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We analysed DNA methylation data from 30 datasets comprising 3474 individuals, 19 tissues and 8 ethnicities at CpGs covered by the Illumina450K array. We identified 4143 hypervariable CpGs (‘hvCpGs’) with methylation in the top 5% most variable sites across multiple tissues and ethnicities. hvCpG methylation was influenced but not determined by genetic variation, and was not linked to probe reliability, epigenetic drift, age, sex or cell heterogeneity effects. hvCpG methylation tended to covary across tissues derived from different germ-layers and hvCpGs were enriched for proximity to ERV1 and ERVK retrovirus elements. hvCpGs were also enriched for loci previously associated with periconceptional environment, parent-of-origin-specific methylation, and distinctive methylation signatures in monozygotic twins. Together, these properties position hvCpGs as strong candidates for studying how stochastic and/or environmentally influenced DNA methylation states which are established in the early embryo and maintained stably thereafter can influence life-long health and disease.

In humans, DNA methylation marks inherited from gametes are largely erased following fertilisation, prior to construction of the embryonic methylome. Exploiting a natural experiment of seasonal variation including changes in diet and nutritional status in rural Gambia, we analysed three datasets covering two independent child cohorts and identified 259 CpGs showing consistent associations between season of conception (SoC) and DNA methylation. SoC effects were most apparent in early infancy, with evidence of attenuation by mid-childhood. SoC-associated CpGs were enriched for metastable epialleles, parent-of-origin-specific methylation and germline differentially methylated regions, supporting a periconceptional environmental influence. Many SoC-associated CpGs overlapped enhancers or sites of active transcription in H1 embryonic stem cells and fetal tissues. Half were influenced but not determined by measured genetic variants that were independent of SoC. Environmental ‘hotspots’ providing a record of environmental influence at periconception constitute a valuable resource for investigating epigenetic mechanisms linking early exposures to lifelong health and disease.

Background The prevalence of cardiometabolic disease (CMD) is rising globally, with environmentally induced epigenetic changes suggested to play a role. Few studies have investigated epigenetic associations with CMD risk factors in children from low- and middle-income countries. We sought to identify associations between DNA methylation (DNAm) and CMD risk factors in children from India and The Gambia. Results Using the Illumina Infinium HumanMethylation 850 K Beadchip array, we interrogated DNAm in 293 Gambian (7–9 years) and 698 Indian (5–7 years) children. We identified differentially methylated CpGs (dmCpGs) associated with systolic blood pressure, fasting insulin, triglycerides and LDL-Cholesterol in the Gambian children; and with insulin sensitivity, insulinogenic index and HDL-Cholesterol in the Indian children. There was no overlap of the dmCpGs between the cohorts. Meta-analysis identified dmCpGs associated with insulin secretion and pulse pressure that were different from cohort-specific dmCpGs. Several differentially methylated regions were associated with diastolic blood pressure, insulin sensitivity and fasting glucose, but these did not overlap with the dmCpGs. We identified significant cis-methQTLs at three LDL-Cholesterol-associated dmCpGs in Gambians; however, methylation did not mediate genotype effects on the CMD outcomes. Conclusion This study identified cardiometabolic biomarkers associated with differential DNAm in Indian and Gambian children. Most associations were cohort specific, potentially reflecting environmental and ethnic differences.

Description, Child PAX8 methylation is associated with thyroid volume and function and influenced by maternal periconceptional nutrition., PAX8 is a key thyroid transcription factor implicated in thyroid gland differentiation and function, and PAX8 gene methylation is reported to be sensitive to the periconceptional environment. Using a novel recall-by-epigenotype study in Gambian children, we found that PAX8 hypomethylation at age 2 years is associated with a 21% increase in thyroid volume and an increase in free thyroxine (T4) at 5 to 8 years, the latter equivalent to 8.4% of the normal range. Free T4 was associated with a decrease in DXA-derived body fat and bone mineral density. Furthermore, offspring PAX8 methylation was associated with periconceptional maternal nutrition, and methylation variability was influenced by genotype, suggesting that sensitivity to environmental exposures may be under partial genetic control. Together, our results demonstrate a possible link between early environment, PAX8 gene methylation and thyroid gland development and function, with potential implications for early embryonic programming of thyroid-related health and disease.

