An epigenome-wide association meta-analysis of prenatal maternal stress in neonates: A model approach for replication

Prenatal maternal stress exposure has been associated with neonatal differential DNA methylation. However, the available evidence in humans is largely based on candidate gene methylation studies, where only a few CpG sites were evaluated. The aim of this study was to examine the association between prenatal exposure to maternal stress and offspring genome-wide cord blood methylation using different methods. First, we conducted a meta-analysis and follow-up pathway analyses. Second, we used novel region discovery methods [i.e., differentially methylated regions (DMRs) analyses]. To this end, we used data from two independent population-based studies, the Generation R Study (n = 912) and the Avon Longitudinal Study of Parents and Children (ALSPAC, n = 828), to (i) measure genome-wide DNA methylation in cord blood and (ii) extract a prenatal maternal stress composite. The meta-analysis (n_total = 1,740) revealed no epigenome-wide (meta P <1.00e-07) associations of prenatal maternal stress exposure with neonatal differential DNA methylation. Follow-up analyses of the top hits derived from our epigenome-wide meta-analysis (meta P <1.00e-04) indicated an over-representation of the methyltransferase activity pathway. We identified no Bonferroni-corrected (P <1.00e-06) DMRs associated with prenatal maternal stress exposure. Combining data from two independent population-based samples in an epigenome-wide meta-analysis, the current study indicates that there are no large effects of prenatal maternal stress exposure on neonatal DNA methylation. Such replication efforts are essential in the search for robust associations, whether derived from candidate gene methylation or epigenome-wide studies.     

Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood

DNA methylation at cytosine-phosphate-guanine (CpG) dinucleotides changes as a function of age in humans and animal models, a process that may contribute to chronic disease development. Recent studies have investigated the role of an oxidized form of DNA methylation – 5-hydroxymethylcytosine (5hmC) – in the epigenome, but its contribution to age-related DNA methylation remains unclear. We tested the hypothesis that 5hmC changes with age, but in a direction opposite to 5-methylcytosine (5mC), potentially playing a distinct role in aging. To characterize epigenetic aging, genome-wide 5mC and 5hmC were measured in longitudinal blood samples (2, 4, and 10 months of age) from isogenic mice using two sequencing methods – enhanced reduced representation bisulfite sequencing and hydroxymethylated DNA immunoprecipitation sequencing. Examining the epigenome by age, we identified 28,196 unique differentially methylated CpGs (DMCs) and 8,613 differentially hydroxymethylated regions (DHMRs). Mouse blood showed a general pattern of epigenome-wide hypermethylation and hypo-hydroxymethylation with age. Comparing age-related DMCs and DHMRs, 1,854 annotated genes showed both differential 5mC and 5hmC, including one gene – Nfic – at five CpGs in the same 250 bp chromosomal region. At this region, 5mC and 5hmC levels both decreased with age. Reflecting these age-related epigenetic changes, Nfic RNA expression in blood decreased with age, suggesting that age-related regulation of this gene may be driven by 5hmC, not canonical DNA methylation. Combined, our genome-wide results show age-related differential 5mC and 5hmC, as well as some evidence that changes in 5hmC may drive age-related DNA methylation and gene expression.     

Imputation of missing covariate values in epigenome-wide analysis of DNA methylation data

DNA methylation is a widely studied epigenetic mechanism and alterations in methylation patterns may be involved in the development of common diseases. Unlike inherited changes in genetic sequence, variation in site-specific methylation varies by tissue, developmental stage, and disease status, and may be impacted by aging and exposure to environmental factors, such as diet or smoking. These non-genetic factors are typically included in epigenome-wide association studies (EWAS) because they may be confounding factors to the association between methylation and disease. However, missing values in these variables can lead to reduced sample size and decrease the statistical power of EWAS. We propose a site selection and multiple imputation (MI) method to impute missing covariate values and to perform association tests in EWAS. Then, we compare this method to an alternative projection-based method. Through simulations, we show that the MI-based method is slightly conservative, but provides consistent estimates for effect size. We also illustrate these methods with data from the Atherosclerosis Risk in Communities (ARIC) study to carry out an EWAS between methylation levels and smoking status, in which missing cell type compositions and white blood cell counts are imputed.     

