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Genetic risk variants that have been identified in genome-wide association studies of complex diseases are primarily non-coding1. Translating these risk variants into mechanistic insights requires detailed maps of gene regulation in disease-relevant cell types2. Here we combined two approaches: a genome-wide association study of type 1 diabetes (T1D) using 520,580 samples, and the identification of candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cells using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC–seq) of 131,554 nuclei. Risk variants for T1D were enriched in cCREs that were active in T cells and other cell types, including acinar and ductal cells of the exocrine pancreas. Risk variants at multiple T1D signals overlapped with exocrine-specific cCREs that were linked to genes with exocrine-specific expression. At the CFTR locus, the T1D risk variant rs7795896 mapped to a ductal-specific cCRE that regulated CFTR; the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in the pathogenesis of T1D and highlight the power of large-scale genome-wide association studies and single-cell epigenomics for understanding the cellular origins of complex disease. A genome-wide association study combined with single-cell epigenomics identifies risk loci for type 1 diabetes.

The response of beta cells to inflammatory cytokines contributes to type 1 diabetes (T1D) risk, but the specific genes that underlie this response and mediate beta cell survival remain largely unknown. In this study we used a suite of functional genomics assays combined with human genetics, to map cis-regulatory programs in beta cells and to identify T1D genes that affect beta cell survival upon exposure to the pro-inflammatory cytokines IL1β, IFNγ and TNFα. We mapped 38,931 cytokine-responsive candidate cis­­ -regulatory elements (cCREs) active in beta cells using ATAC-seq and single nuclear ATAC-seq (snATAC-seq) , and linked cytokine-responsive beta cell cCREs to putative target genes using single cell co-accessibility and HiChIP. We performed a genome-wide pooled CRISPR loss-of-function screen in EndoC-βH1 cells, which identified 867 genes affecting cytokine-induced beta cell loss. Genes that promoted beta cell survival and had up-regulated expression in cytokine exposure were specifically enriched at T1D loci, and these genes were preferentially involved in inhibiting inflammatory response, ubiquitin-mediated proteolysis, mitophagy and autophagy. We identified 2,229 variants in cytokine-responsive beta cell cCREs altering transcription factor (TF) binding using high-throughput SNP-SELEX, and variants altering binding of TF families regulating stress, inflammation and apoptosis were broadly enriched for T1D association. Finally, through integration with genetic fine mapping, we identified T1D risk variants regulating beta cell survival in cytokine exposure. At the 16p13 locus, a T1D variant affected TF binding in a cytokine-induced beta cell cCRE that physically interacted with the SOCS1 promoter, and increased SOCS1 activity promoted beta cell survival in cytokine exposure. Together our findings reveal processes and genes acting in beta cells during cytokine exposure that intrinsically modulate risk of T1D. Disclosure P.Benaglio: None. M.Donovan: None. J.Chiou: Employee; Pfizer Inc. J.M.Newsome ashmus: None. J.Kaur: None. S.Corban: None. B.Ren: Stock/Shareholder; Arima Genomics, Epigenome Technologies. K.Frazer: n/a. M.Sander: None. K.J.Gaulton: Consultant; Genentech, Inc., Stock/Shareholder; Neurocrine Biosciences, Inc., Vertex Pharmaceuticals Incorporated. H.Zhu: None. M.Okino: None. J.Yan: None. R.Elgamal: None. N.Nariai: None. E.Beebe: None. K.Korgaonkar: None. Y.Qiu: None. Funding NIH DK122607

The Major Histocompatibility Complex (MHC) locus regulates the adaptive immune response and self-recognition and is the largest contributor to genetic risk of type 1 diabetes (T1D) . Protein-coding alleles in MHC class II and class I genes have long been known to contribute substantial T1D risk, yet whether additional variants affect T1D risk at this locus particularly in the non-coding genome has not been exhaustively evaluated. We therefore performed high-resolution fine-mapping of the MHC locus to identify additional coding and non-coding variants that contribute to T1D risk. We imputed genotypes for 39,270 samples from six European or American cohorts of T1D cases and controls into the T1DGC reference panel using SNP2HLA. We then performed two analyses to fine-map causal variants, first, stepwise conditional analysis using Firth bias-corrected logistic likelihood ratio test and second, iterative Bayesian stepwise selection analysis using the Sum of Single Effects (SuSiE) method. We identified 33 signals in stepwise conditional analysis with p Disclosure C.Mcgrail: None. J.Chiou: Employee; Pfizer Inc. P.Benaglio: None. K.J.Gaulton: Consultant; Genentech, Inc., Stock/Shareholder; Neurocrine Biosciences, Inc., Vertex Pharmaceuticals Incorporated. Funding National Institutes of Health (DK120429) National Institutes of Health (DK122607)

