Illumina Infinium MethylationEPIC BeadChip Research Articles

Integrated sequence-based genomic, transcriptomic, and methylation characterization of the susceptibility to tuberculosis in monozygous twins

BackgroundTuberculosis (TB) is a complex disease with a spectrum of outcomes for more than six decades; however, the genomic and epigenetic mechanisms underlying the highly heritable susceptibility to TB remain unclear. MethodsIntegrated sequence-based genomic, transcriptomic, and methylation analyses were conducted to identity the genetic factors associated with susceptibility to TB in two pairs of Mongolian monozygous twins. In this study, whole-genome sequencing was employed to analyze single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), and copy number variations (CNVs). Gene expression was assessed through RNA sequencing, and methylation patterns were examined using the Illumina Infinium Methylation EPIC BeadChip. The gene–gene interaction network was analyzed using differentially expressed genes. ResultsOur study revealed no significant difference in SNP and InDel profiles between participants with and without TB. Genes with CNVs were involved in human immunity (human leukocyte antigen [HLA] family and interferon [IFN] pathway) and the inflammatory response. Different DNA methylation patterns and mRNA expression profiles were observed in genes participating in immunity (HLA family) and inflammatory responses (IFNA, interleukin 10 receptor [IL-10R], IL-12B, Toll-like receptor, and IL-1B). ConclusionsThe results of this study suggested that susceptibility to TB is associated with transcriptional and epigenetic alternations of genes involved in immune and inflammatory responses. The genes in the HLA family (HLA-A, HLA-B, and HLA-DRB1) and IFN pathway (IFN-α and IFN-γ) may play major roles in susceptibility to TB.

Abstract P521: Sleep Timing and Duration in Relation to DNA Methylation in Adolescents: An Epigenome-Wide Analysis

Background: Accumulating evidence suggests that both sleep duration and sleep timing can impact metabolic health of adolescents, potentially through epigenetic modification. The objective of this study was to investigate the relationship between sleep duration and timing and DNA methylation, one type of epigenetic modification that may be responsive to changes in sleep. Methods: Cross-sectional epigenome-wide analysis of DNA methylation was conducted to identify sleep-related CpG sites in 269 females and 233 males (14.4±2.1 years on average) participating in the Early Life Exposure in Mexico to ENvironmental Toxicants (ELEMENT) cohort study. Sleep duration and midpoint on weekdays and weekends were assessed using7-day wrist actigraphy (Actigraph GTX-BT), and DNA methylation in blood leukocytes was measured using the Illumina Infinium Methylation EPIC BeadChip. Linear regression was conducted to assess the relationship between sleep variables and DNA methylation at each locus on the array (using beta values), adjusting for sex, age, maternal education, batch effects, monocytes, natural killer cells, B lymphocyte, granulocyte, and naïve CD8 T cells. A false discovery rate (FDR) using the Benjamini-Hochberg method was applied to correct for multiple testing. Sex-stratified associations were also examined. Results: The average sleep duration was 8.5±1.3 hours on weekdays and 9.1±1.3 hours on weekends, and the average sleep midpoint was 3:48AM±1.4 hours on weekdays and 4.48AM±1.3 hours on weekends. At false discovery rate <0.05, there were no significant associations at any of the 762,657 CpG sites. However, at a false discovery rate of <0.2, there were positive associations between weekday sleep duration and 4 CpG sites (i.e., longer sleep duration associated with higher % DNA methylation), including a site in or near CpG islands annotated to the gene DENND2C. Sex-stratified analyses revealed associations were more evident in males. Conclusion: Epigenome-wide analysis identified possible associations between sleep duration and multiple CpG sites, and in a sex-specific manner. Findings suggest that sleep duration may impact the epigenome in a distinct manner from sleep timing.

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 (>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<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.

