Increased DNA methylation variability in type 1 diabetes across three immune effector cell types

The incidence of type 1 diabetes (T1D) has substantially increased over the past decade, suggesting a role for non-genetic factors such as epigenetic mechanisms in disease development. Here we present an epigenome-wide association study across 406,365 CpGs in 52 monozygotic twin pairs discordant for T1D in three immune effector cell types. We observe a substantial enrichment of differentially variable CpG positions (DVPs) in T1D twins when compared with their healthy co-twins and when compared with healthy, unrelated individuals. These T1D-associated DVPs are found to be temporally stable and enriched at gene regulatory elements. Integration with cell type-specific gene regulatory circuits highlight pathways involved in immune cell metabolism and the cell cycle, including mTOR signalling. Evidence from cord blood of newborns who progress to overt T1D suggests that the DVPs likely emerge after birth. Our findings, based on 772 methylomes, implicate epigenetic changes that could contribute to disease pathogenesis in T1D.This work was funded by the EU-FP7 project BLUEPRINT (282510) and the Wellcome Trust (99148). We thank all twins for taking part in this study; Kerra Pearce and Mark Kristiansen (UCL Genomics) for processing the Illumina Infinium HumanMethylation450 BeadChips; Rasmus Bennet for technical assistance; and Laura Phipps for proofreading the manuscript. The BMBF Pediatric Diabetes Biobank recruits patients from the National Diabetes Patient Documentation System (DPV), and is financed by the German Ministry of Education and Research within the German Competence Net Diabetes Mellitus (01GI1106 and 01GI1109B). It was integrated into the German Center for Diabetes Research in January 2015. We thank the Swedish Research Council and SUS Funds for support. We gratefully acknowledge the participation of all NIHR Cambridge BioResource volunteers, and thank the Cambridge BioResource staff for their help with volunteer recruitment. We thank members of the Cambridge BioResource SAB and Management Committee for their support of our study and the NIHR Cambridge Biomedical Research Centre for funding. The Cardiovascular Epidemiology Unit is supported by the UK Medical Research Council (G0800270), BHF (SP/09/002), and NIHR Cambridge Biomedical Research Centre. Research in the Ouwehand laboratory is supported by the NIHR, BHF (PG-0310-1002 and RG/09/12/28096) and NHS Blood and Transplant. K.D. is funded as a HSST trainee by NHS Health Education England. M.F. is supported by the BHF Cambridge Centre of Excellence (RE/13/6/30180). A.D., E.L., L.C. and P.F. receive additional support from the European Molecular Biology Laboratory. A.K.S. is supported by an ADA Career Development Award (1-14-CD-17). B.O.B. and R.D.L. acknowledge support from the Deutsche Forschungsgemeinschaft (DFG) and European Federation for the Study of Diabetes, respectively

Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14$^{+}$ monocytes, CD16$^{+}$ neutrophils, and naive CD4$^{+}$ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of $\textit{cis}$-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.This work was predominantly funded by the EU FP7 High Impact Project BLUEPRINT (HEALTH-F5-2011-282510) and the Canadian Institutes of Health Research (CIHR EP1-120608). The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 282510 (BLUEPRINT), the European Molecular Biology Laboratory, the Max Planck society, the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208 and Spanish National Bioinformatics Institute (INB-ISCIII) PT13/0001/0021 co-funded by FEDER "“Una Manera de hacer Europa”. D.G. is supported by a “la Caixa”-Severo Ochoa pre-doctoral fellowship, M.F. was supported by the BHF Cambridge Centre of Excellence [RE/13/6/30180], K.D. is funded as a HSST trainee by NHS Health Education England, S.E. is supported by a fellowship from La Caixa, V.P. is supported by a FEBS long-term fellowship and N.S.'s research is supported by the Wellcome Trust (Grant Codes WT098051 and WT091310), the EU FP7 (EPIGENESYS Grant Code 257082 and BLUEPRINT Grant Code HEALTH-F5-2011-282510) and the NIHR BRC. The Blood and Transplant Unit (BTRU) in Donor Health and Genomics is part of and funded by the National Institute for Health Research (NIHR) and is a partnership between the University of Cambridge and NHS Blood and Transplant (NHSBT) in collaboration with the University of Oxford and the Wellcome Trust Sanger Institute. The T-cell data was produced by the McGill Epigenomics Mapping Centre (EMC McGill). It is funded under the Canadian Epigenetics, Environment, and Health Research Consortium (CEEHRC) by the Canadian Institutes of Health Research and by Genome Quebec (CIHR EP1-120608), with additional support from Genome Canada and FRSQ. T.P. holds a Canada Research Chair

