Fine mapping of candidate effector genes for heart rate.

An elevated resting heart rate (RHR) is associated with increased cardiovascular mortality. Genome-wide association studies (GWAS) have identified > 350 loci. Uniquely, in this study we applied genetic fine-mapping leveraging tissue specific chromatin segmentation and colocalization analyses to identify causal variants and candidate effector genes for RHR. We used RHR GWAS summary statistics from 388,237 individuals of European ancestry from UK Biobank and performed fine mapping using publicly available genomic annotation datasets. High-confidence causal variants (accounting for > 75% posterior probability) were identified, and we collated candidate effector genes using a multi-omics approach that combined evidence from colocalisation with molecular quantitative trait loci (QTLs), and long-range chromatin interaction analyses. Finally, we performed druggability analyses to investigate drug repurposing opportunities. The fine mapping pipeline indicated 442 distinct RHR signals. For 90 signals, a single variant was identified as a high-confidence causal variant, of which 22 were annotated as missense. In trait-relevant tissues, 39 signals colocalised with cis-expression QTLs (eQTLs), 3 with cis-protein QTLs (pQTLs), and 75 had promoter interactions via Hi-C. In total, 262 candidate genes were highlighted (79% had promoter interactions, 15% had a colocalised eQTL, 8% had a missense variant and 1% had a colocalised pQTL), and, for the first time, enrichment in nervous system pathways. Druggability analyses highlighted ACHE, CALCRL, MYT1 and TDP1 as potential targets. Our genetic fine-mapping pipeline prioritised 262 candidate genes for RHR that warrant further investigation in functional studies, and we provide potential therapeutic targets to reduce RHR and cardiovascular mortality.

Epigenomic insights into common human disease pathology.

The epigenome-the chemical modifications and chromatin-related packaging of the genome-enables the same genetic template to be activated or repressed in different cellular settings. This multi-layered mechanism facilitates cell-type specific function by setting the local sequence and 3D interactive activity level. Gene transcription is further modulated through the interplay with transcription factors and co-regulators. The human body requires this epigenomic apparatus to be precisely installed throughout development and then adequately maintained during the lifespan. The causal role of the epigenome in human pathology, beyond imprinting disorders and specific tumour suppressor genes, was further brought into the spotlight by large-scale sequencing projects identifying that mutations in epigenomic machinery genes could be critical drivers in both cancer and developmental disorders. Abrogation of this cellular mechanism is providing new molecular insights into pathogenesis. However, deciphering the full breadth and implications of these epigenomic changes remains challenging. Knowledge is accruing regarding disease mechanisms and clinical biomarkers, through pathogenically relevant and surrogate tissue analyses, respectively. Advances include consortia generated cell-type specific reference epigenomes, high-throughput DNA methylome association studies, as well as insights into ageing-related diseases from biological 'clocks' constructed by machine learning algorithms. Also, 3rd-generation sequencing is beginning to disentangle the complexity of genetic and DNA modification haplotypes. Cell-free DNA methylation as a cancer biomarker has clear clinical utility and further potential to assess organ damage across many disorders. Finally, molecular understanding of disease aetiology brings with it the opportunity for exact therapeutic alteration of the epigenome through CRISPR-activation or inhibition.

Integration of genetic fine-mapping and multi-omics data reveals candidate effector genes for hypertension.

Genome-wide association studies of blood pressure (BP) have identified >1,000 loci, but the effector genes and biological pathways at these loci are mostly unknown. Using published association summary statistics, we conducted annotation-informed fine-mapping incorporating tissue-specific chromatin segmentation and colocalization to identify causal variants and candidate effector genes for systolic BP, diastolic BP, and pulse pressure. We observed 532 distinct signals associated with ≥2 BP traits and 84 with all three. For >20% of signals, a single variant accounted for >75% posterior probability, 65 were missense variants in known (SLC39A8, ADRB2, and DBH) and previously unreported BP candidate genes (NRIP1 and MMP14). In disease-relevant tissues, we colocalized >80 and >400 distinct signals for each BP trait with cis-eQTLs and regulatory regions from promoter capture Hi-C, respectively. Integrating mouse, human disorder, gene expression and tissue abundance data, and literature review, we provide consolidated evidence for 436 BP candidate genes for future functional validation and discover several potential drug targets.