Twin and family studies are a useful tool in epigenetic research on obesity as they can help to elucidate and control for the effects of genetic variation and may also reveal the effects of intergenerational exposures. Twin and family studies have identified novel regions with differential DNA methylation and distinct miRNA profiles associated with obesity and metabolically adverse fat distribution. These studies provide useful insights into the pathogenesis of obesity and metabolic disease. Most research focuses on changes in DNA methylation, and family studies have revealed numerous parental exposures, both environmental and health related, associated with methylation changes, and with adiposity, weight-related and metabolic phenotypes in the offspring. Further work is required to replicate these findings and understand the causal pathways involved.

Background DNA methylation is thought to be an important determinant of human phenotypic variation, but its inherent cell type specificity has impeded progress on this question. At exceptional genomic regions, interindividual variation in DNA methylation occurs systemically. Like genetic variants, systemic interindividual epigenetic variants are stable, can influence phenotype, and can be assessed in any easily biopsiable DNA sample. We describe an unbiased screen for human genomic regions at which interindividual variation in DNA methylation is not tissue-specific. Results For each of 10 donors from the NIH Genotype-Tissue Expression (GTEx) program, CpG methylation is measured by deep whole-genome bisulfite sequencing of genomic DNA from tissues representing the three germ layer lineages: thyroid (endoderm), heart (mesoderm), and brain (ectoderm). We develop a computational algorithm to identify genomic regions at which interindividual variation in DNA methylation is consistent across all three lineages. This approach identifies 9926 correlated regions of systemic interindividual variation (CoRSIVs). These regions, comprising just 0.1% of the human genome, are inter-correlated over long genomic distances, associated with transposable elements and subtelomeric regions, conserved across diverse human ethnic groups, sensitive to periconceptional environment, and associated with genes implicated in a broad range of human disorders and phenotypes. CoRSIV methylation in one tissue can predict expression of associated genes in other tissues. Conclusions In addition to charting a previously unexplored molecular level of human individuality, this atlas of human CoRSIVs provides a resource for future population-based investigations into how interindividual epigenetic variation modulates risk of disease. Electronic supplementary material The online version of this article (10.1186/s13059-019-1708-1) contains supplementary material, which is available to authorized users.

The prenatal environment can alter an individual’s developmental trajectory with long-lasting effects on health. Animal models demonstrate that the impact of the early life environment extends to subsequent generations, but there is a paucity of data from human populations on intergenerational transmission of environmentally induced phenotypes. Here we investigated the association of parental exposure to energy and nutrient restriction in utero on their children’s growth in rural Gambia. In a Gambian cohort with infants born between 1972 and 2011, we used multiple regression to test whether parental season of birth predicted offspring birth weight (n = 2097) or length (n = 1172), height-for-age z score (HAZ), weight-for-height z score (WHZ), and weight-for-age z score (WAZ) at 2 yr of age (n = 923). We found that maternal exposure to seasonal energy restriction in utero was associated with reduced offspring birth length (crude:−4.2 mm, P = 0.005; adjusted: −4.0 mm, P = 0.02). In contrast, paternal birth season predicted offspring HAZ at 24 mo (crude: −0.21, P = 0.005; adjusted: −0.22, P = 0.004) but had no discernible impact at birth. Our results indicate that periods of nutritional restriction in a parent’s fetal life can have intergenerational consequences in human populations. Fetal growth appears to be under matriline influence, and postnatal growth appears to be under patriline intergenerational influences.—Eriksen, K. G., Radford, E. J., Silver, M. J., Fulford, A. J. C., Wegmüller, R., Prentice, A. M. Influence of intergenerational in utero parental energy and nutrient restriction on offspring growth in rural Gambia.