Associations of environmental cadmium exposure with kidney damage: Exploring mediating DNA methylation sites in Chinese adults

Environmental exposure is widely recognized as the primary sources of Cadmium (Cd) in the human body, and exposure to Cd is associated with kidney damage in adults. Nevertheless, the role of DNA methylation in Cd-induced kidney damage remains unclear. This study aimed to investigate the epigenome-wide association of environmental Cd-related DNA methylation changes with kidney damage. We included 300 non-smoking adults from the China in 2019. DNA methylation profiles were measured with Illumina Infinium MethylationEPIC BeadChip array. Linear mixed-effect model was employed to estimate the effects of urinary Cd with DNA methylation. Differentially methylated positions (DMPs) associated with urinary Cd were then tested for the association with kidney damage indicators. The mediation analysis was further applied to explore the potential DNA methylation based mediators. The prediction model was developed using a logistic regression model, and used 1000 bootstrap resampling for the internal validation. We identified 27 Cd-related DMPs mapped to 20 genes after the adjustment of false-discovery-rate for multiple testing among non-smoking adults. 17 DMPs were found to be associated with both urinary Cd and kidney damage, and 14 of these DMPs were newly identified within the Chinese. Mediation analysis revealed that DNA methylation of cg26907612 and cg16848624 mediated the Cd-related reduced kidney damage. In addition, ten variables were selected using the LASSO regression analysis and were utilized to develop the prediction model. It found that the nomogram model predicted the risk of kidney damage caused by environmental Cd with a corrected C-index of 0.779. Our findings revealed novel DMPs associated with both environmental Cd exposure and kidney damage among non-smoking adults, and developed an easy-to-use nomogram-illustrated model using these novel DMPs. These findings could provide a theoretical basis for formulating prevention and control strategies for kidney damage from the perspective of environmental pollution and epigenetic regulation.

Comparing feature selection and machine learning approaches for predicting CYP2D6 methylation from genetic variation.

Pharmacogenetics currently supports clinical decision-making on the basis of a limited number of variants in a few genes and may benefit paediatric prescribing where there is a need for more precise dosing. Integrating genomic information such as methylation into pharmacogenetic models holds the potential to improve their accuracy and consequently prescribing decisions. Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic gene conventionally associated with the metabolism of commonly used drugs and endogenous substrates. We thus sought to predict epigenetic loci from single nucleotide polymorphisms (SNPs) related to CYP2D6 in children from the GUSTO cohort. Buffy coat DNA methylation was quantified using the Illumina Infinium Methylation EPIC beadchip. CpG sites associated with CYP2D6 were used as outcome variables in Linear Regression, Elastic Net and XGBoost models. We compared feature selection of SNPs from GWAS mQTLs, GTEx eQTLs and SNPs within 2 MB of the CYP2D6 gene and the impact of adding demographic data. The samples were split into training (75%) sets and test (25%) sets for validation. In Elastic Net model and XGBoost models, optimal hyperparameter search was done using 10-fold cross validation. Root Mean Square Error and R-squared values were obtained to investigate each models' performance. When GWAS was performed to determine SNPs associated with CpG sites, a total of 15 SNPs were identified where several SNPs appeared to influence multiple CpG sites. Overall, Elastic Net models of genetic features appeared to perform marginally better than heritability estimates and substantially better than Linear Regression and XGBoost models. The addition of nongenetic features appeared to improve performance for some but not all feature sets and probes. The best feature set and Machine Learning (ML) approach differed substantially between CpG sites and a number of top variables were identified for each model. The development of SNP-based prediction models for CYP2D6 CpG methylation in Singaporean children of varying ethnicities in this study has clinical application. With further validation, they may add to the set of tools available to improve precision medicine and pharmacogenetics-based dosing.

Epigenome-wide Association Study for Tic Disorders in Children: A Preliminary Study in Korean Population.

