MethylationEPIC BeadChip Research Articles

Abstract TP382: Genome-wide DNA Methylation Profiling Reveals Low Methylation Variability in Moyamoya Disease

Background: Moyamoya disease (MMD) is a chronic cerebrovascular disorder that can affect both children and adults. It is characterized by chronic and progressive narrowing of the cerebral arteries and the formation of fragile collateral blood vessels. The narrowed arteries can lead to lead to stroke and neurological dysfunctions. Considering that MMD is mostly sporadic and gene-environment interactions play an important in various diseases, we sought to investigate epigenetic modifications in MMD, focusing specifically on changes in DNA methylation magnitude and variability. Methods: We performed genome-wide DNA methylation using Illumina 850K Methylation EPIC BeadChip, in two racially distinct adult female cohorts: A Non-Asian cohort (13 MMD patients and 7 healthy controls) and an Asian cohort (14 MMD patients and 3 healthy controls). An additional external cohort with both sexes (females: 5 MMD patients and 5 healthy controls, males: 5 MMD patients and 5 healthy controls) was included for validation. Results: Our findings revealed subtle changes in the magnitude of DNA methylation, but a strikingly low DNA methylation variability between MMD patients and healthy controls across both female MMD cohorts. In the Non-Asian cohort, only 6 probes showed increased variability, compared to 647 probes that showed decreased variability. Similarly, in the Asian cohort, the MMD group also displayed a reduced methylation variability across all 2845 probes. Further analysis showed that these differentially variable probes are associated with genes involved in key biological processes such as methylation and transcription, DNA repair, cytoskeletal remodeling, natural killer cell signaling, cellular growth, and migration. Conclusions: These findings mark the first observation of low methylation variability in any disease, contrasting with the high variability observed in other disorders. This reduced methylation variability in MMD may impair patients’ adaptability to environmental changes, such as hemodynamic stress, thereby disrupting vascular homeostasis and contributing to MMD pathology. These findings offer new insights into the mechanisms of MMD and suggest potential avenues for treatment strategies.

The Impact of Air Pollution on Sperm DNA Methylation.

A number of environmental factors have been shown to impact the sperm epigenome. Air pollution is one of the largest health and environmental hazards in the world today and has been implicated in many modern diseases. Recently, air pollution has been shown to alter methylation signatures in some body tissues, indicating that air pollution may also affect the sperm epigenome. The present experiment was conducted to analyze how seasonal air pollution in the Salt Lake Valley may impact DNA methylation patterns in sperm and to establish a relationship between air pollution and sperm epigenetic health as measured by DNA methylation. Sperm DNA methylation patterns were assessed in 74 individuals, who presented at the University of Utah Andrology Clinic for semen analysis, using the Illumina Human MethylationEPIC BeadChip array. Each semen sample collected, as per the fifth edition of WHO reference values for human semen characterization, was deemed normal. Two sample groups from the Salt Lake Valley, Urban Winter (UW, n=20), Urban Summer (US, n=21), and two sample groups east of the Wasatch mountains, Rural Winter (RW, n=19) and Rural Summer (RS, n=14), were compared to assess the effect of air pollution on sperm DNA methylation patterns. Due to seasonal inversions, urban winters are characterized by increased air pollution compared to summer months. Therefore, the UW sample group was designated as treatment and the three remaining groups (US, RW, RS) were designated as control. We conducted multiple differential methylation analyses using a sliding window approach which utilized the USeq software package. A sliding window analysis of UW versus US was conducted first, followed by a confirmatory analysis comparing UW versus RW and RS. Outputs from the USeq analysis were assessed using several tools including the Stanford GREAT analysis and an analysis of methylation instability at key promoter regions in sperm. The sliding window analysis identified six differentially methylated regions (DMRs) between the UW and US groups (Wilcoxon FDR ≥ 40, corresponding p-value of ~0.0001). Three of these six regions were confirmed with the second confirmatory analysis of UW versus RS/RW (Wilcoxon FDR ≥ 20, p-value<0.01). According to a GREAT analysis, each of the identified regions exhibited multiple gene ontology associations. Air pollution subtly alters DNA methylation in sperm, indicating that certain regions of the sperm epigenome may be susceptible to air pollution-induced modification with possible implications for reproductive and offspring health.

