Blood DNA Methylation Research Articles

Hypomethylation of DYRK4 in peripheral blood is associated with increased lung cancer risk.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. It is urgent to identify new biomarkers for the early detection of LC. DNA methylation in peripheral blood has been reported to be associated with cancers. We conducted two independent case-control studies and a nested case-control study (168 LC cases and 167 controls in study Ⅰ, 677 LC cases and 833 controls in study Ⅱ, 147 precancers and 21 controls in the nested case-control study). The methylation levels of DYRK4 CpG sites were measured using mass spectrometry and their correlations with LC were analyzed by logistic regression and nonparametric tests. Bonferroni correction was used for the multiple comparisons. LC-related decreased DYRK4 methylation was discovered in Study I and validated in Study II (the odds ratios [ORs] for the lowest vs. highest quartile of all three DYRK4 CpG sites ranged from 1.64 to 2.09, all p < 0.001). Combining the two studies, hypomethylation of DYRK4 was observed in stage I cases (ORs per -10% methylation ranged from 1.16 to 1.38, all p < 5.9E-04), and could be enhanced by male gender (ORs ranged from 1.77 to 4.17 via interquartile analyses, all p < 0.017). Hypomethylation of DYRK4_A_CpG_2 was significantly correlated with tumor size, length, and stage (p = 0.034, 0.002, and 0.002, respectively) in LC cases. Our study disclosed the association between DYRK4 hypomethylation in peripheral blood and LC, suggesting the feasibility of blood-based DNA methylation as new biomarker for LC detection.

Epigenetic aging in cows is accelerated by milk production

ABSTRACT DNA methylation has proven to be the most promising age-predictive biomarker in mammals resulting in the emergence of ‘epigenetic clocks’ that describe the relationship between methylation levels and age. Using Targeted bisulfite Sequencing, we evaluated blood DNA-methylation data from 96 domesticated cows (Bos Taurus) of which 88 were adults and 8 were calves. This allowed us to measure DNA methylation across three thousand regions in the genome that were conserved across mammals. The significant association of age with the changes in DNA methylation enabled us to construct an epigenetic clock that predicts the age of cows to within nine months. We also investigated whether factors exist that moderate the association between epigenetic age and actual age and found that milk production levels significantly increase the rate of epigenetic ageing, suggesting that the stress of excessive milk production might be accelerating epigenetic ageing in cows.

Correlation Between MMP9 Promoter Methylation and Transient Ischemic Attack/Mild Ischemic Stroke with Early Cognitive Impairment.

Dyskinesia caused by transient ischemic attack (TIA) and mild ischemic stroke (MIS) is mild and short-lived; however, cognitive impairment (CI) can occur in the acute phase and be easily overlooked. DNA methylation is an epigenetic phenomenon that can affect gene expression through gene silencing. Blood levels of matrix metalloproteinase (MMP) 9 are elevated in ischemic stroke patients and is associated with the destruction of the blood-brain barrier and the occurrence of CI. No studies have investigated the relationship between MMP9 gene methylation and TIA/MIS with early cognitive impairment (ECI). As such, the purpose of the present study was to investigate the correlation between MMP9 gene methylation and TIA/MIS with ECI. Data from 112 subjects were collected, including 84 with TIA/MIS (National Institutes of Health Stroke Scale <5 points) and 28 non-stroke control subjects. Patients were evaluated within 7 days of TIA/MIS onset according to four single-domain cognitive scales. Whole blood DNA methylation was detected using MethylTarget sequencing technology. Comparison of MMP9 gene methylation levels among subgroups was performed using statistical methods. The site S33-79 in the TIA/MIS group was hypomethylated compared with the control group, and sites S33-25 and S33-30 in TIA/MIS with ECI was hypomethylated compared with TIA/MIS without ECI. Compared with the small artery occlusion group, MMP9 gene, S33-25, 30, 39, 53, 58, 73, 79, 113 and 131 sites in the large artery atherosclerosis group were hypomethylated. MMP9 gene hypomethylation sites were associated with TIA/MIS and TIA/MIS with ECI, and there was a strong correlation between MMP9 gene hypomethylation and atherosclerotic TIA/MIS. MMP9 gene methylation can reflect the severity of TIA/MIS. MMP9 gene hypomethylation sites may be used as potential biomarkers and therapeutic targets for TIA/MIS and TIA/MIS with ECI.

Genome-Scale Methylation Analysis Identifies Immune Profiles and Age Acceleration Associations with Bladder Cancer Outcomes.