: Tic disorders can affect the quality of life in both childhood and adolescence. Many factors are involved in the etiology of tic disorders, and the genetic and epigenetic factors of tic disorders are considered complex and heterogeneous. : In this study, the differentially methylated regions (DMRs) between normal controls (n = 24; aged 6-15; 7 females) and patients with tic disorders (n = 16; aged 6-15; 5 females) were analyzed. We performed an epigenome-wide association study of tic disorders in Korean children. The tics were assessed using Yale Global Tic Severity Scale. The DNA methylation data consisted of 726,945 cytosine phosphate guanine (CpG) sites, assessed using the Illumina Infinium MethylationEPIC (850k) BeadChip. The DNA methylation data of the 40 participants were retrieved, and DMRs between the four groups based on sex and tic disorder were identified. From 28 male and 16 female samples, 37 and 38 DMRs were identified, respectively. We analyzed the enriched terms and visualized the network, heatmap, and upset plot. : In male, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed hypomethylated patterns in the ligand, receptor, and second signal transductors of the PI3K-Akt and MAPK signaling pathway (most cells were indicated as green color), and in female, the opposite patterns were revealed (most cells were indicated as red color). Five mental disorder-related enriched terms were identified in the network analysis. : Here, we provide insights into the epigenetic mechanisms of tic disorders. Abnormal DNA methylation patterns are associated with mental disorder-related symptoms.

Methylation and transcriptomic expression profiles of HUVEC in the oxygen and glucose deprivation model and its clinical implications in AMI patients.

The obstructed coronary artery undergoes a series of pathological changes due to ischemic-hypoxic shocks during acute myocardial infarction (AMI). However, the altered DNA methylation levels in endothelial cells under these conditions and their implication for the etiopathology of AMI have not been investigated in detail. This study aimed to explore the relationship between DNA methylation and pathologically altered gene expression profile in human umbilical vein endothelial cells (HUVECs) subjected to oxygen-glucose deprivation (OGD), and its clinical implications in AMI patients. The Illumina Infinium MethylationEPIC BeadChip assay was used to explore the genome-wide DNA methylation profile using the Novaseq6000 platform for mRNA sequencing in 3 pairs of HUVEC-OGD and control samples. GO and KEGG pathway enrichment analyses, as well as correlation, causal inference test (CIT), and protein-protein interaction (PPI) analyses identified 22 hub genes that were validated by MethylTarget sequencing as well as qRT-PCR. ELISA was used to detect four target molecules associated with the progression of AMI. A total of 2,524 differentially expressed genes (DEGs) and 22,148 differentially methylated positions (DMPs) corresponding to 6,642 differentially methylated genes (DMGs) were screened (|Δβ|>0.1 and detection p < 0.05). After GO, KEGG, correlation, CIT, and PPI analyses, 441 genes were filtered. qRT-PCR confirmed the overexpression of VEGFA, CCL2, TSP-1, SQSTM1, BCL2L11, and TIMP3 genes, and downregulation of MYC, CD44, BDNF, GNAQ, RUNX1, ETS1, NGFR, MME, SEMA6A, GNAI1, IFIT1, and MEIS1. DNA fragments BDNF_1_ (r = 0.931, p < 0.0001) and SQSTM1_2_NEW (r = 0.758, p = 0.0043) were positively correlated with the expressions of corresponding genes, and MYC_1_ (r = -0.8245, p = 0.001) was negatively correlated. Furthermore, ELISA confirmed TNFSF10 and BDNF were elevated in the peripheral blood of AMI patients (p = 0.0284 and p = 0.0142, respectively). Combined sequencing from in vitro cellular assays with clinical samples, aiming to establish the potential causal chain of the causal factor (DNA methylation) - mediator (mRNA)-cell outcome (endothelial cell ischemic-hypoxic injury)-clinical outcome (AMI), our study identified promising OGD-specific genes, which provided a solid basis for screening fundamental diagnostic and prognostic biomarkers of coronary endothelial cell injury of AMI. Moreover, it furnished the first evidence that during ischemia and hypoxia, the expression of BNDF was regulated by DNA methylation in endothelial cells and elevated in peripheral blood.

Epigenetic variation in neonatal tissues in infants conceived using capacitation-in vitro maturation vs. in vitro fertilization