Many sequence variants have been linked to complex human traits and diseases1, but deciphering their biological functions remains challenging, as most of them reside in noncoding DNA. Here we have systematically assessed the binding of 270 human transcription factors to 95,886 noncoding variants in the human genome using an ultra-high-throughput multiplex protein-DNA binding assay, termed single-nucleotide polymorphism evaluation by systematic evolution of ligands by exponential enrichment (SNP-SELEX). The resulting 828 million measurements of transcription factor-DNA interactions enable estimation of the relative affinity of these transcription factors to each variant in vitro and evaluation of the current methods to predict the effects of noncoding variants on transcription factor binding. We show that the position weight matrices of most transcription factors lack sufficient predictive power, whereas the support vector machine combined with the gapped k-mer representation show much improved performance, when assessed on results from independent SNP-SELEX experiments involving a new set of 61,020 sequence variants. We report highly predictive models for 94 human transcription factors and demonstrate their utility in genome-wide association studies and understanding of the molecular pathways involved in diverse human traits and diseases.

Publisher Copyright: © 2022 We combined functional genomics and human genetics to investigate processes that affect type 1 diabetes (T1D) risk by mediating beta cell survival in response to proinflammatory cytokines. We mapped 38,931 cytokine-responsive candidate cis-regulatory elements (cCREs) in beta cells using ATAC-seq and snATAC-seq and linked them to target genes using co-accessibility and HiChIP. Using a genome-wide CRISPR screen in EndoC-βH1 cells, we identified 867 genes affecting cytokine-induced survival, and genes promoting survival and up-regulated in cytokines were enriched at T1D risk loci. Using SNP-SELEX, we identified 2,229 variants in cytokine-responsive cCREs altering transcription factor (TF) binding, and variants altering binding of TFs regulating stress, inflammation, and apoptosis were enriched for T1D risk. At the 16p13 locus, a fine-mapped T1D variant altering TF binding in a cytokine-induced cCRE interacted with SOCS1, which promoted survival in cytokine exposure. Our findings reveal processes and genes acting in beta cells during inflammation that modulate T1D risk.

Beta cells intrinsically contribute to the pathogenesis of type 1 diabetes (T1D), but the genes and molecular processes that mediate beta cell survival in T1D remain largely unknown. We combined high throughput functional genomics and human genetics to identify T1D risk loci regulating genes affecting beta cell survival in response to the proinflammatory cytokines IL-1β, IFNγ, and TNFα. We mapped 38,931 cytokine-responsive candidate cis-regulatory elements (cCREs) active in beta cells using ATAC-seq and single nuclear ATAC-seq (snATAC-seq), and linked cytokine-responsive beta cell cCREs to putative target genes using single cell co-accessibility and HiChIP. We performed a genome-wide pooled CRISPR loss-of-function screen in EndoC-βH1 cells, which identified 867 genes affecting cytokine-induced beta cell loss. Genes that promoted beta cell survival and had up-regulated expression in cytokine exposure were specifically enriched at T1D loci, and these genes were preferentially involved in inhibiting inflammatory response, ubiquitin-mediated proteolysis, mitophagy and autophagy. We identified 2,229 variants in cytokine-responsive beta cell cCREs altering transcription factor (TF) binding using high-throughput SNP-SELEX, and variants altering binding of TF families regulating stress, inflammation and apoptosis were broadly enriched for T1D association. Finally, through integration with genetic fine mapping, we annotated T1D loci regulating beta cell survival in cytokine exposure. At the 16p13 locus, a T1D variant affected TF binding in a cytokine-induced beta cell cCRE that physically interacted with the SOCS1 promoter, and increased SOCS1 activity promoted beta cell survival in cytokine exposure. Together our findings reveal processes and genes acting in beta cells during cytokine exposure that intrinsically modulate risk of T1D.

