scholarly journals Regulatory dissection of the severe COVID-19 risk locus introgressed by Neanderthals

2021 ◽  
Author(s):  
Evelyn Jagoda ◽  
Davide Marnetto ◽  
Francesco Montinaro ◽  
Daniel Richard ◽  
Luca Pagani ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Past Research ◽  
Massively Parallel ◽  
Risk Haplotype ◽  
Reporter Assay ◽  
Biological Mechanisms ◽  
Related Cell ◽  
Regulatory Potential ◽  
Genomics Tools ◽  
Massively Parallel Reporter Assay

Individuals infected with the SARS-CoV-2 virus present with a wide variety of phenotypes ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe COVID-19 phenotype. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the severe COVID-19 phenotype. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform an locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify 4 introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. These variants likely drive the locus impact on severity by putatively modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

scholarly journals Identification of functional variants for plateletCD36expression by Massively Parallel Reporter Assay

2019 ◽  
Author(s):  
Namrata Madan ◽  
Andrew R. Ghazi ◽  
Xianguo Kong ◽  
Edward S. Chen ◽  
Chad A. Shaw ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Massively Parallel ◽  
Low Density ◽  
Reporter Assay ◽  
Lipid Levels ◽  
Oxidized Low Density Lipoprotein ◽  
Cd36 Expression ◽  
Massively Parallel Reporter Assay

AbstractCD36 is a platelet membrane glycoprotein whose engagement with oxidized low-density lipoprotein (oxLDL) results in platelet activation. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk by genome-wide association studies. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative. We screened 81 expression quantitative trait loci (eQTL) single nucleotide polymorphisms (SNPs) associated with plateletCD36expression by a Massively Parallel Reporter Assay (MPRA) and analyzed the results with a novel Bayesian statistical method. Ten eQTLs located in a 35kb region upstream of theCD36transcriptional start site demonstrated significant transcription shifts between their minor and major allele in the MPRA assay. Of these, rs2366739 and rs1194196, separated by only 20bp, were confirmed by luciferase assay to alter transcriptional regulation. In addition, electromobility shift assays demonstrated differential DNA:protein complex formation between the two alleles of this locus. Furthermore, deletion of the genomic locus by CRISPR/Cas9 in K562 cells results in upregulation of CD36 transcription. These data indicate that we have identified a variant that regulates expression ofCD36, which in turn affects platelet function. To assess the clinical relevance of our findings we used the PhenoScanner tool, which aggregates large scale GWAS findings; the results reinforce the clinical relevance of our variants and the utility of the MPRA assay. The study demonstrates a generalizable paradigm for functional testing of genetic variants to inform mechanistic studies, support patient management and develop precision therapies.Author SummaryPlatelets are anucleate cells that are best known as regulators of vascular hemostasis and thrombosis but also play important roles in cancer, angiogenesis, and inflammation. CD36 is a platelet surface marker that can activate platelet in response to oxidized low density lipoprotein (oxLDL). CD36 has been associated with numerous cardiovascular traits in human including blood lipid levels, platelet count, and cardiovascular disease prevalence in human genetic studies. Human variability in platelet CD36 levels are associated with the platelet response to oxLDL. However, the genetic mechanisms responsible for the variability of CD36 levels are unknown. We examined 81 genetic variants associated withCD36levels for functionality using a high-throughput assay. Of the ten variants that were identified in that assay, one doublet, rs2366739 and rs1194196, were confirmed using additional molecular and cellular assays. Deletion of the genomic region containing rs2366739 and rs1194196 resulted in overexpression ofCD36in a cell culture system. This finding indicates a control locus which can serve as a potential target in modulating CD36 expression and altering platelet function in cardiovascular disease.

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 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 High-throughput identification of RNA nuclear enrichment sequences

2017 ◽  
Author(s):  
Chinmay J Shukla ◽  
Alexandra L McCorkindale ◽  
Chiara Gerhardinger ◽  
Keegan D Korthauer ◽  
Moran N Cabili ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Localization ◽  
Single Molecule ◽  
Noncoding Rnas ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Sequence Elements ◽  
Massively Parallel Reporter Assay ◽  
Insight Into

