A systematic review of methylome-wide associations with anxiety disorders.

Anxiety disorders are a prevalent public health burden that significantly impair daily functioning and decrease quality of life. A growing body of research suggests DNA methylation (DNAm), an epigenetic modification that can impact gene expression, may be altered in anxiety disorders. The current review used a systematic approach to identify and synthesize the literature regarding epigenome-wide association studies (EWASs) of anxiety disorders in humans. We screened 804 articles returned by a search in PubMed in May 2025 and identified 12 studies for inclusion. All included studies examined DNAm in blood. In total, 2023 DNAm sites corresponding to 985 genes were significantly associated with anxiety disorders. Three DNAm sites significantly replicated across studies and four nominally replicated, meeting methylome-wide significance in one study and nominal significance (p < 0.05) in at least one other. This low replicability is likely a result of phenotypic heterogeneity, small sample sizes, the use of different multiple testing correction methods, and inconsistent adjustment for relevant comorbidities. Findings suggest DNAm associated with anxiety disorders may promote dysregulation of immune and inflammatory processes, some possibly sex-dependent. Collectively, the findings from studies included in this review provide preliminary evidence of DNAm alterations related to anxiety in whole blood and multiple blood cell-types. Future EWASs of anxiety disorders could benefit from using more ethnically diverse participants and longitudinal study designs.

Early patterning of DNA methylation (DNAm) may play an important role in later disease development. To better understand intergenerational epigenetic inheritance, we investigated the correlation between DNAm in blood in mother-newborn and in father-newborn pairs in the Isle of Wight (IoW) birth cohort. For parent-newborn pairs (n = 48), offspring DNAm was measured in cord blood and the parent’s DNAm in whole blood. Mothers’ DNAm was analyzed at birth (Guthrie card), age 18, early and late pregnancy respectively, and fathers’ DNAm was measured during the mother’s pregnancy. Linear regressions were applied to assess the intergenerational correlation of parental DNAm with that of offspring. Among various pairs of mother-newborn and father-newborn DNAm, the pairs where the mothers’ DNAm was measured at age 18 years exhibited the highest number of CpGs with significant intergenerational correlation in DNAm, with 1829 CpGs (0.54%) of the 338,526 CpGs studied (FDR < 0.05). Amongst these 1829 CpGs, 986 (54%) are known quantitative trait loci (QTL) for CpG methylation (methQTL). When the mother’s DNAm was assessed at early pregnancy, the number of CpGs showing intergenerational correlation was the smallest (384 CpGs, 0.11%). The second smallest number of such CpGs (559 CpGs, 0.17%) was found when investigating DNAm in offspring cord blood and father pairs. The low proportions of intergenerationally correlated CpGs suggest that epigenetic inheritance is limited.

Traffic-related air pollution (TRAP) exposure is associated with negative health outcomes. Changes in DNA methylation level may be an important mechanism through which air pollution can induce its effects. The objective of this study was to evaluate the association between DNA methylation in blood and personally measured TRAP exposure. Global DNA methylation in whole blood was analyzed with HPLC in a population of 55 healthy adults (average age 41 years). TRAP was assessed for each participant with a portable aethalometer measuring black carbon (BC). Exposure measurements were collected during one typical working week. These data were used in combination with ambient levels measured at a reference site to derive subchronic BC exposure. Urinary trans,trans-muconic acid (t,t-MA), a metabolite of benzene, was used as an internal proxy of traffic exposure. DNA methylation levels were associated with short- and subchronic BC exposure. An IQR increase in BC exposure on lag 24 h (477 ng/m3), lag 48 h (491 ng/m3), lag 1 week (314 ng/m3) and subchronic exposure (618 ng/m3) were associated with a decrease in DNA methylation levels of respectively 0.0020% (− 0.0040 to − 0.0001, p = 0.047), 0.0028% (− 0.0054 to − 0.0001, p = 0.043), 0.0024% (− 0.0043 to − 0.0005, p = 0.019), and 0.025% (− 0.048 to − 0.0015, p = 0.044). In addition, an IQR increase in t,t-MA (0.135 mg/l) was associated with a 0.0021% (− 0.0033 to − 0.0008, p = 0.0019) decrease in global DNA methylation levels. Analysis of a panel of cytokines in blood samples failed to demonstrate an association between inflammatory and oxidative stress biomarkers and TRAP or DNA methylation. In a panel of healthy adults, we found negative associations between total DNA methylation and markers of TRAP exposure. Considering that change in DNA methylation concentration is a biological marker connecting environmental and lifestyle exposures and disease development trajectories, our results warrant further study.

