Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders.
- Authors
- Rao, Xi; Thapa, Kriti S; Chen, Andy B; Lin, Hai; Gao, Hongyu; Reiter, Jill L; Hargreaves, Katherine A; Ipe, Joseph; Lai, Dongbing; Xuei, Xiaoling; Wang, Yue; Gu, Hongmei; Kapoor, Manav; Farris, Sean P; Tischfield, Jay; Foroud, Tatiana; Goate, Alison M; Skaar, Todd C; Mayfield, R Dayne; Edenberg, Howard J; Liu, Yunlong
- Year
- 2021
- Journal
- Molecular psychiatry
- PMID
- 31477794
- DOI
- 10.1038/s41380-019-0508-z
- PMCID
- PMC7050407
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.
Schematic representation of the PASSPORT-seq assay. A pool of oligonucleotides flanking both alleles of 437 SNPs were cloned in parallel into the 3′UTR of the luciferase gene in pIS-0 vector. Colonies were pooled and DNA purified. The resulting plasmid library was transiently transfected into two neuroblastoma cells lines, SH-SY5Y and SK-N-BE(2). The cDNAs were synthesized from the total RNA. The target sequences were amplified from the cDNAs and the plasmid DNA extracted from the transfected cells using two-step-PCR with unique barcodes for each cell line and each biological replicate. Following next-generation sequencing, the reads were aligned to the reference library consisting of all the test sequences and ASE was measured for each SNPs. ss single-stranded, ds double-stranded
Allele-specific expression in the postmortem brain samples from subjects with and without AUD. a Volcano plot comparing the adjusted log2 fold change (adj log2 FC) and false discovery rate (FDR) of the percentage of alternative alleles between AUD subjects and controls. SNPs with FDR < 0.05 and adjusted log2(fold change) >1 or <−1 were color-coded by brain region. BLA basolateral nucleus of the amygdala, CE central nucleus of the amygdala, NAC nucleus accumbens, and SFC superior frontal cortex; alt alternative. b Alternative allele frequency box plot and a scatter-plot of the number of reference and alternative reads for one significant SNP from each brain region (symbols noted above). alt freq alternative allele frequency; Ctl = social/non-drinking control subjects; Alc = AUD subjects; ref = reference. c Consistency in the adj log2 FC in different brain regions. SNPs with FDR < 0.05 in BLA and p < 0.05 in another brain region are plotted by adjusted fold change, color-coded by the other brain region. Of the 24 SNPs, 20 had consistent fold change direction in the two brain regions. d Heatmap of adjusted fold change of SNPs with FDR < 0.05 in at least one brain region shows consistency in fold change among all four brain regions. Dark and light colors indicate genome-wide significant (FDR < 0.05), or borderline significant (FDR > 0.05 but p < 0.05), respectively. Red and blue indicate increased and decreased percentage of alternative allele in the AUD brain comparing to control group. e Ingenuity pathway analysis results of genes enriched in nervous system development and function
PASSPORT-seq results in SH-SY5Y and SK-N-BE(2) cell lines. a Plot of the adjusted log2(fold change) of alternative allele frequency between RNA and DNA in SH-SY5Y [SH] and SK-N-BE(2) [SK] cell lines in the four brain regions. SNPs with FDR < 0.05 in both cell lines were color-coded by the brain region. b Alternative allele frequency and read depth for significant SNPs derived from each brain region
ASE, PASSPORT-seq, and ethanol treatment results for selected SNPs. Three SNPs (rs2950846, rs1968676, rs45474901) that had alternative allele frequency significantly different in PASSPORT-seq results (FDR < 0.05) and significantly different across 0, 10, and 20 mM ethanol treatment dosages in SK-N-BE(2) cells (p < 0.05) were identified. The differences in the alternative allele frequency in the brains of heterozygous social/non-drinkers (Ctl) and AUD subjects (Alc) are also shown. SK = SK-N-BE(2) cells
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| Bulk and single-cell transcriptomic brain data identify overlapping processes and cell-types with human AUD and mammalian models of alcohol use. | Huggett SB et al. | — | 2026 | → |
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