ObjectiveTo investigate alterations of the global DNA methylation profile in placenta, cord blood and neonatal buccal smears in infants conceived using in vitro maturation (IVM) with a prematuration step (capacitation-IVM [CAPA-IVM] versus in vitro fertilization (IVF). DesignAnalysis of data from the offspring of participants in a randomized controlled trial. SettingPrivate clinic. PatientsForty-six women with polycystic ovary syndrome and/or high antral follicle count and their offspring (58 newborns). Intervention(s)Women with polycystic ovary syndrome and/or a high antral follicle count participating in the clinical trial were randomized to undergo CAPA-IVM or conventional IVF. Main Outcome Measure(s)At delivery, biological samples including cord blood, placental tissue, and neonatal buccal smear were collected. Genome-wide DNA methylation was determined by Illumina Infinium MethylationEPIC BeadChip. Variability in methylation was also considered and mean variances for the two treatment categories were compared. ResultsIn neonatal buccal smears, there were no significant differences between the CAPA-IVM and conventional IVF groups based on CpG probe after linear regression analysis using a significant cut-off of false discovery rate <0.05 and |Δβ|≥0.05. In cord blood, only one CpG site showed a significant gain of methylation in the CAPA-IVM group. In placenta, CAPA-IVM was significantly associated with changes in methylation at five CpG sites. Significantly more variable DNA methylation was found in five probes in placenta, 54 in cord blood and two in buccal smears after IVM of oocytes. In cord blood samples, 20 CpG sites had more variable methylation in the conventional IVF versus IVM group. Isolated CpG sites showing differences in methylation in cord blood were not associated with changes in gene expression of the overlapping genes. Conclusion(s)CAPA-IVM appeared to be associated with only a small amount of epigenetic variation in cord blood, placental tissue and neonate buccal smears.

High overall copy number variation burden by genome-wide methylation profiling holds negative prognostic value in surgically treated pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer with an overall 5-year survival of around 10 %. New prognostic tools to stratify patients are needed. Our main aim was to evaluate the prognostic value of overall copy number variation (CNV) burden in surgically treated PDAC. DNA extracted from 108 surgical PDAC specimens was examined to collect data on the genome-wide DNA methylation status of >850,000 CpG sites in promoter, gene body, and enhancer regions (Illumina Infinium Methylation EPIC BeadChip Kit). CNV profiles were obtained and all PDACs were stratified into one of three groups: Low, moderate, or high overall CNV burden. Tumors histologically showing a dominant conventional and/or tubulopapillary pattern in 60 %–100 % and 0–59 % were categorized as Group A and Group B as per Kalimuthu. We also performed targeted next-generation sequencing (NGS) and immunohistochemistry. High overall CNV burden held independent negative prognostic value with poor survival (HR 4.01 (95%CI 1.96–8.19), p = 0.00014) and was more frequent in Group B (p = 0.0003). Most frequent chromosomal arm-level aberrations were gains of 8q (29 %) and 1q (19 %) and losses of 17p (55 %), 18q (43 %), 6q (37 %), 9p (36 %), 6p (26 %), 19p (26 %), and 8p (25 %). Most frequent mutations found were in KRAS (95 %), TP53 (62 %), CDKN2A (24 %), SMAD4 (23 %), ATM (9 %), ARID1A (7 %), RNF43 (7 %), GNAS (6 %), and KDM6A (6 %). Group A PDACs showed more frequently KRAS variants other than Gly12Val and Gly12Asp (p = 0.012). Our data indicate that overall CNV burden using genome-wide methylation profiling may be a useful prognostic tool in surgically treated PDAC. Importantly, our approach, using data from genome-wide methylation profiling for analysis of overall CNV burden, can be performed on formalin-fixed and paraffin embedded PDAC tissues. Future studies should examine the prognostic value of overall CNV burden in unresectable PDAC.

Placental DNA methylation analysis of selective fetal growth restriction in monochorionic twins reveals aberrant methylated CYP11A1 gene for fetal growth restriction.