Genome-wide DNA methylation associations with spontaneous preterm birth in US blacks: findings in maternal and cord blood samples

Preterm birth (PTB) affects one in six Black babies in the United States. Epigenetics is believed to play a role in PTB; however, only a limited number of epigenetic studies of PTB have been reported, most of which have focused on cord blood DNA methylation (DNAm) and/or were conducted in white populations. Here we conducted, by far, the largest epigenome-wide DNAm analysis in 300 Black women who delivered early spontaneous preterm (sPTB, n = 150) or full-term babies (n = 150) and replicated the findings in an independent set of Black mother-newborn pairs from the Boston Birth Cohort. DNAm in maternal blood and/or cord blood was measured using the Illumina HumanMethylation450 BeadChip. We identified 45 DNAm loci in maternal blood associated with early sPTB, with a false discovery rate (FDR) <5%. Replication analyses confirmed sPTB associations for cg03915055 and cg06804705, located in the promoter regions of the CYTIP and LINC00114 genes, respectively. Both loci had comparable associations with early sPTB and early medically-indicated PTB, but attenuated associations with late sPTB. These associations could not be explained by cell composition, gestational complications, and/or nearby maternal genetic variants. Analyses in the newborns of the 110 Black women showed that cord blood methylation levels at both loci had no associations with PTB. The findings from this study underscore the role of maternal DNAm in PTB risk, and provide a set of maternal loci that may serve as biomarkers for PTB. Longitudinal studies are needed to clarify temporal relationships between maternal DNAm and PTB risk.     

DNA methylation in imprinted genes IGF2 and GNASXL is associated with prenatal maternal stress

Epigenetic regulation of imprinted genes during embryonic development is influenced by the prenatal environment. Our aim was to examine the effect of maternal emotional stress and cortisol levels during pregnancy on methylation of imprinted genes, insulin‐like growth factor 2 (IGF2) and guanine nucleotide‐binding protein, alpha stimulating extra‐large (GNASXL), using umbilical cord blood DNA. Maternal depressed mood (Edinburgh Depression Scale; EDS), pregnancy‐related anxiety questionnaire (PRAQ) and cortisol day profiles were assessed throughout pregnancy. At birth, a cord blood sample (n = 80) was taken to study DNA methylation of IGF2 DMR0 (differentially methylated region), IGF2 anti‐sense (IGF2AS) and GNASXL using Sequenom Epi TYPER. Linear mixed models were used to examine the relationship between DNA methylation and maternal stress, while correcting for confounders. We also studied the association of DNA methylation with the child ponderal index at birth. We found a cytosine–guanine dinucleotide (CpG)‐specific association of PRAQ subscales with IGF2 DMR0 (CpG5, P < 0.0001) and GNASXL (CpG11, P = 0.0003), while IGF2AS was associated with maternal EDS scores (CpG33, P = 0.0003) and cortisol levels (CpG33, P = 0.0006; CpG37‐38, P = 0.0005). However, there was no association of methylation with ponderal index at birth. In conclusion, maternal stress during pregnancy, as defined by cortisol measurements, EDS and PRAQ scores, is associated with DNA methylation of imprinted genes IGF2 and GNASXL. Our results provide further evidence that prenatal adversity can influence imprinted gene methylation, although future studies are needed to unravel the exact mechanisms.     

Differential DNA methylation and PM2.5 species in a 450K epigenome-wide association study