Epigenetics in health and disease: heralding the EWAS era

Influence of ambient air pollution on global DNA methylation in healthy adults: A seasonal follow-up

ABSTRACTAnniston, Alabama was home to a major polychlorinated biphenyl (PCB) production facility from 1929 until 1971. The Anniston Community Health Survey I and II (ACHS-I 2005–2007, ACHS-II 2013–2014) were conducted to explore the effects of PCB exposures. In this report we examined associations between PCB exposure and DNA methylation in whole blood using EPIC arrays (ACHS-I, n = 518; ACHS-II, n = 299). For both cohorts, 35 PCBs were measured in serum. We modelled methylation versus PCB wet-weight concentrations for: the sum of 35 PCBs, mono-ortho substituted PCBs, di-ortho substituted PCBs, tri/tetra-ortho substituted PCBs, oestrogenic PCBs, and antiestrogenic PCBs. Using robust multivariable linear regression, we adjusted for age, race, sex, smoking, total lipids, and six blood cell-type percentages. We carried out a two-stage analysis; discovery in ACHS-I followed by replication in ACHS-II. In ACHS-I, we identified 28 associations (17 unique CpGs) at p ≤ 6.70E-08 and 369 associations (286 unique CpGs) at FDR p ≤ 5.00E-02. A large proportion of the genes have been observed to interact with PCBs or dioxins in model studies. Among the 28 genome-wide significant CpG/PCB associations, 14 displayed replicated directional effects in ACHS-II; however, only one in ACHS-II was statistically significant at p ≤ 1.70E-04. While we identified many novel CpGs significantly associated with PCB exposures in ACHS-I, the differential methylation was modest and the effect was attenuated seven years later in ACHS-II, suggesting a lack of persistence of the associations between PCB exposures and altered DNA methylation in blood cells.

BackgroundBetween 1962 and 1971, the US Air Force sprayed Agent Orange across Vietnam, exposing many soldiers to this dioxin-containing herbicide. Several negative health outcomes have been linked to Agent Orange exposure, but data is lacking on the effects this chemical has on the genome. Therefore, we sought to characterize the impact of Agent Orange exposure on DNA methylation in the whole blood and adipose tissue of veterans enrolled in the Air Force Health Study (AFHS).MethodsWe received adipose tissue (n = 37) and whole blood (n = 42) from veterans in the AFHS. Study participants were grouped as having low, moderate, or high TCDD body burden based on their previously measured serum levels of dioxin. DNA methylation was assessed using the Illumina 450 K platform.ResultsEpigenome-wide analysis indicated that there were no FDR-significantly methylated CpGs in either tissue with TCDD burden. However, 3 CpGs in the adipose tissue (contained within SLC9A3, LYNX1, and TNRC18) were marginally significantly (q < 0.1) hypomethylated, and 1 CpG in whole blood (contained within PTPRN2) was marginally significantly (q < 0.1) hypermethylated with high TCDD burden. Analysis for differentially methylated DNA regions yielded SLC9A3, among other regions in adipose tissue, to be significantly differentially methylated with higher TCDD burden. Comparing whole blood data to a study of dioxin exposed adults from Alabama identified a CpG within the gene SMO that was hypomethylated with dioxin exposure in both studies.ConclusionWe found limited evidence of dioxin associated DNA methylation in adipose tissue and whole blood in this pilot study of Vietnam War veterans. Nevertheless, loci in the genes of SLC9A3 in adipose tissue, and PTPRN2 and SMO in whole blood, should be included in future exposure analyses.