Fetal growth restriction (FGR) is associated with increased susceptibility to perinatal morbidity and mortality. Evidence suggests that epigenetic changes play critical roles in the regulation of fetal growth. We sought to present a comprehensive analysis of the associations between placental DNA methylation and selective fetal growth restriction (sFGR), which is a severe complication of monochorionic twin pregnancies, characterized by one fetus experiencing restricted growth. Genome-wide methylation analysis was performed on 24 placental samples obtained from 12 monochorionic twins with sFGR (Cohort 1) using Illumina Infinium MethylationEPIC BeadChip. Integrative analysis of our EPIC data and two previous placental methylation studies of sFGR (a total of 30 placental samples from 15 sFGR twins) was used to identify convincing differential promoter methylation. Validation analysis was performed on the placentas from 15 sFGR twins (30 placental samples), 15 FGR singletons, and 14 control singletons (Cohort 2) using pyrosequencing, quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry (IHC). A globe shift toward hypomethylation was identified in the placentas of growth-restricted fetuses compared with the placentas of normal fetuses in monochorionic twins, including 5625 hypomethylated CpGs and 452 hypermethylated CpGs, especially in the regions of CpG islands, gene-body and promoters. The analysis of pathways revealed dysregulation primarily in steroid hormone biosynthesis, metabolism, cell adhesion, signaling transduction, and immune response. Integrative analysis revealed a differentially methylated promoter region in the CYP11A1 gene, encoding a rate-limiting enzyme of steroidogenesis converting cholesterol to pregnenolone. The CYP11A1 gene was validated to have hypomethylation and higher mRNA expression in sFGR twins and FGR singletons. In conclusion, our findings suggested that the changes in placental DNA methylation pattern in sFGR may have functional implications for differentially methylated genes and regulatory regions. The study provides reliable evidence for identifying abnormally methylated CYP11A1 gene in the placenta of sFGR.

KS03.7.A INDEPENDENT PROGNOSTIC ROLE OF METHYLATION CLASS IN ATYPICAL AND MALIGNANT MENINGIOMA UNDERGOING ADJUVANT HIGH-DOSE RADIOTHERAPY: COMPREHENSIVE MOLECULAR ANALYSIS OF EORTC TRIAL 22042-26042

Abstract BACKGROUND In EORTC trial 22042-2604 WHO grade 2 and 3 meningioma patients underwent adjuvant high dose radiotherapy. The current study investigated clinicopathological correlations and the prognostic impact of genetic and epigenetic molecular factors in EORTC 22042-26042 trial patients. METHODS For patients that gave consent for additional molecular analysis of the resected tumor specimens (53 of 78 enrolled patients), Illumina Infinium Methylation EPIC BeadChip DNA methylation arrays and next generation sequencing (NGS) panels were performed to investigate the meningioma methylation class, and the presence of mutations in AKT1, TRAF7, BAP1, NF2 and the TERT-promotor as well as loss of chromosomes 1p and 22q and/or homozygous loss of CDKN2A/B. The cohort included 50 atypical and 3 malignant meningiomas per local assessment according to WHO classification at the time of patient enrollment. Factors significant at 10% significance in the univariate and interaction factor analyses were included in multivariate analyses with stepwise selection at the same significance. RESULTS The Cox multivariate analyses identified meningioma methylation class as a significant independent prognostic factor for progression-free survival (PFS) and overall survival (OS) measured from patient registration in the trial, respectively Chromosome 1p loss was significantly associated with PFS but not with OS. Age was significantly associated with OS but not with PFS. Due to small number of cases and/or events the prognostic value of homozygous deletion of CDKN2A/B (n=2) and TERT-promotor mutation (n=2) could not be adequately studied. CONCLUSIONS In this cohort of WHO grade 2 and 3 meningioma undergoing high-dose adjuvant radiotherapy, meningioma methylation class was identified as an independent prognostic factor for PFS and OS. Chromosome 1p loss was associated with reduced PFS but not OS. Larger cohorts are required to validate our findings.

Association between acetaminophen metabolites and CYP2E1 DNA methylation level in neonate cord blood in the Boston Birth Cohort