Background Infant DNA methylation profiles are associated with their mother’s periconceptional nutritional status. DNA methylation relies on nutritional inputs for one-carbon metabolic pathways, including the efficient recycling of homocysteine. This randomised controlled trial in nonpregnant women in rural Gambia tests the efficacy of a novel nutritional supplement designed to improve one-carbon-related nutrient status by reducing plasma homocysteine, and assesses its potential future use in preconception trials. Methods and findings We designed a novel drink powder based on determinants of plasma homocysteine in the target population and tested it in a three-arm, randomised, controlled trial. Nonpregnant women aged between 18 and 45 from the West Kiang region of The Gambia were randomised in a 1:1:1 allocation to 12 weeks daily supplementation of either (a) a novel drink powder (4 g betaine, 800 μg folic acid, 5.2 μg vitamin B12, and 2.8 mg vitamin B2), (b) a widely used multiple micronutrient tablet (United Nations Multiple Micronutrient Preparation [UNIMMAP]) containing 15 micronutrients, or (c) no intervention. The trial was conducted between March and July 2018. Supplementation was observed daily. Fasted venepuncture samples were collected at baseline, midline (week 5), and endline (week 12) to measure plasma homocysteine. We used linear regression models to determine the difference in homocysteine between pairs of trial arms at midline and endline, adjusted for baseline homocysteine, age, and body mass index (BMI). Blood pressure and pulse were measured as secondary outcomes. Two hundred and ninety-eight eligible women were enrolled and randomised. Compliance was >97.8% for both interventions. At endline (our primary endpoint), the drink powder and UNIMMAP reduced mean plasma homocysteine by 23.6% (−29.5 to −17.1) and 15.5% (−21.2 to −9.4), respectively (both p < 0.001), compared with the controls. Compared with UNIMMAP, the drink powder reduced mean homocysteine by 8.8% (−15.8 to −1.2; p = 0.025). The effects were stronger at midline. There was no effect of either intervention on blood pressure or pulse compared with the control at endline. Self-reported adverse events (AEs) were similar in both intervention arms. There were two serious AEs reported over the trial duration, both in the drink powder arm, but judged to be unrelated to the intervention. Limitations of the study include the use of a single targeted metabolic outcome, homocysteine. Conclusions The trial confirms that dietary supplements can influence metabolic pathways that we have shown in previous studies to predict offspring DNA methylation. Both supplements reduced homocysteine effectively and remain potential candidates for future epigenetic trials in pregnancy in rural Gambia. Trial registration Clinicaltrials.gov Reference NCT03431597., Philip Thomas James and colleagues reveal a potential, unique link between mother’ nutritional input and an offspring’s methylation status., Author summary Why was this study done? At conception, the methylation marks on DNA from sperm and egg are erased and then re-established in the very early embryo. This DNA methylation relies on nutritional inputs for one-carbon metabolic pathways, including the efficient recycling of homocysteine, an absence of which might interfere with normal fetal development. We sought to test nutritional supplements that could reduce homocysteine and thereby optimise methylation pathways in women at the time of conception. What did the researchers do and find? We designed a novel drink powder containing betaine, folic acid, and vitamins B12 and B2 and tested its efficacy in reducing plasma homocysteine. Efficacy was tested in nonpregnant rural Gambian women in a randomised controlled trial, in which we also tested the UNIMMAP supplement that contains 15 micronutrients but no betaine. Both our novel supplement and UNIMMAP reduced homocysteine after 5 and 12 weeks of supplementation, with the novel drink powder showing a significantly enhanced efficacy. What do these findings mean? This trial demonstrates that it is feasible to lower homocysteine levels even in women whose homocysteine levels are only marginally elevated. Our novel supplement would be a potential candidate for future trials of preconceptional supplements aimed at optimising offspring DNA methylation.