The maternal epigenome may be responsive to prenatal metals exposures. We tested whether metals are associated with concurrent differential maternal whole blood DNA methylation. In the Early Autism Risk Longitudinal Investigation cohort, we measured first or second trimester maternal blood metals concentrations (cadmium, lead, mercury, manganese, and selenium) using inductively coupled plasma mass spectrometry. DNA methylation in maternal whole blood was measured on the Illumina 450 K array. A subset sample of 97 women had both measures available for analysis, all of whom did not report smoking during pregnancy. Linear regression was used to test for site-specific associations between individual metals and DNA methylation, adjusting for cell type composition and confounding variables. Discovery gene ontology analysis was conducted on the top 1,000 sites associated with each metal. We observed hypermethylation at 11 DNA methylation sites associated with lead (FDR False Discovery Rate q-value <0.1), near the genes CYP24A1, ASCL2, FAT1, SNX31, NKX6-2, LRC4C, BMP7, HOXC11, PCDH7, ZSCAN18, and VIPR2. Lead-associated sites were enriched (FDR q-value <0.1) for the pathways cell adhesion, nervous system development, and calcium ion binding. Manganese was associated with hypermethylation at four DNA methylation sites (FDR q-value <0.1), one of which was near the gene ARID2. Manganese-associated sites were enriched for cellular metabolism pathways (FDR q-value<0.1). Effect estimates for DNA methylation sites associated (p < 0.05) with cadmium, lead, and manganese were highly correlated (Pearson ρ > 0.86). DNA methylation sites associated with lead and manganese may be potential biomarkers of exposure or implicate downstream gene pathways.

Calorie restriction (CR) increases healthy life span and is accompanied by slowing or reversal of aging-associated DNA methylation (DNAm) changes in animal models. In the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIETM) human trial, we evaluated associations of CR and changes in whole-blood DNAm. CALERIETM randomized 220 healthy, nonobese adults in a 2:1 allocation to 2 years of CR or ad libitum (AL) diet. The average CR in the treatment group through 24 months of follow-up was 12%. Whole blood (baseline, 12, and 24 months) DNAm profiles were measured. Epigenome-wide association study (EWAS) analysis tested CR-induced changes from baseline to 12 and 24 months in the n = 197 participants with available DNAm data. CR treatment was not associated with epigenome-wide significant (false discovery rate [FDR] < 0.05) DNAm changes at the individual-CpG-site level. Secondary analysis of sets of CpG sites identified in published EWAS revealed that CR induced DNAm changes opposite to those associated with higher body mass index and cigarette smoking (p < .003 at 12- and 24-month follow-ups). In contrast, CR altered DNAm at chronological-age-associated CpG sites in the direction of older age (p < .003 at 12- and 24-month follow-ups). Although individual CpG site DNAm changes in response to CR were not identified, analyses of sets CpGs identified in prior EWAS revealed CR-induced changes to blood DNAm. Altered CpG sets were enriched for insulin production, glucose tolerance, inflammation, and DNA-binding and DNA-regulation pathways, several of which are known to be modified by CR. DNAm changes may contribute to CR effects on aging.

Folate is a key source of one‐carbon groups for DNA methylation; but studies on DNA methylation response to folic acid (FA) supplementation show inconsistent results. We hypothesize that measuring DNA methylation in whole blood (WB), which contains a mixture of distinct leukocyte cell types, may have confounded the results of previous studies. Therefore, the aim of this pilot study was to determine if a single cell type could serve as a more reliable epigenetic reporter. The study was performed in normal weight (BMI 18.5 – 24.9 kg/m2) women (18 – 35 y; n = 12), with blood samples taken before and after eight weeks of supplementation with 800 ug/day of FA. CD16+ cells (CD16) were isolated using antibody‐bound magnetic beads. DNA methylation patterns from WB and CD16 cells were measured across &gt;485,000 CpG sites using the Infinium Human Methylation450 Bead Chip. For each CpG site, the proportion of DNA methylation that changed over the 8 week intervention was evaluated by fitting a separate linear model. Genome‐wide analysis indicated changes in 7887 vs. 8482 CpG sites in response to supplementation in WB and CD16, respectively; with DNA methylation decreased in 77.2% (WB) or 66.3% (CD16) of these sites. Results suggest that there are differences in methylation response in WB and CD16 after FA supplementation. The implication of these methylation differences are being explored in our on‐going gene enrichment analyses.Grant Funding Source: Funded in part by HATCH #GEO00706, #GEO00707 and the Obesity Initiative at the University of Georgia

