Epigenome-wide Association Studies Research Articles

Effects of highly active antiretroviral therapy initiation on epigenomic DNA methylation in persons living with HIV

Introduction: Highly active antiretroviral therapy (HAART) helps improve some measures of accelerated epigenetic aging in persons living with HIV (PLWH), but its overall impact on the epigenome is not fully understood.Methods: In this study, we analyzed the DNA methylation profiles of PLWH (n = 187) shortly before and approximately 2–3 years after they started HAART, as well as matched seronegative (SN) controls (n = 187), taken at two time intervals. Our aim was to identify specific CpGs and biologic pathways associated with HIV infection and initiation of HAART. Additionally, we attempted to identify epigenetic changes associated with HAART initiation that were independent of HIV-associated changes, using matched HIV seronegative (SN) controls (matched on age, hepatitis C status, and interval between visits) to identify CpGs that did not differ between PLWH and SN pre-HAART but were significantly associated with HAART initiation while being unrelated to HIV viral load. Epigenome-wide association studies (EWAS) on >850,000 CpG sites were performed using pre- and post-HAART samples from PLWH. The results were then annotated using the Genomic Regions Enrichment of Annotations Tool (GREAT).Results: When only pre- and post-HAART visits in PLWH were compared, gene ontologies related to immune function and diseases related to immune function were significant, though with less significance for PLWH with detectable HIV viral loads (>50 copies/mL) at the post-HAART visit. To specifically elucidate the effects of HAART separately from HIV-induced methylation changes, we performed EWAS of HAART while also controlling for HIV viral load, and found gene ontologies associated with transplant rejection, transplant-related diseases, and other immunologic signatures. Additionally, we performed a more focused analysis that examined CpGs reaching genome-wide significance (p < 1 × 10−7) from the viral load-controlled EWAS that did not differ between all PLWH and matched SN controls pre-HAART. These CpGs were found to be near genes that play a role in retroviral drug metabolism, diffuse large B cell lymphoma proliferation, and gastric cancer metastasis.Discussion: Overall, this study provides insight into potential biological functions associated with DNA methylation changes induced by HAART initiation in persons living with HIV.

#2056 Epigenetic profiles differ by level of kidney function in idiopathic nephrotic syndrome

Abstract Background and Aims DNA methylation (DNAm) is an epigenetic mechanism which can therefore induce changes in gene expression without any change to the underlying DNA sequence. Although generally quite stable, DNAm patterns in whole blood do vary across individuals, with DNAm levels associating with age and responding to environmental factors. We aimed to investigate whether peripheral blood cell DNAm associates with level of kidney function in patients with Idiopathic Nephrotic Syndrome (INS). Method Peripheral blood cell DNAm values were generated using the Illumina MethylationEPIC Beadchip (>850,000 CpG sites) for 293 INS patients recruited to the National Unified Renal Translational Research Enterprise–National Study of INS (NURTuRE- INS and NephroS). All patients were younger than 30 years old at INS diagnosis. An Epigenome Wide Association Study (EWAS) was performed using generalised linear models to evaluate associations between DNAm sites and estimated glomerular filtration rate (eGFR). Regions of differentially methylated sites were identified using the ‘dmrff’ R package. Sex chromosomes were excluded from the analyses and all analyses were adjusted for estimated cell type proportions, age, sex and technical variation. A Bonferroni adjusted p value of 5.88 × 10−8 was used as a significance threshold to adjust for multiple tests. Results Of the selected 293 INS patients, 173 (59%) were male, 213 (73%) were white and the median age at INS diagnosis was 4 years old (IQR 2-10). One hundred and forty-three (48%) patients were steroid responsive at diagnosis and the remainder were steroid resistant. Sixty-eight of the steroid resistant patients had pathogenic NS variants. We identified 6 CpG sites associated with eGFR (p < 5.88 × 10−8) in this cohort of INS patients, one replicating a previously reported association. We identified 3 differentially methylated regions, one coinciding with the transcriptional start site of an RNA binding protein which regulates alternative splicing, and another located in the body of a transcription factor regulator containing genetic variants associated with chronic kidney disease. Conclusion We have demonstrated variation in peripheral blood DNAm by level of kidney function in children and young adults with INS. Associations between DNAm and eGFR have previously been demonstrated in diabetic kidney disease and heterogenous chronic kidney disease cohorts, including one of the sites which we have identified in our study. Further work is underway to determine if these differences in DNAm are likely to be a cause or consequence of reduced kidney function, which will help to determine their potential clinical utility.

