Abstract 18374: Targeted Sequencing and Massively Parallel Reporter Assay Identify the Functional Variation Underlying the 4q25 Locus for Atrial Fibrillation

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Nathan R Tucker ◽  
Jiangchuan Ye ◽  
Honghuang Lin ◽  
Michael A McLellan ◽  
Emelia J Benjamin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Association Studies ◽  
Targeted Sequencing ◽  
Massively Parallel ◽  
Genome Wide Association Studies ◽  
Sequencing Analysis ◽  
Reporter Assay ◽  
Enhancer Activity ◽  
Functional Variants ◽  
Massively Parallel Reporter Assay

Introduction: Genome-wide association studies have identified 14 independent loci for atrial fibrillation (AF). The 4q25 locus upstream of the left-right asymmetry gene PITX2 is, by far, the strongest association signal for AF. However, as with most GWAS loci, the functional variants are noncoding, presumed to be regulatory, and remain unknown. We therefore sought to rapidly identify the functional variants at an AF locus by combining high throughput sequencing and massively parallel reporter assays. Methods and Results: We sequenced a ~750kb region encompassing the PITX2 locus in 462 individuals with early-onset AF from the MGH AF Study and 464 referents from the Framingham Heart Study. The SNP most significantly associated with AF in our sequenced sample was rs2129983, which is 140kb from PITX2 (OR=2.43, P =8.9X10 -16 ). rs2129983 is approximately 1.7kb from the most significantly associated SNP in a prior AF GWAS, rs6817105 (r 2 =0.52). From the targeted sequencing analysis, we identified 262 SNVs with a MAF >0.5% within a genomic region bounded by SNPs with an r2 greater than 0.4 with the top variant. To identify functional variants, we then utilized a massively parallel reporter assay (MPRA) in order to measure enhancer activity at each SNP across the entire AF locus. In both HL-1 and C2C12 myoblasts, MPRA identified many distinct SNP regions with differential enhancer activity. Using AF-association status as a standard, we were able to identify a series of variants that have both differential activity in either cell line tested and also a high level of association (rs17042076, rs4469143). Mechanistically, these functional SNPs are predicted to alter transcription factor binding. Conclusions: We have comprehensively identified the AF-associated variation at 4q25 and determined which of these variants are functional through differential enhancer activity. Here, in addition to identifying the causative variation for AF at 4q25, we provide a generalizable pathway for translating this work to other loci, a method that could expedite the identification of causative genetic variants at other disease loci.

scholarly journals Atrial fibrillation associated common risk variants in SYNE2 lead to lower expression of nesprin-2α1 and increased nuclear stiffness

2019 ◽  
Author(s):  
Nana Liu ◽  
Jeffrey Hsu ◽  
Gautam Mahajan ◽  
Han Sun ◽  
John Barnard ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Association Studies ◽  
Dominant Negative ◽  
Genome Wide Association Studies ◽  
Nucleotide Polymorphisms ◽  
Gene Encoding ◽  
Enhancer Activity ◽  
Risk Alleles ◽  
Risk Variants ◽  
Lower Expression