Immune profiles have been associated with bladder cancer outcomes and may have clinical applications for prognosis. However, associations of detailed immune cell subtypes with patient outcomes remain underexplored and may contribute crucial prognostic information for better managing bladder cancer recurrence and survival. Bladder cancer case peripheral blood DNA methylation was measured using the Illumina HumanMethylationEPIC array. Extended cell-type deconvolution quantified 12 immune cell-type proportions, including memory, naïve T and B cells, and granulocyte subtypes. DNA methylation clocks determined biological age. Cox proportional hazards models tested associations of immune cell profiles and age acceleration with bladder cancer outcomes. The partDSA algorithm discriminated 10-year overall survival groups from clinical variables and immune cell profiles, and a semi-supervised recursively partitioned mixture model (SS-RPMM) with DNA methylation data was applied to identify a classifier for 10-year overall survival. Higher CD8T memory cell proportions were associated with better overall survival [HR = 0.95, 95% confidence interval (CI) = 0.93-0.98], while higher neutrophil-to-lymphocyte ratio (HR = 1.36, 95% CI = 1.23-1.50), CD8T naïve (HR = 1.21, 95% CI = 1.04-1.41), neutrophil (HR = 1.04, 95% CI = 1.03-1.06) proportions, and age acceleration (HR = 1.06, 95% CI = 1.03-1.08) were associated with worse overall survival in patient with bladder cancer. partDSA and SS-RPMM classified five groups of subjects with significant differences in overall survival. We identified associations between immune cell subtypes and age acceleration with bladder cancer outcomes. The findings of this study suggest that bladder cancer outcomes are associated with specific methylation-derived immune cell-type proportions and age acceleration, and these factors could be potential prognostic biomarkers.

Peripheral Blood DNA Methylation Signatures and Response to Tofacitinib in Moderate-to-severe Ulcerative Colitis.

Predictive biomarkers for treatment efficacy of ulcerative colitis [UC] treatments are lacking. Here, we performed a longitudinal study investigating the association and potential predictive power of genome-wide peripheral blood [PB] DNA methylation signatures and response to tofacitinib treatment in UC. We recruited moderate-to-severe UC patients starting tofacitinib treatment, and measured PB DNA methylation profiles at baseline [T1], after 8 weeks [T2], and in a subset [n = 8] after a median of 20 weeks [T3] using the Illumina Infinium HumanMethylation EPIC BeadChip. After 8 weeks, we distinguished responders [R] from non-responders [NR] based on a centrally read endoscopic response [decrease in endoscopic Mayo score ≥1 or Ulcerative Colitis Endoscopic Index of Severity ≥2] combined with corticosteroid-free clinical and/or biochemical response. T1 PB samples were used for biomarker identification, and T2 and publicly available intraclass correlation [ICC] data were used for stability analyses. RNA-sequencing was performed to understand the downstream effects of the predictor CpG loci. In total, 16 R and 15 NR patients, with a median disease duration of 7 [4-12] years and overall comparable patient characteristics at baseline, were analysed. We identified a panel of 53 differentially methylated positions [DMPs] associated with response to tofacitinib [AUROC 0.74]. Most DMPs [77%] demonstrated both short- and long-term hyperstability [ICC ≥0.90], irrespective of inflammatory status. Gene expression analysis showed lower FGFR2 [pBH = 0.011] and LRPAP1 [pBH = 0.020], and higher OR2L13 [pBH = 0.016] expression at T1 in R compared with NR. Our observations demonstrate the utility of genome-wide PB DNA methylation signatures to predict response to tofacitinib.

Circulating Cell-Free SEPT9 DNA Methylation in Blood Is a Biomarker for Minimal Residual Disease Detection in Head and Neck Squamous Cell Carcinoma Patients.

Tumorous SEPT9 (septin 9, SEPTIN9) circulating cell-free DNA (ccfDNA) methylation in blood plasma is a powerful biomarker for diagnosis, molecular staging, prognosis, and recurrence monitoring in head and neck squamous cell carcinoma (HNSCC) patients. The present study aimed to evaluate the clinical performance of SEPT9 ccfDNA methylation to detect post-surgical minimal residual disease (MRD) in patients with localized or locally advanced HNSCC treated with curative intent. We applied quasi-digital methylation-specific real-time PCR to quantify SEPT9 ccfDNA methylation levels 2 to 30 days post-surgically in plasma from n = 219 prospectively enrolled HNSCC patients. We tested the associations of SEPT9 ccfDNA methylation with clinicopathological parameters and used Kaplan-Meier and Cox proportional hazards analyses for univariate, pairwise bivariate, and multivariate analyses of disease-free survival. Of 219 patients, 26.5% (58/219) were post-surgically SEPT9 ccfDNA methylation positive. SEPT9 ccfDNA methylation positivity was significantly associated with tumor site, American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC; 8th edition) tumor stage, nodal category and extracapsular extension, lymphatic and vascular invasion, and surgical margin. Bivariate Cox proportional hazards analysis proved post-surgical SEPT9 ccfDNA methylation positivity to be an independent prognostic factor tested together with AJCC/UICC tumor stage (SEPT9: hazard ratio [HR] = 2.43, 95% CI, 1.37-4.30, P = 0.002; AJCC/UICC stage: HR = 1.48, 95% CI, 1.11-1.98, P = 0.008). Post-surgical SEPT9 ccfDNA methylation may aid to identify high-risk HNSCC patients who could benefit from an intensified adjuvant treatment and surveillance.

