CHROMATIN ACCESSIBILITY CHANGES AND GENOMIC INTEGRATION IDENTIFY GENETIC REGULATORY MECHANISMS

Cardiovascular disease, type 2 diabetes, and related cardiometabolic traits are responsible for substantial mortality and economic costs globally. Cardiometabolic traits are common complex phenotypes with both genetic and environmental components and are influenced by several tissues, including adipose, liver, skeletal muscle, and pancreas. Genome-wide association studies (GWAS) have identified thousands of loci associated with cardiometabolic traits, but most of these loci are in noncoding regions of the genome and their molecular functions are not well annotated. Variants at GWAS loci are often found within transcriptional regulatory elements and/or are associated with gene expression in trait-relevant tissues, suggesting that GWAS variants frequently alter gene regulation. Transcriptional regulatory elements vary by genotype, tissue, and cellular state, but regulatory element annotation in many of these disease-relevant contexts is lacking. A more complete annotation of regulatory elements may uncover mechanisms for GWAS loci for cardiometabolic traits. To this end, I profiled chromatin accessibility, a marker of regulatory elements, in adipose tissue, liver tissue, and multiple stages of adipocyte differentiation. The accessible chromatin landscape in adipose tissue was underannotated and our profiles helped identify GWAS variants that may alter adipose gene regulation. I identified accessible chromatin regions that vary by genotype in liver tissue, providing suggestive evidence that these variants alter regulatory element activity. The accessible chromatin regions that differ between stages of adipocyte differentiation suggest specific cellular states in which GWAS variants may alter gene regulation. I integrated accessible chromatin regions with multiple genomic data types to predict functional variants, disrupted TF binding motifs, and target genes at cardiometabolic GWAS loci. Variants at several loci showed allelic differences in transcriptional reporter and protein binding assays, providing further evidence of regulatory function. My findings contribute to the understanding of which variants, regulatory elements, and genes influence cardiometabolic traits. These predicted functional variants, regulatory elements, and target genes are strong candidates for testing in functional assays and may help guide therapeutic strategies for cardiometabolic diseases.Doctor of Philosoph

A survey of microRNA single nucleotide polymorphisms identifies novel breast cancer susceptibility loci in a case-control, population-based study of African-American women

Abstract Background MicroRNAs (miRNAs) regulate gene expression and influence cancer. Primary transcripts of miRNAs (pri-miRNAs) are poorly annotated and little is known about the role of germline variation in miRNA genes and breast cancer (BC). We sought to identify germline miRNA variants associated with BC risk and tumor subtype among African-American (AA) women. Methods Under the African American Breast Cancer Epidemiology and Risk (AMBER) Consortium, genotyping and imputed data from four studies on BC in AA women were combined into a final dataset containing 224,188 miRNA gene single nucleotide polymorphisms (SNPs) for 8350 women: 3663 cases and 4687 controls. The primary miRNA sequence was identified for 566 miRNA genes expressed in Encyclopedia of DNA Elements (ENCODE) Tier 1 cell types and human pancreatic islets. Association analysis was conducted using logistic regression for BC status overall and by tumor subtype. Results A novel BC signal was localized to an 8.6-kb region of 17q25.3 by four SNPs (rs9913477, rs1428882938, rs28585511, and rs7502931) and remained statistically significant after multiple test correction (odds ratio (OR) = 1.44, 95% confidence interval (CI) = 1.26–1.65; p = 3.15 × 10−7; false discovery rate (FDR) = 0.03). These SNPs reside in a genomic location that includes both the predicted primary transcript of the noncoding miRNA gene MIR3065 and the first intron of the gene for brain-specific angiogenesis inhibitor 1-associated protein 2 (BAIAP2). Furthermore, miRNA-associated SNPs on chromosomes 1p32.3, 5q32, and 3p25.1 were the strongest signals for hormone receptor, luminal versus basal-like, and HER2 enrichment status, respectively. A second phase of genotyping (1397 BC cases, 2418 controls) that included two SNPs in the 8.6-kb region was used for validation and meta-analysis. While neither rs4969239 nor rs9913477 was validated, when meta-analyzed with the original dataset their association with BC remained directionally consistent (OR = 1.29, 95% CI = 1.16–1.44 (p = 4.18 × 10–6) and OR = 1.33, 95% CI = 1.17–1.51 (p = 1.6 × 10–5), respectively). Conclusion Germline genetic variation indicates that MIR3065 may play an important role in BC development and heterogeneity among AA women. Further investigation to determine the potential functional effects of these SNPs is warranted. This study contributes to our understanding of BC risk in AA women and highlights the complexity in evaluating variation in gene-dense regions of the human genome.https://deepblue.lib.umich.edu/bitstream/2027.42/144216/1/13058_2018_Article_964.pd