A DNA methylation signature in the stress driver gene Fkbp5 indicates a neuropathic component in chronic pain.

BACKGROUND: Epigenetic changes can bring insight into gene regulatory mechanisms associated with disease pathogenicity, including chronicity and increased vulnerability. To date, we are yet to identify genes sensitive to epigenetic regulation that contribute to the maintenance of chronic pain and with an epigenetic landscape indicative of the susceptibility to persistent pain. Such genes would provide a novel opportunity for better pain management, as their epigenetic profile could be targeted for the treatment of chronic pain or used as an indication of vulnerability for prevention strategies. Here, we investigated the epigenetic profile of the gene Fkbp5 for this potential, using targeted bisulphite sequencing in rodent pre-clinical models of chronic and latent hypersensitive states. RESULTS: The Fkbp5 promoter DNA methylation (DNAm) signature in the CNS was significantly different between models of persistent pain, and there was a significant correlation between CNS and peripheral blood Fkbp5 DNAm, indicating that further exploration of Fkbp5 promoter DNAm as an indicator of chronic pain pathogenic origin is warranted. We also found that maternal separation, which promotes the persistency of inflammatory pain in adulthood, was accompanied by long-lasting reduction in Fkbp5 DNAm, suggesting that Fkbp5 DNAm profile may indicate the increased vulnerability to chronic pain in individuals exposed to trauma in early life. CONCLUSIONS: Overall, our data demonstrate that the Fkbp5 promoter DNAm landscape brings novel insight into the differing pathogenic origins of chronic pain, may be able to stratify patients and predict the susceptibility to chronic pain.

CRISPR/dCas9 DNA methylation editing is heritable during human hematopoiesis and shapes immune progeny.

Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.

DNA Methylome Alterations Are Associated with Airway Macrophage Differentiation and Phenotype during Lung Fibrosis.

Rationale: Airway macrophages (AMs) are key regulators of the lung environment and are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal respiratory disease with no cure. However, knowledge about the epigenetics of AMs in IPF is limited. Objectives: To assess the role of epigenetic regulation of AMs during lung fibrosis. Methods: We undertook DNA methylation (DNAm) profiling by using Illumina EPIC (850k) arrays in sorted AMs from healthy donors (n = 14) and donors with IPF (n = 30). Cell-type deconvolution was performed by using reference myeloid-cell DNA methylomes. Measurements and Main Results: Our analysis revealed that epigenetic heterogeneity was a key characteristic of IPF AMs. DNAm "clock" analysis indicated that epigenetic alterations in IPF AMs were not associated with accelerated aging. In differential DNAm analysis, we identified numerous differentially methylated positions (n = 11) and differentially methylated regions (n = 49) between healthy and IPF AMs, respectively. Differentially methylated positions and differentially methylated regions encompassed genes involved in lipid (LPCAT1 [lysophosphatidylcholine acyltransferase 1]) and glucose (PFKFB3 [6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3]) metabolism, and importantly, the DNAm status was associated with disease severity in IPF. Conclusions: Collectively, our data identify that changes in the epigenome are associated with the development and function of AMs in the IPF lung.

The genomic loci of specific human tRNA genes exhibit ageing-related DNA hypermethylation.

