Accelerated biological aging six decades after prenatal famine exposure.

To test the hypothesis that early-life adversity accelerates the pace of biological aging, we analyzed data from the Dutch Hunger Winter Families Study (DHWFS, N = 951). DHWFS is a natural-experiment birth-cohort study of survivors of in-utero exposure to famine conditions caused by the German occupation of the Western Netherlands in Winter 1944 to 1945, matched controls, and their siblings. We conducted DNA methylation analysis of blood samples collected when the survivors were aged 58 to quantify biological aging using the DunedinPACE, GrimAge, and PhenoAge epigenetic clocks. Famine survivors had faster DunedinPACE, as compared with controls. This effect was strongest among women. Results were similar for GrimAge, although effect-sizes were smaller. We observed no differences in PhenoAge between survivors and controls. Famine effects were not accounted for by blood-cell composition and were similar for individuals exposed early and later in gestation. Findings suggest in-utero undernutrition may accelerate biological aging in later life.

Maternal educational attainment in pregnancy and epigenome-wide DNA methylation changes in the offspring from birth until adolescence.

Maternal educational attainment (MEA) shapes offspring health through multiple potential pathways. Differential DNA methylation may provide a mechanistic understanding of these long-term associations. We aimed to quantify the associations of MEA with offspring DNA methylation levels at birth, in childhood and in adolescence. Using 37 studies from high-income countries, we performed meta-analysis of epigenome-wide association studies (EWAS) to quantify the associations of completed years of MEA at the time of pregnancy with offspring DNA methylation levels at birth (n = 9 881), in childhood (n = 2 017), and adolescence (n = 2 740), adjusting for relevant covariates. MEA was found to be associated with DNA methylation at 473 cytosine-phosphate-guanine sites at birth, one in childhood, and four in adolescence. We observed enrichment for findings from previous EWAS on maternal folate, vitamin-B12 concentrations, maternal smoking, and pre-pregnancy BMI. The associations were directionally consistent with MEA being inversely associated with behaviours including smoking and BMI. Our findings form a bridge between socio-economic factors and biology and highlight potential pathways underlying effects of maternal education. The results broaden our understanding of bio-social associations linked to differential DNA methylation in multiple early stages of life. The data generated also offers an important resource to help a more precise understanding of the social determinants of health.

DNA methylation differences at birth after conception through ART.

STUDY QUESTION: Is there a relation between ART and DNA methylation (DNAm) patterns in cord blood, including any differences between IVF and ICSI? SUMMARY ANSWER: DNAm at 19 CpGs was associated with conception via ART, with no difference found between IVF and ICSI. WHAT IS KNOWN ALREADY: Prior studies on either IVF or ICSI show conflicting outcomes, as both widespread effects on DNAm and highly localized associations have been reported. No study on both IVF and ICSI and genome-wide neonatal DNAm has been performed. STUDY DESIGN, SIZE, DURATION: This was a cross-sectional study comprising 87 infants conceived with IVF or ICSI and 70 conceived following medically unassisted conception. The requirement for inclusion in the study was an understanding of the Swedish language and exclusion was the use of donor gametes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were from the UppstART study, which was recruited from fertility and reproductive health clinics, and the Born into Life cohort, which is recruited from the larger LifeGene study. We measured DNAm from DNA extracted from cord blood collected at birth using a micro-array (450k array). Group differences in DNAm at individual CpG dinucleotides (CpGs) were determined using robust linear models and post-hoc Tukey's tests. MAIN RESULTS AND THE ROLE OF CHANCE: We found no association of ART conception with global methylation levels, imprinted loci and meta-stable epialleles. In contrast, we identify 19 CpGs at which DNAm was associated with being conceived via ART (effect estimates: 0.5-4.9%, PFDR < 0.05), but no difference was found between IVF and ICSI. The associated CpGs map to genes related to brain function/development or genes connected to the plethora of conditions linked to subfertility, but functional annotation did not point to any likely functional consequences. LIMITATIONS, REASONS FOR CAUTION: We measured DNAm in cord blood and not at later ages or in other tissues. Given the number of tests performed, our study power is limited and the findings need to be replicated in an independent study. WIDER IMPLICATIONS OF THE FINDINGS: We find that ART is associated with DNAm differences in cord blood when compared to non-ART samples, but these differences are limited in number and effect size and have unknown functional consequences in adult blood. We did not find indications of differences between IVF and ICSI. STUDY FUNDING/COMPETING INTEREST(S): E.W.T. was supported by a VENI grant from the Netherlands Organization for Scientific Research (91617128) and JPI-H2020 Joint Programming Initiative a Healthy Diet for a Healthy Life (JPI HDHL) under proposal number 655 (PREcisE Project) through ZonMw (529051023). Financial support was provided from the European Union's Seventh Framework Program IDEAL (259679), the Swedish Research Council (K2011-69X-21871-01-6, 2011-3060, 2015-02434 and 2018-02640) and the Strategic Research Program in Epidemiology Young Scholar Awards, Karolinska Institute (to A.N.I.) and through the Swedish Initiative for Research on Microdata in the Social And Medical Sciences (SIMSAM) framework grant no 340-2013-5867, grants provided by the Stockholm County Council (ALF-projects), the Strategic Research Program in Epidemiology at Karolinska Institutet and the Swedish Heart-Lung Foundation and Danderyd University Hospital (Stockholm, Sweden). The funders had no role in study design, data collection, analysis, decision to publish or preparation of the manuscript. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Tea and coffee consumption in relation to DNA methylation in four European cohorts.