Epigenome-wide association study of objectively measured physical activity in peripheral blood leukocytes

BackgroundFew studies have explored the association between DNA methylation and physical activity. The aim of this study was to evaluate the association of objectively measured hours of sedentary behavior (SB) and moderate physical activity (MPA) with DNA methylation. We further aimed to explore the association between SB or MPA related CpG sites and cardiometabolic traits, gene expression, and genetic variation.ResultsFor discovery, we performed cross sectional analyses in pregnant women from the Epigenetics in pregnancy (EPIPREG) sample with both DNA methylation (Illumina MethylationEPIC BeadChip) and objectively measured physical activity data (SenseWear™ Pro 3 armband) (European = 244, South Asian = 109). For EWAS of SB and MPA, two main models were designed: model (1) a linear mixed model adjusted for age, smoking, blood cell composition, including ancestry as random intercept, and model (2) which was additionally adjusted for the total number of steps per day. In model 1, we did not identify any CpG sites associated with neither SB nor MPA. In model 2, SB was positively associated (false discovery rate, FDR < 0.05) with two CpG sites within the VSX1 gene. Both CpG sites were positively associated with BMI and were associated with several genetic variants in cis. MPA was associated with 122 significant CpG sites at FDR < 0.05 (model 2). We further analyzed the ten most statistically significant MPA related CpG sites and found that they presented opposite associations with sedentary behavior and BMI. We were not able to replicate the SB and MPA-related CpG sites in the Avon Longitudinal Study of Parents and Children (ALSPAC). ALSPAC had available objectively measured physical activity data from Actigraph (without steps/day available) and leucocyte DNA methylation data collected during adolescence (n = 408, European).ConclusionThis study suggests associations of objectively measured SB and MPA with maternal DNA methylation in peripheral blood leukocytes, that needs to be confirmed in larger samples of similar study design.

Longitudinal DNA methylation profiles in saliva of offspring from mothers with gestational diabetes: associations with early childhood growth patterns

BackgroundThe prevalence of obesity and type 2 diabetes mellitus (T2DM) is rising globally, particularly among children exposed to adverse intrauterine environments, such as those associated with gestational diabetes mellitus (GDM). Epigenetic modifications, specifically DNA methylation, have emerged as mechanisms by which early environmental exposures can predispose offspring to metabolic diseases. This study aimed to investigate DNA methylation differences in children born to mothers with GDM compared to non-GDM mothers, using saliva samples, and to assess the association of these epigenetic patterns with early growth measurements.MethodsThis study analyzed saliva DNA methylation patterns in 30 children (15 born to GDM mothers and 15 to non-GDM mothers) from the EPIDG cohort. Samples were collected at two time points: 8–10 weeks postpartum and at one year of age. Epigenome-wide analysis of over 850,000 CpG sites was conducted using the Illumina Methylation EPIC Bead Chip. Differential methylation positions (DMPs) were identified with the limma package, using a significance threshold of p < 0.01 and delta β ≥ 5%. Correlation analysis examined associations between methylation and growth variables (weight, height, BMI and annual growth) using Spearman tests.ResultsWe identified 6,968 DMPs at the postpartum stage and 5,132 after one year, with 50 sites remaining differentially methylated over time, 16 of which maintained consistent methylation directionality. Functional analysis linked several of these DMPs to genes involved in inflammation and metabolic processes, including CYTH3 and FARP2, both implicated in growth and metabolic pathways. Significant correlations were found between specific CpG sites and growth-related variables such as weight, head circumference, height, and BMI.ConclusionsThis study’s longitudinal design reveals stable DNA methylation patterns in saliva samples that differentiate GDM-exposed children from controls across the first year of life, highlighting the feasibility of saliva as a minimally invasive biomarker source. The persistence of these epigenetic signatures underscores their potential as early indicators of metabolic risk, offering valuable insights into the long-term impact of maternal GDM on child health. Although the use of saliva offers a practical and non-invasive tool for pediatric epigenetic research, further studies are necessary to validate these findings in larger populations.

Genome-Wide DNA Methylation Confirms Oral Squamous Cell Carcinomas in Proliferative Verrucous Leukoplakia as a Distinct Oral Cancer Subtype: A Case-Control Study.

Oral cancers in patients with proliferative verrucous leukoplakia (PVL-OSCC) exhibit different clinical and prognostic outcomes from those seen in conventional oral squamous cell carcinomas (cOSSCs). The aim of the present study is to compare the genome-wide DNA methylation signatures in fresh frozen tissues between oral squamous cell carcinomas in patients with PVL and cOSCC using the Illumina Infinium MethylationEPIC BeadChip. This case-control study was carried out at the Stomatology and Maxillofacial Surgery Department of the General University Hospital of Valencia. For the epigenomic study, unsupervised exploratory bioinformatic analyses were performed using principal component and heatmap analysis. Supervised differential methylation analyses were conducted using a rank-based regression model and a penalized logistic regression model to identify potential prognostic biomarkers. The unsupervised analyses of the global methylation profiles did not allow us to differentiate between the distinct oral cancer groups. However, the two supervised analyses confirmed the existence of two oral carcinoma phenotypes. We identified 21 differentially methylated CpGs corresponding to 14 genes. Among them, three CpGs had not been previously assigned to any known gene, and the remaining were associated with genes unrelated to oral cancer. The AGL, WRB, and ARL15 genes were identified as potential prognostic biomarkers. This study emphasizes the significant role of epigenetic dysregulation in OSCC, particularly in cases preceded by PVL. We have provided data on differential methylation genes that could be involved in the molecular carcinogenesis of PVL-OSCC.