Exploring the cross-cancer effect of smoking and its fingerprints in blood DNA methylation on multiple cancers: A Mendelian randomization study.

Aberrant smoking-related DNA methylation has been widely investigated as a carcinogenesis mechanism, but whether the cross-cancer epigenetic pathways exist remains unclear. We conducted two-sample Mendelian randomization (MR) analyses respectively on smoking behaviors (age of smoking initiation, smoking initiation, smoking cessation, and lifetime smoking index [LSI]) and smoking-related DNA methylation to investigate their effect on 15 site-specific cancers, based on a genome-wide association study (GWAS) of 1.2 million European individuals and an epigenome-WAS (EWAS) of 5907 blood samples of Europeans for smoking and 15 GWASs of European ancestry for multiple site-specific cancers. Significantly identified CpG sites were further used for colocalization analysis, and those with cross-cancer effect were validated by overlapping with tissue-specific eQTLs. In the genomic MR, smoking measurements of smoking initiation, smoking cessation and LSI were suggested to be casually associated with risk of seven types of site-specific cancers, among which cancers at lung, cervix and colorectum were provided with strong evidence. In the epigenetic MR, methylation at 75 CpG sites were reported to be significantly associated with increased risks of multiple cancers. Eight out of 75 CpG sites were observed with cross-cancer effect, among which cg06639488 (EFNA1), cg12101586 (CYP1A1) and cg14142171 (HLA-L) were validated by eQTLs at specific cancer sites, and cg07932199 (ATXN2) had strong evidence to be associated with cancers of lung (coefficient, 0.65, 95% confidence interval [CI], 0.31-1.00), colorectum (0.90 [0.61, 1.18]), breast (0.31 [0.20, 0.43]) and endometrium (0.98 [0.68, 1.27]). These findings highlight the potential practices targeting DNA methylation-involved cross-cancer pathways.

Smoking, blood DNA methylation sites and lung cancer risk

Altered DNA methylation (DNAm) might be a biological intermediary in the pathway from smoking to lung cancer. In this study, we investigated the contribution of differential blood DNAm to explain the association between smoking and lung cancer incidence. Blood DNAm was measured in 2321 Strong Heart Study (SHS) participants. Incident lung cancer was assessed as time to event diagnoses. We conducted mediation analysis, including validation with DNAm and paired gene expression data from the Framingham Heart Study (FHS). In the SHS, current versus never smoking and pack-years single-mediator models showed, respectively, 29 and 21 differentially methylated positions (DMPs) for lung cancer with statistically significant mediated effects (14 of 20 available, and five of 14 available, positions, replicated, respectively, in FHS). In FHS, replicated DMPs showed gene expression downregulation largely in trans, and were related to biological pathways in cancer. The multimediator model identified that DMPs annotated to the genes AHRR and IER3 jointly explained a substantial proportion of lung cancer. Thus, the association of smoking with lung cancer was partly explained by differences in baseline blood DNAm at few relevant sites. Experimental studies are needed to confirm the biological role of identified eQTMs and to evaluate potential implications for early detection and control of lung cancer.

GWAS of biological aging to find longevity genes in schizophrenia.

Schizophrenia (SCZ) is a severe psychotic disorder associated with premature mortality and aging. Moreover, the symptoms and progression of psychiatric disorders in general are associated with decreased lifespan, biological aging, and poorer medical outcomes. In this study, we investigated the relationship between several epigenetic clocks and scanned the entire genome for association in a cohort of SCZ individuals (n = 107). Biological age was computed from blood DNA methylation (DNAm) and tested for association against common variants across thegenomeusinggeneral linear models. Genes affecting epigenetic age acceleration in our cohort were found mainly when using the telomeric lengthclock rather than the other biological clocks. These findings pair with existing evidence that there are some genes associated with longevity and suggest further investigations of putative biological mechanisms for morbidity and premature mortality, not only in patients with SCZ but also in the general population.

BMI change during childhood, DNA methylation change at TXNIP, and glucose change during midlife.