Prenatal arsenic exposure is associated with increased risk of disease in adulthood. This has led to considerable interest in arsenic’s ability to disrupt fetal programming. Many studies report that arsenic exposure alters DNA methylation in whole blood but these studies did not adjust for cell mixture. In this study, we examined the relationship between arsenic in maternal drinking water collected ≤ 16 weeks gestational age and DNA methylation in cord blood (n = 44) adjusting for leukocyte-tagged differentially methylated regions. DNA methylation was quantified using the Infinium HumanMethylation 450 BeadChip array. Recursively partitioned mixture modeling examined the relationship between arsenic and methylation at 473,844 CpG sites. Median arsenic concentration in water was 12 µg/L (range < 1- 510 µg/L). Log10 arsenic was associated with altered DNA methylation across the epigenome (P = 0.002); however, adjusting for leukocyte distributions attenuated this association (P = 0.013). We also observed that arsenic had a strong effect on the distribution of leukocytes in cord blood. In adjusted models, every log10 increase in maternal drinking water arsenic exposure was estimated to increase CD8+ T cells by 7.4% (P = 0.0004) and decrease in CD4+ T cells by 9.2% (P = 0.0002). These results show that prenatal exposure to arsenic had an exposure-dependent effect on specific T cell subpopulations in cord blood and altered DNA methylation in cord blood. Future research is needed to determine if these small changes in DNA methylation alter gene expression or are associated with adverse health effects.

&lt;div&gt;Abstract&lt;p&gt;DNA methylation in peripheral blood is a potential biomarker of gastric cancer risk which could be used for early detection. We conducted a prospective case–control study nested within the Melbourne Collaborative Cohort Study. Genomic DNA was prepared from blood samples collected a median of 12 years before diagnosis for cases (&lt;i&gt;N&lt;/i&gt; = 168). Controls (&lt;i&gt;N&lt;/i&gt; = 163) were matched to cases on sex, year of birth, country of birth, and blood sample type using incidence density sampling. Genome-wide DNA methylation was measured using the Infinium HumanMethylation450K Beadchip. Global measures of DNA methylation were defined as the median methylation M value, calculated for each of 13 CpG subsets representing genomic function, mean methylation and location, and reliability of measurement. Conditional logistic regression was conducted to assess associations between these global measures of methylation and gastric cancer risk, adjusting for &lt;i&gt;Helicobacter pylori&lt;/i&gt; and other potential confounders. We tested nonlinear associations using quintiles of the global measure distribution. A genome-wide association study of DNA methylation and gastric cancer risk was also conducted (&lt;i&gt;N&lt;/i&gt; = 484,989 CpGs) using conditional logistic regression, adjusting for potential confounders. Differentially methylated regions (DMR) were investigated using the R package &lt;i&gt;DMRcate&lt;/i&gt;. We found no evidence of associations with gastric cancer risk for individual CpGs or DMRs (&lt;i&gt;P&lt;/i&gt; &gt; 7.6 × 10&lt;sup&gt;−6&lt;/sup&gt;). No evidence of association was observed with global measures of methylation (OR 1.07 per SD of overall median methylation; 95% confidence interval, 0.80–1.44; &lt;i&gt;P&lt;/i&gt; = 0.65). We found no evidence that blood DNA methylation is prospectively associated with gastric cancer risk.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Prevention Relevance:&lt;/b&gt; We studied DNA methylation in blood to try and predict who was at risk of gastric cancer before symptoms developed, by which stage survival is poor. We did not find any such markers, but the importance of early diagnosis in gastric cancer remains, and the search for markers continues.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;DNA methylation in peripheral blood is a potential biomarker of gastric cancer risk which could be used for early detection. We conducted a prospective case–control study nested within the Melbourne Collaborative Cohort Study. Genomic DNA was prepared from blood samples collected a median of 12 years before diagnosis for cases (&lt;i&gt;N&lt;/i&gt; = 168). Controls (&lt;i&gt;N&lt;/i&gt; = 163) were matched to cases on sex, year of birth, country of birth, and blood sample type using incidence density sampling. Genome-wide DNA methylation was measured using the Infinium HumanMethylation450K Beadchip. Global measures of DNA methylation were defined as the median methylation M value, calculated for each of 13 CpG subsets representing genomic function, mean methylation and location, and reliability of measurement. Conditional logistic regression was conducted to assess associations between these global measures of methylation and gastric cancer risk, adjusting for &lt;i&gt;Helicobacter pylori&lt;/i&gt; and other potential confounders. We tested nonlinear associations using quintiles of the global measure distribution. A genome-wide association study of DNA methylation and gastric cancer risk was also conducted (&lt;i&gt;N&lt;/i&gt; = 484,989 CpGs) using conditional logistic regression, adjusting for potential confounders. Differentially methylated regions (DMR) were investigated using the R package &lt;i&gt;DMRcate&lt;/i&gt;. We found no evidence of associations with gastric cancer risk for individual CpGs or DMRs (&lt;i&gt;P&lt;/i&gt; &gt; 7.6 × 10&lt;sup&gt;−6&lt;/sup&gt;). No evidence of association was observed with global measures of methylation (OR 1.07 per SD of overall median methylation; 95% confidence interval, 0.80–1.44; &lt;i&gt;P&lt;/i&gt; = 0.65). We found no evidence that blood DNA methylation is prospectively associated with gastric cancer risk.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Prevention Relevance:&lt;/b&gt; We studied DNA methylation in blood to try and predict who was at risk of gastric cancer before symptoms developed, by which stage survival is poor. We did not find any such markers, but the importance of early diagnosis in gastric cancer remains, and the search for markers continues.&lt;/p&gt;&lt;/div&gt;