Glucocorticoids are key regulators of glucose homeostasis and pancreatic islet function, but the gene regulatory programs driving responses to glucocorticoid signaling in islets and the contribution of these programs to diabetes risk are unknown. In this study we used ATAC-seq and RNA-seq to map chromatin accessibility and gene expression from eleven primary human islet samples cultured in vitro with the glucocorticoid dexamethasone at multiple doses and durations. We identified thousands of accessible chromatin sites and genes with significant changes in activity in response to glucocorticoids. Chromatin sites up-regulated in glucocorticoid signaling were prominently enriched for glucocorticoid receptor binding sites and up-regulated genes were enriched for ion transport and lipid metabolism, whereas down-regulated chromatin sites and genes were enriched for inflammatory, stress response and proliferative processes. Genetic variants associated with glucose levels and T2D risk were enriched in glucocorticoid-responsive chromatin sites, including fine-mapped variants at 51 known signals. Among fine-mapped variants in glucocorticoid-responsive chromatin, a likely casual variant at the 2p21 locus had glucocorticoid-dependent allelic effects on beta cell enhancer activity and affected SIX2 and SIX3 expression. Our results provide a comprehensive map of islet regulatory programs in response to glucocorticoids through which we uncover a role for islet glucocorticoid signaling in mediating genetic risk of T2D., Author summary Glucocorticoids regulate inflammation and metabolism and are widely used in the treatment of immune disorders, although prolonged exposure to glucocorticoids can lead to the development of diabetes. In this study we determined the response of primary pancreatic islets, which are central to the development of diabetes, to the glucocorticoid dexamethasone at multiple doses and durations. We observed widespread changes in pancreatic islets after glucocorticoid treatment at glucocorticoid receptor binding sites, as well as at key genes involved in islet function and processes related to steroid and lipid metabolism, ion channel activity, inflammation, and growth. Genetic variants affecting type 2 diabetes and glucose levels were located in sites affected by glucocorticoids at many genomic regions, and highlighted genes regulated by these sites through which glucocorticoid signaling may contribute directly to the development of diabetes. Together these results provide key insight into how glucocorticoid treatment affects pancreatic islet function and risk of diabetes.

Translating genome-wide association studies (GWAS) of complex disease into mechanistic insight requires a comprehensive understanding of risk variant effects on disease-relevant cell types. To uncover cell type-specific mechanisms of type 1 diabetes (T1D) risk, we combined genetic association mapping and single cell epigenomics. We performed the largest to-date GWAS of T1D in 489,679 samples imputed into 59.2M variants, which identified 74 novel association signals including several large-effect rare variants. Fine-mapping of 141 total signals substantially improved resolution of causal variant credible sets, which primarily mapped to non-coding sequence. To annotate cell type-specific regulatory mechanisms of T1D risk variants, we mapped 448,142 candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cell types using snATAC-seq of 131,554 nuclei. T1D risk variants were enriched in cCREs active in CD4+ T cells as well as several additional cell types including pancreatic exocrine acinar and ductal cells. High-probability T1D risk variants at multiple signals mapped to exocrine-specific cCREs including novel loci near CEL, GP2 and CFTR. At the CFTR locus, the likely causal variant rs7795896 mapped in a ductal-specific distal cCRE which regulated CFTR and the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in T1D pathogenesis and highlight the power of combining large-scale GWAS and single cell epigenomics to provide insight into the cellular origins of complex disease.