BackgroundAcetaminophen is a commonly used medication by pregnant women and is known to cross the placenta. However, little is known about the biological mechanisms that regulate acetaminophen in the developing offspring. Cytochrome 2E1 (CYP2E1) is the primary enzyme responsible for the conversion of acetaminophen to its toxic metabolite. Ex vivo studies have shown that the CYP2E1 gene expression in human fetal liver and placenta is largely controlled by DNA methylation (DNAm) at CpG sites located in the gene body of CYP2E1 at the 5’ end. To date, no population studies have examined the association between acetaminophen metabolite and fetal DNAm of CYP2E1 at birth.MethodsWe utilized data from the Boston Birth Cohort (BBC) which represents an urban, low-income, racially and ethnically diverse population in Boston, Massachusetts. Acetaminophen metabolites were measured in the cord plasma of newborns enrolled in BBC between 2003 and 2013 using liquid chromatography-tandem mass spectrometry. DNAm at 28 CpG sites of CYP2E1 was measured by Illumina Infinium MethylationEPIC BeadChip. We used linear regression to identify differentially methylated CpG sites and the “DiffVar” method to identify differences in methylation variation associated with the detection of acetaminophen, adjusting for cell heterogeneity and batch effects. The false discovery rate (FDR) was calculated to account for multiple comparisons.ResultsAmong the 570 newborns included in this study, 96 (17%) had detectable acetaminophen in cord plasma. We identified 7 differentially methylated CpGs (FDR < 0.05) associated with the detection of acetaminophen and additional 4 CpGs showing a difference in the variation of methylation (FDR < 0.05). These CpGs were all located in the gene body of CYP2E1 at the 5’ end and had a 3–6% lower average methylation level among participants with detectable acetaminophen compared to participants without. The CpG sites we identified overlap with previously identified DNase hypersensitivity and open chromatin regions in the ENCODE project, suggesting potential regulatory functions.ConclusionsIn a US birth cohort, we found detection of cord biomarkers of acetaminophen was associated with DNAm level of CYP2E1 in cord blood. Our findings suggest that DNA methylation of CYP2E1 may be an important regulator of acetaminophen levels in newborns.

Excessive Gestational Weight Gain Alters DNA Methylation and Influences Foetal and Neonatal Body Composition.

Changes in body weight are associated with the regulation of DNA methylation (DNAm). In this study, we investigated the associations between maternal gestational weight gain-related DNAm and foetal and neonatal body composition. Brazilian pregnant women from the Araraquara Cohort Study were followed up during pregnancy, delivery, and after hospital discharge. Women with normal pre-pregnancy BMI were allocated into two groups: adequate gestational weight gain (AGWG, n = 45) and excessive gestational weight gain (EGWG, n = 30). Foetal and neonatal body composition was evaluated via ultrasound and plethysmography, respectively. DNAm was assessed in maternal blood using Illumina Infinium MethylationEPIC BeadChip arrays. Linear regression models were used to explore the associations between DNAm and foetal and neonatal body composition. Maternal weight, GWG, neonatal weight, and fat mass were higher in the EGWG group. Analysis of DNAm identified 46 differentially methylated positions and 11 differentially methylated regions (DMRs) between the EGWG and AGWG groups. Nine human phenotypes were enriched for these 11 DMRs located in 13 genes (EMILIN1, HOXA5, CPT1B, CLDN9, ZFP57, BRCA1, POU5F1, ANKRD33, HLA-B, RANBP17, ZMYND11, DIP2C, TMEM232), highlighting the terms insulin resistance, and hyperglycaemia. Maternal DNAm was associated with foetal total thigh and arm tissues and subcutaneous thigh and arm fat, as well as with neonatal fat mass percentage and fat mass. The methylation pattern in the EGWG group indicated a risk for developing chronic diseases and involvement of maternal DNAm in foetal lean and fat mass and in neonatal fat mass.

Genome-wide analysis of DNA methylation and its relationship with serum homocysteine levels in patients with hypertension.

Homocysteine (Hcy) is an independent risk factor for cardiovascular diseases, and elevated plasma Hcy levels could aggravate vascular injury in hypertension. Hyperhomocysteinemia can change the methylation status of global DNA and specific genes. In the present study, we aim to examine the comprehensive influence of Hcy levels on DNA methylation status in patients with hypertension. Epigenome-wide methylation profiles of the peripheral leukocyte DNA of 218 patients with hypertension were analyzed using the Illumina Infinium Methylation EPIC BeadChip. Differentially methylated positions (DMPs) associated with serum Hcy levels were identified by mixed linear regression with the adjustment of potential confounders. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were conducted to determine the potential functions of the identified DMPs. The association between the methylation level of DMPs and carotid-femoral pulse wave velocity (Cf-PWV) was also analyzed. Five DMPs at cg13169662, cg03179312, cg21976560, cg25262698, and cg09433843 showed significant association with serum Hcy levels (false discovery rate-corrected P < 0.05). An additional six CpG sites met the threshold for suggestive significance ( P < 1 × 10 -6 ), among which three DMPs (cg25781123, cg26463106, and cg06679221) were annotated to THUMPD3 . Furthermore, the methylation levels of cg13169662 and cg25262698 (RPRD1A) were significantly associated with Cf-PWV. Our results suggest that Hcy could induce DNA methylation alteration in patients with hypertension. Further functional research is warranted to elucidate the concrete role of DMPs in hypertension.