Lifestyle factors, such as food choices and exposure to chemicals, can alter DNA methylation and lead to changes in gene activity. Two such exposures with pharmacologically active components are coffee and tea consumption. Both coffee and tea have been suggested to play an important role in modulating disease-risk in humans by suppressing tumour progression, decreasing inflammation and influencing estrogen metabolism. These mechanisms may be mediated by changes in DNA methylation. To investigate if DNA methylation in blood is associated with coffee and tea consumption, we performed a genome-wide DNA methylation study for coffee and tea consumption in four European cohorts (N = 3,096). DNA methylation was measured from whole blood at 421,695 CpG sites distributed throughout the genome and analysed in men and women both separately and together in each cohort. Meta-analyses of the results and additional regional-level analyses were performed. After adjusting for multiple testing, the meta-analysis revealed that two individual CpG-sites, mapping to DNAJC16 and TTC17, were differentially methylated in relation to tea consumption in women. No individual sites were associated with men or with the sex-combined analysis for tea or coffee. The regional analysis revealed that 28 regions were differentially methylated in relation to tea consumption in women. These regions contained genes known to interact with estradiol metabolism and cancer. No significant regions were found in the sex-combined and male-only analysis for either tea or coffee consumption.

DNA Methylation Landscapes of Human Fetal Development.

Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality.

MethylAid: visual and interactive quality control of large Illumina 450k datasets.

UNLABELLED: The Illumina 450k array is a frequently used platform for large-scale genome-wide DNA methylation studies, i.e. epigenome-wide association studies. Currently, quality control of 450k data can be performed with Illumina's GenomeStudio and is part of a limited number 450k analysis pipelines. However, GenomeStudio cannot handle large-scale studies, and existing pipelines provide limited options for quality control and neither support interactive exploration by the user. To aid the detection of bad-quality samples in large-scale genome-wide DNA methylation studies as flexible and transparent as possible, we have developed MethylAid; a visual and interactive Web application using RStudio's shiny package. Bad-quality samples are detected using sample-dependent and sample-independent quality control probes present on the array and user-adjustable thresholds. In-depth exploration of bad-quality samples can be performed using several interactive diagnostic plots. Furthermore, plots can be annotated with user-provided metadata, for example, to identify outlying batches. Our new tool makes quality assessment of 450k array data interactive, flexible and efficient and is, therefore, expected to be useful for both data analysts and core facilities. AVAILABILITY AND IMPLEMENTATION: MethylAid is implemented as an R/Bioconductor package (www.bioconductor.org/packages/3.0/bioc/html/MethylAid.html). A demo application is available from shiny.bioexp.nl/MethylAid.