Background Adult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary characterized by elevated estrogen levels. aGCTs ubiquitously harbor a somatic mutation in FOXL2 gene, Cys134Trp (c.402C

BackgroundAdult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary characterized by elevated estrogen levels. aGCTs ubiquitously harbor a somatic mutation in FOXL2 gene, Cys134Trp (c.402CC134W, its partner SMAD3 and its antagonist FOXO1 in cellular models of aGCT.MethodsIn this work, seeking more comprehensive profiling of FOXL2C134W transcriptomic effects, we performed an RNA-seq analysis comparing the effect of FOXL2WT/SMAD3 and FOXL2C143W/SMAD3 overexpression in an established human GC line (HGrC1), which is not luteinized, and bears normal alleles of FOXL2.ResultsOur data shows that FOXL2C143W/SMAD3 overexpression alters the expression of 717 genes. These genes include known and novel FOXL2 targets (TGFB2, SMARCA4, HSPG2, MKI67, NFKBIA) and are enriched for neoplastic pathways (Proteoglycans in Cancer, Chromatin remodeling, Apoptosis, Tissue Morphogenesis, Tyrosine Kinase Receptors). We additionally expressed the FOXL2 antagonistic Forkhead protein, FOXO1. Surprisingly, overexpression of FOXO1 mitigated 40% of the altered genome-wide effects specifically related to FOXL2C134W, suggesting it can be a new target for aGCT treatment.Conclusionsour transcriptomic data provide novel insights into potential genes (FOXO1 regulated) that could be used as biomarkers of efficacy in aGCT patients.

Gene regulation is highly cell type-specific and understanding the function of non-coding genetic variants associated with complex traits requires molecular phenotyping at cell type resolution. In this study we performed single nucleus ATAC-seq (snATAC-seq) and genotyping in peripheral blood mononuclear cells from 10 individuals. Clustering chromatin accessibility profiles of 66,843 total nuclei identified 14 immune cell types and sub-types. We mapped chromatin accessibility QTLs (caQTLs) in each immune cell type and sub-type which identified 6,248 total caQTLs, including those obscured from assays of bulk tissue such as with divergent effects on different cell types. For 3,379 caQTLs we further annotated putative target genes of variant activity using single cell co-accessibility, and caQTL variants were significantly correlated with the accessibility level of linked gene promoters. We fine-mapped loci associated with 16 complex immune traits and identified immune cell caQTLs at 517 candidate causal variants, including those with cell type-specific effects. At the 6q15 locus associated with type 1 diabetes, in line with previous reports, variant rs72928038 was a naïve CD4+ T cell caQTL linked to BACH2 and we validated the allelic effects of this variant on regulatory activity in Jurkat T cells. These results highlight the utility of snATAC-seq for mapping genetic effects on accessible chromatin in specific cell types and provide a resource for annotating complex immune trait loci.

Background: Adult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary. Postmenopausal genital bleeding is the main aGCT clinical sign which is attributed to estrogen excess driven by CYP19 upregulation. Typically, aGCTs that are diagnosed at an initial stage can be treated with surgery. However, recurrences are mostly fatal1. Current studies are focused on finding new molecular markers and targets that aim to treat the aGCTs recurrence. Between 95-97% of aGCTs harbor a somatic mutation in the FOXL2 gene, Cys134Trp (c.402C

The cardiac transcription factor (TF) gene NKX2–5 has been associated with electrocardiographic (EKG) traits through GWAS, but the extent to which differential binding of NKX2–5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from iPSC-derived cardiomyocytes (iPSC-CMs) from seven related individuals and identified ~2,000 single nucleotide variants (SNVs) associated with allele-specific effects (ASE) on NKX2–5 binding. NKX2–5 ASE-SNVs were enriched for altered TF motifs, for heart-specific eQTLs, and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from iPSC-CMs, we prioritized candidate causal variants for EKG traits, many of which were NKX2–5 ASE-SNVs. Experimentally characterizing two NKX2–5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2–5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes., Editorial summary: Analysis of iPSC-derived cardiomyocytes identifies variants associated with allele-specific effects on NKX2–5 binding. Fine-mapping and functional studies suggest that such variants underlie cardiac-specific expression quantitative trait loci and associations with electrocardiographic traits.