An epigenome-wide association study of waist circumference in Chinese monozygotic twins.

Central obesity poses significant health risks because it increases susceptibility to multiple chronic diseases. Epigenetic features such as DNA methylation may be associated with specific obesity traits, which could help us understand how genetic and environmental factors interact to influence the development of obesity. This study aims to identify DNA methylation sites associated with the waist circumference (WC) in Northern Han Chinese population, and to elucidate potential causal relationships. A total of 59 pairs of WC discordant monozygotic twins (ΔWC >0) were selected from the Qingdao Twin Registry in China. Generalized estimated equation model was employed to estimate the methylation levels of CpG sites on WC. Causal relationships between methylation and WC were assessed through the examination of family confounding factors using FAmiliaL CONfounding (ICE FALCON). Additionally, the findings of the epigenome-wide analysis were corroborated in the validation stage. We identified 26 CpG sites with differential methylation reached false discovery rate (FDR) < 0.05 and 22 differentially methylated regions (slk-corrected p < 0.05) strongly linked to WC. These findings provided annotations for 26 genes, with notable emphasis on MMP17, ITGA11, COL23A1, TFPI, A2ML1-AS1, MRGPRE, C2orf82, and NINJ2. ICE FALCON analysis indicated the DNA methylation of ITGA11 and TFPI had a causal effect on WC and vice versa (p < 0.05). Subsequent validation analysis successfully replicated 10 (p < 0.05) out of the 26 identified sites. Our research has ascertained an association between specific epigenetic variations and WC in the Northern Han Chinese population. These DNA methylation features can offer fresh insights into the epigenetic regulation of obesity and WC as well as hints to plausible biological mechanisms.

O03 Genetic impacts on the skin methylome

Abstract Introduction and aims DNA methylation at CpG dinucleotides is a dynamic epigenetic mechanism that has been linked to numerous skin phenotypes and diseases. Previous studies in blood show that a substantial proportion of the human DNA methylome exhibits evidence for genetic influence, and that 15–17% of narrow-sense methylation heritability is explained by genetic variants, or methylation quantitative trait loci (meQTLs). However, no genome-wide meQTL studies have previously been performed in skin. The primary purpose of this work was to identify the genetic basis of DNA methylation levels in whole skin. Methods Utilizing a twin-based heritability model, we estimated DNA methylation heritability using 414 bulk skin tissue samples from healthy older female twins from the TwinsUK cohort, profiled using the Illumina Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA). We then integrated genomic data for 394 of these twins to perform genome-wide meQTL analyses. Finally, we integrated these meQTL Results with skin eQTL Results from an overlapping sample (N = 664). Results Over 8.2% of measured CpG sites showed evidence for strong heritability (h2 &amp;gt; 0.4), with a mean additive heritability estimate of 11.07% across all tested sites. We identified 76 442 CpG sites (18.8% of measured CpG sites) associated with a meQTL in cis (&amp;lt; 1 MB variant-CpG distance) and 1438 (0.35%) in trans. Both CpG sites under meQTL effects, and genetic variants underlying these meQTL effects in skin, are enriched in regions previously identified from genome-wide and epigenome-wide association studies for skin-related phenotypes, including psoriasis, melanoma and vitiligo. Additionally, some meQTLs influence gene expression via mediation of expression quantitative trait loci effects in skin, including in genes linked to melanoma and psoriasis. Conclusions Our study is the first genome-wide DNA methylation heritability and meQTL analysis of whole skin, giving insights into the regulatory landscape of epigenomic variation in skin.

Mitochondrial DNA: Inherent Complexities Relevant to Genetic Analyses.

Mitochondrial DNA (mtDNA) exhibits distinct characteristics distinguishing it from the nuclear genome, necessitating specific analytical methods in genetic studies. This comprehensive review explores the complex role of mtDNA in a variety of genetic studies, including genome-wide, epigenome-wide, and phenome-wide association studies, with a focus on its implications for human traits and diseases. Here, we discuss the structure and gene-encoding properties of mtDNA, along with the influence of environmental factors and epigenetic modifications on its function and variability. Particularly significant are the challenges posed by mtDNA's high mutation rate, heteroplasmy, and copy number variations, and their impact on disease susceptibility and population genetic analyses. The review also highlights recent advances in methodological approaches that enhance our understanding of mtDNA associations, advocating for refined genetic research techniques that accommodate its complexities. By providing a comprehensive overview of the intricacies of mtDNA, this paper underscores the need for an integrated approach to genetic studies that considers the unique properties of mitochondrial genetics. Our findings aim to inform future research and encourage the development of innovative methodologies to better interpret the broad implications of mtDNA in human health and disease.