Underlying molecular mechanisms of DIO2 susceptibility in symptomatic osteoarthritis.

OBJECTIVES: To investigate how the genetic susceptibility gene DIO2 confers risk to osteoarthritis (OA) onset in humans and to explore whether counteracting the deleterious effect could contribute to novel therapeutic approaches. METHODS: Epigenetically regulated expression of DIO2 was explored by assessing methylation of positional CpG-dinucleotides and the respective DIO2 expression in OA-affected and macroscopically preserved articular cartilage from end-stage OA patients. In a human in vitro chondrogenesis model, we measured the effects when thyroid signalling during culturing was either enhanced (excess T3 or lentiviral induced DIO2 overexpression) or decreased (iopanoic acid). RESULTS: OA-related changes in methylation at a specific CpG dinucleotide upstream of DIO2 caused significant upregulation of its expression (ß=4.96; p=0.0016). This effect was enhanced and appeared driven specifically by DIO2 rs225014 risk allele carriers (ß=5.58, p=0.0006). During in vitro chondrogenesis, DIO2 overexpression resulted in a significant reduced capacity of chondrocytes to deposit extracellular matrix (ECM) components, concurrent with significant induction of ECM degrading enzymes (ADAMTS5, MMP13) and markers of mineralisation (ALPL, COL1A1). Given their concurrent and significant upregulation of expression, this process is likely mediated via HIF-2α/RUNX2 signalling. In contrast, we showed that inhibiting deiodinases during in vitro chondrogenesis contributed to prolonged cartilage homeostasis as reflected by significant increased deposition of ECM components and attenuated upregulation of matrix degrading enzymes. CONCLUSIONS: Our findings show how genetic variation at DIO2 could confer risk to OA and raised the possibility that counteracting thyroid signalling may be a novel therapeutic approach.

Epigenetic signature of birth weight discordance in adult twins.

BACKGROUND: A low birth weight has been extensively related to poor adult health outcomes. Birth weight can be seen as a proxy for environmental conditions during prenatal development. Identical twin pairs discordant for birth weight provide an extraordinary model for investigating the association between birth weight and adult life health while controlling for not only genetics but also postnatal rearing environment. We performed an epigenome-wide profiling on blood samples from 150 pairs of adult monozygotic twins discordant for birth weight to look for molecular evidence of epigenetic signatures in association with birth weight discordance. RESULTS: Our association analysis revealed no CpG site with genome-wide statistical significance (FDR < 0.05) for either qualitative (larger or smaller) or quantitative discordance in birth weight. Even with selected samples of extremely birth weight discordant twin pairs, no significant site was found except for 3 CpGs that displayed age-dependent intra-pair differential methylation with FDRs 0.014 (cg26856578, p = 3.42e-08), 0.0256 (cg15122603, p = 1.25e-07) and 0.0258 (cg16636641, p = 2.05e-07). Among the three sites, intra-pair differential methylation increased with age for cg26856578 but decreased with age for cg15122603 and cg16636641. There was no genome-wide statistical significance for sex-dependent effects on intra-pair differential methylation in either the whole samples or the extremely discordant twins. CONCLUSIONS: Genome-wide DNA methylation profiling did not reveal epigenetic signatures of birth weight discordance although some sites displayed age-dependent intra-pair differential methylation in the extremely discordant twin pairs.

Adults prenatally exposed to the Dutch Famine exhibit a metabolic signature associated with a broad spectrum of common diseases.