Hypermethylation of PM20D1 Is Associated With Carotid Bifurcation Intima-Media Thickness in Dominican Republic Families.

Carotid intima-media thickness (IMT) is a measure of atherosclerosis and a predictor of vascular diseases. Traditional vascular risk factors and genetic variants do not completely explain the variation in carotid IMT. We sought to identify epigenetic factors that may contribute to the remaining carotid IMT variability. An epigenome-wide association study was performed in 61 Dominican families with 769 individuals. A cytosine nucleotide that precedes a guanine nucleotide methylation in blood-derived DNA was measured using the Human MethylationEPIC BeadChip. Linear mixed model analyses were performed regressing bifurcation carotid IMT (bIMT) on beta values for cytosine nucleotide that precedes a guanine nucleotide sites, adjusting for covariates, followed by differentially methylated region (DMR) analysis. One-sample Mendelian randomization analysis was conducted to investigate causal associations between DMRs and bIMT. Twenty-five DMRs were associated with bIMT (Sidak P <0.05), with the strongest DMR (Sidak P =2.45×10-17) overlapping with the promoter of PM20D1. All 11 cytosine nucleotides that precede a guanine nucleotide within the PM20D1 DMR were positively associated with bIMT (P=0.0007-0.00006). Methylation of the PM20D1 DMR was associated with cis variants, including rs823154 (β=0.26; P=1.1×10-121). As reported in GTEx (Genotype-Tissue Expression project), rs823154 is an expression quantitative trait locus for PM20D1 in multiple tissues, including the aorta (P=2.3×10-60) and blood (P=4.0×10-73), suggesting that hypermethylation of the PM20D1 DMR directs lower expression of the gene. Mendelian randomization analysis supported a causal role for PM20D1 DMR in bIMT (P=0.049). Our study and previous expression quantitative trait locus studies provide converging evidence that reduced PM20D1 expression via hypermethylation of the promoter is associated with increased atherosclerosis.

Analysis of differentially methylated sites and regions associated with intrauterine transmission of hepatitis B virus in infants.

The goal is to identify methylation sites linked to transmission and their impact on host gene expression and HBV spread, aiming to uncover new molecular targets for preventing and treating intrauterine HBV infection. This study recruited 1205 infants born to HBsAg-positive mothers in Liuzhou City, China, between July 2023 and January 2024. Infants were followed up at 7-12months of age and classified as HBsAg-positive (case, n=5) or HBsAg-negative (control, n=14) based on serological testing. Peripheral blood samples were collected for DNA extraction. DNA methylation profiling was performed using the Illumina Infinium MethylationEPIC BeadChip (850K). Data were processed using the ChAMP package in R, including quality control, normalization, and identification of Differentially Methylated Positions (DMPs) and differentially methylated regions (DMRs). DMPs and DMRs were annotated using ANNOVAR 2018Apr16, and GO enrichment analysis was conducted using DAVID. The study was approved by the Guangxi University of Chinese Medicine Ethics Committee, and informed consent was obtained. We identified 734,978 DMPs and 660 DMRs, with 1813 DMPs and 221 DMRs showing significant differences between groups. HBV-infected infants exhibited lower overall genomic methylation levels, with significant concentrations in gene body regions and CpG islands. GO enrichment analysis indicated that differentially methylated genes were enriched in processes related to cell adhesion and calcium ion binding. Prenatal HBV exposure was associated with significant infant hypomethylation, particularly in regulatory regions like TSS1500, TSS200, and CpG islands, potentially impacting gene expression. Enrichment of immune-related pathways among differentially methylated genes suggests that HBV may alter infant immune development through epigenetic modifications.

Childhood trauma is linked to epigenetic age deceleration in young adults with previous youth residential care placements

ABSTRACT Background: Early adversity increases the risk for mental and physical disorders as well as premature death. Epigenetic processes, and altered epigenetic aging in particular, might mediate these effects. While the literature that examined links between early adversity and epigenetic aging is growing, results have been heterogeneous. Objective: In the current work, we explored the link between early adversity and epigenetic aging in a sample of formerly out-of-home placed young adults. Method: A total of N = 117 young adults (32% women, age mean = 26.3 years, SD = 3.6 years) with previous youth residential care placements completed the Childhood Trauma Questionnaire (CTQ) and the Life Events Checklist (LEC-R) and provided blood samples for the analysis of DNA methylation using the Illumina Infinium MethylationEPIC BeadChip Microarray. Epigenetic age was estimated using Hovarth’s and Hannum’s epigenetic clocks. Furthermore, Hovarth’s and Hannum’s epigenetic age residuals were calculated as a proxy of epigenetic aging by regressing epigenetic age on chronological age. The statistical analysis plan was preregistered (https://osf.io/b9ev8). Results: Childhood trauma (CTQ) was negatively associated with Hannum’s epigenetic age residuals, β = −.23, p = .004 when controlling for sex, BMI, smoking status and proportional white blood cell type estimates. This association was driven by experiences of physical neglect, β = −.25, p = .001. Lifetime trauma exposure (LEC-R) was not a significant predictor of epigenetic age residuals. Conclusion: Childhood trauma, and physical neglect in particular, was associated with decelerated epigenetic aging in our sample. More studies focusing on formerly institutionalized at-risk populations are needed to better understand which factors affect stress-related adaptations following traumatic experiences.