This study investigated whether changes in DNA methylation (DNAm) at TXNIP are associated with glycemic changes and whether such an association differs with early-life adiposity changes. A total of 594 Bogalusa Heart Study participants who had blood DNAm measurements at two time points in midlife were included. Of them, 353 participants had at least four BMI measurements during childhood and adolescence. The incremental area under the curve was calculated as a measure of long-term trends of BMI during childhood and adolescence. Increase in DNAm at TXNIP was significantly associated with decrease in fasting plasma glucose (FPG) independent of covariates (p < 0.001). The study found that the strength of this relationship was significantly modified by a trend of increasing BMI during childhood and adolescence (p-interaction = 0.003). Each 1% increase in DNAm at TXNIP was associated with a 2.90- (0.77) mg/dL decrease in FPG among participants with the highest tertile of BMI incremental area under the curveand a 0.96- (0.38) mg/dL decrease among those with the middle tertile, whereas no association was observed among participants with the lowest tertile. These results indicate that changes in blood DNAm at TXNIP are significantly associated with changes in FPG in midlife, and this association was modified by BMI trendsduring childhood and adolescence.

Brain serotonin 1A receptor binding: relationship to peripheral blood DNA methylation, recent life stress and childhood adversity in unmedicated major depression.

Childhood and lifetime adversity may reduce brain serotonergic (5-HT) neurotransmission by epigenetic mechanisms. We tested the relationships of childhood adversity and recent stress to serotonin 1A (5-HT1A) receptor genotype, DNA methylation of this gene in peripheral blood monocytes and in vivo 5-HT1A receptor binding potential (BPF) determined by positron emission tomography (PET) in 13 a priori brain regions, in participants with major depressive disorder (MDD) and healthy volunteers (controls). Medication-free participants with MDD (n = 192: 110 female, 81 male, 1 other) and controls (n = 88: 48 female, 40 male) were interviewed about childhood adversity and recent stressors and genotyped for rs6295. DNA methylation was assayed at three upstream promoter sites (-1019, -1007, -681) of the 5-HT1A receptor gene. A subgroup (n = 119) had regional brain 5-HT1A receptor BPF quantified by PET. Multi-predictor models were used to test associations between diagnosis, recent stress, childhood adversity, genotype, methylation and BPF. Recent stress correlated positively with blood monocyte methylation at the -681 CpG site, adjusted for diagnosis, and had positive and region-specific correlations with 5-HT1A BPF in participants with MDD, but not in controls. In participants with MDD, but not in controls, methylation at the -1007 CpG site had positive and region-specific correlations with binding potential. Childhood adversity was not associated with methylation or BPF in participants with MDD. These findings support a model in which recent stress increases 5-HT1A receptor binding, via methylation of promoter sites, thus affecting MDD psychopathology.

Maternal glycemia in pregnancy is longitudinally associated with blood DNAm variation at the FSD1L gene from birth to 5 years of age

BackgroundIn utero exposure to maternal hyperglycemia has been associated with an increased risk for the development of chronic diseases in later life. These predispositions may be programmed by fetal DNA methylation (DNAm) changes that persist postnatally. However, although some studies have associated fetal exposure to gestational hyperglycemia with DNAm variations at birth, and metabolic phenotypes in childhood, no study has yet examined how maternal hyperglycemia during pregnancy may be associated with offspring DNAm from birth to five years of age.HypothesisMaternal hyperglycemia is associated with variation in offspring DNAm from birth to 5 years of age.MethodsWe estimated maternal hyperglycemia using the area under the curve for glucose (AUCglu) following an oral glucose tolerance test conducted at 24–30 weeks of pregnancy. We quantified DNAm levels in cord blood (n = 440) and peripheral blood at five years of age (n = 293) using the Infinium MethylationEPIC BeadChip (Illumina). Our total sample included 539 unique dyads (mother–child) with 194 dyads having DNAm at both time-points. We first regressed DNAm M-values against the cell types and child age for each time-point separately to account for the difference by time of measurement for these variables. We then used a random intercept model from the linear mixed model (LMM) framework to assess the longitudinal association between maternal AUCglu and the repeated measures of residuals of DNAm. We adjusted for the following covariates as fixed effects in the random intercept model: maternal age, gravidity, smoking status, child sex, maternal body mass index (BMI) (measured at first trimester of pregnancy), and a binary variable for time-point.ResultsIn utero exposure to higher maternal AUCglu was associated with lower offspring blood DNAm levels at cg00967989 located in FSD1L gene (β = − 0.0267, P = 2.13 × 10–8) in adjusted linear regression mixed models. Our study also reports other CpG sites for which DNAm levels were suggestively associated (P < 1.0 × 10–5) with in utero exposure to gestational hyperglycemia. Two of these (cg12140144 and cg07946633) were found in the promotor region of PRDM16 gene (β: − 0.0251, P = 4.37 × 10–07 and β: − 0.0206, P = 2.24 × 10–06, respectively).ConclusionMaternal hyperglycemia is associated with offspring DNAm longitudinally assessed from birth to 5 years of age.