Background: Exposure to famine in the prenatal period is associated with an increased risk of metabolic disease, including obesity and type-2 diabetes. We employed nuclear magnetic resonance (NMR) metabolomic profiling to provide a deeper insight into the metabolic changes associated with survival of prenatal famine exposure during the Dutch Famine at the end of World War II and explore their link to disease. Methods: NMR metabolomics data were generated from serum in 480 individuals prenatally exposed to famine (mean 58.8 years, 0.5 SD) and 464 controls (mean 57.9 years, 5.4 SD). We tested associations of prenatal famine exposure with levels of 168 individual metabolic biomarkers and compared the metabolic biomarker signature of famine exposure with those of 154 common diseases. Results: Prenatal famine exposure was associated with higher concentrations of branched-chain amino acids ((iso)-leucine), aromatic amino acid (tyrosine), and glucose in later life (0.2-0.3 SD, p < 3x10-3). The metabolic biomarker signature of prenatal famine exposure was positively correlated to that of incident type-2 diabetes (r = 0.77, p = 3x10-27), also when re-estimating the signature of prenatal famine exposure among individuals without diabetes (r = 0.67, p = 1x10-18). Remarkably, this association extended to 115 common diseases for which signatures were available (0.3 ≤ r ≤ 0.9, p < 3.2x10-4). Correlations among metabolic signatures of famine exposure and disease outcomes were attenuated when the famine signature was adjusted for body mass index. Conclusions: Prenatal famine exposure is associated with a metabolic biomarker signature that strongly resembles signatures of a diverse set of diseases, an observation that can in part be attributed to a shared involvement of obesity.

Duration of breastfeeding and gender are associated with methylation of the LEPTIN gene in very young children.

BACKGROUND: Perinatal environmental factors have been associated with the metabolic programming of children and consequent disease risks in later life. Epigenetic modifications that lead to altered gene expression may be involved. Here, we study early life environmental and constitutional factors in association with the DNA methylation of leptin (LEP), a non-imprinted gene implicated in appetite regulation and fat metabolism. METHODS: We investigated maternal education, breastfeeding, and constitutional factors of the child at 17 mo of age. We measured the DNA methylation of LEP in whole blood and the concentration of leptin in serum. RESULTS: Duration of breastfeeding was negatively associated with LEP methylation. Low education (≤12 y of education) was associated with higher LEP methylation. Boys had higher birth weight and lower LEP methylation than girls. An inverse association was established between birth weight per SD increase (+584 g) and LEP methylation. High BMI and leptin concentration were associated with lower methylation of LEP. CONCLUSION: The early life environment and constitutional factors of the child are associated with epigenetic variations in LEP. Future studies must reveal whether breastfeeding and the associated decrease in LEP methylation is an epigenetic mechanism contributing to the protective effect of breastfeeding against obesity.

Accelerated biological aging six decades after prenatal famine exposure.

To test the hypothesis that early-life adversity accelerates the pace of biological aging, we analyzed data from the Dutch Hunger Winter Families Study (DHWFS, N=951). DHWFS is a natural-experiment birth-cohort study of survivors of in-utero exposure to famine conditions caused by the German occupation of the Western Netherlands in Winter 1944-5, matched controls, and their siblings. We conducted DNA methylation analysis of blood samples collected when the survivors were aged 58 to quantify biological aging using the DunedinPACE, GrimAge, and PhenoAge epigenetic clocks. Famine survivors had faster DunedinPACE, as compared with controls. This effect was strongest among women. Results were similar for GrimAge, although effect-sizes were smaller. We observed no differences in PhenoAge between survivors and controls. Famine effects were not accounted for by blood-cell composition and were similar for individuals exposed early and later in gestation. Findings suggest in-utero undernutrition may accelerate biological aging in later life.

Epigenome-Wide Meta-analysis Reveals Associations Between Dietary Glycemic Index and Glycemic Load and DNA Methylation in Children and Adolescents of Different Body Sizes.