Epigenome-wide association studies identify novel DNA methylation sites associated with PTSD: a meta-analysis of 23 military and civilian cohorts

BackgroundThe occurrence of post-traumatic stress disorder (PTSD) following a traumatic event is associated with biological differences that can represent the susceptibility to PTSD, the impact of trauma, or the sequelae of PTSD itself. These effects include differences in DNA methylation (DNAm), an important form of epigenetic gene regulation, at multiple CpG loci across the genome. Moreover, these effects can be shared or specific to both central and peripheral tissues. Here, we aim to identify blood DNAm differences associated with PTSD and characterize the underlying biological mechanisms by examining the extent to which they mirror associations across multiple brain regions.MethodsAs the Psychiatric Genomics Consortium (PGC) PTSD Epigenetics Workgroup, we conducted the largest cross-sectional meta-analysis of epigenome-wide association studies (EWASs) of PTSD to date, involving 5077 participants (2156 PTSD cases and 2921 trauma-exposed controls) from 23 civilian and military studies. PTSD diagnosis assessments were harmonized following the standardized guidelines established by the PGC-PTSD Workgroup. DNAm was assayed from blood using Illumina HumanMethylation450 or MethylationEPIC (850 K) BeadChips. Within each cohort, DNA methylation was regressed on PTSD, sex (if applicable), age, blood cell proportions, and ancestry. An inverse variance-weighted meta-analysis was performed. We conducted replication analyses in tissue from multiple brain regions, neuronal nuclei, and a cellular model of prolonged stress.ResultsWe identified 11 CpG sites associated with PTSD in the overall meta-analysis (1.44e − 09 < p < 5.30e − 08), as well as 14 associated in analyses of specific strata (military vs civilian cohort, sex, and ancestry), including CpGs in AHRR and CDC42BPB. Many of these loci exhibit blood–brain correlation in methylation levels and cross-tissue associations with PTSD in multiple brain regions. Out of 9 CpGs annotated to a gene expressed in blood, methylation levels at 5 CpGs showed significant correlations with the expression levels of their respective annotated genes.ConclusionsThis study identifies 11 PTSD-associated CpGs and leverages data from postmortem brain samples, GWAS, and genome-wide expression data to interpret the biology underlying these associations and prioritize genes whose regulation differs in those with PTSD.

Impact of combined exercise on blood DNA methylation and physical health in older women with obesity.

This study examined the effects of a 14-week combined exercise program on blood DNA methylation (DNAm) and its potential biological pathways in normal-weight, overweight, and obese older women. A total of 41 participants were assessed at baseline, 7 weeks, and 14 weeks into the training. Their whole-blood DNAm profiles were measured using the Infinitum MethylationEPIC BeadChip, alongside physical and biochemical health evaluations. The results showed notable health improvements, with decreases in blood pressure and cholesterol levels in the overweight and obese groups. Blood triglycerides were reduced only in the overweight group. Physical performance also improved across all groups. At 14 weeks, 1,043 differentially methylated positions (DMPs) were identified, affecting 744 genes. The genes were linked to biological processes, such as cellular metabolism, with significant pathway enrichment related to oxidative phosphorylation and chemical carcinogenesis. Additionally, the overweight group experienced significant reductions in methylation levels at eight lipogenesis-related genes. Protein EpiScore analysis revealed decreased levels of CCL11, VEGFA, and NTRK3 proteins at 14 weeks compared to baseline. Despite these significant molecular changes, there was no observable difference in DNAm age after the intervention. This study highlights how combined exercise can modify DNAm patterns in older women, particularly in lipogenesis-related genes, but suggests that further research is needed to understand the full implications for biological ageing.

Genome-wide methylome-based molecular pathologies associated with depression and suicide.