ABSTRACTRationaleAtrial fibrillation (AF) genome-wide association studies (GWAS) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding the nesprin-2 protein that connects the nuclear membrane with the cytoskeletonObjectiveDetermine the effects of the AF-associated rs1152591 and rs1152595, two linked intronic single nucleotide polymorphisms (SNPs), on SYNE2 expression and investigate the mechanisms for their association with AF.Methods and ResultsRNA sequencing of human left atrial appendage (LAA) tissues indicated that rs1152591 and rs1152595 were significantly associated with the expressions of SYNE2α1, a short mRNA isoform, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 mRNA uses an alternative transcription start site and encodes an N-terminal deleted 62 kDa nesprin-2α1 isoform, which can act as a dominant-negative on nuclear-cytoskeleton connectivity. Western blot and qPCR assays confirmed that AF risk alleles of both SNPs were associated with lower expression of nesprin-2α1 in human LAA tissues. Reporter gene transfections demonstrated that the risk vs. reference alleles of rs1152591 and rs1152595 had decreased enhancer activity. SYNE2 siRNA knockdown (KD) or nesprin-2α1 overexpression studies in human stem cell-derived induced cardiomyocytes (iCMs) resulted in ~12.5 % increases in the nuclear area compared to controls (p<0.001). Atomic force microscopy demonstrated that SYNE2 KD or nesprin-2α1 overexpression led to 57.5% or 33.2% decreases, respectively, in nuclear stiffness compared to controls (p< 0.0001).ConclusionsAF-associated SNPs rs1152591 and rs1152595 downregulate the expression of SYNE2α1, increasing nuclear-cytoskeletal connectivity and nuclear stiffness. The resulting increase in mechanical stress may play a role in the development of AF.

scholarly journals A Human Accelerated Region is a Leydig cell GLI2 Enhancer that Affects Male-Typical Behavior

2021 ◽  
Author(s):  
Andrew R. Norman ◽  
Ann H. Ryu ◽  
Kirsty Jamieson ◽  
Sean Thomas ◽  
Yin Shen ◽  
...  
Keyword(s):  
Cell Line ◽  
Reproductive Biology ◽  
Leydig Cells ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Enhancer Activity ◽  
Human Male ◽  
Enhancer Function ◽  
Massively Parallel Reporter Assay ◽  
Human Accelerated Regions

ABSTRACTHuman accelerated regions (HARs) are sequences that have evolved at an accelerated rate in the human lineage. Some HARs are developmental enhancers. We used a massively parallel reporter assay (MPRA) to identify HARs with enhancer activity in a mammalian testis cell line. A subset of HARs exhibited differential activity between the human and chimpanzee orthologs, representing candidates for underlying unique human male reproductive biology. We further characterized one of these candidate testis enhancers, 2xHAR.238. CRISPR/Cas9-mediated deletion in a testis cell line and mice revealed that 2xHAR.238 enhances expression of Gli2, encoding a Hedgehog pathway effector, in testis Leydig cells. 4C-seq revealed that 2xHAR.238 contacts the Gli2 promoter, consistent with enhancer function. In adult male mice, deletion of 2xHAR.238 disrupted mouse male-typical behavior and male interest in female odor. Combined, our work identifies a HAR that promotes the expression of Gli2 in Leydig cells and may have contributed to the evolution of human male reproductive biology.

scholarly journals A functional genomics pipeline identifies pleiotropy and cross-tissue effects within obesity-associated GWAS loci

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Amelia C. Joslin ◽  
Débora R. Sobreira ◽  
Grace T. Hansen ◽  
Noboru J. Sakabe ◽  
Ivy Aneas ◽  
...  
Keyword(s):  
Association Studies ◽  
Complex Model ◽  
Genome Wide Association Studies ◽  
Enhancer Activity ◽  
Chromatin Interactions ◽  
Genome Wide ◽  
Causal Variants ◽  
Cell Type Specific ◽  
Gene Regulatory ◽  
Massively Parallel Reporter Assay

AbstractGenome-wide association studies (GWAS) have identified many disease-associated variants, yet mechanisms underlying these associations remain unclear. To understand obesity-associated variants, we generate gene regulatory annotations in adipocytes and hypothalamic neurons across cellular differentiation stages. We then test variants in 97 obesity-associated loci using a massively parallel reporter assay and identify putatively causal variants that display cell type specific or cross-tissue enhancer-modulating properties. Integrating these variants with gene regulatory information suggests genes that underlie obesity GWAS associations. We also investigate a complex genomic interval on 16p11.2 where two independent loci exhibit megabase-range, cross-locus chromatin interactions. We demonstrate that variants within these two loci regulate a shared gene set. Together, our data support a model where GWAS loci contain variants that alter enhancer activity across tissues, potentially with temporally restricted effects, to impact the expression of multiple genes. This complex model has broad implications for ongoing efforts to understand GWAS.