Selection and validation of a novel set of specific differential methylation markers and construction of a random forestprediction model for the accurate tissue origin identifications of body fluids involving young and middle-aged group of Chinese Han population.

The identification of tissue origin of body fluid is helpful to the determination of the case nature and the reproduction of the case process. It has been confirmed that tissue-specific differential methylation markers could be used to identify the tissue origins of different body fluids. To select suitable tissue-specific differential methylation markers and establish the efficient typing system which could be applied to the identifications of body fluids in forensic cases involving Chinese Han individuals of young and middle-aged group, a total of 125 body fluids (venous blood, semen, vaginal fluid, saliva, and menstrual blood) were collected from healthy Chinese Han volunteers aged 20-45years old. After genome-wide explorations of DNA methylation patterns in these five kinds of body fluids based on the Illumina Infinium Methylation EPIC BeadChip, 15 novel body fluid-specific differential CpGs were selected and verified based on the pyrosequencing method. And these identification efficiencies for target body fluids were verified by ROC curves. The pyrosequencing results indicated that the average methylation rates of nine CpGs were consistent with those of DNA methylation chip detection results, and the other five CpGs (except for cg12152558) were still helpful for the tissue origin identifications of target body fluids. Finally, a random forest classification prediction model based on these 14 CpGs was constructed to successfully identify five kinds of body fluids, and the tested accuracy rates all reached 100%.

RNautophagic regulation of DNMT3a-dependent DNA methylation by Linc00942 enhances chemoresistance in gastric cancer.

Energy balance has long been known to extend lifespans and inhibit carcinogenesis in multiple species by slowing age-related epigenetic changes while the underlying mechanisms remain largely unknown. Herein, we found that starvation activated autophagy to remodel the DNA methylation profile by inhibiting DNMT3a expression. Illumina Infinium MethylationEPIC BeadChip and dot blot assay were performed to quantify the global DNA methylation level. Protein-RNA interactions were validated through RNA immunoprecipitation and RNA pull-down assay. In vitro and in vivo experiments were carried out to testify the effect of DNMT3a on chemoresistance. Autophagy is impaired in chemoresistance which was associated with differential DNA methylation and could be reversed by DNMT3a inhibition. Autophagy activation decreases the expression of DNMT3a mRNA, accompanied with the downregulation of chemoresistance-related Linc00942. Knockdown of Linc00942 reduces DNMT3a expression and genome-wide DNA methylation while Linc00942 overexpression increased DNMT3a expression and correlated hypermethylation in cancer cells and primary tumour tissues. Mechanistically, Linc00942 recruits RNA methyltransferase METTL3 to stimulate N6-methyladenosine (m6A) deposit on DNMT3a transcripts, triggering IGF2BP3/HuR to recognize modified mRNA for reinforced stability. SQSTM1/p62 recruits Linc00942 for autophagic degradation which can be abrogated after autophagy inhibition by p62 knockdown or chloroquine treatment. Inhibition of autophagy increases Linc00942 expression to promote chemoresistance and autophagy activation or hypomethylating agent decitabine restores chemosensitivity by reducing global DNA methylation. Overall, this study identifies a novel methylation cascade linking impaired RNautophagy to global hypermethylation in chemoresistance, and provides a rationale for repurposing decitabine to overcome chemoresistance in cancer treatment.