Major depressive disorder (MDD) is a debilitating disorder. Suicide attempts are 5-times higher in MDD patients than in the general population. Interestingly, not all MDD patients develop suicidal thoughts or complete suicide. Thus, it is important to study the risk factors that can distinguish suicidality among MDD patients. The present study examined if DNA methylation changes can distinguish suicidal behavior among depressed subjects. Genome-wide DNA methylation was examined in the dorsolateral prefrontal cortex of depressed suicide (MDD+S; n = 15), depressed non-suicide (MDD-S; n = 17), and nonpsychiatric control (C; n = 16) subjects using 850 K Infinium Methylation EPIC BeadChip. The significantly differentially methylated genes were used to determine the functional enrichment of genes for ontological clustering and pathway analysis. Based on the number of CpG content and their relative distribution from specific landmark regions of genes, 32,958 methylation sites were identified across 12,574 genes in C vs. MDD+/-S subjects, 30,852 methylation sites across 12,019 genes in C vs. MDD-S, 41,648 methylation sites across 13,941 genes in C vs. MDD+S, and 49,848 methylation sites across 15,015 genes in MDD-S vs. MDD+S groups. A comparison of methylation sites showed 33,129 unique methylation sites and 5451 genes in the MDD-S group compared to the MDD+S group. Functional analysis suggested oxytocin, GABA, VGFA, TNFA, and mTOR pathways associated with suicide in the MDD group. Altogether, our data show a distinct pattern of DNA methylation, the genomic distribution of differentially methylated sites, gene enrichment, and pathways in MDD suicide compared to non-suicide MDD subjects.

Smoking-independent DNA methylation markers for lung cancer risk: External validation in a large population-based cohort study.

Smoking-associated epigenetic changes have been linked to lung cancer (LC) risk; however, the role of epigenetic alterations independent of smoking is yet to be fully understood. This study aimed to validate 16 previously reported CpG sites that are independent of smoking yet associated with LC risk within a population-based prospective cohort. Using the Infinium Methylation EPIC BeadChip kit or the Infinium HumanMethylation450K BeadChip Assay, DNA methylation (DNAm) in whole blood was assessed in four subsets (n = 736, 1027, 997, and 312) of a population-based cohort from Germany. The DNAm levels of the 16 smoking-independent CpG sites were analyzed. Hazard ratios (HRs) and their 95% confidence intervals (95% CIs) were calculated to assess associations of DNAm at the 16 CpG sites with LC risk, adjusting for multiple covariates, including smoking habits and a smoking-associated DNAm score. Over 17 years of follow-up, a total of 199 LCs were observed. Among the 16 CpGs, cg02211449 showed a negative association with LC risk (HR [95% CI] per SD increase, = 0.70 [0.63-0.78]), while cg11385536 (1.04 [1.01-1.07]), cg09736286 (1.64 [1.10-2.44]), cg19907023 (1.64 [1.01-2.66]), and cg22032485 (1.52 [1.04-2.21]) displayed positive associations with LC risk. Five of the 16 suggested smoking-independent CpGs could be externally validated as predictors of LC risk. Further research should address their potential contribution to enhanced LC risk stratification.

Pre-operative DNA methylation marks as predictors of weight loss outcomes after sleeve gastrectomy.

Although DNA methylation has been suggested to be a potential predictor of the progression of obesity and obesity-related diseases, little is known about its potential role as predictive marker of successful weight loss after bariatric surgery. 20 patients who underwent sleeve gastrectomy were classified according to the percentage of excess weight loss (%EWL) 1 year after bariatric surgery, using 60% as the cut-off point. Blood DNA methylation was analyzed prior to surgery using the Infinium Methylation EPIC Bead Chip array-based platform. A total number of 76,559 differentially methylated positions (DMPs) (p<0.05) were found between <60% EWL and >60% EWL groups. Of them, 59,308 DMPs were annotated to genes. KEGG enrichment analysis showed that pathways involved in the signalling of MAPK, Wnt, mTor, FoxO and AMPK, among others, were involved in weight loss trajectory. A stepwise logistic regression using the DMPs with an absolute Δβ >0.2 showed that higher methylation levels in the CpG sites cg02405213 (mapping to JAK2) (OR: 1.20098, [0.9586, 1.5044]) and cg01702330 (OR: 2.4426, [0.5761, 10.3567]), were shown to be associated with a higher probability of achieving >60 %EWL after sleeve gastrectomy, whereas higher methylation levels in the CpG site cg04863892 (mapping to HOXA5) were associated with lower probability of achieving >60 %EWL after sleeve gastrectomy (OR: 0.7966, [0.5637, 1.1259]). Our results show a different pre-surgery methylation pattern according to %EWL. We identified three CpG sites (cg04863892, cg02405213, cg01702330) with potential value as predictor markers of weight loss response to bariatric surgery.

Parental arsenic exposure and tissue-specific DNA methylation in Bangladeshi infants with spina bifida.