The epigenome has been shown to deteriorate with age, potentially impacting on ageing-related disease. tRNA, while arising from only ˜46 kb (<0.002% genome), is the second most abundant cellular transcript. tRNAs also control metabolic processes known to affect ageing, through core translational and additional regulatory roles. Here, we interrogate the DNA methylation state of the genomic loci of human tRNA. We identify a genomic enrichment for age-related DNA hypermethylation at tRNA loci. Analysis in 4,350 MeDIP-seq peripheral-blood DNA methylomes (16-82 years), identifies 44 and 21 hypermethylating specific tRNAs at study-and genome-wide significance, respectively, contrasting with none hypomethylating. Validation and replication (450k array and independent targeted Bisuphite-sequencing) supported the hypermethylation of this functional unit. Tissue-specificity is a significant driver, although the strongest consistent signals, also independent of major cell-type change, occur in tRNA-iMet-CAT-1-4 and tRNA-Ser-AGA-2-6. This study presents a comprehensive evaluation of the genomic DNA methylation state of human tRNA genes and reveals a discreet hypermethylation with advancing age.

Somatostatin receptor 2 expression in nasopharyngeal cancer is induced by Epstein Barr virus infection: impact on prognosis, imaging and therapy.

Nasopharyngeal cancer (NPC), endemic in Southeast Asia, lacks effective diagnostic and therapeutic strategies. Even in high-income countries the 5-year survival rate for stage IV NPC is less than 40%. Here we report high somatostatin receptor 2 (SSTR2) expression in multiple clinical cohorts comprising 402 primary, locally recurrent and metastatic NPCs. We show that SSTR2 expression is induced by the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) via the NF-κB pathway. Using cell-based and preclinical rodent models, we demonstrate the therapeutic potential of SSTR2 targeting using a cytotoxic drug conjugate, PEN-221, which is found to be superior to FDA-approved SSTR2-binding cytostatic agents. Furthermore, we reveal significant correlation of SSTR expression with increased rates of survival and report in vivo uptake of the SSTR2-binding 68Ga-DOTA-peptide radioconjugate in PET-CT scanning in a clinical trial of NPC patients (NCT03670342). These findings reveal a key role in EBV-associated NPC for SSTR2 in infection, imaging, targeted therapy and survival.

Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial-mesenchymal transition and subcellular re-localization of Beta-Catenin.

DNA methyltransferase I plays the central role in maintenance of CpG DNA methylation patterns across the genome and alteration of CpG methylation patterns is a frequent and significant occurrence across many cancers. Cancer cells carrying hypomorphic alleles of Dnmt1 have become important tools for understanding Dnmt1 function and CpG methylation. In this study, we analyse colorectal cancer cells with a homozygous deletion of exons 3 to 5 of Dnmt1, resulting in reduced Dnmt1 activity. Although this cell model has been widely used to study the epigenome, the effects of the Dnmt1 hypomorph on cell signalling pathways and the wider proteome are largely unknown. In this study, we perform the first quantitative proteomic analysis of this important cell model and identify multiple signalling pathways and processes that are significantly dysregulated in the hypomorph cells. In Dnmt1 hypomorph cells, we observed a clear and unexpected signature of increased Epithelial-to-Mesenchymal transition (EMT) markers as well as reduced expression and sub-cellular re-localization of Beta-Catenin. Expression of wild-type Dnmt1 in hypomorph cells or knock-down of wild-type Dnmt1 did not recapitulate or rescue the observed protein profiles in Dnmt1 hypomorph cells suggesting that hypomorphic Dnmt1 causes changes not solely attributable to Dnmt1 protein levels. In summary, we present the first comprehensive proteomic analysis of the widely studied Dnmt1 hypomorph colorectal cancer cells and identify redistribution of Dnmt1 and its interaction partner Beta-Catenin.

DNA methylation aging clocks: challenges and recommendations.

Epigenetic clocks comprise a set of CpG sites whose DNA methylation levels measure subject age. These clocks are acknowledged as a highly accurate molecular correlate of chronological age in humans and other vertebrates. Also, extensive research is aimed at their potential to quantify biological aging rates and test longevity or rejuvenating interventions. Here, we discuss key challenges to understand clock mechanisms and biomarker utility. This requires dissecting the drivers and regulators of age-related changes in single-cell, tissue- and disease-specific models, as well as exploring other epigenomic marks, longitudinal and diverse population studies, and non-human models. We also highlight important ethical issues in forensic age determination and predicting the trajectory of biological aging in an individual.