Although there is growing evidence that exposure to ambient particulate matter is associated with global DNA methylation and gene-specific methylation, little is known regarding epigenome-wide changes in DNA methylation in relation to particles and, especially, particle components. Using the Illumina Infinium HumanMethylation450 BeadChip, we examined the relationship between one-year moving averages of PM_2.5 species (Al, Ca, Cu, Fe, K, Na, Ni, S, Si, V, and Zn) and DNA methylation at 484,613 CpG probes in a longitudinal cohort that included 646 subjects. Bonferroni correction was applied to adjust for multiple comparisons. Bioinformatics analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was also performed. We observed 20 Bonferroni significant (P-value < 9.4× 10^?9) CpGs for Fe, 8 for Ni, and 1 for V. Particularly, methylation at Schlafen Family Member 11 (SLFN11) cg10911913 was positively associated with measured levels of all 3 species. The SLFN11 gene codes for an interferon-induced protein that inhibits retroviruses and sensitizes cancer cells to DNA-damaging agents. Bioinformatics analysis suggests that gene targets may be relevant to pathways including cancers, signal transduction, and cell growth and death. Ours is the first study to examine the epigenome-wide association between ambient particles species and DNA methylation. We found that long-term exposures to specific components of ambient particle pollution, especially particles emitted during oil combustion, were associated with methylation changes in genes relevant to immune responses. Our findings provide insight into potential biologic mechanisms on an epigenetic level.     

DNA methylation and obesity traits: An epigenome-wide association study. The REGICOR study

Obesity is associated with increased risk of several diseases and has become epidemic. Obesity is highly heritable but the genetic variants identified by genome-wide association studies explain only limited variability. Epigenetics could contribute to explain the missing variability. The study aim was to discover differential methylation patterns related to obesity. We designed an epigenome-wide association study with a discovery phase in a subsample of 641 REGICOR study participants, validated by analysis of 2,515 participants in the Framingham Offspring Study. Blood DNA methylation was assessed using Illumina HumanMethylation450 BeadChip. Next, we meta-analyzed the data using the fixed effects method and performed a functional and pathway analysis using the Ingenuity Pathway Analysis software. We were able to validate 94 CpGs associated with body mass index (BMI) and 49 CpGs associated with waist circumference, located in 95 loci. In addition, we newly discovered 70 CpGs associated with BMI and 33 CpGs related to waist circumference. These CpGs explained 25.94% and 29.22% of the variability of BMI and waist circumference, respectively, in the REGICOR sample. We also evaluated 65 of the 95 validated loci in the GIANT genome-wide association data; 10 of them had Tag SNPs associated with BMI. The top-ranked diseases and functions identified in the functional and pathway analysis were neurologic, psychological, endocrine, and metabolic.     

Impact of maternal dietary counseling in the first year of life on DNA methylation in a cohort of children

Epigenetic modifications established during prenatal and early life, including DNA methylation, have been suggested as potential mediators of the interaction between environmental exposures during the perinatal period and adult metabolic health adverse outcomes, especially cardiometabolic complications and overweight. The effect of a dietary intervention in the first year of life on global methylation levels in leukocyte samples from a cohort of children born between 2001 and 2002 in southern Brazil was examined. Overall methylation measurements were performed using enzyme-linked immunosorbent assays on DNA samples from 237 children at 4 years old. Mean methylation values were higher in the intervention group (mean: 2.20 ± 1.31%) than in the control group (mean: 1.65 ± 1.11%; P = 0.001). It was observed that nutritional counseling in the first year increased breastfeeding duration and stimulated the development of healthier eating habits. Therefore, these factors might have contributed to increase global DNA methylation. The findings of the present study reinforce the notion that performing nutritional interventions in the early stages of life is important and provide further evidence of the interaction between the environment and epigenetic traits.     

Maternal caregiving and DNA methylation in human infants and children: Systematic review

The integration of behavioral epigenetics' principles (eg, DNA methylation) into the study of human infants' development has mainly focused on the effects of early adverse exposures, paying less attention to protective caregiving experiences. The present review focused on DNA methylation linked to variations in maternal behavior in human infants and children. Literature search occurred on three databases (PubMed, Scopus and Web of Science) and 11 records were selected. Key variables were abstracted from each article including: sample size and characteristics, time and type of maternal caregiving behavior exposure, time and locus of methylation biomarker, presence/absence, time and type of adverse exposure. Six out of eleven records documented the predictive effect of maternal caregiving on DNA methylation, whereas the remaining five reported on the role of maternal behavior as an influencing factor of the adversity‐to‐methylation link. Consistent with evidence from the animal model, the quality of maternal caregiving in humans (a) might be associated with variations in DNA methylation status of specific genes involved in socio‐emotional development and (b) might partially buffer the association between early adversities and epigenetic variations in infants and children. Current evidence suggests that the quality of maternal caregiving can contribute to behavioral development trajectories of human infants and children at least partially through epigenetic regulation. Open questions and methodological aspects are discussed to guide future human developmental research in behavioral epigenetics.     