Abstract 18865: Identification of a Functional SNP Regulating PRRX1 at the 1q24 Locus for Atrial Fibrillation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Elena Dolmatova ◽  
Nathan R Tucker ◽  
Honghuang Lin ◽  
Rebecca R Cooper ◽  
Jiangchuan Ye ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Action Potential ◽  
Action Potential Duration ◽  
Association Studies ◽  
Physical Interaction ◽  
Eqtl Analysis ◽  
Genome Wide Association Studies ◽  
Enhancer Activity ◽  
Atrial Tissue ◽  
Functional Snp

Introduction: Genome-wide association studies have identified 9 genomic loci associated with atrial fibrillation (AF). Hypothesis: We sought to identify the functional variant at the 1q24 locus for AF, located upstream of the paired related homeobox 1 gene ( PRRX1 ). Methods: We used morpholino-mediated knockdown in zebrafish to assess the role of PRRX1 in cardiac function and development. To identify potential enhancers at the PRRX1 locus we analyzed DNase hypersensitivity, histone methylation, and mammalian conservation data from ENCODE. Tissue-specific enhancer activity was evaluated by microinjection of eGFP reporter constructs for each putative enhancer into zebrafish and luciferase assays in a mouse atrial myocyte (HL-1) cell line. To determine physical interaction between the AF-associated enhancer and PRRX1 promoter we analyzed available Hi-C data and performed chromatin conformation capture (3C). The functional SNP was localized using luciferase assays in HL-1 cells. The effect of the functional SNP on gene expression in human left atrial tissue was measured by qPCR. Results: Knockdown of the PRRX1 ortholog in zebrafish resulted in atrial dilation and shortening of atrial action potential duration (APD 80 : 114.8±2.2ms vs 126±1.5ms in controls, p=0.0004). Of the 4 regions tested at the 1q24 locus, 2 adjacent regions exhibited enhancer activity in the zebrafish myocardium. 3C demonstrated an increased interaction frequency between the enhancer and PRRX1 promoter regions in cells of cardiac lineage when compared to controls (103±57%, p=0.038). Screening for functional SNPs within these regions revealed that the AF risk allele (C) at SNP rs577676 associated with ~4 fold increased enhancer activity as compared to the non-risk (T) allele in HL-1 cells. Finally, regional eQTL analysis of human atrial tissue showed that rs577676 correlated with PRRX1 expression. Conclusions: We have implicated PRRX1 in cardiac electrophysiology by demonstrating that knockdown of the gene results in atrial dilation and shortening of atrial action potential duration. Further, we have found that SNP rs577676 modifies an enhancer regulating PRRX1 expression.

scholarly journals Transcriptional-regulatory convergence across functional MDD risk variants identified by massively parallel reporter assays

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bernard Mulvey ◽  
Joseph D. Dougherty
Keyword(s):  
Transcription Factors ◽  
Retinoic Acid ◽  
Association Studies ◽  
Massively Parallel ◽  
Genome Wide Association Studies ◽  
Retinoid Receptor ◽  
Functional Snps ◽  
Functional Variants ◽  
Transcriptional Regulatory ◽  
Reporter Assays