Background\ud \ud The consequences of preterm birth are a major public health concern with high rates of ensuing multisystem morbidity, and uncertain biological mechanisms. Common genetic variation may mediate vulnerability to the insult of prematurity and provide opportunities to predict and modify risk.\ud \ud Objective\ud \ud To gain novel biological and therapeutic insights from the integrated analysis of magnetic resonance imaging and genetic data, informed by prior knowledge.\ud \ud Methods\ud \ud We apply our previously validated pathway-based statistical method and a novel network-based method to discover sources of common genetic variation associated with imaging features indicative of structural brain damage.\ud \ud Results\ud \ud Lipid pathways were highly ranked by Pathways Sparse Reduced Rank Regression in a model examining the effect of prematurity, and PPAR (peroxisome proliferator-activated receptor) signaling was the highest ranked pathway once degree of prematurity was accounted for. Within the PPAR pathway, five genes were found by Graph Guided Group Lasso to be highly associated with the phenotype: aquaporin 7 (AQP7), malic enzyme 1, NADP(+)-dependent, cytosolic (ME1), perilipin 1 (PLIN1), solute carrier family 27 (fatty acid transporter), member 1 (SLC27A1), and acetyl-CoA acyltransferase 1 (ACAA1). Expression of four of these (ACAA1, AQP7, ME1, and SLC27A1) is controlled by a common transcription factor, early growth response 4 (EGR-4).\ud \ud Conclusions\ud \ud This suggests an important role for lipid pathways in influencing development of white matter in preterm infants, and in particular a significant role for interindividual genetic variation in PPAR signaling.

The estimated heritability of human BMI is close to 75%, but identified genetic variants explain only a small fraction of interindividual body-weight variation. Inherited epigenetic variants identified in mouse models named “metastable epialleles” could in principle explain this “missing heritability.” We provide evidence that methylation in a variably methylated region (VMR) in the pro-opiomelanocortin gene (POMC), particularly in postmortem human laser-microdissected melanocyte-stimulating hormone (MSH)-positive neurons, is strongly associated with individual BMI. Using cohorts from different ethnic backgrounds, including a Gambian cohort, we found evidence suggesting that methylation of the POMC VMR is established in the early embryo and that offspring methylation correlates with the paternal somatic methylation pattern. Furthermore, it is associated with levels of maternal one-carbon metabolites at conception and stable during postnatal life. Together, these data suggest that the POMC VMR may be a human metastable epiallele that influences body-weight regulation.

Choline is an essential nutrient, and the amount needed in the diet is modulated by several factors. Given geographical differences in dietary choline intake and disparate frequencies of single-nucleotide polymorphisms (SNPs) in choline metabolism genes between ethnic groups, we tested the hypothesis that 3 SNPs that increase dependence on dietary choline would be under negative selection pressure in settings where choline intake is low: choline dehydrogenase (CHDH) rs12676, methylenetetrahydrofolate reductase 1 (MTHFD1) rs2236225, and phosphatidylethanolamine-N-methyltransferase (PEMT) rs12325817. Evidence of negative selection was assessed in 2 populations: one in The Gambia, West Africa, where there is historic evidence of a choline-poor diet, and the other in the United States, with a comparatively choline-rich diet. We used 2 independent methods, and confirmation of our hypothesis was sought via a comparison with SNP data from the Maasai, an East African population with a genetic background similar to that of Gambians but with a traditional diet that is higher in choline. Our results show that frequencies of SNPs known to increase dependence on dietary choline are significantly reduced in the low-choline setting of The Gambia. Our findings suggest that adequate intake levels of choline may have to be reevaluated in different ethnic groups and highlight a possible approach for identifying novel functional SNPs under the influence of dietary selective pressure.—Silver, M. J., Corbin, K. D., Hellenthal, G., da Costa, K.-A., Dominguez-Salas, P., Moore, S. E., Owen, J., Prentice, A. M., Hennig, B. J., Zeisel, S. H. Evidence for negative selection of gene variants that increase dependence on dietary choline in a Gambian cohort.