Neuroimaging and epigenetic analysis reveal novel epigenetic loci in major depressive disorder.

Epigenetic modifications, such as DNA methylation, contribute to the pathophysiology of major depressive disorder (MDD). This study aimed to identify novel MDD-associated epigenetic loci using DNA methylation profiles and explore the correlations between epigenetic loci and cortical thickness changes in patients with MDD. A total of 350 patients with MDD and 161 healthy controls (HCs) were included in the epigenome-wide association studies (EWAS). We analyzed methylation, copy number alteration (CNA), and gene network profiles in the MDD group. A total of 234 patients with MDD and 135 HCs were included in neuroimaging methylation analysis. Pearson's partial correlation analysis was used to estimate the correlation between cortical thickness of brain regions and DNA methylation levels of the loci. In total, 2018 differentially methylated probes (DMPs) and 351 differentially methylated regions (DMRs) were identified. DMP-related genes were enriched in two networks involved in the central nervous system. In neuroimaging analysis, patients with MDD showed cortical thinning in the prefrontal regions and cortical thickening in several occipital regions. Cortical thickness of the left ventrolateral prefrontal cortex (VLPFC, i.e. pars triangularis) was negatively correlated with eight DMPs associated with six genes (EML6, ZFP64, CLSTN3, KCNMA1, TAOK2, and NT5E). Through combining DNA methylation and neuroimaging analyses, negative correlations were identified between the cortical thickness of the left VLPFC and DNA methylation levels of eight DMPs. Our findings could improve our understanding of the pathophysiology of MDD.

Epigenome-wide association study of lung cancer among never smokers in two prospective cohorts in Shanghai, China

BackgroundThe aetiology of lung cancer among individuals who never smoked remains elusive, despite 15% of lung cancer cases in men and 53% in women worldwide being unrelated to smoking. Epigenetic...

Placental DNA methylation signatures of prenatal air pollution exposure and potential effects on birth outcomes: an analysis of three prospective cohorts.

Pregnancy air pollution exposure (PAPE) has been linked to a wide range of adverse birth and childhood outcomes, but there is a paucity of data on its influence on the placental epigenome, which can regulate the programming of physiological functions and affect child development. This study aimed to investigate the association between prenatal air pollutant exposure concentrations and changes in placental DNA methylation patterns, and to explore the potential windows of susceptibility and sex-specific alterations. This multi-site study used three prospective population-based mother-child cohorts: EDEN, PELAGIE, and SEPAGES, originating from four French geographical regions (Nancy, Poitiers, Brittany, and Grenoble). Pregnant women were included between 2003 and 2006 for EDEN and PELAGIE, and between 2014 and 2017 for SEPAGES. The main eligibility criteria were: being older than 18 years, having a singleton pregnancy, and living and planning to deliver in one of the maternity clinics in one of the study areas. A total of 1539 mother-child pairs were analysed, measuring placental DNA methylation using Illumina BeadChips. We used validated spatiotemporally resolved models to estimate PM2·5, PM10, and NO2 exposure over each trimester of pregnancy at the maternal residential address. We conducted a pooled adjusted epigenome-wide association study to identify differentially methylated 5'-C-phosphate-G-3' (CpG) sites and regions (assessed using the Infinium HumanMethylationEPIC BeadChip array, n=871), including sex-specific and sex-linked alterations, and independently validated our results (assessed using the Infinium HumanMethylation450 BeadChip array, n=668). We identified four CpGs and 28 regions associated with PAPE in the total population, 469 CpGs and 87 regions in male infants, and 150 CpGs and 66 regions in female infants. We validated 35% of the CpGs available. More than 30% of the identified CpGs were related to one (or more) birth outcome and most significant alterations were enriched for neural development, immunity, and metabolism related genes. The 28 regions identified for both sexes overlapped with imprinted genes (four genes), and were associated with neurodevelopment (nine genes), immune system (seven genes), and metabolism (five genes). Most associations were observed for the third trimester for female infants (134 of 150 CpGs), and throughout pregnancy (281 of 469 CpGs) and the first trimester (237 of 469 CpGs) for male infants. These findings highlight the molecular pathways through which PAPE might affect child health in a widespread and sex-specific manner, identifying the genes involved in the major physiological functions of a developing child. Further studies are needed to elucidate whether these epigenetic changes persist and affect health later in life. French Agency for National Research, Fondation pour la Recherche Médicale, Fondation de France, and the Plan Cancer.