Non-genetic variability in human induced pluripotent stem cell (iPSC) lines impacts their differentiation outcome, limiting their utility for genetic studies and clinical applications. Despite the importance of understanding how non-genetic molecular variability influences iPSC differentiation outcome, large-scale studies capable of addressing this question have not yet been conducted. Here, we performed 258 directed differentiations of 191 iPSC lines using established protocols to generate iPSC-derived cardiovascular progenitor cells (iPSC-CVPCs). We observed cellular heterogeneity across the iPSC-CVPC samples due to varying fractions of two cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Analyzing the transcriptomes of CM-fated and EPDC-fated iPSCs discovered that 91 signature genes and X chromosome dosage differences influence WNT inhibition response during differentiation and are associated with cardiac fate. Analysis of an independent set of 39 iPSCs differentiated to the cardiac lineage confirmed shared sex and transcriptional differences that impact cardiac fate outcome. The scale and systematic approach of our study enabled novel insights into how iPSC transcriptional and X chromosome gene dosage differences influence WNT signaling during differentiation and hence cardiac cell fate.

SummaryWhile genetic variation at chromatin loops is relevant for human disease, the relationships between loop strength, genetics, gene expression, and epigenetics are unclear. Here, we quantitatively interrogate this relationship using Hi-C and molecular phenotype data across cell types and haplotypes. We find that chromatin loops consistently form across multiple cell types and quantitatively vary in strength, instead of exclusively forming within only one cell type. We show that large haplotype loop imbalance is primarily associated with imprinting and copy number variation, rather than genetically driven traits such as allele-specific expression. Finally, across cell types and haplotypes, we show that subtle changes in chromatin loop strength are associated with large differences in other molecular phenotypes, with a 2-fold change in looping corresponding to a 100-fold change in gene expression. Our study suggests that regulatory genetic variation could mediate its effects on gene expression through subtle modification of chromatin loop strength.

Genetic variation affecting the binding of transcription factors (TFs) has been proposed as a major mechanism underlying susceptibility to common disease. NKX2-5, a key cardiac development TF, has been associated with electrocardiographic (EKG) traits through GWAS, but the extent to which differential binding of NKX2-5 contributes to these traits has not yet been studied. Here, we analyzed transcriptomic and epigenomic data generated from iPSC-derived cardiomyocyte lines (iPSC-CMs) from seven whole-genome sequenced individuals in a three-generational family. We identified ~2,000 single nucleotide variants (SNVs) associated with allele-specific effects (ASE) on NKX2-5 binding. These ASE-SNVs were enriched for altered TF motifs (both cognate and other cardiac TFs), and were positively correlated with changes in H3K27ac in iPSC-CMs, suggesting they impact cardiac enhancer activity. We found that NKX2-ASE-SNVs were significantly enriched for being heart-specific eQTLs and EKG GWAS variants, suggesting that altered NKX2-5 binding at multiple sites across the genome influences EKG traits. We used a fine-mapping approach to integrate iPSC-CM molecular phenotype data with a GWAS for heart rate, and determined that NKX2-5 ASE variants are likely causal for numerous known, as well as previously unidentified, heart rate loci. Analyzing Hi-C and gene expression data from iPSC-CMs at these heart rate loci, we identified several genes likely to be causally involved in heart rate variability. Our study demonstrates that differential binding of NKX2-5 is a common mechanism underlying genetic association with EKG traits, and shows that characterizing variants associated with differential binding of development TFs in iPSC-derived cell lines can identify novel loci and mechanisms influencing complex traits.

SummaryTo understand the mutational burden of human induced pluripotent stem cells (iPSCs), we whole genome sequenced 18 fibroblast-derived iPSC lines and identified different classes of somatic mutations based on structure, origin and frequency. Copy number alterations affected 295 kb in each sample and strongly impacted gene expression. UV-damage mutations were present in ~45% of the iPSCs and accounted for most of the observed heterogeneity in mutation rates across lines. Subclonal mutations (not present in all iPSCs within a line) composed 10% of point mutations, and compared with clonal variants, showed an enrichment in active promoters and increased association with altered gene expression. Our study shows that, by combining WGS, transcriptome and epigenome data, we can understand the mutational burden of each iPSC line on an individual basis and suggests that this information could be used to prioritize iPSC lines for models of specific human diseases and/or transplantation therapy.