Type 2 diabetes mellitus in relation to global LINE-1 DNA methylation in peripheral blood: A cohort study

In the last years, epigenetic processes have emerged as a promising area of complex diseases research. DNA methylation measured in Long Interspersed Nucleotide Element 1 (LINE-1) sequences has been considered a surrogate marker for global genome methylation. New findings have suggested the potential involvement of epigenetic mechanisms in Type 2 diabetes (T2DM) as a crucial interface between the effects of genetic predisposition and environmental influences. Our study evaluated whether global DNA methylation predicted increased risk from T2DM or other carbohydrate metabolism disorders in a cohort study. We used a prospective cohort intervention study and a control group. We collected phenotypic, anthropometric, biochemical, and nutritional information from all subjects. Global LINE-1 DNA methylation was quantified by pyrosequencing technology. Subjects that did not improve their carbohydrate metabolism status showed lower levels of global LINE-1 DNA methylation (63.9 ± 1.7 vs. 64.7 ± 2.4) and they practiced less intense physical activity (5.8% vs. 21.5%). Logistic regression analyses showed a significant association between LINE-1 DNA methylation and metabolic status after adjustment for sex, age, BMI, and physical activity. Our study showed that lower LINE-1 DNA methylation levels were associated with a higher risk metabolic status worsening, independent of other classic risk factors. This finding highlights the potential role for epigenetic biomarkers as predictors of T2DM risk or other related metabolic disorders.     

Type 2 diabetes mellitus in relation to global LINE-1 DNA methylation in peripheral blood: A cohort study

In the last years, epigenetic processes have emerged as a promising area of complex diseases research. DNA methylation measured in Long Interspersed Nucleotide Element 1 (LINE-1) sequences has been considered a surrogate marker for global genome methylation. New findings have suggested the potential involvement of epigenetic mechanisms in Type 2 diabetes (T2DM) as a crucial interface between the effects of genetic predisposition and environmental influences. Our study evaluated whether global DNA methylation predicted increased risk from T2DM or other carbohydrate metabolism disorders in a cohort study. We used a prospective cohort intervention study and a control group. We collected phenotypic, anthropometric, biochemical, and nutritional information from all subjects. Global LINE-1 DNA methylation was quantified by pyrosequencing technology. Subjects that did not improve their carbohydrate metabolism status showed lower levels of global LINE-1 DNA methylation (63.9 ± 1.7 vs. 64.7 ± 2.4) and they practiced less intense physical activity (5.8% vs. 21.5%). Logistic regression analyses showed a significant association between LINE-1 DNA methylation and metabolic status after adjustment for sex, age, BMI, and physical activity. Our study showed that lower LINE-1 DNA methylation levels were associated with a higher risk metabolic status worsening, independent of other classic risk factors. This finding highlights the potential role for epigenetic biomarkers as predictors of T2DM risk or other related metabolic disorders.     

Locus-specific DNA methylation in the placenta is associated with levels of pro-inflammatory proteins in cord blood and they are both independently affected by maternal smoking during pregnancy

We investigated the impact of maternal smoking during pregnancy on placental DNA methylation and how this may mediate the association between maternal smoking and pro-inflammatory proteins in cord blood. The study population consisted of 27 individuals exposed to maternal smoking throughout pregnancy, 32 individuals exposed during a proportion of the pregnancy, and 61 unexposed individuals. Methylation of 11 regions within 6 genes in placenta tissue was assessed by pyrosequencing. Levels of 7 pro-inflammatory proteins in cord blood were assessed by electrochemiluminescence. Differential methylation was observed in the CYP1A1 promoter and AHRR gene body regions between women who smoked throughout pregnancy and non-smokers on the fetal-side of the placenta and in the GFI1 promoter between women who quit smoking while pregnant and non-smokers on the maternal-side of the placenta. Maternal smoking resulted in elevated levels of IL-8 protein in cord blood, which was not mediated by DNA methylation of our candidate regions at either the maternal or the fetal side of the placenta. Placental DNA methylation was associated with levels of inflammatory proteins in cord blood. Our observations suggest that maternal smoking during pregnancy affects both placental DNA methylation and the neonate's immune response.     