OBJECTIVE: Dietary glycemic index (GI) and glycemic load (GL) are associated with cardiometabolic health in children and adolescents, with potential distinct effects in people with increased BMI. DNA methylation (DNAm) may mediate these effects. Thus, we conducted meta-analyses of epigenome-wide association studies (EWAS) between dietary GI and GL and blood DNAm of children and adolescents. RESEARCH DESIGN AND METHODS: We calculated dietary GI and GL and performed EWAS in children and adolescents (age range: 4.5-17 years) from six cohorts (N = 1,187). We performed stratified analyses of participants with normal weight (n = 801) or overweight or obesity (n = 386). We performed look-ups for the identified cytosine-phosphate-guanine (CpG) sites (false discovery rate [FDR] <0.05) with tissue-specific gene expression of 832 blood and 223 subcutaneous adipose tissue samples from children and adolescents. RESULTS: Dietary GL was positively associated with DNAm of cg20274553 (FDR <0.05), annotated to WDR27. Several CpGs were identified in the normal-weight (GI: 85; GL: 17) and overweight or obese (GI: 136; GL: 298; FDR <0.05) strata, and none overlapped between strata. In participants with overweight or obesity, identified CpGs were related to RNA expression of genes associated with impaired metabolism (e.g., FRAT1, CSF3). CONCLUSIONS: We identified 537 associations between dietary GI and GL and blood DNAm, mainly in children and adolescents with overweight or obesity. High-GI and/or -GL diets may influence epigenetic gene regulation and thereby promote metabolic derangements in young people with increased BMI.

Analysis of DNA methylation at birth and in childhood reveals changes associated with season of birth and latitude.

BACKGROUND: Seasonal variations in environmental exposures at birth or during gestation are associated with numerous adult traits and health outcomes later in life. Whether DNA methylation (DNAm) plays a role in the molecular mechanisms underlying the associations between birth season and lifelong phenotypes remains unclear. METHODS: We carried out epigenome-wide meta-analyses within the Pregnancy And Childhood Epigenetic Consortium to identify associations of DNAm with birth season, both at differentially methylated probes (DMPs) and regions (DMRs). Associations were examined at two time points: at birth (21 cohorts, N = 9358) and in children aged 1-11 years (12 cohorts, N = 3610). We conducted meta-analyses to assess the impact of latitude on birth season-specific associations at both time points. RESULTS: We identified associations between birth season and DNAm (False Discovery Rate-adjusted p values < 0.05) at two CpGs at birth (winter-born) and four in the childhood (summer-born) analyses when compared to children born in autumn. Furthermore, we identified twenty-six differentially methylated regions (DMR) at birth (winter-born: 8, spring-born: 15, summer-born: 3) and thirty-two in childhood (winter-born: 12, spring and summer: 10 each) meta-analyses with few overlapping DMRs between the birth seasons or the two time points. The DMRs were associated with genes of known functions in tumorigenesis, psychiatric/neurological disorders, inflammation, or immunity, amongst others. Latitude-stratified meta-analyses [higher (≥ 50°N), lower (< 50°N, northern hemisphere only)] revealed differences in associations between birth season and DNAm by birth latitude. DMR analysis implicated genes with previously reported links to schizophrenia (LAX1), skin disorders (PSORS1C, LTB4R), and airway inflammation including asthma (LTB4R), present only at birth in the higher latitudes (≥ 50°N). CONCLUSIONS: In this large epigenome-wide meta-analysis study, we provide evidence for (i) associations between DNAm and season of birth that are unique for the seasons of the year (temporal effect) and (ii) latitude-dependent variations in the seasonal associations (spatial effect). DNAm could play a role in the molecular mechanisms underlying the effect of birth season on adult health outcomes.

Environmentally induced DNA methylation is inherited across generations in an aquatic keystone species.

Transgenerational inheritance of environmentally induced epigenetic marks can have significant impacts on eco-evolutionary dynamics, but the phenomenon remains controversial in ecological model systems. We used whole-genome bisulfite sequencing of individual water fleas (Daphnia magna) to assess whether environmentally induced DNA methylation is transgenerationally inherited. Genetically identical females were exposed to one of three natural stressors, or a de-methylating drug, and their offspring were propagated clonally for four generations under control conditions. We identified between 70 and 225 differentially methylated CpG positions (DMPs) in F1 individuals whose mothers were exposed to a natural stressor. Roughly half of these environmentally induced DMPs persisted until generation F4. In contrast, treatment with the drug demonstrated that pervasive hypomethylation upon exposure is reset almost completely after one generation. These results suggest that environmentally induced DNA methylation is non-random and stably inherited across generations in Daphnia, making epigenetic inheritance a putative factor in the eco-evolutionary dynamics of freshwater communities.