An emerging hypothesis linking arsenic toxicity involves altered epigenetic mechanisms, such as DNA methylation. In this study, we examined the relationship between parents' arsenic exposure and DNA methylation in tissues obtained from 28 infants with spina bifida from Bangladesh. We analyzed arsenic in parents' toenails using inductively coupled plasma mass spectrometry (ICP-MS). DNA methylation was measured in infants' dural tissue, buccal swabs, and whole blood using the Illumina Infinium MethylationEPIC BeadChip. We performed epigenome-wide association analyses (EWAS) and tested differentially methylated regions (DMRs). In EWAS, DNA methylation at cg24039697 in dural tissue was positively associated (β = 0.59, p = 7.6 × 10-9) with father's toenail arsenic concentrations, adjusting for covariates. We did not identify any CpG sites related to father's arsenic exposure in the other tissues, or any CpG sites related to mother's arsenic exposure. Gene ontology analysis identified many biological pathways of interest, including the Wnt signaling pathways. We identified several DMRs across the tissues related to arsenic exposure that included probes mapping to genes that have previously been identified in studies of neural tube defects. This study emphasizes the potential impact of arsenic exposure in fathers, often understudied in epidemiological studies, on DNA methylation in a unique neurological tissue specific to spina bifida.

Accelerated midlife epigenetic age associates with poor later life brain health in the 1946 British Birth Cohort

AbstractBackgroundAccelerated epigenetic ageing has been associated with various age‐related health outcomes, but its relevance for dementia risk prediction is unclear. We investigated whether accelerated midlife epigenetic age associates with poor later‐life brain health.MethodsParticipants were 230 individuals from Insight 46, drawn from the 1946 British Birth Cohort, a population‐based study of individuals born in the first week of March 1946. DNA methylation was measured at 53 years using Infinium MethylationEPIC BeadChip and GrimAge and PhenoAge were calculated. ‘Age acceleration’ was derived for each clock (AgeAccelGrim and AgeAccelPheno), as previously. At 69‐71 years, we measured MRI whole brain and white matter hyperintensity volume, amyloid‐PET burden, plasma neurofilament light, and MRI Brain Age (with chronological age subtracted to produce the Brain‐Predicted Age Difference/Brain‐PAD). For n=176 with follow‐up MRI aged 72‐75 (n=176), whole brain atrophy was calculated using the boundary shift integral. CSF p‐tau181 was measured in n=64.ResultsDespite participants’ highly similar chronological age (mean=53.4 years, SD=0.2), there was considerable variation in AgeAccelGrim (SD=4.8yr) and AgeAccelPheno (SD=5.6yr). Linear regression revealed that accelerated GrimAge was associated with smaller later‐life whole brain volume, (b=‐1.9ml, p=0.010), whilst accelerated PhenoAge was associated with greater whole brain atrophy (b=0.403, p=0.001). Neither GrimAge nor PhenoAge acceleration was associated with amyloid‐PET burden. Accelerated PhenoAge was associated with higher white matter hyperintensity burden (b=0.03, p=0.04). Acceleration in both GrimAge (b=0.4pg/ml, p=0.001) and PhenoAge (b=0.4pg/ml, p=0.001) was linked to higher plasma neurofilament light, and both AgeAccelGrim (r=0.32, p=0.010) and AgeAccelPheno (r=0.35, p=0.005) were positively correlated with CSF p‐Tau181. MRI brain age (Brain‐PAD) was significantly correlated with AgeAccelGrim (r=0.15, p=0.02) but not AgeAccelPheno (r=0.08, p=0.2).ConclusionAcceleration of midlife epigenetic age was associated with a wide range of features of poor brain health ∼20 years later, in a population‐based sample. Epigenetic clocks may hold promise as tools for future brain health prediction.

War Exposure and DNA Methylation in Syrian Refugee Children and Adolescents

Exposure to war is associated with poor mental health outcomes. Adverse and traumatic experiences can lead to long-lasting DNA methylation changes, potentially mediating the link between adversity and mental health. To date, limited studies have investigated the impact of war on DNA methylation in children or adolescents, hampering our understanding of the biological impact of war exposure. To identify salivary DNA methylation differences associated with war exposure in refugee children and adolescents. This cohort study included Syrian refugee children and adolescents, and their primary caregiver were recruited from tented settlements in Lebanon. Data collection was carried out in 2 waves, 1 year apart, from October 2017 to January 2018 and October 2018 to January 2019. Children and their caregiver were interviewed, and children provided saliva samples for DNA extraction. Data analysis was conducted in 2022, 2023, and 2024. War exposure assessed by interviewing children and their caregiver using the War Events Questionnaire. Salivary DNA methylation levels were assayed with the Infinium MethylationEPIC BeadChip (Illumina). Epigenetic aging acceleration was estimated using a set of preexisting epigenetic aging clocks. A literature search was conducted to identify previously reported DNA methylation correlates of childhood trauma. The study population included 1507 children and adolescents (mean [SD] age, 11.3 [2.4] years; age range, 6-19 years; 793 female [52.6%]). A total of 1449 children provided saliva samples for DNA extraction in year 1, and 872 children provided samples in year 2. Children who reported war events had a number of differentially methylated sites and regions. Enrichment analyses indicated an enrichment of gene sets associated with transmembrane transport, neurotransmission, and intracellular movement in genes that exhibited differential methylation. Sex-stratified analyses found a number of sex-specific DNA methylation differences associated with war exposure. Only 2 of 258 (0.8%) previously reported trauma-associated DNA methylation sites were associated with war exposure (B = -0.004; 95% CI, -0.005 to -0.003; Bonferroni P = .04 and B = -0.005; 95% CI, -0.006 to -0.004; Bonferroni P = .03). Any war exposure or bombardment was nominally associated with decreased epigenetic age using the Horvath multitissue clock (B = -0.39; 95% CI, -0.63 to -0.14; P = .007 and B = -0.42; 95% CI, -0.73 to -0.11; P = .002). In this cohort of Syrian refugee children and adolescents, war exposure was associated with a small number of distinct differences in salivary DNA methylation.