Genome- and epigenome-wide association studies identify susceptibility of CpG sites and regions for metabolic syndrome in a Korean population

BackgroundWhile multiple studies have investigated the relationship between metabolic syndrome (MetS) and its related traits (fasting glucose, triglyceride, HDL cholesterol, blood pressure, waist circumference) and DNA methylation, our understanding of the epigenetic mechanisms in MetS remains limited. Therefore, we performed an epigenome-wide meta-analysis of blood DNA methylation to identify differentially methylated probes (DMPs) and differentially methylated regions (DMRs) associated with MetS and its components using two independent cohorts comprising a total of 2,334 participants. We also investigated the specific genetic effects on DNA methylation, identified methylation quantitative trait loci (meQTLs) through genome-wide association studies and further utilized Mendelian randomization (MR) to assess how these meQTLs subsequently influence MetS status.ResultsWe identified 40 DMPs and 27 DMRs that are significantly associated with MetS. In addition, we identified many novel DMPs and DMRs underlying inflammatory and steroid hormonal processes. The most significant associations were observed in 3 DMPs (cg19693031, cg26974062, cg02988288) and a DMR (chr1:145440444–145441553) at the TXNIP, which are involved in lipid metabolism. These CpG sites were identified as coregulators of DNA methylation in MetS, TG and FAG levels. We identified a total of 144 cis-meQTLs, out of which only 13 were found to be associated with DMPs for MetS. Among these, we confirmed the identified causal mediators of genetic effects at CpG sites cg01881899 at ABCG1 and cg00021659 at the TANK genes for MetS.ConclusionsThis study observed whether specific CpGs and methylated regions act independently or are influenced by genetic effects for MetS and its components in the Korean population. These associations between the identified DNA methylation and MetS, along with its individual components, may serve as promising targets for the development of preventive interventions for MetS.

Associations of Heavy Metals with Cognitive Function: An Epigenome-Wide View of DNA Methylation and Mediation Analysis.

Exposure to heavy metals has been reported to be associated with impaired cognitive function, but the underlying mechanisms remain unclear. This pilot study aimed to identify key heavy metal elements associated with cognitive function and further explore the potential mediating role of metal-related DNA methylation. Blood levels of arsenic, cadmium, lead, copper, manganese, and zinc and genome-wide DNA methylations were separately detected in peripheral blood in 155 older adults. Cognitive function was evaluated using the Mini-Mental State Examination (MMSE). Least absolute shrinkage and selection operator penalized regression and Bayesian kernel machine regression were used to identify metals associated with cognitive function. An epigenome-wide association study examined the DNA methylation profile of the identified metal, and mediation analysis investigated its mediating role. The MMSE scores showed a significant decrease of 1.61 (95% confidence interval [CI]: -2.64, -0.59) with each 1 standard deviation increase in ln-transformed arsenic level; this association was significant in multiple-metal models and dominated the overall negative effect of 6 heavy metal mixture on cognitive function. Seventy-three differentially methylated positions were associated with blood arsenic (p < 1.0 × 10-5). The methylation levels at cg05226051 (annotated to TDRD3) and cg18886932 (annotated to GAL3ST3) mediated 24.8% and 25.5% of the association between blood arsenic and cognitive function, respectively (all p < 0.05). Blood arsenic levels displayed a negative association with the cognitive function of older adults. This finding shows that arsenic-related DNA methylation alterations are critical partial mediators that may serve as potential biomarkers for further mechanism-related studies. ANN NEUROL 2024.

The association between neighborhood deprivation and DNA methylation in an autopsy cohort.

Previous research has found that living in a disadvantaged neighborhood is associated with poor health outcomes. Living in disadvantaged neighborhoods may alter inflammation and immune response in the body, which could be reflected in epigenetic mechanisms such as DNA methylation (DNAm). We used robust linear regression models to conduct an epigenome-wide association study examining the association between neighborhood deprivation (Area Deprivation Index; ADI), and DNAm in brain tissue from 159 donors enrolled in the Emory Goizueta Alzheimer's Disease Research Center (Georgia, USA). We found one CpG site (cg26514961, gene PLXNC1) significantly associated with ADI after controlling for covariates and multiple testing (p-value=5.0e-8). Effect modification by APOE ε4 was statistically significant for the top ten CpG sites from the EWAS of ADI, indicating that the observed associations between ADI and DNAm were mainly driven by donors who carried at least one APOE ε4 allele. Four of the top ten CpG sites showed a significant concordance between brain tissue and tissues that are easily accessible in living individuals (blood, buccal cells, saliva), including DNAm in cg26514961 (PLXNC1). Our study identified one CpG site (cg26514961, PLXNC1 gene) that was significantly associated with neighborhood deprivation in brain tissue. PLXNC1 is related to immune response, which may be one biological pathway how neighborhood conditions affect health. The concordance between brain and other tissues for our top CpG sites could make them potential candidates for biomarkers in living individuals.

