P2-143: USING MASSIVELY PARALLEL REPORTER ASSAYS TO IDENTIFY PROTECTIVE FUNCTIONAL VARIANTS IN THE APOE REGION

AbstractMassively parallel reporter assays (MPRAs) have enabled the study of transcriptional regulatory mechanisms at an unprecedented scale and with high quantitative resolution. However, this realm lacks models that can discover sequence-specific signals de novo from the data and integrate them in a mechanistic way. We present MuSeAM (Multinomial CNNs for Sequence Activity Modeling), a convolutional neural network that overcomes this gap. MuSeAM utilizes multinomial convolutions that directly model sequence-specific motifs of protein-DNA binding. We demonstrate that MuSeAM fits MPRA data with high accuracy and generalizes over other tasks such as predicting chromatin accessibility and prioritizing potentially functional variants.

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

Translational Psychiatry ◽

10.1038/s41398-021-01493-6 ◽

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.

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Massively parallel reporter assays combined with cell-type specific eQTL informed multiple melanoma loci and identified a pleiotropic function of HIV-1 restriction gene,MX2, in melanoma promotion

10.1101/625400 ◽

2019 ◽

Cited By ~ 3

Author(s):

Jiyeon Choi ◽

Tongwu Zhang ◽

Andrew Vu ◽

Julien Ablain ◽

Matthew M Makowski ◽

...

Keyword(s):

Transcriptional Activity ◽

Susceptibility Genes ◽

Massively Parallel ◽

Chromosome Band ◽

Cell Type ◽

Functional Variants ◽

Reporter Assays ◽

Cell Type Specific ◽

Hiv 1 ◽

Pleiotropic Function

AbstractGenome-wide association studies (GWAS) have identified ∼20 melanoma susceptibility loci. To identify susceptibility genes and variants simultaneously from multiple GWAS loci, we integrated massively-parallel reporter assays (MPRA) with cell type-specific epigenomic data as well as melanocyte-specific expression quantitative trait loci (eQTL) profiling. Starting from 16 melanoma loci, we selected 832 variants overlapping active regions of chromatin in cells of melanocytic lineage and identified 39 candidate functional variants displaying allelic transcriptional activity by MPRA. For four of these loci, we further identified four colocalizing melanocytecis-eQTL genes (CTSS,CASP8,MX2, andMAFF) matching the allelic activity of MPRA functional variants. Among these, we further characterized the locus encompassing the HIV-1 restriction gene,MX2, on chromosome band Chr21q22.3 and validated a functional variant, rs398206, among multiple high LD variants. rs398206 mediates allelic transcriptional activity via binding of the transcription factor, YY1. This allelic transcriptional regulation is consistent with a significantcis-eQTL ofMX2in primary human melanocytes, where the melanoma risk-associated A allele of rs398206 is correlated with higherMX2levels. Melanocyte-specific transgenic expression of humanMX2in a zebrafish model demonstrated accelerated melanoma formation in aBRAFV600Ebackground. Thus, using an efficient scalable approach to streamline GWAS follow-up functional studies, we identified multiple candidate melanoma susceptibility genes and variants, and uncovered a pleiotropic function ofMX2in melanoma susceptibility.

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

10.1101/2021.03.05.434177 ◽

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.

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A semisupervised model to predict regulatory effects of genetic variants at single nucleotide resolution using massively parallel reporter assays

Bioinformatics ◽

10.1093/bioinformatics/btab040 ◽

2021 ◽

Author(s):

Zikun Yang ◽

Chen Wang ◽

Stephanie Erjavec ◽

Lynn Petukhova ◽

Angela Christiano ◽

...

Keyword(s):

Genetic Variants ◽

Prediction Models ◽

Statistical Prediction ◽

Massively Parallel ◽

Supplementary Information ◽

Functional Variants ◽

Increased Risk ◽

Regulatory Variant ◽

Reporter Assays ◽

Regulatory Effects

Abstract Motivation Predicting regulatory effects of genetic variants is a challenging but important problem in functional genomics. Given the relatively low sensitivity of functional assays, and the pervasiveness of class imbalance in functional genomic data, popular statistical prediction models can sharply underestimate the probability of a regulatory effect. We describe here the presence-only model (PO-EN), a type of semisupervised model, to predict regulatory effects of genetic variants at sequence-level resolution in a context of interest by integrating a large number of epigenetic features and massively parallel reporter assays (MPRAs). Results Using experimental data from a variety of MPRAs we show that the presence-only model produces better calibrated predicted probabilities and has increased accuracy relative to state-of-the-art prediction models. Furthermore, we show that the predictions based on pretrained PO-EN models are useful for prioritizing functional variants among candidate eQTLs and significant SNPs at GWAS loci. In particular, for the costimulatory locus, associated with multiple autoimmune diseases, we show evidence of a regulatory variant residing in an enhancer 24.4 kb downstream of CTLA4, with evidence from capture Hi-C of interaction with CTLA4. Furthermore, the risk allele of the regulatory variant is on the same risk increasing haplotype as a functional coding variant in exon 1 of CTLA4, suggesting that the regulatory variant acts jointly with the coding variant leading to increased risk to disease. Availability and implementation The presence-only model is implemented in the R package ‘PO.EN’, freely available on CRAN. A vignette describing a detailed demonstration of using the proposed PO-EN model can be found on github at https://github.com/Iuliana-Ionita-Laza/PO.EN/ Supplementary information Supplementary data are available at Bioinformatics online.

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Identification of Functional Variants in the FAM13A Chronic Obstructive Pulmonary Disease Genome-Wide Association Study Locus by Massively Parallel Reporter Assays

American Journal of Respiratory and Critical Care Medicine ◽

10.1164/rccm.201802-0337oc ◽

2019 ◽

Vol 199 (1) ◽

pp. 52-61 ◽

Cited By ~ 13

Author(s):

Peter J. Castaldi ◽

Feng Guo ◽

Dandi Qiao ◽

Fei Du ◽

Zun Zar Chi Naing ◽

...

Keyword(s):

Chronic Obstructive Pulmonary Disease ◽

Association Study ◽

Genome Wide Association Study ◽

Genome Wide Association ◽

Massively Parallel ◽

Chronic Obstructive ◽

Obstructive Pulmonary Disease ◽

Functional Variants ◽

Genome Wide ◽

Reporter Assays

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

Circulation ◽

10.1161/circ.132.suppl_3.18374 ◽

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.

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