A survey of microRNA single nucleotide polymorphisms identifies novel breast cancer susceptibility loci in a case-control, population-based study of African-American women

Abstract Background MicroRNAs (miRNAs) regulate gene expression and influence cancer. Primary transcripts of miRNAs (pri-miRNAs) are poorly annotated and little is known about the role of germline variation in miRNA genes and breast cancer (BC). We sought to identify germline miRNA variants associated with BC risk and tumor subtype among African-American (AA) women. Methods Under the African American Breast Cancer Epidemiology and Risk (AMBER) Consortium, genotyping and imputed data from four studies on BC in AA women were combined into a final dataset containing 224,188 miRNA gene single nucleotide polymorphisms (SNPs) for 8350 women: 3663 cases and 4687 controls. The primary miRNA sequence was identified for 566 miRNA genes expressed in Encyclopedia of DNA Elements (ENCODE) Tier 1 cell types and human pancreatic islets. Association analysis was conducted using logistic regression for BC status overall and by tumor subtype. Results A novel BC signal was localized to an 8.6-kb region of 17q25.3 by four SNPs (rs9913477, rs1428882938, rs28585511, and rs7502931) and remained statistically significant after multiple test correction (odds ratio (OR) = 1.44, 95% confidence interval (CI) = 1.26–1.65; p = 3.15 × 10−7; false discovery rate (FDR) = 0.03). These SNPs reside in a genomic location that includes both the predicted primary transcript of the noncoding miRNA gene MIR3065 and the first intron of the gene for brain-specific angiogenesis inhibitor 1-associated protein 2 (BAIAP2). Furthermore, miRNA-associated SNPs on chromosomes 1p32.3, 5q32, and 3p25.1 were the strongest signals for hormone receptor, luminal versus basal-like, and HER2 enrichment status, respectively. A second phase of genotyping (1397 BC cases, 2418 controls) that included two SNPs in the 8.6-kb region was used for validation and meta-analysis. While neither rs4969239 nor rs9913477 was validated, when meta-analyzed with the original dataset their association with BC remained directionally consistent (OR = 1.29, 95% CI = 1.16–1.44 (p = 4.18 × 10–6) and OR = 1.33, 95% CI = 1.17–1.51 (p = 1.6 × 10–5), respectively). Conclusion Germline genetic variation indicates that MIR3065 may play an important role in BC development and heterogeneity among AA women. Further investigation to determine the potential functional effects of these SNPs is warranted. This study contributes to our understanding of BC risk in AA women and highlights the complexity in evaluating variation in gene-dense regions of the human genome

Proceedings of the 13th annual conference of INEBRIA

CITATION: Watson, R., et al. 2016. Proceedings of the 13th annual conference of INEBRIA. Addiction Science & Clinical Practice, 11:13, doi:10.1186/s13722-016-0062-9.The original publication is available at https://ascpjournal.biomedcentral.comENGLISH SUMMARY : Meeting abstracts.https://ascpjournal.biomedcentral.com/articles/10.1186/s13722-016-0062-9Publisher's versio

Developmentally regulated impairment of parvalbumin interneuron synaptic transmission in an experimental model of Dravet syndrome

Dravet syndrome is a neurodevelopmental disorder characterized by epilepsy, intellectual disability, and sudden death due to pathogenic variants in SCN1A with loss of function of the sodium channel subunit Nav1.1. Nav1.1-expressing parvalbumin GABAergic interneurons (PV-INs) from young Scn1a+/− mice show impaired action potential generation. An approach assessing PV-IN function in the same mice at two time points shows impaired spike generation in all Scn1a+/− mice at postnatal days (P) 16–21, whether deceased prior or surviving to P35, with normalization by P35 in surviving mice. However, PV-IN synaptic transmission is dysfunctional in young Scn1a+/− mice that did not survive and in Scn1a+/− mice ≥ P35. Modeling confirms that PV-IN axonal propagation is more sensitive to decreased sodium conductance than spike generation. These results demonstrate dynamic dysfunction in Dravet syndrome: combined abnormalities of PV-IN spike generation and propagation drives early disease severity, while ongoing dysfunction of synaptic transmission contributes to chronic pathology

Liver regulatory mechanisms of noncoding variants at lipid and metabolic trait loci