Blood DNA Methylation Analysis Reveals a Distinctive Epigenetic Signature of Vasospasm in Aneurysmal Subarachnoid Hemorrhage.

Vasospasm is a potentially preventable cause of poor prognosis in patients with aneurysmal subarachnoid hemorrhage (aSAH). Epigenetics might provide insight on its molecular mechanisms. We aimed to analyze the association between differential DNA methylation (DNAm) and development of vasospasm. We conducted an epigenome-wide association study in 282 patients with aSAH admitted to our hospital. DNAm was assessed with the EPIC Illumina chip (> 850K CpG sites) in whole-blood samples collected at hospital admission. We identified differentially methylated positions (DMPs) at the CpG level using Cox regression models adjusted for potential confounders, and then we used the DMP results to find differentially methylated regions (DMRs) and enriched biological pathways. A total of 145 patients (51%) experienced vasospasm. In the DMP analysis, we identified 31 CpGs associated with vasospasm at p-value < 10-5. One of them (cg26189827) was significant at the genome-wide level (p-value < 10-8), being hypermethylated in patients with vasospasm and annotated to SUGCT gene, mainly expressed in arteries. Region analysis revealed 13 DMRs, some of them annotated to interesting genes such as POU5F1, HLA-DPA1, RUFY1, and CYP1A1. Functional enrichment analysis showed the involvement of biological processes related to immunity, inflammatory response, oxidative stress, endothelial nitric oxide, and apoptosis. Our findings show, for the first time, a distinctive epigenetic signature of vasospasm in aSAH, establishing novel links with essential biological pathways, including inflammation, immune responses, and oxidative stress. Although further validation is required, our results provide a foundation for future research into the complex pathophysiology of vasospasm.

Assessing the effect of childbearing on blood DNA methylation through comparison of parous and nulliparous females

BackgroundPregnancy and childbirth have been connected to modified risk of a wide variety of conditions in later life, including neurodegenerative disorders and cancers. The presence, extent, and direction of the effect that childbearing status has on decreasing or increasing the risk of these conditions differs depending on the disease. The mechanisms by which pregnancy and childbirth modify the risk of diseases are still unknown. DNA methylation (DNAm) alterations that occur during pregnancy and persist after childbirth may help us understand this phenomenon.ResultsBlood DNAm was available from 89 women (28 parous; 61 nulliparous) at ages 18 and 26 years in the Isle of Wight birth cohort; no significant differences in the population characteristics were present between the analyzed population and the full cohort. We performed an epigenome-wide association study on 389,355 CpGs and identified 184 CpGs to be significantly differentially methylated between parous and nulliparous women after adjusting for confounders and multiple testing. Of these CpGs, 105 had regression coefficients in the same direction in an independent Mexico City based ELEMENT cohort, of which 13 were significant (replication P < 0.05). These 13 CpGs were associated with 16 unique genes. DNAm levels tracked with gene expression in 3 of the replicated genes, one of which (TM2D3) was differentially expressed in parous vs nulliparous women. Gene disease association analysis identified a network of parous-associated diseases.ConclusionsOur results suggest that pregnancy and childbirth lead to DNAm changes in parous women and these changes persist at least 6 months and up to 8 years postpartum. Parous-related CpG sites may play a role in how childbearing status modifies risk of later life diseases in women. Further studies are needed to explore the linkage and mechanism.

Neuronal-specific methylome and hydroxymethylome analysis reveal significant loci associated with alcohol use disorder.