Impaired one carbon metabolism and DNA methylation in alcohol toxicity

Excessive alcohol consumption is a prominent problem and one of the major causes of mortality and morbidity around the world. Long‐term, heavy alcohol consumption is associated with a number of deleterious health consequences, such as cancer, heart and liver disease, a variety of neurological, cognitive, and behavioral deficits. Alcohol consumption is also associated with developmental defects. The causes of alcohol‐induced toxicity are presently unclear. One of the mechanisms underlying alcohol toxicity has to do with its interaction with folic acid/homocysteine or one‐carbon metabolism (OCM). OCM is a major donor of methyl groups for methylation, particularly DNA methylation critical for epigenetic regulation of gene expression, and its disturbance may compromise DNA methylation, thereby affecting gene expression. OCM disturbance mediated by nutrient deficits is a well‐known risk factor for various disorders and developmental defects (e.g., neural tube defects). In this review, we summarize the role of OCM disturbance and associated epigenetic aberrations in chronic alcohol‐induced toxicity.

     

Placental mitochondrial methylation and exposure to airborne particulate matter in the early life environment: An ENVIRONAGE birth cohort study

Most research to date has focused on epigenetic modifications in the nuclear genome, with little attention devoted to mitochondrial DNA (mtDNA). Placental mtDNA content has been shown to respond to environmental exposures that induce oxidative stress, including airborne particulate matter (PM). Damaged or non-functioning mitochondria are specifically degraded through mitophagy, exemplified by lower mtDNA content, and could be primed by epigenetic modifications in the mtDNA. We studied placental mtDNA methylation in the context of the early life exposome. We investigated placental tissue from 381 mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. We determined mtDNA methylation by bisulfite-pyrosequencing in 2 regions, i.e., the D-loop control region and 12S rRNA (MT-RNR1), and measured mtDNA content by qPCR. PM_2.5 exposure was calculated for each participant''s home address using a dispersion model. An interquartile range (IQR) increment in PM_2.5 exposure over the entire pregnancy was positively associated with mtDNA methylation (MT-RNR1: +0.91%, P = 0.01 and D-loop: +0.21%, P = 0.05) and inversely associated with mtDNA content (relative change of −15.60%, P = 0.001) in placental tissue. mtDNA methylation was estimated to mediate 54% [P = 0.01 (MT-RNR1)] and 27% [P = 0.06 (D-loop)] of the inverse association between PM_2.5 exposure and mtDNA content. This study provides new insight into the mechanisms of altered mitochondrial function in the early life environment. Epigenetic modifications in the mitochondrial genome, especially in the MT-RNR1 region, substantially mediate the association between PM_2.5 exposure during gestation and placental mtDNA content, which could reflect signs of mitophagy and mitochondrial death.     

Differentiation of trophoblast lineage is associated with DNA methylation and demethylation.

Our previous study has shown that the placenta and kidney had different genomic methylation patterns regarding CpG island loci detected by restriction landmark genomic scanning (RLGS). To investigate whether differentiation involves changes in DNA methylation, we analyzed the rat Rcho-1 cell line, which retains trophoblast cell features and differentiates from stem cells into trophoblast giant cells in vitro. By RLGS, a total of 1,232 spots were identified in the Rcho-1 stem and differentiated giant cells. Four spots (0.3%) were detected only in giant cells, implying that the loci were originally methylated, but became demethylated during differentiation. Another four spots (0.3%) were detected only in stem cells, implying that these loci, originally unmethylated, became methylated during differentiation. DNAs from three loci that became methylated during differentiation were cloned and sequenced. All showed high homologies with expressed sequence tags (ESTs) or with genomic DNA of other species, suggesting that these loci are biologically important. Thus, the eight differentially methylated loci should be good tools to study epigenetic modification specific to differentiation of trophoblast giant cells.     