Gestational Vitamin D Supplementation Leads to Reduced Perinatal RXRA DNA Methylation: Results From the MAVIDOS Trial.

We have previously demonstrated inverse associations between maternal 25(OH)-vitamin D status and perinatal DNA methylation at the retinoid-X-receptor-alpha (RXRA) locus and between RXRA methylation and offspring bone mass. In this study, we used an existing randomized trial to test the hypothesis that maternal gestational vitamin D supplementation would lead to reduced perinatal RXRA locus DNA methylation. The Maternal Vitamin D Osteoporosis Study (MAVIDOS) was a multicenter, double-blind, randomized, placebo-controlled trial of 1000 IU/day cholecalciferol or matched placebo from 14 weeks' gestation until delivery. Umbilical cord (fetal) tissue was collected at birth and frozen at -80°C (n = 453). Pyrosequencing was used to undertake DNA methylation analysis at 10 CpG sites within the RXRA locus (identified previously). T tests were used to assess differences between treatment groups in methylation at the three most representative CpG sites. Overall, methylation levels were significantly lower in the umbilical cord from offspring of cholecalciferol-supplemented mothers, reaching statistical significance at four CpG sites, represented by CpG5: mean difference in % methylation between the supplemented and placebo groups was -1.98% (95% CI, -3.65 to -0.32, p = 0.02). ENCODE (Encyclopedia of DNA Elements) evidence supports the functionality of this locus with strong DNase hypersensitivity and enhancer chromatin within biologically relevant cell types including osteoblasts. Enrichment of the enhancer-related H3K4me1 histone mark is also seen in this region, as are binding sites for a range of transcription factors with roles in cell proliferation, response to stress, and growth factors. Our findings are consistent with previous observational results and provide new evidence that maternal gestational supplementation with cholecalciferol leads to altered perinatal epigenetic marking, informing mechanistic understanding of early life mechanisms related to maternal vitamin D status, epigenetic marks, and bone development. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Integration of human pancreatic islet genomic data refines regulatory mechanisms at Type 2 Diabetes susceptibility loci.

Human genetic studies have emphasised the dominant contribution of pancreatic islet dysfunction to development of Type 2 Diabetes (T2D). However, limited annotation of the islet epigenome has constrained efforts to define the molecular mechanisms mediating the, largely regulatory, signals revealed by Genome-Wide Association Studies (GWAS). We characterised patterns of chromatin accessibility (ATAC-seq, n = 17) and DNA methylation (whole-genome bisulphite sequencing, n = 10) in human islets, generating high-resolution chromatin state maps through integration with established ChIP-seq marks. We found enrichment of GWAS signals for T2D and fasting glucose was concentrated in subsets of islet enhancers characterised by open chromatin and hypomethylation, with the former annotation predominant. At several loci (including CDC123, ADCY5, KLHDC5) the combination of fine-mapping genetic data and chromatin state enrichment maps, supplemented by allelic imbalance in chromatin accessibility pinpointed likely causal variants. The combination of increasingly-precise genetic and islet epigenomic information accelerates definition of causal mechanisms implicated in T2D pathogenesis.

Obligatory and facilitative allelic variation in the DNA methylome within common disease-associated loci.