Due to its convenience, the blood is commonly used in epigenomic studies, but its heterogeneous nature leads to interpretation difficulties, given the now widely recognized potential for confounding by cell composition effects. Many recent publications have reported significant associations between DNA methylation and a variety of health conditions or exposures. In this review, we summarize many of these recent publications, highlighting the findings in the context of potential cell composition effects, particularly findings that are indicative of immune response or inflammation. While there is substantial evidence for confounding by cell composition, there is nevertheless also evidence for differential DNA methylation suggestive of processes that are not cell mediated. We conclude that important biological insights still may be gained from studying DNA methylation in whole blood, either by investigating the cell composition effects themselves or processes that demonstrate associations even after adjusting for cell composition effects.

ContextDNA methylation has been proposed to play a critical role in many cellular and biological processes.ObjectiveTo examine the influence of Roux-en-Y gastric bypass (RYGB) surgery on genome-wide promoter-specific DNA methylation in obese patients. Promoters are involved in the initiation and regulation of gene transcription.MethodsPromoter-specific DNA methylation in whole blood was measured in 11 obese patients (presurgery BMI >35 kg/m2, 4 females), both before and 6 months after RYGB surgery, as well as once only in a control group of 16 normal-weight men. In addition, body weight and fasting plasma glucose were measured after an overnight fast.ResultsThe mean genome-wide distance between promoter-specific DNA methylation of obese patients at six months after RYGB surgery and controls was shorter, as compared to that at baseline (p<0.001). Moreover, postsurgically, the DNA methylation of 51 promoters was significantly different from corresponding values that had been measured at baseline (28 upregulated and 23 downregulated, P<0.05 for all promoters, Bonferroni corrected). Among these promoters, an enrichment for genes involved in metabolic processes was found (n = 36, P<0.05). In addition, the mean DNA methylation of these 51 promoters was more similar after surgery to that of controls, than it had been at baseline (P<0.0001). When controlling for the RYGB surgery-induced drop in weight (-24% of respective baseline value) and fasting plasma glucose concentration (-16% of respective baseline value), the DNA methylation of only one out of 51 promoters (~2%) remained significantly different between the pre-and postsurgery time points.ConclusionsEpigenetic modifications are proposed to play an important role in the development of and predisposition to metabolic diseases, including type II diabetes and obesity. Thus, our findings may form the basis for further investigations to unravel the molecular effects of gastric bypass surgery.Clinical TrialClinicalTrials.gov NCT01730742