Background: Alcohol use disorder (AUD) is a complex condition associated with adverse health consequences that affect millions of individuals worldwide. Epigenetic modifications, including DNA methylation (5mC), have been associated with AUD and other alcohol-related traits. Epigenome-wide association studies (EWAS) have identified differentially methylated genes associated with AUD in human peripheral and brain tissue. More recently, epigenetic studies of AUD have also evaluated DNA hydroxymethylation (5hmC) in the human brain. However, most of the epigenetic work in postmortem brain tissue has examined bulk tissue. In this study, we investigated neuronal-specific 5mC and 5hmC alterations at CpG sites associated with AUD in the human orbitofrontal cortex (OFC). Methods: Neuronal nuclei from the OFC were evaluated in 34 human postmortem brain samples (10 AUD, 24 non-AUD). Reduced representation oxidative bisulfite sequencing was used to assess 5mC and 5hmC at the genome-wide level. Differential 5mC and 5hmC were evaluated using the methylKit R package and significance was set at false discovery rate < 0.05 and differential methylation > 2. Functional enrichment analyses were performed, and gene-level convergence was evaluated in an independent dataset that assessed 5mC and 5hmC of AUD in bulk cortical tissue. Results: We identified 417 5mC and 3635hmC significant differential CpG sites associated with AUD, with 59% in gene promoters. Some of the identified genes have been previously implicated in alcohol consumption, including SYK, DNMT3A for 5mC, GAD1, DLX1, DLX2, for 5hmC and GATA4 in both. Convergence with a previous AUD 5mC and 5hmC study was observed for 28 genes. We also identified 5 and 35 differential regions for 5mC and 5hmC, respectively. Lastly, GWAS enrichment analysis showed an association with AUD for differential 5mC genes. Discussion: This study reveals neuronal-specific methylome and hydroxymethylome dysregulation associated with AUD, identifying both previously reported and potentially novel gene associations with AUD. Our findings provide new insights into the epigenomic dysregulation of AUD in the human brain.

Epigenome-wide association study identifies DNA methylation loci associated with handgrip strength in Chinese monozygotic twins.

Background: The decline in muscle strength and function with aging is well recognized, but remains poorly characterized at the molecular level. Here, we report the epigenetic relationship between genome-wide DNA methylation and handgrip strength (HGS) among Chinese monozygotic (MZ) twins. Methods: DNA methylation (DNAm) profiling was conducted in whole blood samples through Reduced Representation Bisulfite Sequencing method. Generalized estimating equation was applied to regress the DNAm of each CpG with HGS. The Genomic Regions Enrichment of Annotations Tool was used to perform enrichment analysis. Differentially methylated regions (DMRs) were detected using comb-p. Causal inference was performed using Inference about Causation through Examination of Familial Confounding method. Finally, we validated candidate CpGs in community residents. Results: We identified 25 CpGs reaching genome-wide significance level. These CpGs located in 9 genes, especially FBLN1, RXRA, and ABHD14B. Many enriched terms highlighted calcium channels, neuromuscular junctions, and skeletal muscle organ development. We identified 21 DMRs of HGS, with several DMRs within FBLN1, SLC30A8, CST3, and SOCS3. Causal inference indicated that the DNAm of 16 top CpGs within FBLN1, RXRA, ABHD14B, MFSD6, and TYW1B might influence HGS, while HGS influenced DNAm at two CpGs within FBLN1 and RXRA. In validation analysis, methylation levels of six CpGs mapped to FLBN1 and one CpG mapped to ABHD14B were negatively associated with HGS weakness in community population. Conclusion: Our study identified multiple DNAm variants potentially related to HGS, especially CpGs within FBLN1 and ABHD14B. These findings provide new clues to the epigenetic modification underlying muscle strength decline.

Critical evaluation of the reliability of DNA methylation probes on the Illumina MethylationEPIC v1.0 BeadChip microarrays

ABSTRACT DNA methylation (DNAm) plays a crucial role in a number of complex diseases. However, the reliability of DNAm levels measured using Illumina arrays varies across different probes. Previous research primarily assessed probe reliability by comparing duplicate samples between the 450k-450k or 450k-EPIC platforms, with limited investigations on Illumina EPIC v1.0 arrays. We conducted a comprehensive assessment of the EPIC v1.0 array probe reliability using 69 blood DNA samples, each measured twice, generated by the Alzheimer’s Disease Neuroimaging Initiative study. We observed higher reliability in probes with average methylation beta values of 0.2 to 0.8, and lower reliability in type I probes or those within the promoter and CpG island regions. Importantly, we found that probe reliability has significant implications in the analyses of Epigenome-wide Association Studies (EWAS). Higher reliability is associated with more consistent effect sizes in different studies, the identification of differentially methylated regions (DMRs) and methylation quantitative trait locus (mQTLs), and significant correlations with downstream gene expression. Moreover, blood DNAm measurements obtained from probes with higher reliability are more likely to show concordance with brain DNAm measurements. Our findings, which provide crucial reliability information for probes on the EPIC v1.0 array, will serve as a valuable resource for future DNAm studies.