1461-P: HbA1c-Independent Association between DNA Methylation (DNAm) and 28-Year Risk of Microvascular Complications in Type 1 Diabetes (T1D)

DNAm has been associated with microvascular disease but the few epigenome-wide association studies (EWAS) of complications in T1D were largely cross-sectional with limited data on glycemic exposure. We thus performed separate EWAS of 28-yr overt nephropathy (ON) and proliferative retinopathy (PR) risk to determine whether DNAm associations are independent of concurrent HbA1c or differ by long-term HbA1c trajectory in the Pittsburgh Epidemiology of Diabetes Complications study of childhood onset (&amp;lt;17 yrs) T1D (baseline mean age 29, T1D duration 21 yrs). After quality control, baseline whole blood DNAm (Illumina EPIC array) at 683,597 CpGs was analyzed in Cox models for time-to-ON (albumin excretion rate &amp;gt;200µg/min in ≥2 of 3 timed urines, n=306 free of ON at baseline) and PR (ETDRS grade≥60 or laser photocoagulation, n=265 free of PR), adjusting for T1D duration, sex, smoking pack-years, est. blood cell composition, and technical covariates. Additional EWAS further adjusted for baseline HbA1c. CpGs with false discovery rate (FDR) &amp;lt;0.20 were examined by previously derived latent HbA1c trajectory (Low: stable 7-8%, Improved: 10% decreasing to 7%, Intermediate-High: stable 9%-11%). Over 28 yrs, 65 (21.2%) developed ON; 139 (52.5%) PR. For ON, DNAm at cg19693031 (TXNIP) was associated (β per 5% DNAm=−0.43±0.09, p=2.84E-07, FDR=0.19) but attenuated in a baseline HbA1c-adjusted EWAS (β=−0.32±0.08, p=7.76E-05, FDR=0.98). For PR, 3 CpGs (cg27512687 [KIF16B]; cg04202206 [ISCA1]; cg14678509 [intergenic]) had FDR&amp;lt;0.20; only cg27512687 remained so after HbA1c adjustment (β=−2.6±0.49, p=8.46E-08, FDR=0.06). There was no interaction with baseline HbA1c (p=0.49); the magnitude of the cg27512687-PR association was similar regardless of HbA1c trajectory. Thus, further study of cg27512687 in KIF16B, a locus encoding a kinesin involved in receptor recycling/degradation, is warranted as it may make an HbA1c-independent contribution to PR risk T1D. Disclosure R.G.Miller: None. J.Mychaleckyj: None. S.Onengut-gumuscu: None. T.J.Orchard: None. T.Costacou: None. Funding American Diabetes Association (1-19-JDF-109 to R.G.M.); National Institute of Diabetes and Digestive and Kidney Diseases (R01DK034818)

45-OR: Newborn Adiposity and Cord Blood DNA Methylation—An Epigenome-Wide Association Study in HAPO with Validation in Gen3G

Differential cord blood DNA methylation (cb DNAm) in response to hyperglycemia in pregnancy may be associated with neonatal adiposity, a risk factor for childhood obesity. The Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study enrolled a multiethnic cohort to evaluate glycemia in pregnancy and newborn adiposity measured by skinfold thicknesses. We conducted an epigenome-wide association study using HAPO as a discovery sample and performed a replication study in an independent cohort, Gen3G. We analyzed cb DNAm with Illumina EPIC arrays (791,359 CpG sites after QC) on 2712 HAPO samples. We assessed the association between sum of 3 skinfolds and cb DNAm using linear regression models adjusted for maternal and offspring covariates and cell counts. We identified 90 CpG sites after accounting for multiple testing (Bonferroni-adjusted p&amp;lt;0.05). Using these sites identified in HAPO, we conducted similar regression analyses in 139 Gen3G cb DNAm samples to replicate findings. Similar associations at 7 CpG sites were identified including loci near KLF7, which encodes a regulator of cell proliferation and inhibitor of adipogenesis, and IGF1R, which encodes a transmembrane receptor involved in cell growth and survival that binds IGF1 and insulin, the latter especially during fetal life. These findings support epigenetic mechanisms in the developmental origins of neonatal adiposity. Disclosure J.L.Josefson: None. A.Kuang: None. D.Scholtens: None. C.Allard: None. W.Lowe: None. M.Hivert: None. Funding National Institutes of Health (R01HD034243, R01DK118403)

Stage 2 Registered Report: Epigenetic Intergenerational Transmission: Mothers’ Adverse Childhood Experiences and DNA Methylation