SummaryOne of the biggest surprises since the sequencing of the human genome has been the discovery of thousands of long noncoding RNAs (lncRNAs)1–6. Although lncRNAs and mRNAs are similar in many ways, they differ with lncRNAs being more nuclear-enriched and in several cases exclusively nuclear7,8. Yet, the RNA-based sequences that determine nuclear localization remain poorly understood9–11. Towards the goal of systematically dissecting the lncRNA sequences that impart nuclear localization, we developed a massively parallel reporter assay (MPRA). Unlike previous MPRAs12–15 that determine motifs important for transcriptional regulation, we have modified this approach to identify sequences sufficient for RNA nuclear enrichment for 38 human lncRNAs. Using this approach, we identified 109 unique, conserved nuclear enrichment regions, originating from 29 distinct lncRNAs. We also discovered two shorter motifs within our nuclear enrichment regions. We further validated the sufficiency of several regions to impart nuclear localization by single molecule RNA fluorescence in situ hybridization (smRNA-FISH). Taken together, these results provide a first systematic insight into the sequence elements responsible for the nuclear enrichment of lncRNA molecules.

scholarly journals Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay

2013 ◽  
Vol 23 (5) ◽  
pp. 800-811 ◽  
Author(s):  
P. Kheradpour ◽  
J. Ernst ◽  
A. Melnikov ◽  
P. Rogov ◽  
L. Wang ◽  
...  
Keyword(s):  
Massively Parallel ◽  
Reporter Assay ◽  
Regulatory Motifs ◽  
Massively Parallel Reporter Assay

SA131A MASSIVELY PARALLEL REPORTER ASSAY FOR VARIANTS ASSOCIATED WITH SCHIZOPHRENIA AND ALZHEIMER'S DISEASE

2019 ◽  
Vol 29 ◽  
pp. S1260-S1261
Author(s):  
Dimitrios Avramopoulos ◽  
Leslie Myint ◽  
Kasper Hansen ◽  
Ruihua Wang ◽  
Leandros Boukas ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Massively Parallel Reporter Assay

scholarly journals Development of a massively parallel reporter assay to identify functional regulatory variants in the PICALM Alzheimer disease associated locus

2020 ◽  
Vol 16 (S2) ◽  
Author(s):  
Karen Nuytemans ◽  
Derek Van Booven ◽  
Derek M. Dykxhoorn ◽  
Jeffery M. Vance ◽  
Margaret A. Pericak‐Vance ◽  
...  
Keyword(s):  
Alzheimer Disease ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Regulatory Variants ◽  
Massively Parallel Reporter Assay

scholarly journals Massively parallel analysis of human 3′ UTRs reveals that AU-rich element length and registration predict mRNA destabilization

2021 ◽  
Author(s):  
David A Siegel ◽  
Olivier Le Tonqueze ◽  
Anne Biton ◽  
Noah Zaitlen ◽  
David J Erle
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Stem Loop ◽  
Mrna Destabilization ◽  
The Stability ◽  
Massively Parallel Reporter Assay ◽  
Simple Sequence

Abstract AU-rich elements (AREs) are 3′ UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3′ UTR sequences that conform to these motifs affect their function. Here we use functional annotation of sequences from 3′ UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3′ UTR sequence fragments from 4,653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3′ UTRs.

scholarly journals Predicting mean ribosome load for 5’UTR of any length using deep learning

2021 ◽  
Vol 17 (5) ◽  
pp. e1008982
Author(s):  
Alexander Karollus ◽  
Žiga Avsec ◽  
Julien Gagneur
Keyword(s):  
Translational Control ◽  
Genetic Variants ◽  
Mrna Translation ◽  
Beta Thalassemia ◽  
Regulatory Sequences ◽  
Control Mechanisms ◽  
Reporter Assay ◽  
Translation Rate ◽  
Network Operation ◽  
Massively Parallel Reporter Assay

The 5’ untranslated region plays a key role in regulating mRNA translation and consequently protein abundance. Therefore, accurate modeling of 5’UTR regulatory sequences shall provide insights into translational control mechanisms and help interpret genetic variants. Recently, a model was trained on a massively parallel reporter assay to predict mean ribosome load (MRL)—a proxy for translation rate—directly from 5’UTR sequence with a high degree of accuracy. However, this model is restricted to sequence lengths investigated in the reporter assay and therefore cannot be applied to the majority of human sequences without a substantial loss of information. Here, we introduced frame pooling, a novel neural network operation that enabled the development of an MRL prediction model for 5’UTRs of any length. Our model shows state-of-the-art performance on fixed length randomized sequences, while offering better generalization performance on longer sequences and on a variety of translation-related genome-wide datasets. Variant interpretation is demonstrated on a 5’UTR variant of the gene HBB associated with beta-thalassemia. Frame pooling could find applications in other bioinformatics predictive tasks. Moreover, our model, released open source, could help pinpoint pathogenic genetic variants.

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