Summary: Genome-wide association studies (GWASs) have identified hundreds of risk loci for liver disease and lipid-related metabolic traits, although identifying their target genes and molecular mechanisms remains challenging. We predicted target genes at GWAS signals by integrating them with molecular quantitative trait loci for liver gene expression (eQTL) and liver chromatin accessibility QTL (caQTL). We predicted specific regulatory caQTL variants at four GWAS signals located near EFHD1, LITAF, ZNF329, and GPR180. Using transcriptional reporter assays, we determined that caQTL variants rs13395911, rs11644920, rs34003091, and rs9556404 exhibit allelic differences in regulatory activity. We also performed a protein binding assay for rs13395911 and found that FOXA2 differentially interacts with the alleles of rs13395911. For variants rs13395911 and rs11644920 in putative enhancer regulatory elements, we used CRISPRi to demonstrate that repression of the enhancers altered the expression of the predicted target and/or nearby genes. Repression of the element at rs13395911 reduced the expression of EFHD1, and repression of the element at rs11644920 reduced the expression of LITAF, SNN, and TXNDC11. Finally, we showed that EFHD1 is a metabolically active gene in HepG2 cells. Together, these results provide key steps to connect genetic variants with cellular mechanisms and help elucidate the causes of liver disease

Chromatin accessibility and gene expression during adipocyte differentiation identify context-dependent effects at cardiometabolic GWAS loci.

Chromatin accessibility and gene expression in relevant cell contexts can guide identification of regulatory elements and mechanisms at genome-wide association study (GWAS) loci. To identify regulatory elements that display differential activity across adipocyte differentiation, we performed ATAC-seq and RNA-seq in a human cell model of preadipocytes and adipocytes at days 4 and 14 of differentiation. For comparison, we created a consensus map of ATAC-seq peaks in 11 human subcutaneous adipose tissue samples. We identified 58,387 context-dependent chromatin accessibility peaks and 3,090 context-dependent genes between all timepoint comparisons (log2 fold change>1, FDR<5%) with 15,919 adipocyte- and 18,244 preadipocyte-dependent peaks. Adipocyte-dependent peaks showed increased overlap (60.1%) with Roadmap Epigenomics adipocyte nuclei enhancers compared to preadipocyte-dependent peaks (11.5%). We linked context-dependent peaks to genes based on adipocyte promoter capture Hi-C data, overlap with adipose eQTL variants, and context-dependent gene expression. Of 16,167 context-dependent peaks linked to a gene, 5,145 were linked by two or more strategies to 1,670 genes. Among GWAS loci for cardiometabolic traits, adipocyte-dependent peaks, but not preadipocyte-dependent peaks, showed significant enrichment (LD score regression P<0.005) for waist-to-hip ratio and modest enrichment (P < 0.05) for HDL-cholesterol. We identified 659 peaks linked to 503 genes by two or more approaches and overlapping a GWAS signal, suggesting a regulatory mechanism at these loci. To identify variants that may alter chromatin accessibility between timepoints, we identified 582 variants in 454 context-dependent peaks that demonstrated allelic imbalance in accessibility (FDR<5%), of which 55 peaks also overlapped GWAS variants. At one GWAS locus for palmitoleic acid, rs603424 was located in an adipocyte-dependent peak linked to SCD and exhibited allelic differences in transcriptional activity in adipocytes (P = 0.003) but not preadipocytes (P = 0.09). These results demonstrate that context-dependent peaks and genes can guide discovery of regulatory variants at GWAS loci and aid identification of regulatory mechanisms

Open Chromatin Profiling in Adipose Tissue Marks Genomic Regions with Functional Roles in Cardiometabolic Traits

Identifying the regulatory mechanisms of genome-wide association study (GWAS) loci affecting adipose tissue has been restricted due to limited characterization of adipose transcriptional regulatory elements. We profiled chromatin accessibility in three frozen human subcutaneous adipose tissue needle biopsies and preadipocytes and adipocytes from the Simpson Golabi-Behmel Syndrome (SGBS) cell strain using an assay for transposase-accessible chromatin (ATAC-seq). We identified 68,571 representative accessible chromatin regions (peaks) across adipose tissue samples (FDR < 5%). GWAS loci for eight cardiometabolic traits were enriched in these peaks (P < 0.005), with the strongest enrichment for waist-hip ratio. Of 110 recently described cardiometabolic GWAS loci colocalized with adipose tissue eQTLs, 59 loci had one or more variants overlapping an adipose tissue peak. Annotated variants at the SNX10 waist-hip ratio locus and the ATP2A1-SH2B1 body mass index locus showed allelic differences in regulatory assays. These adipose tissue accessible chromatin regions elucidate genetic variants that may alter adipose tissue function to impact cardiometabolic traits