Individual differences in risk for mental disorders over the lifespan are shaped by forces acting before the individual is born-in utero, but likely even earlier, during the mother's own childhood. The environmental epigenetics hypothesis proposes that sustained effects of environmental conditions on gene expression are mediated by epigenetic mechanisms. Recent human studies have shown that adversities in childhood are correlated with DNA methylation (DNAm) in adulthood. In the current study, we tested the following pre-registered hypotheses: Mothers' adverse childhood experiences (ACEs) are correlated with DNAm in peripheral blood during pregnancy (hypothesis 1) and in cord blood samples from newborn infants (hypothesis 2), and women's depression and anxiety symptoms during pregnancy mediate the association between mothers' ACE exposure and prenatal/neonatal DNA methylation (hypothesis 3). Data were from the Avon Longitudinal Study of Parents and Children Accessible Resource for Integrated Epigenomic Studies substudy. Women provided retrospective self-reports during pregnancy of ACE exposure. We conducted an epigenome-wide association study testing whether mothers' ACE exposure, cumulative score (0-10), was associated with DNAm in maternal antenatal blood and infant cord blood in more than 450,000 CpG (point on DNA sequence where cytosine and guanine base pairs are linked by a phosphate, where methylation usually occurs) sites on the Illumina 450K BeadChip. Analyses for cord blood were separated by infant sex, a pre-registered analysis. Hypothesis 1: In 896 mother-infant pairs with available methylation and ACE exposure data, there were no significant associations between mothers' ACE score and DNAm from antenatal peripheral blood, after controlling for covariates. Hypothesis 2: In infant cord blood, there were 5 CpG sites significantly differentially methylated in relation to mothers' ACEs (false discovery rate [FDR]< .05), but only in male offspring. Effect sizes were medium, with partial eta squared values ranging from 0.060 to 0.078. CpG sites were in genes related to mitochondrial function and neuronal development in the cerebellum. Hypothesis 3: There was no mediation by maternal anxiety/depression symptoms found between mothers' ACEs score and DNAm in the significant CpG sites in male cord blood. Mediation was not tested in antenatal peripheral blood, because no direct association between mothers' ACE score and antenatal peripheral blood was found. Our results show that mothers' ACE exposure is associated with DNAm in male offspring, supporting the notion that DNAm could be a marker of intergenerational biological embedding of mothers' childhood adversity. Epigenetic Intergenerational Transmission: Mothers' Adverse Childhood Experiences and DNA Methylation; https://doi.org/10.1016/j.jaac.2020.03.008.

No impact of prenatal paracetamol and folic acid exposure on cord blood DNA methylation in children with attention-deficit/hyperactivity disorder.

Pharmacoepigenetic studies are important to understand the mechanisms through which medications influence the developing fetus. For instance, we and others have reported associations between prenatal paracetamol exposure and offspring DNA methylation (DNAm). Additionally, folic acid (FA) intake during pregnancy has been associated with DNAm in genes linked to developmental abnormalities. In this study, we aimed to: (i) expand on our previous findings showing differential DNAm associated with long-term prenatal paracetamol exposure in offspring with attention-deficit/hyperactivity disorder (ADHD), and (ii) examine if there is an interaction effect of FA and paracetamol on DNAm in children with ADHD. We used data from the Norwegian Mother, Father and Child Cohort Study (MoBa) and the Medical Birth Registry of Norway (MBRN). We did not identify any impact of paracetamol or any interaction effect of paracetamol and FA on cord blood DNAm in children with ADHD. Our results contribute to the growing literature on prenatal pharmacoepigenetics, but should be replicated in other cohorts. Replication of pharmacoepigenetic studies is essential to ensure robust findings and to increase the clinical relevance of such studies.

Association between the timing of childhood adversity and epigenetic patterns across childhood and adolescence: findings from the Avon Longitudinal Study of Parents and Children (ALSPAC) prospective cohort