Abstract 7018: Application of HiTIMED and cell type adjusted epigenome-wide models identifies driver tumor DNA methylation alterations in multiple cancers

Abstract DNA methylation (DNAm) is an epigenetic mark crucial in lineage specification and cellular differentiation, resulting in distinct patterns by cell type. These patterns can serve as biomarkers in deconvolution approaches inferring cell proportions from composite DNAm signals in biospecimens like blood and tumor tissue. Adjusting for cellular heterogeneity is critical to avoid confounding by cell type. The HiTIMED (hierarchical tumor immune microenvironment epigenetic deconvolution) algorithm provides high-resolution tumor microenvironment profiling, up to 17 cell types, including tumor, myeloid, and angiogenic compartments. We analyzed bladder, prostate, and uterine cancers, applying HiTIMED on the tumor (n=1,341) and normal adjacent tissue (n=105), adjusting for age, sex, and cell types. DNAm data from The Cancer Genome Atlas (TCGA) was used, and HiTIMED was applied to deconvolute various cell proportions. Linear model comparisons to identify significantly differentiated loci with/without cell type adjustment are summarized in Table 1. We demonstrate greater than 99% reduction in both hypermethylated and hypomethylated loci in all cancer types after adjustment for cell type. Our study demonstrates HiTIMED's applicability to deconvolve the tumor microenvironment in multiple cancer types and demonstrates the importance of adjusting for cell type in epigenome-wide association studies to identify differentially methylated loci, offering greater biological insight into the etiology of specific cancer types. Significant differentially methylated loci in tumor versus normal adjacent tissue, FDR &amp;lt;0.05 Cancer type Linear model Hypermethylated in tumor (logFC &amp;gt;2.0) Hypomethylated in tumor (logFC &amp;lt; -2.0) Bladder cancer n = 21, normal adjacent tissue n = 413 tumor Model 1 (Cancer Status, Age, Sex) 4495 19,936 Model 2: (Cancer Status, Sex, age, six cell types) 27 39 Prostate adenocarcinoman = 50, normal adjacent tissuen = 499 tumor Model 1 (Cancer Status, Age) 8988 2207 Model 2: (Cancer Status, age, six cell types) 2 4 Uterine carcinoman = 34 normal adjacent tissuen = 429 tumor Model 1 (Cancer Status, Age) 11,698 16,249 Model 2: (Cancer Status, age, six cell types) 250 8245 Citation Format: Irma M. Vlasac, Ze Zhang, Lucas A. Salas, Brock C. Christensen. Application of HiTIMED and cell type adjusted epigenome-wide models identifies driver tumor DNA methylation alterations in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7018.

Abstract P524: Prolonged Mild Sleep Restriction Results in Specific Alterations in Genome-Wide DNA Methylation: A Randomized Crossover Trial

Introduction: Short sleep duration is associated with increased cardiometabolic risk. DNA methylation plays a critical role in the regulation of gene expression and studies suggest that sleep deprivation may alter DNA-methylation patterns. However, most findings are from acute sleep deprivation or short duration studies. Hypothesis: Prolonged sleep restriction study that mimics real-life situations is associated with differentially methylated CpG loci (DML) in the core clock candidate genes and across the epigenome in an epigenome-wide association study (EWAS) Methods: Sixty participants (65% women) aged 21-73 y were included in randomized crossover studies with 2 phases of 6 weeks each. Phases differed in sleep duration: either adequate sleep (AS; sleep ≥7h/night) or sleep restriction (SR; -1.5h/night relative to AS). DNA was isolated from whole blood at wk 0 and wk 6 of each phase. DNA methylation (DNAm) was quantified the Illumina Infinium MethylationEPIC BeadChip array v.2.0 (&gt;900K methylation sites) and Partek Genomic Suite v.720.0831. After quality control and normalization, several global and stratified EWASs were performed to test the impact of sleep condition on DNAm. Analyses were adjusted for week, phase, sex, age, BMI and cell type composition. We also focused on methylation changes of the selected core-clock candidate genes ( RORA, CRY1, NR1D2, TIMELESS, NPAS2, NFIL3, RORB, PER3, CSNK1E, ARNTL2, CRY2, CLOCK, ARNTL, RORC, PER2, BHLHE40, NR1D1, CSNK1D, BHLHE41 and PER1 ). Results: In the candidate gene approach, we detected several significant DML. Top-ranked DML in selected core clock genes with significant interactions between condition and time were: cg02394126 at ARNTL (p&lt;0.001), cg23506964 at CLOCK (p=0.001), cg03701037 at NPAS2 (p=0.009), cg06606972 at NPAS2 (p=0.009), and cg13576304 at RORA (p=0.008). Hypermethylation was primarily observed after 6 wk of SR vs AS for cg02394126, cg23506964, cg03701037, and cg06606972, while hypomethylation was observed cg13576304 ( RORA ). In our EWAS, we detected some suggestive significant condition x time interactions in cg23738833 at SNHG3-RCC1 (p=1,34E-06), cg13280380 in the FAF1 gene (p=2,25E-05), cg03179866 in the MMP12 gene (p=2,78E-05), and cg13063696 (p=3,21E-05). All but cg23738833 ( SNHG3-RCC1 ) showed hypermethylation after 6 wk of SR vs AS. Conclusion: Six weeks of mild SR was associated with changes in DNAm in core-clock candidate genes and in other genes in the EWAS. However, further studies are needed to confirm these findings and determine the functional pathways.