Maternal Dietary Glycemic Index and Glycemic Load in Pregnancy and Offspring Cord Blood DNA Methylation.

OBJECTIVE: Suboptimal nutrition in pregnancy is associated with worse offspring cardiometabolic health. DNA methylation may be an underlying mechanism. We meta-analyzed epigenome-wide association studies (EWAS) of maternal dietary glycemic index and load with cord blood DNA methylation. RESEARCH DESIGN AND METHODS: We calculated maternal glycemic index and load from food frequency questionnaires and ran EWAS on cord blood DNA methylation in 2,003 mother-offspring pairs from three cohorts. Analyses were additionally stratified by maternal BMI categories. We looked-up the findings in EWAS of maternal glycemic traits and BMI as well as in EWAS of birth weight and child BMI. We examined associations with gene expression in child blood in the online Human Early Life Exposome eQTM catalog and in 223 adipose tissue samples. RESULTS: Maternal glycemic index and load were associated with cord blood DNA methylation at 41 cytosine-phosphate-guanine sites (CpGs, P < 1.17 × 10-7), mostly in mothers with overweight/obesity. We did not observe overlap with CpGs associated with maternal glycemic traits, BMI, or child birth weight or BMI. Only DNA methylation at cg24458009 and cg23347399 was associated with expression of PCED1B and PCDHG, respectively, in child blood, and DNA methylation at cg27193519 was associated with expression of TFAP4, ZNF500, PPL, and ANKS3 in child subcutaneous adipose tissue. CONCLUSIONS: We observed multiple associations of maternal glycemic index and load during pregnancy with cord blood DNA methylation, mostly in mothers with overweight/obesity; some of these CpGs were associated with gene expression. Additional studies are required to further explore functionality, uncover causality, and study pathways to offspring health.

Maternal Glycemic Dysregulation During Pregnancy and Neonatal Blood DNA Methylation: Meta-analyses of Epigenome-Wide Association Studies.

OBJECTIVE: Maternal glycemic dysregulation during pregnancy increases the risk of adverse health outcomes in her offspring, a risk thought to be linearly related to maternal hyperglycemia. It is hypothesized that changes in offspring DNA methylation (DNAm) underline these associations. RESEARCH DESIGN AND METHODS: To address this hypothesis, we conducted fixed-effects meta-analyses of epigenome-wide association study (EWAS) results from eight birth cohorts investigating relationships between cord blood DNAm and fetal exposure to maternal glucose (Nmaximum = 3,503), insulin (Nmaximum = 2,062), and area under the curve of glucose (AUCgluc) following oral glucose tolerance tests (Nmaximum = 1,505). We performed lookup analyses for identified cytosine-guanine dinucleotides (CpGs) in independent observational cohorts to examine associations between DNAm and cardiometabolic traits as well as tissue-specific gene expression. RESULTS: Greater maternal AUCgluc was associated with lower cord blood DNAm at neighboring CpGs cg26974062 (ß [SE] -0.013 [2.1 × 10-3], P value corrected for false discovery rate [PFDR] = 5.1 × 10-3) and cg02988288 (ß [SE]-0.013 [2.3 × 10-3], PFDR = 0.031) in TXNIP. These associations were attenuated in women with GDM. Lower blood DNAm at these two CpGs near TXNIP was associated with multiple metabolic traits later in life, including type 2 diabetes. TXNIP DNAm in liver biopsies was associated with hepatic expression of TXNIP. We observed little evidence of associations between either maternal glucose or insulin and cord blood DNAm. CONCLUSIONS: Maternal hyperglycemia, as reflected by AUCgluc, was associated with lower cord blood DNAm at TXNIP. Associations between DNAm at these CpGs and metabolic traits in subsequent lookup analyses suggest that these may be candidate loci to investigate in future causal and mediation analyses.

DNA methylation differences after exposure to prenatal famine are common and timing- and sex-specific.