Background Interindividual epigenetic variation that occurs systemically must be established prior to gastrulation in the very early embryo and, because it is systemic, can be assessed in easily biopsiable tissues. We employ two independent genome-wide approaches to search for such variants. Results First, we screen for metastable epialleles by performing genomewide bisulfite sequencing in peripheral blood lymphocyte (PBL) and hair follicle DNA from two Caucasian adults. Second, we conduct a genomewide screen for genomic regions at which PBL DNA methylation is affected by season of conception in rural Gambia. Remarkably, both approaches identify the genomically imprinted VTRNA2-1 as a top environmentally responsive epiallele. We demonstrate systemic and stochastic interindividual variation in DNA methylation at the VTRNA2-1 differentially methylated region in healthy Caucasian and Asian adults and show, in rural Gambians, that periconceptional environment affects offspring VTRNA2-1 epigenotype, which is stable over at least 10 years. This unbiased screen also identifies over 100 additional candidate metastable epialleles, and shows that these are associated with cis genomic features including transposable elements. Conclusions The non-coding VTRNA2-1 transcript (also called nc886) is a putative tumor suppressor and modulator of innate immunity. Thus, these data indicating environmentally induced loss of imprinting at VTRNA2-1 constitute a plausible causal pathway linking early embryonic environment, epigenetic alteration, and human disease. More broadly, the list of candidate metastable epialleles provides a resource for future studies of epigenetic variation and human disease. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0660-y) contains supplementary material, which is available to authorized users.

Choline is an essential dietary nutrient and, along with its metabolites, has profound implications in cell signaling, methyl group donation, neurotransmitter synthesis, structural integrity of cel...

Standard approaches to data analysis in genome-wide association studies (GWAS) ignore any potential functional relationships between gene variants. In contrast gene pathways analysis uses prior information on functional structure within the genome to identify pathways associated with a trait of interest. In a second step, important single nucleotide polymorphisms (SNPs) or genes may be identified within associated pathways. The pathways approach is motivated by the fact that genes do not act alone, but instead have effects that are likely to be mediated through their interaction in gene pathways. Where this is the case, pathways approaches may reveal aspects of a trait's genetic architecture that would otherwise be missed when considering SNPs in isolation. Most pathways methods begin by testing SNPs one at a time, and so fail to capitalise on the potential advantages inherent in a multi-SNP, joint modelling approach. Here, we describe a dual-level, sparse regression model for the simultaneous identification of pathways and genes associated with a quantitative trait. Our method takes account of various factors specific to the joint modelling of pathways with genome-wide data, including widespread correlation between genetic predictors, and the fact that variants may overlap multiple pathways. We use a resampling strategy that exploits finite sample variability to provide robust rankings for pathways and genes. We test our method through simulation, and use it to perform pathways-driven gene selection in a search for pathways and genes associated with variation in serum high-density lipoprotein cholesterol levels in two separate GWAS cohorts of Asian adults. By comparing results from both cohorts we identify a number of candidate pathways including those associated with cardiomyopathy, and T cell receptor and PPAR signalling. Highlighted genes include those associated with the L-type calcium channel, adenylate cyclase, integrin, laminin, MAPK signalling and immune function., Author Summary Genes do not act in isolation, but interact in complex networks or pathways. By accounting for such interactions, pathways analysis methods hope to identify aspects of a disease or trait's genetic architecture that might be missed using more conventional approaches. Most existing pathways methods take a univariate approach, in which each variant within a pathway is separately tested for association with the phenotype of interest. These statistics are then combined to assess pathway significance. As a second step, further analysis can reveal important genetic variants within significant pathways. We have previously shown that a joint-modelling approach using a sparse regression model can increase the power to detect pathways influencing a quantitative trait. Here we extend this approach, and describe a method that is able to simultaneously identify pathways and genes that may be driving pathway selection. We test our method using simulations, and apply it to a study searching for pathways and genes associated with high-density lipoprotein cholesterol in two separate East Asian cohorts.