AbstractFamily and population studies indicate clear heritability of major depressive disorder (MDD), though its underlying biology remains unclear. The majority of single-nucleotide polymorphism (SNP) linkage blocks associated with MDD by genome-wide association studies (GWASes) are believed to alter transcriptional regulators (e.g., enhancers, promoters) based on enrichment of marks correlated with these functions. A key to understanding MDD pathophysiology will be elucidation of which SNPs are functional and how such functional variants biologically converge to elicit the disease. Furthermore, retinoids can elicit MDD in patients and promote depressive-like behaviors in rodent models, acting via a regulatory system of retinoid receptor transcription factors (TFs). We therefore sought to simultaneously identify functional genetic variants and assess retinoid pathway regulation of MDD risk loci. Using Massively Parallel Reporter Assays (MPRAs), we functionally screened over 1000 SNPs prioritized from 39 neuropsychiatric trait/disease GWAS loci, selecting SNPs based on overlap with predicted regulatory features—including expression quantitative trait loci (eQTL) and histone marks—from human brains and cell cultures. We identified >100 SNPs with allelic effects on expression in a retinoid-responsive model system. Functional SNPs were enriched for binding sequences of retinoic acid-receptive transcription factors (TFs), with additional allelic differences unmasked by treatment with all-trans retinoic acid (ATRA). Finally, motifs overrepresented across functional SNPs corresponded to TFs highly specific to serotonergic neurons, suggesting an in vivo site of action. Our application of MPRAs to screen MDD-associated SNPs suggests a shared transcriptional-regulatory program across loci, a component of which is unmasked by retinoids.

scholarly journals Transcriptional-Regulatory Convergence Across Functional MDD Risk Variants Identified by Massively Parallel Reporter Assays

2021 ◽  
Author(s):  
Bernard Mulvey ◽  
Joseph D. Dougherty
Keyword(s):  
Transcription Factors ◽  
Retinoic Acid ◽  
Association Studies ◽  
Massively Parallel ◽  
Genome Wide Association Studies ◽  
Retinoid Receptor ◽  
Functional Snps ◽  
Functional Variants ◽  
Transcriptional Regulatory ◽  
Reporter Assays

ABSTRACTFamily and population studies indicate clear heritability of major depressive disorder (MDD), though its underlying biology remains unclear. The majority of single-nucleotide polymorphism (SNP) linkage blocks associated with MDD by genome-wide association studies (GWASes) are believed to alter transcriptional regulators (e.g., enhancers, promoters), based on enrichment of marks correlated with these functions. A key to understanding MDD pathophysiology will be elucidation of which SNPs are functional and how such functional variants biologically converge to elicit the disease. Furthermore, retinoids can elicit MDD in patients, and promote depressive behaviors in rodent models, acting via a regulatory system of retinoid receptor transcription factors (TFs). We therefore sought to simultaneously identify functional genetic variants and assess retinoid pathway regulation of MDD risk loci. Using Massively Parallel Reporter Assays (MPRAs), we functionally screened over 1 000 SNPs prioritized from 39 neuropsychiatric trait/disease GWAS loci, with SNPs selected based on overlap with predicted regulatory features—including expression quantitative trait loci (eQTL) and histone marks—from human brains and cell cultures. We identified >100 SNPs with allelic effects on expression in a retinoid-responsive model system. Further, functional SNPs were enriched for binding sequences of retinoic acid-receptive transcription factors (TFs); with additional allelic differences unmasked by treatment with all-trans retinoic acid (ATRA). Finally, motifs overrepresented across functional SNPs corresponded to TFs highly specific to serotonergic neurons, suggesting an in vivo site of action. Our application of MPRAs to screen MDD-associated SNPs suggests a shared transcriptional regulatory program across loci, a subset of which are unmasked by retinoids.

scholarly journals High-Throughput Screening and CRISPR-Cas9 Modeling of Causal Lipid-Associated Expression Quantitative Trait Locus Variants

2016 ◽  
Author(s):  
Avanthi Raghavan ◽  
Xiao Wang ◽  
Peter Rogov ◽  
Li Wang ◽  
Xiaolan Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Quantitative Trait ◽  
Genome Wide Association ◽  
Massively Parallel ◽  
Genome Wide Association Studies ◽  
Reporter Assay ◽  
Genome Wide ◽  
Dna Variants ◽  
Massively Parallel Reporter Assay