Prenatal famine in humans has been associated with various later-life consequences, depending on the gestational timing of the insult and the sex of the exposed individual. Epigenetic mechanisms have been proposed to underlie these associations. Indeed, animal studies and our early human data on the imprinted IGF2 locus indicated a link between prenatal nutritional and DNA methylation. However, it remains unclear how common changes in DNA methylation are and whether they are sex- and timing-specific paralleling the later-life consequences of prenatal famine exposure. To this end, we investigated the methylation of 15 loci implicated in growth and metabolic disease in individuals who were prenatally exposed to a war-time famine in 1944-45. Methylation of INSIGF was lower among individuals who were periconceptionally exposed to the famine (n = 60) compared with their unexposed same-sex siblings (P = 2 x 10(-5)), whereas methylation of IL10, LEP, ABCA1, GNASAS and MEG3 was higher (all P < 10(-3)). A significant interaction with sex was observed for INSIGF, LEP and GNASAS. Next, methylation of eight representative loci was compared between 62 individuals exposed late in gestation and their unexposed siblings. Methylation was different for GNASAS (P = 1.1 x 10(-7)) and, in men, LEP (P = 0.017). Our data indicate that persistent changes in DNA methylation may be a common consequence of prenatal famine exposure and that these changes depend on the sex of the exposed individual and the gestational timing of the exposure.

Variation, patterns, and temporal stability of DNA methylation: considerations for epigenetic epidemiology.

The prospect of finding epigenetic risk factors for complex diseases would be greatly enhanced if DNA from existing biobanks, which is generally extracted from whole blood, could be used to perform epigenetic association studies. We characterized features of DNA methylation at 16 candidate loci, 8 of which were imprinted, in DNA samples from the Netherlands Twin Register biobank. Except for unmethylated or fully methylated sites, CpG methylation varied considerably in a sample of 30 unrelated individuals. This variation remained after accounting for the cellular heterogeneity of blood. Methylation of CpG sites was correlated within loci and, for 4 imprinted loci, across chromosomes. In 34 additional individuals, we investigated the DNA methylation of 8 representative loci in 2 longitudinal blood and 2 longitudinal buccal cell samples (follow-up 11-20 and 2-8 yr, respectively). Five of 8 loci were stable over time (rho>0.75) in both tissues, indicating that prospective epigenetic studies may be possible. For 4 loci, the DNA methylation in blood (mesoderm) correlated with that in the buccal cells (ectoderm) (rho>0.75). Our data suggest that epigenetic studies on complex diseases may be feasible for a proportion of genomic loci provided that they are carefully designed.

Persistent epigenetic differences associated with prenatal exposure to famine in humans.

Extensive epidemiologic studies have suggested that adult disease risk is associated with adverse environmental conditions early in development. Although the mechanisms behind these relationships are unclear, an involvement of epigenetic dysregulation has been hypothesized. Here we show that individuals who were prenatally exposed to famine during the Dutch Hunger Winter in 1944-45 had, 6 decades later, less DNA methylation of the imprinted IGF2 gene compared with their unexposed, same-sex siblings. The association was specific for periconceptional exposure, reinforcing that very early mammalian development is a crucial period for establishing and maintaining epigenetic marks. These data are the first to contribute empirical support for the hypothesis that early-life environmental conditions can cause epigenetic changes in humans that persist throughout life.

Selective Survival of Embryos Can Explain DNA Methylation Signatures of Adverse Prenatal Environments.

An adverse intrauterine environment is associated with long-term physiological changes in offspring. These are believed to be mediated by epigenomic marks, including DNA methylation (DNAm). Changes in DNAm are often interpreted as damage or plastic responses of the embryo. Here, we propose that stochastic DNAm variation, generated during remodeling of the epigenome after fertilization, contributes to DNAm signatures of prenatal adversity through differential survival of embryos. Using a mathematical model of re-methylation in the early embryo, we demonstrate that selection, but not plasticity, will generate a characteristic reduction in DNAm variance at loci that contribute to survival. Such a reduction in DNAm variance was apparent in a human cohort prenatally exposed to the Dutch famine, illustrating that it is possible to detect a signature of selection on epigenomic variation. Selection should be considered as a possible mechanism linking prenatal adversity to subsequent health and may have implications when evaluating interventions.