Allele-Specific Expression and High-Throughput Reporter Assay Reveal Functional Variants in Human Brains with Alcohol Use Disorders

Rao, Xin; Ks, Thapa; Ab, Chen; Lin, Han; H, Gao; Reiter, Jill L.; Ka, Hargreaves; J, Ipe; Lai, Dehui; Xuei, Xiaoling; Gu, Heng; Kapoor, Manav; Sp, Farris; Tischfield, Jay A.; Foroud, Tatiana; Goate, Alison M.; Skaar, Todd C.; Mayfield, RD; Hj, Edenberg; Y, Liu

doi:10.1101/514992

Cited by 7 publications

(8 citation statements)

References 33 publications

(38 reference statements)

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“…To understand one potential mechanism of action underlying loci associated with AD and related phenotypes, we selected 296 SNPs that showed some evidence of association (p ≤ 0.001) and were located in 3 0 UTRs, and simultaneously tested whether they affected gene expression using a highthroughput assay, PASSPORT-seq (Rao et al, 2019). Both alleles of 84 and 86% of the SNPs were detected in both DNA and RNA in every replicate in SH-SY5Y and SK-N-BE(2) cells, respectively (Table S3).…”

Section: Resultsmentioning

confidence: 99%

“…This is done by transfecting a pool of expression vectors containing both alleles at all chosen SNPs, and examining biases in allelic expression by comparing at each SNP the ratio of alleles in the RNA to their ratio in the expression vectors transfected into the cell. The PASSPORT-seq assay was conducted as previously described in detail (Rao et al, 2019). Briefly, the assay involves (i) synthesis of a pool of oligonucleotides that includes both alleles of 296 SNPs, each flanked by 25 nt of their genomic sequence (Oligomix ® , LC Sciences, Houston, TX; Table S2), (ii) cloning them in bulk into reporter plasmid pIS-0 (12178, Addgene, Cambridge, MA) and preparing DNA, (iii) transient transfection of the pooled DNA into cells, (iv) extraction and purification of DNA and RNA from the cells, and (v) quantitation of the relative amounts of RNA and DNA for each oligonucleotide by NGS.…”

Section: Selection Of 3 0 Utr Snpsmentioning

confidence: 99%

“…A very sensitive way to detect the effects of a variant in the 3 0 UTR is to examine differential expression of each allele of a single nucleotide polymorphism (SNP) inserted into a reporter gene (allele-biased expression). We recently developed a high-throughput assay, PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing; Ipe et al, 2018;Rao et al, 2019), that can simultaneously and efficiently detect whether hundreds of SNPs are cis-regulators. This assay combines pooled synthesis of oligonucleotides, bulk cloning into a reporter vector, transfection of the pool into mammalian cells, and next-generation sequencing (NGS) to measure differential expression.…”

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confidence: 99%

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

Thapa

Chen

Lai

et al. 2020

Alcoholism Clin & Exp Res

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Selection Of 3 0 Utr Snpsmentioning

confidence: 99%

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confidence: 99%

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

Thapa

Chen

Lai

et al. 2020

Alcoholism Clin & Exp Res

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“…Notably, ERASE uses all ASEs rather than summarizing information across a gene, thus retaining ASEs generated due to splicing QTLs. Given the growing popularity of ASE analyses [59][60][61][62] and the increased interest in splicing, ERASE's ability to detect enrichment in small ASE datasets and multiple annotations has the potential to add significantly to biological knowledge. This accounts for biases in ASE discovery due to high read depth and exonic structure.…”

Section: Discussionmentioning

confidence: 99%

ERASE: Extended Randomization for assessment of annotation enrichment in ASE datasets