AbstractGenome-wide association studies have identified a number of novel genetic loci linked to serum cholesterol and triglyceride levels. The causal DNA variants at these loci and the mechanisms by which they influence phenotype and disease risk remain largely unexplored. Expression quantitative trait locus analyses of patient liver and fat biopsies indicate that many lipid-associated variants influence gene expression in a cis-regulatory manner. However, linkage disequilibrium among neighboring SNPs at a genome-wide association study-implicated locus makes it challenging to pinpoint the actual variant underlying an association signal. We used a methodological framework for causal variant discovery that involves high-throughput identification of putative disease-causal loci through a functional reporter-based screen, the massively parallel reporter assay, followed by validation of prioritized variants in genome-edited human pluripotent stem cell models generated with CRISPR-Cas9. We complemented the stem cell models with CRISPR interference experiments in vitro and in knock-in mice in vivo. We provide validation for two high-priority SNPs, rs2277862 and rs10889356, being causal for lipid-associated expression quantitative trait loci. We also highlight the challenges inherent in modeling common genetic variation with these experimental approaches.Author SummaryGenome-wide association studies have identified numerous loci linked to a variety of clinical phenotypes. It remains a challenge to identify and validate the causal DNA variants in these loci. We describe the use of a high-throughput technique called the massively parallel reporter assay to analyze thousands of candidate causal DNA variants for their potential effects on gene expression. We use a combination of genome editing in human pluripotent stem cells, “CRISPR interference” experiments in other cultured human cell lines, and genetically modified mice to analyze the two highest-priority candidate DNA variants to emerge from the massively parallel reporter assay, and we confirm the relevance of the variants to nearby gene expression. These findings highlight a methodological framework with which to identify and functionally validate causal DNA variants.

scholarly journals Genetics and Epigenetics of Atrial Fibrillation

2020 ◽  
Vol 21 (16) ◽  
pp. 5717 ◽  
Author(s):  
Estefanía Lozano-Velasco ◽  
Diego Franco ◽  
Amelia Aranega ◽  
Houria Daimi
Keyword(s):  
Atrial Fibrillation ◽  
Regulatory Networks ◽  
Association Studies ◽  
Functional Characterization ◽  
The Elderly ◽  
Supraventricular Arrhythmia ◽  
Genome Wide Association Studies ◽  
Genome Wide ◽  
Transcriptional Regulatory ◽  
Transcriptional Regulatory Mechanisms

Atrial fibrillation (AF) is known to be the most common supraventricular arrhythmia affecting up to 1% of the general population. Its prevalence exponentially increases with age and could reach up to 8% in the elderly population. The management of AF is a complex issue that is addressed by extensive ongoing basic and clinical research. AF centers around different types of disturbances, including ion channel dysfunction, Ca2+-handling abnormalities, and structural remodeling. Genome-wide association studies (GWAS) have uncovered over 100 genetic loci associated with AF. Most of these loci point to ion channels, distinct cardiac-enriched transcription factors, as well as to other regulatory genes. Recently, the discovery of post-transcriptional regulatory mechanisms, involving non-coding RNAs (especially microRNAs), DNA methylation, and histone modification, has allowed to decipher how a normal heart develops and which modifications are involved in reshaping the processes leading to arrhythmias. This review aims to provide a current state of the field regarding the identification and functional characterization of AF-related epigenetic regulatory networks

scholarly journals Upregulation of c-MYC in cis through a Large Chromatin Loop Linked to a Cancer Risk-Associated Single-Nucleotide Polymorphism in Colorectal Cancer Cells

2010 ◽  
Vol 30 (6) ◽  
pp. 1411-1420 ◽  
Author(s):  
Jason B. Wright ◽  
Seth J. Brown ◽  
Michael D. Cole
Keyword(s):  
Cancer Risk ◽  
Cancer Cells ◽  
Association Studies ◽  
Beta Catenin ◽  
Genome Wide Association Studies ◽  
Chromatin Loop ◽  
Nucleotide Polymorphisms ◽  
Distal Enhancer ◽  
Enhancer Activity ◽  
Single Nucleotide