Concordance in hippocampal and fecal Nr3c1 methylation is moderated by maternal behavior in the mouse

Recent advances in genomic technologies now enable a reunion of molecular and evolutionary biology. Researchers investigating naturally living animal populations are thus increasingly able to capitalize upon genomic technologies to connect molecular findings with multiple levels of biological organization. Using this vertical approach in the laboratory, epigenetic gene regulation has emerged as an important mechanism integrating genotype and phenotype. To connect phenotype to population fitness, however, this same vertical approach must now be applied to naturally living populations. A major obstacle to studying epigenetics in noninvasive samples is tissue specificity of epigenetic marks. Here, using the mouse as a proof‐of‐principle model, we present the first known attempt to validate an epigenetic assay for use in noninvasive samples. Specifically, we compare DNA methylation of the NGFI‐A (nerve growth factor‐inducible protein A) binding site in the promoter of the glucocorticoid receptor (Nr3c1) gene between central (hippocampal) and peripheral noninvasive (fecal) tissues in juvenile Balb/c mice that had received varying levels of postnatal maternal care. Our results indicate that while hippocampal DNA methylation profiles correspond to maternal behavior, fecal DNA methylation levels do not. Moreover, concordance in methylation levels between these tissues within individuals only emerges after accounting for the effects of postnatal maternal care. Thus, although these findings may be specific to the Nr3c1 gene, we urge caution when interpreting DNA methylation patterns from noninvasive tissues, and offer suggestions for further research in this field.     

Effect of maternal serum on viability and function of early human placental explants

Summary Fetal bovine serum (FBS) is frequently used to supplement chemically defined media such as Ham’s F10 when studying placental explant cultures. However in vitro production of hormones is usually declining by the 2nd or 3rd day and is short-lived (7 to 10 days). In this study we explored the use of human maternal serum (HMS) from early gestation as the medium supplement to Ham’s F10. Early placental hormone production was compared using two concentrations of FBS and HMS. On Day 3 of incubation, progesterone production in 10% HMS was 12-fold increased over that in 10% FBS, estradiol production was increased 10-fold, and βhCG production more than 3-fold. When the serum concentrations were increased to 40%, the results in all cases were similar to those at 10%. Preliminary characterization studies revealed that the stimulatory activity of HMS is heat-labile, neither extractable into organic solvent (diethyl ether) nor dialyzable, suggesting that it is protein in nature. In a long-term incubation, compared with FBS (7 days), HMS permitted survival of culture up to 30 days, judged both histologically and biochemically. We conclude that HMS provides substance(s), probably protein in nature, not present in FBS or non-pregnant human serum, which are important for human placental viability and function in vivo.     

Improved reporting of DNA methylation data derived from studies of the human placenta

Epigenetic variation is increasingly hypothesized as a mechanism underlying the effect of the in utero environment on long-term postnatal health; however, there is currently little clear data to support this in humans. A number of biological and technical factors provide challenges for the design of clinical epigenetic studies: from the type of cells or tissues that are available to the large range of predicted confounders that may impact findings. The human placenta, in addition to other neonatal tissues and whole blood, is commonly sampled for the study of epigenetic modifications. However there is little conformity for the most appropriate methods for study design, data analysis, and importantly, data interpretation. Here we present general recommendations for the reporting of DNA methylation in biological samples, with specific focus on the placenta. We outline key guidelines for: (1) placental sampling, (2) data analysis and presentation, and (3) interpretation of DNA methylation data. We emphasize the need to consider methodological noise, increase statistical power and to ensure appropriate adjustment for biological covariates. Finally, we highlight that epigenetic changes may be non-pathological and not necessarily translate into disease-associated changes. Improved reporting of DNA methylation data will be critical to identify epigenetic-based effects and to better understand the full phenotypic impact of these widely-reported epigenomic changes.