A Common and Unstable Copy Number Variant Is Associated with Differences in Glo1 Expression and Anxiety-Like Behavior

Glyoxalase 1 (Glo1) has been implicated in anxiety-like behavior in mice and in multiple psychiatric diseases in humans. We used mouse Affymetrix exon arrays to detect copy number variants (CNV) among inbred mouse strains and thereby identified a ∼475 kb tandem duplication on chromosome 17 that includes Glo1 (30,174,390–30,651,226 Mb; mouse genome build 36). We developed a PCR-based strategy and used it to detect this duplication in 23 of 71 inbred strains tested, and in various outbred and wild-caught mice. Presence of the duplication is associated with a cis-acting expression QTL for Glo1 (LOD>30) in BXD recombinant inbred strains. However, evidence for an eQTL for Glo1 was not obtained when we analyzed single SNPs or 3-SNP haplotypes in a panel of 27 inbred strains. We conclude that association analysis in the inbred strain panel failed to detect an eQTL because the duplication was present on multiple highly divergent haplotypes. Furthermore, we suggest that non-allelic homologous recombination has led to multiple reversions to the non-duplicated state among inbred strains. We show associations between multiple duplication-containing haplotypes, Glo1 expression and anxiety-like behavior in both inbred strain panels and outbred CD-1 mice. Our findings provide a molecular basis for differential expression of Glo1 and further implicate Glo1 in anxiety-like behavior. More broadly, these results identify problems with commonly employed tests for association in inbred strains when CNVs are present. Finally, these data provide an example of biologically significant phenotypic variability in model organisms that can be attributed to CNVs

Transcriptional, epigenetic and metabolic signatures in cardiometabolic syndrome defined by extreme phenotypes

This is the final version. Available on open access from BMC via the DOI in this recordAvailability of data and materials: The datasets generated during this study are available at EGA under study ID EGAS00001003780. The codes generated during this study and all supplementary tables are available at GitLab https://gitlab.com/dseyres/extremephenotype.Background: This work is aimed at improving the understanding of cardiometabolic syndrome pathophysiology and its relationship with thrombosis by generating a multi-omic disease signature. Methods/Results: We combined classic plasma biochemistry and plasma biomarkers with the transcriptional and epigenetic characterisation of cell types involved in thrombosis, obtained from two extreme phenotype groups (morbidly obese and lipodystrophy) and lean individuals to identify the molecular mechanisms at play, highlighting patterns of abnormal activation in innate immune phagocytic cells. Our analyses showed that extreme phenotype groups could be distinguished from lean individuals, and from each other, across all data layers. The characterisation of the same obese group, six months after bariatric surgery revealed the loss of the abnormal activation of innate immune cells previously observed. However, rather than reverting to the gene expression landscape of lean individuals, this occurred via the establishment of novel gene expression landscapes. Netosis and its control mechanisms emerge amongst the pathways that show an improvement after surgical intervention. Conclusions: We showed that the morbidly obese and lipodystrophy groups, despite some differences, shared a common cardiometabolic syndrome signature. We also showed that this could be used to discriminate, amongst the normal population, those individuals with a higher likelihood of presenting with the disease, even when not displaying the classic features.British Heart FoundationMedical Research Council (MRC)Wellcome TrustNational Institute for Health Research (NIHR)Isaac Newton fellowshipJohn and Lucille Van Geest Foundatio

Polygenic transmission disequilibrium confirms that common and rare variation act additively to create risk for autism spectrum disorders