ABSTRACT Genome-wide association studies have mapped many single-nucleotide polymorphisms (SNPs) that are linked to cancer risk, but the mechanism by which most SNPs promote cancer remains undefined. The rs6983267 SNP at 8q24 has been associated with many cancers, yet the SNP falls 335 kb from the nearest gene, c-MYC. We show that the beta-catenin-TCF4 transcription factor complex binds preferentially to the cancer risk-associated rs6983267(G) allele in colon cancer cells. We also show that the rs6983267 SNP has enhancer-related histone marks and can form a 335-kb chromatin loop to interact with the c-MYC promoter. Finally, we show that the SNP has no effect on the efficiency of chromatin looping to the c-MYC promoter but that the cancer risk-associated SNP enhances the expression of the linked c-MYC allele. Thus, cancer risk is a direct consequence of elevated c-MYC expression from increased distal enhancer activity and not from reorganization/creation of the large chromatin loop. The findings of these studies support a mechanism for intergenic SNPs that can promote cancer through the regulation of distal genes by utilizing preexisting large chromatin loops.

A Variant Noncoding Region Regulates Prrx1 and Predisposes to Atrial Arrhythmias

2021 ◽  
Author(s):  
Fernanda M Bosada ◽  
Mathilde R Rivaud ◽  
Jae-Sun Uhm ◽  
Sander Verheule ◽  
Karel van Duijvenboden ◽  
...  
Keyword(s):  
Sodium Current ◽  
Association Studies ◽  
Noncoding Region ◽  
Specific Gene ◽  
Genome Wide Association Studies ◽  
Enhancer Activity ◽  
Atrial Cardiomyocytes ◽  
The Impact ◽  
Lineage Specific Gene

Rationale: Atrial Fibrillation (AF) is the most common cardiac arrhythmia diagnosed in clinical practice. Genome-wide association studies have identified AF-associated common variants across 100+ genomic loci, but the mechanism underlying the impact of these variant loci on AF susceptibility in vivo has remained largely undefined. One such variant region, highly associated with AF, is found at 1q24, close to PRRX1, encoding the Paired Related Homeobox 1 transcription factor. Objective: To identify the mechanistic link between the variant region at 1q24 and AF predisposition. Methods and Results: The mouse orthologue of the noncoding variant genomic region (R1A) at 1q24 was deleted using CRISPR genome editing. Among the genes sharing the topologically associated domain with the deleted R1A region (Kifap3, Prrx1, Fmo2, Prrc2c), only the broadly expressed gene Prrx1 was downregulated in mutants, and only in cardiomyocytes. Expression and epigenetic profiling revealed that a cardiomyocyte lineage-specific gene program (Mhrt, Myh6, Rbm20, Tnnt2, Ttn, Ckm) was upregulated in R1A-/- atrial cardiomyocytes, and that Mef2 binding motifs were significantly enriched at differentially accessible chromatin sites. Consistently, Prrx1 suppressed Mef2-activated enhancer activity in HL-1 cells. Mice heterozygous or homozygous for the R1A deletion were susceptible to atrial arrhythmia induction, had atrial conduction slowing and more irregular RR intervals. Isolated R1A-/- mouse left atrial cardiomyocytes showed lower action potential upstroke velocities and sodium current, as well as increased systolic and diastolic calcium concentrations compared to controls. Conclusions: The noncoding AF variant region at 1q24 modulates Prrx1 expression in cardiomyocytes. Cardiomyocyte-specific reduction of Prrx1 expression upon deletion of the noncoding region leads to a profound induction of a cardiac lineage-specific gene program and to propensity for AF. These data indicate that AF-associated variants in humans may exert AF predisposition through reduced PRRX1 expression in cardiomyocytes.

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