Summary To understand the mutational burden of human induced pluripotent stem cells (iPSCs), we sequenced genomes of 18 fibroblast-derived iPSC lines and identified different classes of somatic mutations based on structure, origin, and frequency. Copy-number alterations affected 295 kb in each sample and strongly impacted gene expression. UV-damage mutations were present in ∼45% of the iPSCs and accounted for most of the observed heterogeneity in mutation rates across lines. Subclonal mutations (not present in all iPSCs within a line) composed 10% of point mutations and, compared with clonal variants, showed an enrichment in active promoters and increased association with altered gene expression. Our study shows that, by combining WGS, transcriptome, and epigenome data, we can understand the mutational burden of each iPSC line on an individual basis and suggests that this information could be used to prioritize iPSC lines for models of specific human diseases and/or transplantation therapy.

We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ~446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103 kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a 'core' region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220 kb region and a prophage that drastically change the host metabolic capacity and survivability.

In this study, we used whole genome sequencing and gene expression profiling of 215 human induced pluripotent stem cell (iPSC) lines from different donors to identify genetic variants associated with RNA expression for 5,746 genes. We were able to predict causal variants for these expression quantitative trait loci (eQTLs) that disrupt transcription factor binding and validated a subset of them experimentally. We also identified copy number variant (CNV) eQTLs, including some that appear to affect gene expression by altering the copy number of intergenic regulatory regions. In addition, we were able to identify effects on gene expression of rare genic CNVs and regulatory single nucleotide variants, and found that reactivation of gene expression on the X chromosome depends on gene chromosomal position. Our work highlights the value of iPSCs for genetic association analyses and provides a unique resource for investigating the genetic regulation of gene expression in pluripotent cells.

With the widespread availability of high-throughput sequencing technologies, sequencing projects have become pervasive in the molecular life sciences. The huge bulk of data generated daily must be analyzed further by biologists with skills in bioinformatics and by “embedded bioinformaticians,” i.e., bioinformaticians integrated in wet lab research groups. Thus, students interested in molecular life sciences must be trained in the main steps of genomics: sequencing, assembly, annotation and analysis. To reach that goal, a practical course has been set up for master students at the University of Lausanne: the “Sequence a genome” class. At the beginning of the academic year, a few bacterial species whose genome is unknown are provided to the students, who sequence and assemble the genome(s) and perform manual annotation. Here, we report the progress of the first class from September 2010 to June 2011 and the results obtained by seven master students who specifically assembled and annotated the genome of Estrella lausannensis, an obligate intracellular bacterium related to Chlamydia. The draft genome of Estrella is composed of 29 scaffolds encompassing 2,819,825 bp that encode for 2233 putative proteins. Estrella also possesses a 9136 bp plasmid that encodes for 14 genes, among which we found an integrase and a toxin/antitoxin module. Like all other members of the Chlamydiales order, Estrella possesses a highly conserved type III secretion system, considered as a key virulence factor. The annotation of the Estrella genome also allowed the characterization of the metabolic abilities of this strictly intracellular bacterium. Altogether, the students provided the scientific community with the Estrella genome sequence and a preliminary understanding of the biology of this recently-discovered bacterial genus, while learning to use cutting-edge technologies for sequencing and to perform bioinformatics analyses.

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37–1.73]; combined P =2.58 · 10 −13 ). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25–1.44; P =1.032 · 10 −14 ). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16–3.66) for systolic and 1.40 (95% CI: 0.25–2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

The gene SNRNP200 is composed of 45 exons and encodes a protein essential for pre-mRNA splicing, the 200 kDa helicase hBrr2. Two mutations in SNRNP200 have recently been associated with autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease, in two families from China. In this work we analyzed the entire 35-Kb SNRNP200 genomic region in a cohort of 96 unrelated North American patients with adRP. To complete this large-scale sequencing project, we performed ultra high-throughput sequencing of pooled, untagged PCR products. We then validated the detected DNA changes by Sanger sequencing of individual samples from this cohort and from an additional one of 95 patients. One of the two previously known mutations (p.S1087L) was identified in 3 patients, while 4 new missense changes (p.R681C, p.R681H, p.V683L, p.Y689C) affecting highly conserved codons were identified in 6 unrelated individuals, indicating that the prevalence of SNRNP200-associated adRP is relatively high. We also took advantage of this research to evaluate the pool-and-sequence method, especially with respect to the generation of false positive and negative results. We conclude that, although this strategy can be adopted for rapid discovery of new disease-associated variants, it still requires extensive validation to be used in routine DNA screenings.