DNA methylation biomarker panels for differentiating various liver adenocarcinomas, including hepatocellular carcinoma, cholangiocarcinoma, colorectal liver metastases and pancreatic adenocarcinoma liver metastases

BackgroundDNA methylation biomarkers are one of the most promising tools for the diagnosis and differentiation of adenocarcinomas of the liver, which are among the most common malignancies worldwide. Their differentiation is important because of the different prognoses and treatment options. This study aimed to validate previously identified DNA methylation biomarkers that successfully differentiate between liver adenocarcinomas, including the two most common primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), as well as two common metastatic liver cancers, colorectal liver metastases (CRLM) and pancreatic ductal adenocarcinoma liver metastases (PCLM), and translate them to the methylation-sensitive high-resolution melting (MS-HRM) and digital PCR (dPCR) platforms.MethodsOur study included a cohort of 149 formalin-fixed, paraffin-embedded tissue samples, including 19 CRLMs, 10 PCLMs, 15 HCCs, 15 CCAs, 15 colorectal adenocarcinomas (CRCs), 15 pancreatic ductal adenocarcinomas (PDACs) and their paired normal tissue samples. The methylation status of the samples was experimentally determined by MS-HRM and methylation-specific dPCR. Previously determined methylation threshold were adjusted according to dPCR data and applied to the same DNA methylation array datasets (provided by The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO)) used to originally identify the biomarkers for the included cancer types and additional CRLM projects. The sensitivities, specificities and diagnostic accuracies of the panels for individual cancer types were calculated.ResultsIn the dPCR experiment, the DNA methylation panels identified HCC, CCA, CRC, PDAC, CRLM and PCLM with sensitivities of 100%, 66.7%, 100%, 86.7%, 94.7% and 80%, respectively. The panels differentiate between HCC, CCA, CRLM, PCLM and healthy liver tissue with specificities of 100%, 100%, 97.1% and 94.9% and with diagnostic accuracies of 100%, 94%, 97% and 93%, respectively. Reevaluation of the same bioinformatic data with new additional CRLM projects demonstrated that the lower dPCR methylation threshold still effectively differentiates between the included cancer types. The bioinformatic data achieved sensitivities for HCC, CCA, CRC, PDAC, CRLM and PCLM of 88%, 64%, 97.4%, 75.5%, 80% and 84.6%, respectively. Specificities between HCC, CCA, CRLM, PCLM and healthy liver tissue were 98%, 93%, 86.6% and 98.2% and the diagnostic accuracies were 94%, 91%, 86% and 98%, respectively. Moreover, we confirmed that the methylation of the investigated promoters is preserved from primary CRC and PDAC to their liver metastases.ConclusionsThe cancer-specific methylation biomarker panels exhibit high sensitivities, specificities and diagnostic accuracies and enable differentiation between primary and metastatic adenocarcinomas of the liver using methylation-specific dPCR. High concordance was achieved between MS-HRM, dPCR and bioinformatic data, demonstrating the successful translation of bioinformatically identified methylation biomarkers from the Illumina Infinium HumanMethylation450 BeadChip (HM450) and lllumina MethylationEPIC BeadChip (EPIC) platforms to the simpler MS-HRM and dPCR platforms.

DNA Methylation Signatures Characterize Gene Expression Modulation in Lung Cancer Patients Affected by Anorexia.