Sex effects on DNA methylation affect discovery in epigenome-wide association study of schizophrenia.

Sex differences in the epidemiology and clinical characteristics of schizophrenia are well-known; however, the molecular mechanisms underlying these differences remain unclear. Further, the potential advantages of sex-stratified meta-analyses of epigenome-wide association studies (EWAS) of schizophrenia have not been investigated. Here, we performed sex-stratified EWAS meta-analyses to investigate whether sex stratification improves discovery, and to identify differentially methylated regions (DMRs) in schizophrenia. Peripheral blood-derived DNA methylation data from 1519 cases of schizophrenia (male n = 989, female n = 530) and 1723 controls (male n = 997, female n = 726) from three publicly available datasets, and the TOP cohort were meta-analyzed to compare sex-specific, sex-stratified, and sex-adjusted EWAS. The predictive power of each model was assessed by polymethylation score (PMS). The number of schizophrenia-associated differentially methylated positions identified was higher for the sex-stratified model than for the sex-adjusted one. We identified 20 schizophrenia-associated DMRs in the sex-stratified analysis. PMS from sex-stratified analysis outperformed that from sex-adjusted analysis in predicting schizophrenia. Notably, PMSs from the sex-stratified and female-only analyses, but not those from sex-adjusted or the male-only analyses, significantly predicted schizophrenia in males. The findings suggest that sex-stratified EWAS meta-analyses improve the identification of schizophrenia-associated epigenetic changes and highlight an interaction between sex and schizophrenia status on DNA methylation. Sex-specific DNA methylation may have potential implications for precision psychiatry and the development of stratified treatments for schizophrenia.

Abstract P141: T2D-DNA Methylation Score and Changes in Glycemic Traits in Response to Weight-Loss Dietary Interventions: The MACRO Trial

Introduction: Epigenome-wide association studies (EWAS) have identified several CpGs associated with type 2 diabetes (T2D). A previous study has shown that a DNA methylation score provided more comprehensive information related to T2D risk than individual CpGs. However, whether such T2D-DNA methylation score is associated with changes in glycemic traits in response to weight loss dietary interventions remains unknown. Hypothesis: We hypothesized that a T2D-related DNA methylation score was associated with changes in glycemic traits in response to weight-loss dietary interventions; and may modify the effect of the interventions. Method: The current study included 147 participants from the MACRO trial, who were randomly assigned to a low-fat diet or a low-carbohydrate diet. A weighted DNA methylation score was created by 5 CpGs (cg19693031 at TXNIP , cg11024682 at SREBF1 , cg02650017 at PHOSPHO1 , cg18181703 and SOCS3 , and cg06500161 at ABCG1 ) from a previous EWAS at baseline. One-year changes in glycemic traits were calculated. Results: We found that a higher DNA methylation score was significantly associated with greater reductions in glucose (β: -3.20, SE: 1.08, p=0.003), insulin (β: -2.29, SE: 0.86, p=0.009), and homeostasis model assessments of insulin resistance (HOMA-IR, β: -0.86, SE: 0.30, p=0.005). The DNA methylation score was more strongly associated with changes in glycemic traits than any individual CpG sites ( Figure ). In addition, DNA methylation score significantly interacted with dietary interventions (low-fat vs. low-carbohydrate). The beneficial associations appeared to be stronger among participants with the low-carbohydrate diet (p-interaction=0.009 for glucose, 0.003 for insulin, and 0.004 for HOMA-IR). Conclusions: Our data indicate that a higher DNA methylation score was significantly associated with greater reductions in glycemic traits in response to weight-loss dietary interventions; such beneficial associations were stronger among those who consumed a low-carbohydrate diet.