Childhood adversity is a potent determinant of health across development and is associated with altered DNA methylation signatures, which might be more common in children exposed during sensitive periods in development. However, it remains unclear whether adversity has persistent epigenetic associations across childhood and adolescence. We aimed to examine the relationship between time-varying adversity (defined through sensitive period, accumulation of risk, and recency life course hypotheses) and genome-wide DNA methylation, measured three times from birth to adolescence, using data from a prospective, longitudinal cohort study. We first investigated the relationship between the timing of exposure to childhood adversity between birth and 11 years and blood DNA methylation at age 15 years in the Avon Longitudinal Study of Parents and Children (ALSPAC) prospective cohort study. Our analytic sample included ALSPAC participants with DNA methylation data and complete childhood adversity data between birth and 11 years. We analysed seven types of adversity (caregiver physical or emotional abuse, sexual or physical abuse [by anyone], maternal psychopathology, one-adult households, family instability, financial hardship, and neighbourhood disadvantage) reported by mothers five to eight times between birth and 11 years. We used the structured life course modelling approach (SLCMA) to identify time-varying associations between childhood adversity and adolescent DNA methylation. Top loci were identified using an R2 threshold of 0·035 (ie, ≥3·5% of DNA methylation variance explained by adversity). We attempted to replicate these associations using data from the Raine Study and Future of Families and Child Wellbeing Study (FFCWS). We also assessed the persistence of adversity-DNA methylation associations we previously identified from age 7 blood DNA methylation into adolescence and the influence of adversity on DNA methylation trajectories from ages 0-15 years. Of 13 988 children in the ALSPAC cohort, 609-665 children (311-337 [50-51%] boys and 298-332 [49-50%] girls) had complete data available for at least one of the seven childhood adversities and DNA methylation at 15 years. Exposure to adversity was associated with differences in DNA methylation at 15 years for 41 loci (R2 ≥0·035). Sensitive periods were the most often selected life course hypothesis by the SLCMA. 20 (49%) of 41 loci were associated with adversities occurring between age 3 and 5 years. Exposure to one-adult households was associated with differences in DNA methylation at 20 [49%] of 41 loci, exposure to financial hardship was associated with changes at nine (22%) loci, and physical or sexual abuse was associated with changes at four (10%) loci. We replicated the direction of associations for 18 (90%) of 20 loci associated with exposure to one-adult household using adolescent blood DNA methylation from the Raine Study and 18 (64%) of 28 loci using saliva DNA methylation from the FFCWS. The directions of effects for 11 one-adult household loci were replicated in both cohorts. Differences in DNA methylation at 15 years were not present at 7 years and differences identified at 7 years were no longer apparent by 15 years. We also identified six distinct DNA methylation trajectories from these patterns of stability and persistence. These findings highlight the time-varying effect of childhood adversity on DNA methylation profiles across development, which might link exposure to adversity to potential adverse health outcomes in children and adolescents. If replicated, these epigenetic signatures could ultimately serve as biological indicators or early warning signs of initiated disease processes, helping identify people at greater risk for the adverse health consequences of childhood adversity. Canadian Institutes of Health Research, Cohort and Longitudinal Studies Enhancement Resources, EU's Horizon 2020, US National Institute of Mental Health.

Association of Adverse Childhood Experiences With Accelerated Epigenetic Aging in Midlife

Adverse childhood experiences (ACEs) are associated with the risk of poorer health, and identifying molecular mechanisms may lay the foundation for health promotion in people with ACEs. To investigate the associations of ACEs with changes in epigenetic age acceleration (EAA), a biomarker associated with various health outcomes in middle-aged adults, in a population with balanced race and sex demographics. Data for this cohort study were from the Coronary Artery Risk Development in Young Adults (CARDIA) study. Participants in CARDIA underwent 8 follow-up exams from baseline (year 0 [Y0]; 1985-1986) to Y30 (2015-2016), and participant blood DNA methylation information was obtained at Y15 (2000-2001) and Y20 (2005-2006). Individuals from Y15 and Y20 with available DNA methylation data and complete variables for ACEs and covariates were included. Data were analyzed from September 2021 to August 2022. Participant ACEs (general negligence, emotional negligence, physical violence, physical negligence, household substance abuse, verbal and emotional abuse, and household dysfunction) were obtained at Y15. The primary outcome consisted of results from 5 DNA methylation-based EAA measurements known to be associated with biological aging and long-term health: intrinsic EAA (IEAA), extrinsic EAA (EEAA), PhenoAge acceleration (PhenoAA), GrimAge acceleration (GrimAA), and Dunedin Pace of Aging Calculated From the Epigenome (DunedinPACE), measured at Y15 and Y20. Linear regression and generalized estimating equations were used to assess associations of the burden of ACEs (≥4 vs <4 ACEs) with EAA adjusting for demographics, health-related behaviors, and early life and adult socioeconomic status. A total of 895 participants for Y15 (mean [SD] age, 40.4 [3.5] years; 450 males [50.3%] and 445 females [49.7%]; 319 Black [35.6%] and 576 White [64.4%]) and 867 participants for Y20 (mean [SD] age, 45.4 [3.5] years; 432 males [49.8%] and 435 females [50.2%]; 306 Black [35.3%] and 561 White [64.7%]) were included after excluding participants with missing data. There were 185 participants with (20.7%) vs 710 participants without (79.3%) 4 or more ACEs at Y15 and 179 participants with (20.6%) vs 688 participants without (79.4%) 4 or more ACEs at Y20. Having 4 or more ACEs was positively associated with EAA in years at Y15 (EEAA: β = 0.60 years; 95% CI, 0.18-1.02 years; PhenoAA: β = 0.62 years; 95% CI = 0.13-1.11 years; GrimAA: β = 0.71 years; 95% CI, 0.42-1.00 years; DunedinPACE: β = 0.01; 95% CI, 0.01-0.02) and Y20 (IEAA: β = 0.41 years; 95% CI, 0.05-0.77 years; EEAA: β = 1.05 years; 95% CI, 0.66-1.44 years; PhenoAA: β = 0.57 years; 95% CI, 0.08-1.05 years; GrimAA: β = 0.57 years; 95% CI, 0.28-0.87 years; DunedinPACE: β = 0.01; 95% CI, 0.01-0.02) after adjusting for demographics, health-related behaviors, and socioeconomic status. In this cohort study, ACEs were associated with EAA among middle-aged adults after controlling for demographics, behavior, and socioeconomic status. These findings of the associations between early life experience and the biological aging process in midlife may contribute to health promotion in a life course perspective.