Integrating epigenetic data with genome-wide association study (GWAS) results can reveal disease mechanisms. The genome sequence itself also shapes the epigenome, with CpG density and transcription factor binding sites (TFBSs) strongly encoding the DNA methylome. Therefore, genetic polymorphism impacts on the observed epigenome. Furthermore, large genetic variants alter epigenetic signal dosage. Here, we identify DNA methylation variability between GWAS-SNP risk and non-risk haplotypes. In three subsets comprising 3128 MeDIP-seq peripheral-blood DNA methylomes, we find 7173 consistent and functionally enriched Differentially Methylated Regions. 36.8% can be attributed to common non-SNP genetic variants. CpG-SNPs, as well as facilitative TFBS-motifs, are also enriched. Highlighting their functional potential, CpG-SNPs strongly associate with allele-specific DNase-I hypersensitivity sites. Our results demonstrate strong DNA methylation allelic differences driven by obligatory or facilitative genetic effects, with potential direct or regional disease-related repercussions. These allelic variations require disentangling from pure tissue-specific modifications, may influence array studies, and imply underestimated population variability in current reference epigenomes.

Microelectrode generator-collector systems for electrolytic titration: theoretical and practical considerations.

Electochemical generator-collector systems, where one electrode is used to generate a reagent, have a potentially large field of application in sensing and measurement. We present a new theoretical description for coplanar microelectrode disc-disc systems where the collector is passive (such as a potentiometric sensor) and the generator is operating at constant flux. This solution is then used to develop a leading order solution for such a system where the reagent reacts reversibly in solution, such as in acid-base titration, where a hydrogen ion flux is generated by electrolysis of water. The principal novel result of the theory is that such devices are constrained by a maximum reagent flux. The hydrogen ion concentration at the collector will only reflect the buffer capacity of the bulk solution if this constraint is met. Both mathematical solutions are evaluated with several microfabricated devices and reasonable agreement with theory is demonstrated.

The Epigenomic Analysis of Human Obesity.

OBJECTIVE: Analysis of the epigenome-the chemical modifications and packaging of the genome that can influence or indicate its activity-enables molecular insight into cell type-specific machinery. It can, therefore, reveal the pathophysiological mechanisms at work in disease. Detected changes can also represent physiological responses to adverse environmental exposures, thus enabling the epigenetic mark of DNA methylation to act as an epidemiological biomarker, even in surrogate tissue. This makes epigenomic analysis an attractive prospect to further understand the pathobiology and epidemiological aspects of obesity. Furthermore, integrating epigenomic data with known obesity-associated common genetic variation can aid in deciphering their molecular mechanisms. METHODS AND CONCLUSIONS: This review primarily examines epidemiological or population-based studies of epigenetic modifications in relation to adiposity traits, as opposed to animal or cell models. It discusses recent work exploring the epigenome with respect to human obesity, which to date has predominately consisted of array-based studies of DNA methylation in peripheral blood. It is of note that highly replicated BMI DNA methylation associations are not causal, but strongly driven by coassociations for more precisely measured intertwined outcomes and factors, such as hyperlipidemia, hyperglycemia, and inflammation. Finally, the potential for the future exploration of the epigenome in obesity and related disorders is considered.

Perinatal DNA Methylation at CDKN2A Is Associated With Offspring Bone Mass: Findings From the Southampton Women's Survey.

Poor intrauterine and childhood growth has been linked with the risk of osteoporosis in later life, a relationship that may in part be mediated through altered epigenetic regulation of genes. We previously identified a region within the promoter of the long non-coding RNA ANRIL encoded by the CDKN2A locus, at which differential DNA methylation at birth showed correlations with offspring adiposity. Given the common lineage of adipocytes and osteoblasts, we investigated the relationship between perinatal CDKN2A methylation and bone mass at ages 4 and 6 years. Using sodium bisulfite pyrosequencing, we measured the methylation status of the 9 CpGs within this region in umbilical cord samples from discovery (n = 332) and replication (n = 337) cohorts of children from the Southampton Women's Survey, whose bone mass was assessed by dual-energy X-ray absorptiomietry (DXA; Hologic Discovery). Inverse associations were found between perinatal CDKN2A methylation and whole-body minus head bone area (BA), bone mineral content (BMC), and areal bone mineral density (BMD). This was confirmed in replication and combined data sets (all p < 0.01), with each 10% increase in methylation being associated with a decrease in BMC of 4 to 9 g at age 4 years (p ≤ 0.001). Relationships were similar with 6-year bone mass. Functional investigation of the differentially methylated region in the SaOS-2 osteosarcoma cell line showed that transcription factors bound to the identified CpGs in a methylation-specific manner and that CpG mutagenesis modulated ANRIL expression. In conclusion, perinatal methylation at CDKN2A is associated with childhood bone development and has significance for cell function. © 2017 American Society for Bone and Mineral Research.

