以鉴定与 AD 和相关表型相关的变异中那些导致神经细胞系中差异表达的变异。基于合作酒精遗传研究中对非裔美国人和欧洲裔美国人的酒精相关特征的荟萃分析，我们测试了 296 个单核苷酸多态性（荟萃分析 p 值≤0.001 的 SNP），这些 SNP 位于 3'UTR 中。
我们鉴定出了 60 个影响 SH-SY5Y 细胞基因表达（错误发现率[FDR] < 0.05）的 SNP 和 92 个影响 SK-N-BE(2)细胞表达的 SNP。其中，30 个 SNP 在两种细胞系中改变了 RNA 水平的同一方向。这些 SNP 中的许多位于 miRNA 和 RNA 结合蛋白的结合位点，并且是包括 KIF6、FRMD4A、CADM2、ADD2、PLK2 和 GAS7 在内的基因的表达数量性状基因座。
PASSPORT-seq 检测到的 SNP 是功能变异，可能影响 AD 和相关表型的风险。我们的研究提供了 AD 基因调控的见解，并证明了 PASSPORT-seq 作为一种筛选 GWAS 基因座中遗传变异的工具的价值，用于潜在的作用机制。