Associations of four biological age markers with child development: A multi-omic analysis in the European HELIX cohort.

While biological age in adults is often understood as representing general health and resilience, the conceptual interpretation of accelerated biological age in children and its relationship to development remains unclear. We aimed to clarify the relationship of accelerated biological age, assessed through two established biological age indicators, telomere length and DNA methylation age, and two novel candidate biological age indicators, to child developmental outcomes, including growth and adiposity, cognition, behavior, lung function and the onset of puberty, among European school-age children participating in the HELIX exposome cohort. The study population included up to 1173 children, aged between 5 and 12 years, from study centres in the UK, France, Spain, Norway, Lithuania, and Greece. Telomere length was measured through qPCR, blood DNA methylation, and gene expression was measured using microarray, and proteins and metabolites were measured by a range of targeted assays. DNA methylation age was assessed using Horvath's skin and blood clock, while novel blood transcriptome and 'immunometabolic' (based on plasma proteins and urinary and serum metabolites) clocks were derived and tested in a subset of children assessed six months after the main follow-up visit. Associations between biological age indicators with child developmental measures as well as health risk factors were estimated using linear regression, adjusted for chronological age, sex, ethnicity, and study centre. The clock derived markers were expressed as Δ age (i.e. predicted minus chronological age). Transcriptome and immunometabolic clocks predicted chronological age well in the test set (r=0.93 and r=0.84 respectively). Generally, weak correlations were observed, after adjustment for chronological age, between the biological age indicators.Among associations with health risk factors, higher birthweight was associated with greater immunometabolic Δ age, smoke exposure with greater DNA methylation Δ age, and high family affluence with longer telomere length.Among associations with child developmental measures, all biological age markers were associated with greater BMI and fat mass, and all markers except telomere length were associated with greater height, at least at nominal significance (p<0.05). Immunometabolic Δ age was associated with better working memory (p=4 e-3) and reduced inattentiveness (p=4 e-4), while DNA methylation Δ age was associated with greater inattentiveness (p=0.03) and poorer externalizing behaviors (p=0.01). Shorter telomere length was also associated with poorer externalizing behaviors (p=0.03). In children, as in adults, biological aging appears to be a multi-faceted process and adiposity is an important correlate of accelerated biological aging. Patterns of associations suggested that accelerated immunometabolic age may be beneficial for some aspects of child development while accelerated DNA methylation age and telomere attrition may reflect early detrimental aspects of biological aging, apparent even in children. UK Research and Innovation (MR/S03532X/1); European Commission (grant agreement numbers: 308333; 874583).

DNA methylation-based biomarkers for cardiometabolic-related traits and their importance for risk stratification.

The prevalence of cardiometabolic syndrome in adults is increasing worldwide, highlighting the importance of biomarkers for individuals' classification based on their health status. Although cardiometabolic risk scores and diagnostic criteria have been developed aggregating adverse health effects of individual conditions on the overall syndrome, none of them has gained unanimous acceptance. Therefore, novel molecular biomarkers have been developed to better understand the risk, onset and progression of both individual conditions and the overall cardiometabolic syndrome. Consistent associations between whole blood DNA methylation (DNAm) levels at several single genomic (i.e. CpG) sites and both individual and aggregated cardiometabolic conditions supported the creation of second-generation DNAm-based cardiometabolic-related biomarkers. These biomarkers linearly combine individual DNAm levels from key CpG sites, selected by a two-step machine learning procedures. They can be used, even retrospectively, in populations with extant whole blood DNAm levels and without observed cardiometabolic phenotypes. Here we offer an overview of the second-generation DNAm-based cardiometabolic biomarkers, discussing methodological advancements and implications on the interpretation and generalizability of the findings. We finally emphasize the contribution of DNAm-based biomarkers for risk stratification beyond traditional factors and discuss limitations and future directions of the field.