Higher Nevus Count Exhibits a Distinct DNA Methylation Signature in Healthy Human Skin: Implications for Melanoma.

High nevus count is the strongest risk factor for melanoma, and although gene variants have been discovered for both traits, epigenetic variation is unexplored. We investigated 322 healthy human skin DNA methylomes associated with total body nevi count, incorporating genetic and transcriptomic variation. DNA methylation changes were identified at genes involved in melanocyte biology, such as RAF1 (P = 1.2 × 10-6) and CTC1 (region: P = 6.3 × 10-4), and other genes including ARRDC1 (P = 3.1 × 10-7). A subset exhibited coordinated methylation and transcription changes within the same biopsy. The total analysis was also enriched for melanoma-associated DNA methylation variation (P = 6.33 × 10-6). In addition, we show that skin DNA methylation is associated in cis with known genome-wide association study single nucleotide polymorphisms for nevus count, at PLA2G6 (P = 1.7 × 10-49) and NID1 (P = 6.4 × 10-14), as well as melanoma risk, including in or near MC1R, MX2, and TERT/CLPTM1L (P < 1 × 10-10). Our analysis using a uniquely large dataset comprising healthy skin DNA methylomes identified known and additional regulatory loci and pathways in nevi and melanoma biology. This integrative study improves our understanding of predisposition to nevi and their potential contribution to melanoma pathogenesis.

Insights in human epigenomic dynamics through comparative primate analysis.

Epigenomic analysis gives a molecular insight into cell-specific genomic activity. It provides a detailed functional plan to dissect an organism, tissue by tissue. Therefore comparative epigenomics may increase understanding of human-acquired traits, by revealing regulatory changes in systems such as the neurological, musculoskeletal, and immunological. Enhancer loci evolve fast by hijacking elements from other tissues or rewiring and amplifying existing units for human-specific function. Promoters by contrast often require a CpG dense genetic infrastructure. Specific interplay occurs between the two, but also a shared modality of function, with coordination from global chromatin-modifying enzymes. Changes in specific transcription factor binding sites also facilitate the local epigenetic state. In the case of CTCF, these may further influence 3-dimensional structure and interaction. How these mechanistic units are modulated between tissue and species enables more comprehensive understanding of human processes and pathology. With this information, precise therapeutic targeting of these epigenetic modifications may become possible.

Novel regional age-associated DNA methylation changes within human common disease-associated loci.

BACKGROUND: Advancing age progressively impacts on risk and severity of chronic disease. It also modifies the epigenome, with changes in DNA methylation, due to both random drift and variation within specific functional loci. RESULTS: In a discovery set of 2238 peripheral-blood genome-wide DNA methylomes aged 19-82 years, we identify 71 age-associated differentially methylated regions within the linkage disequilibrium blocks of the single nucleotide polymorphisms from the NIH genome-wide association study catalogue. This included 52 novel regions, 29 within loci not covered by 450 k or 27 k Illumina array, and with enrichment for DNase-I Hypersensitivity sites across the full range of tissues. These age-associated differentially methylated regions also show marked enrichment for enhancers and poised promoters across multiple cell types. In a replication set of 2084 DNA methylomes, 95.7 % of the age-associated differentially methylated regions showed the same direction of ageing effect, with 80.3 % and 53.5 % replicated to p < 0.05 and p < 1.85 × 10-8, respectively. CONCLUSION: By analysing the functionally enriched disease and trait-associated regions of the human genome, we identify novel epigenetic ageing changes, which could be useful biomarkers or provide mechanistic insights into age-related common diseases.