The pathophysiology of cancer anorexia is multifactorial and unclear. Transcriptomic analysis from PBMCs RNA showed diverse patterns of gene expression pathways in anorexic cancer patients. We assessed whether the different transcriptomic signatures are modulated by DNA methylation in lung cancer patients presenting with poor appetite. Lung cancer patients and controls were enrolled, and anorexia was assessed by the FAACT-score questionnaire. Genome-wide DNA methylation was determined by Human Infinium MethylationEPIC BeadChip Kit. Data from genome-wide methylation analysis were merged with those from gene expression analysis, previously obtained by RNA sequencing (NGS). Four groups of genes were identified for each comparison: hypermethylated repressed, hypermethylated induced, hypomethylated repressed, and hypomethylated induced. Cancer patients (n = 16) showed 382 differentially methylated genes when compared with controls (n = 8). Anorexic patients (n = 8) presented 586 hypomethylated and 174 hypermethylated genes compared with controls. In anorexic patients vs. non-anorexic (n = 8), 211 genes were identified as hypomethylated and 90 hypermethylated. When microarray methylation data were merged with transcriptomic data by RNA sequencing, we observed significant differences in anorexic patients vs. controls; a total of 42 genes resulted as hypomethylated and induced, 5 hypermethylated repressed, 10 hypermethylated induced, and 15 hypomethylated repressed. The CG sites analyzed by targeted bisulfite NGS in four genes of interest (FLNA, PGRMC1, GNL3L, and FHL1) resulting as hypomethylated in anorexic vs. controls allowed the validation of the data obtained from DNA methylation. Interestingly, the four genes resulted as hypomethylated in anorexic patients vs. non-anorexic patients and vs. controls (p < 0.0001). Our data support that methylation is implicated in cancer-associated anorexia and nutritional derangements among lung cancer patients.

Epigenetic Study of Cohort of Monozygotic Twins With Hypertrophic Cardiomyopathy Due to MYBPC3 (Cardiac Myosin-Binding Protein C).

Hypertrophic cardiomyopathy is an autosomal dominant cardiac disease. The mechanisms that determine its variable expressivity are poorly understood. Epigenetics could play a crucial role in bridging the gap between genotype and phenotype by orchestrating the interplay between the environment and the genome regulation. In this study we aimed to establish a possible correlation between the peripheral blood DNA methylation patterns and left ventricular hypertrophy severity in patients with hypertrophic cardiomyopathy, evaluating the potential impact of lifestyle variables and providing a biological context to the observed changes. Methylation data were obtained from peripheral blood samples (Infinium MethylationEPIC BeadChip arrays). We employed multiple pair-matched models to extract genomic positions whose methylation correlates with the degree of left ventricular hypertrophy in 3 monozygotic twin pairs carrying the same founder pathogenic variant (MYBPC3 p.Gly263Ter). This model enables the isolation of the environmental influence, beyond age, on DNA methylation changes by removing the genetic background. Our results revealed a more anxious personality among more severely affected individuals. We identified 56 differentially methylated positions that exhibited moderate, proportional changes in methylation associated with left ventricular hypertrophy. These differentially methylated positions were enriched in regions regulated by repressor histone marks and tended to cluster at genes involved in left ventricular hypertrophy development, such as HOXA5, TRPC3, UCN3, or PLSCR2, suggesting that changes in peripheral blood may reflect myocardial alterations. We present a unique pair-matched model, based on 3 monozygotic twin pairs carrying the same founder pathogenic variant and different phenotypes. This study provides further evidence of the pivotal role of epigenetics in hypertrophic cardiomyopathy variable expressivity.

Epigenetic associations with kidney disease in individuals of African ancestry with APOL1 high-risk genotypes and Human Immunodeficiency Virus.

Apolipoprotein L1 (APOL1) high-risk variants are major determinants of chronic kidney disease (CKD) in people of African ancestry. Previous studies have identified epigenetic changes in relation to kidney function and CKD, but not in individuals with APOL1 high-risk genotypes. We conducted an epigenome-wide analysis of CKD and estimated glomerular filtration rate (eGFR) in in people of African ancestry and APOL1 high-risk genotypes with HIV. DNA methylation profiles from peripheral blood mononuclear cells of 119 individuals with APOL1 high-risk genotypes (mean age 48 years, 49% female, median CD4 count 515 cells/mm3, 90% HIV-1 RNA <200 copies/mL, 23% with CKD) were obtained by Illumina MethylationEPIC BeadChip. Differential methylation analysis of CKD considered technical and biological covariates. We also assessed associations with eGFR. Replication was pursued in three independent multi-ancestry cohorts with and without HIV. DNA methylation levels at 14 regions were associated with CKD. The strongest signals were located in SCARB1, DNAJC5B and C4orf50. Seven of the 14 signals also associated with eGFR, and most showed evidence for a genetic basis. Four signals (in SCARB1, FRMD4A, CSRNP1 and RAB38) replicated in other cohorts, and 11 previously reported epigenetic signals for kidney function or CKD replicated in our cohort. We found no significant DNA methylation signals in, or near, the APOL1 promoter region. We report several novel as well as previously reported epigenetic associations with CKD and eGFR in individuals with HIV having APOL1 high-risk genotypes. Further investigation of pathways linking DNA methylation to APOL1 nephropathies is warranted.