Autism spectrum disorder (ASD) risk is influenced by common polygenic and de novo variation. We aimed to clarify the influence of polygenic risk for ASD and to identify subgroups of ASD cases, including those with strongly acting de novo variants, in which polygenic risk is relevant. Using a novel approach called the polygenic transmission disequilibrium test and data from 6,454 families with a child with ASD, we show that polygenic risk for ASD, schizophrenia, and greater educational attainment is over-transmitted to children with ASD. These findings hold independent of proband IQ. We find that polygenic variation contributes additively to risk in ASD cases who carry a strongly acting de novo variant. Lastly, we show that elements of polygenic risk are independent and differ in their relationship with phenotype. These results confirm that the genetic influences on ASD are additive and suggest that they create risk through at least partially distinct etiologic pathways

Steroid receptor coactivator-1 modulates the function of Pomc neurons and energy homeostasis

Hypothalamic neurons expressing the anorectic peptide Pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here, we show that Steroid Receptor Coactivator-1 (SRC-1) interacts with a target of leptin receptor activation, phosphorylated STAT3, to potentiate Pomc transcription. Deletion of SRC-1 in Pomc neurons in mice attenuates their depolarization by leptin, decreases Pomc expression and increases food intake leading to high-fat diet-induced obesity. In humans, fifteen rare heterozygous variants in SRC-1 found in severely obese individuals impair leptin-mediated Pomc reporter activity in cells, whilst four variants found in non-obese controls do not. In a knock-in mouse model of a loss of function human variant (SRC-1L1376P), leptin-induced depolarization of Pomc neurons and Pomc expression are significantly reduced, and food intake and body weight are increased. In summary, we demonstrate that SRC-1 modulates the function of hypothalamic Pomc neurons, and suggest that targeting SRC-1 may represent a useful therapeutic strategy for weight loss.Peer reviewe

De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures.

Next-generation sequencing has been invaluable in the elucidation of the genetic etiology of many subtypes of intellectual disability in recent years. Here, using exome sequencing and whole-genome sequencing, we identified three de novo truncating mutations in WAS protein family member 1 (WASF1) in five unrelated individuals with moderate to profound intellectual disability with autistic features and seizures. WASF1, also known as WAVE1, is part of the WAVE complex and acts as a mediator between Rac-GTPase and actin to induce actin polymerization. The three mutations connected by Matchmaker Exchange were c.1516C>T (p.Arg506Ter), which occurs in three unrelated individuals, c.1558C>T (p.Gln520Ter), and c.1482delinsGCCAGG (p.Ile494MetfsTer23). All three variants are predicted to partially or fully disrupt the C-terminal actin-binding WCA domain. Functional studies using fibroblast cells from two affected individuals with the c.1516C>T mutation showed a truncated WASF1 and a defect in actin remodeling. This study provides evidence that de novo heterozygous mutations in WASF1 cause a rare form of intellectual disability

Identification of common genetic risk variants for autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heritable and heterogeneous group of neurodevelopmental phenotypes diagnosed in more than 1% of children. Common genetic variants contribute substantially to ASD susceptibility, but to date no individual variants have been robustly associated with ASD. With a marked sample-size increase from a unique Danish population resource, we report a genome-wide association meta-analysis of 18,381 individuals with ASD and 27,969 controls that identified five genome-wide-significant loci. Leveraging GWAS results from three phenotypes with significantly overlapping genetic architectures (schizophrenia, major depression, and educational attainment), we identified seven additional loci shared with other traits at equally strict significance levels. Dissecting the polygenic architecture, we found both quantitative and qualitative polygenic heterogeneity across ASD subtypes. These results highlight biological insights, particularly relating to neuronal function and corticogenesis, and establish that GWAS performed at scale will be much more productive in the near term in ASD.Peer reviewe

Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in $\textit{CHM}$ in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.This work was supported by The National Institute for Health Research England (NIHR) for the NIHR BioResource – Rare Diseases project (grant number RG65966). The Moorfields Eye Hospital cohort of patients and clinical and imaging data were ascertained and collected with the support of grants from the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital, National Health Service Foundation Trust, and UCL Institute of Ophthalmology, Moorfields Eye Hospital Special Trustees, Moorfields Eye Charity, the Foundation Fighting Blindness (USA), and Retinitis Pigmentosa Fighting Blindness. M.M. is a recipient of an FFB Career Development Award. E.M. is supported by UCLH/UCL NIHR Biomedical Research Centre. F.L.R. and D.G. are supported by Cambridge NIHR Biomedical Research Centre