D’Sa

Guelfi

et al. 2019

Preprint

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Genome-wide association studies (GWAS) have identified thousands of genetic variants 24 associated with various human phenotypes and many of these loci are thought to act at a molecular 25 level by regulating gene expression. Detection of allele specific expression (ASE), namely 26 preferential usage of an allele at a transcribed locus, is an increasingly important means of studying 27 the genetic regulation of gene expression. However, there are currently a paucity of tools available 28 to link ASE sites with GWAS risk loci. Existing integration methods first use ASE sites to infer 29 cis-acting expression quantitative trait loci (eQTL) and then apply eQTL-based approaches. 30ERASE is a method that assesses the enrichment of risk loci amongst ASE sites directly. 31 Furthermore, ERASE enables additional biological insights to be made through the addition of 32 other SNP level annotations. ERASE is based on a randomization approach and controls for read 33 depth, a significant confounder in ASE analyses. In this paper, we demonstrate that ERASE can 34 efficiently detect the enrichment of eQTLs and risk loci within ASE data and that it remains 35 sensitive even when used with underpowered GWAS datasets. Finally, using ERASE in 36 combination with GWAS data for Parkinson's disease and data on the splicing potential of 37 individual SNPs, we provide evidence to suggest that risk loci for Parkinson's disease are enriched 38 amongst ASEs likely to affect splicing. Thus, we show that ERASE is an important new tool for 39 the integration of ASE and GWAS data, capable of providing novel insights into the 40 pathophysiology of complex diseases. 41 42 43 44 Over the last decade our understanding of complex genetic disorders has been transformed by the 45 use of genome-wide association studies (GWAS) with 71,673 SNP-trait associations reported in 46 the latest release of the NHGRI-EBI GWAS catalogue 1 . Complex neurological and 47 neuropsychiatric disorders are no exception. The most recent GWAS for Parkinson's disease (PD) 48 reported 78 risk loci 2 , while that for schizophrenia 3 reported 145. While there is a lag in terms of 49 understanding the underlying biological processes involved in contributing to disease risk, it is 50 thought that risk loci largely act by regulating gene expression as opposed to changing protein 51 function 4 . This observation has led to an increasing interest in the genetic regulation of gene 52 expression and the identification of expression quantitative trait loci (eQTLs, genetic variants that 53are associated with variation in gene expression) across the genome. eQTL studies have helped in 54 the interpretation of GWAS findings across a range of disorders 5; 6 . However, one limitation of 55 eQTL analyses is the requirement for large sample sizes. Since this can be difficult to achieve for 56 some human tissues, such as specific brain regions, alternative approaches more robust to small 57 sample sets are required. 58Allele specific expression (ASE) analysis is another means of studying ...

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“…expression studies in the prefrontal cortex have identified genes encoding to GABA A receptor subunits or related to mitochondrial function (Buckley et al, 2000;Fan et al, 1999) as well as in functions related to myelination, cell cycling, oxidative stress, and transcription (Farris et al, 2015;Flatscher-Bader et al, 2005, 2006Iwamoto et al, 2004;Kapoor et al, 2019;Lewohl et al, 2000;Mayfield et al, 2002;Ponomarev et al, 2012;Rao et al, 2019;Sutherland et al, 2014;Zhang et al, 2014). Expression studies in nucleus accumbens (NAc) and ventral tegmental area have also revealed gene expression changes related to cell architecture, signaling, vesicle formation, and synaptic transmission (Flatscher-Bader et al, 2006Mamdani et al, 2015;Rao et al, 2019). These findings suggest that there are region-specific susceptibilities and adaptations to chronic alcohol consumption in the brain that are likely to have a distinct effect on the behavioral phenotypes comprising AUD (Flatscher-Bader et al, 2010).…”

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confidence: 99%

Assessing the Role of Long Noncoding RNA in Nucleus Accumbens in Subjects With Alcohol Dependence

Drake

McMichael

Vornholt

et al. 2020

Alcoholism Clin & Exp Res

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Background: Long noncoding RNA (lncRNA) have been implicated in the etiology of alcohol use. Since lncRNA provide another layer of complexity to the transcriptome, assessing their expression in the brain is the first critical step toward understanding lncRNA functions in alcohol use and addiction. Thus, we sought to profile lncRNA expression in the nucleus accumbens (NAc) in a large postmortem alcohol brain sample. Methods: LncRNA and protein-coding gene (PCG) expressions in the NAc from 41 subjects with alcohol dependence (AD) and 41 controls were assessed via a regression model. Weighted gene coexpression network analysis was used to identify lncRNA and PCG networks (i.e., modules) significantly correlated with AD. Within the significant modules, key network genes (i.e., hubs) were also identified. The lncRNA and PCG hubs were correlated via Pearson correlations to elucidate the potential biological functions of lncRNA. The lncRNA and PCG hubs were further integrated with GWAS data to identify expression quantitative trait loci (eQTL). Results: At Bonferroni adj. p-value ≤ 0.05, we identified 19 lncRNA and 5 PCG significant modules, which were enriched for neuronal and immune-related processes. In these modules, we further identified 86 and 315 PCG and lncRNA hubs, respectively. At false discovery rate (FDR) of 10%, the correlation analyses between the lncRNA and PCG hubs revealed 3,125 positive and 1,860 negative correlations. Integration of hubs with genotype data identified 243 eQTLs affecting the expression of 39 and 204 PCG and lncRNA hubs, respectively. Conclusions: Our study identified lncRNA and gene networks significantly associated with AD in the NAc, coordinated lncRNA and mRNA coexpression changes, highlighting potentially regulatory functions for the lncRNA, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD.

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Allele-Specific Expression and High-Throughput Reporter Assay Reveal Functional Variants in Human Brains with Alcohol Use Disorders

Cited by 7 publications

References 33 publications

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

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

ERASE: Extended Randomization for assessment of annotation enrichment in ASE datasets

Assessing the Role of Long Noncoding RNA in Nucleus Accumbens in Subjects With Alcohol Dependence

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