Accelerated epigenetic aging in suicide attempters

Suicide attempts (SA) are associated with excess mortality of natural causes, putatively mediated in part by premature cellular senescence. Epigenetic age (EA) estimators of biological age have been previously demonstrated to strongly predict physiological dysregulation and mortality risk. Herein, we investigate if violent SA with high intent-to-die is predictive of epigenetics-derived estimates of biological aging. The genome-wide methylation pattern was measured using the Illumina Infinium Methylation EPIC BeadChip in whole blood of 88 suicide attempters. Subjects were stratified into two groups (low- [n = 58] and high-risk [n = 30]) based on SA method (violent or non-violent) and/or intent-to-die (high/low). Estimators of intrinsic and extrinsic EA acceleration, one marker optimized to predict physiological dysregulation (DNAmPhenoAge/AgeAccelPheno) and one marker optimized to predict lifespan (DNAmGrimAge/AgeAccelGrim) were investigated for associations to severity of SA, by univariate and multivariate analyses. The study was adequately powered to detect differences of 2.2 years in AgeAccelGrim in relation to SA severity. Baseline DNAmGrimAge exceeded chronological age by 7.3 years on average across all samples, conferring a mean of 24.6% increase in relation to actual age. EA acceleration markers did not differ between high-and low-risk suicide attempters. Thus, SA per se but not severity of SA is related to EA, implicating that excess non-suicidal mortality is unrelated to risk of suicide. Preventative healthcare efforts aimed at curtailing excess mortality after SA may benefit from acting equally powerful to recognize somatic comorbidities irrespective of the severity inherent in the act itself.

Placental microRNA methylome signatures may serve as biomarkers and therapeutic targets for prenatally opioid-exposed infants with neonatal opioid withdrawal syndrome.

Introduction: The neonate exposed to opioids in utero faces a constellation of withdrawal symptoms postpartum commonly called neonatal opioid withdrawal syndrome (NOWS). The incidence of NOWS has increased in recent years due to the opioid epidemic. MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. Epigenetic variations in microRNAs (miRNAs) and their impact on addiction-related processes is a rapidly evolving area of research. Methods: The Illumina Infinium Methylation EPIC BeadChip was used to analyze DNA methylation levels of miRNA-encoding genes in 96 human placental tissues to identify miRNA gene methylation profiles as-sociated with NOWS: 32 from mothers whose prenatally opioid-exposed infants required pharmacologic management for NOWS, 32 from mothers whose prenatally opioid-exposed infants did not require treat-ment for NOWS, and 32 unexposed controls. Results: The study identified 46 significantly differentially methylated (FDR p-value ≤ 0.05) CpGs associated with 47 unique miRNAs, with a receiver operating characteristic (ROC) area under the curve (AUC) ≥0.75 including 28 hypomethylated and 18 hypermethylated CpGs as potentially associated with NOWS. These dysregulated microRNA methylation patterns may be a contributing factor to NOWS pathogenesis. Conclusion: This is the first study to analyze miRNA methylation profiles in NOWS infants and illustrates the unique role miRNAs might have in diagnosing and treating the disease. Furthermore, these data may provide a step toward feasible precision medicine for NOWS babies as well.

GENOME-WIDE DNA METHYLATION ANALYSIS IDENTIFIES SERUM HOMOCYSTEINE-ASSOCIATED CPG SITES IN HYPERTENSIVES

Objective: Homocysteine (Hcy) is associated with DNA methylation in cardiovascular disease (CVD). Moreover, the relationship between Hyperhomocysteine (HHcy) and CVD risk is stronger in individuals with hypertension. However, no research has focused on the influence of Hcy on DNA methylation status in patients with hypertension. We performed an epigenome-wide association study to identify DNA methylation loci associated with Hcy among 218 hypertensive patients using the Illumina Infinium Methylation EPIC BeadChip (850k chip) and further explored their effects on arteriosclerosis. Design and method: Epigenome-wide methylation profiles were assessed using the Illumina Infinium Methylation EPIC BeadChip (850k chip) in peripheral leukocyte DNA from 218 patients with hypertension. Differentially methylated positions (DMPs) associated with serum homocysteine levels were identified using a mixed linear regression with adjustment for potential confounders. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted to explore the possible functions of the identified DMPs. The association of these differentially methylated loci with hypertension- and Hcy- related arteriosclerosis, Cf-PWV (Carotid-femoral pulse wave velocity) was further analyzed. Results: Five DMPs at cg13169662, cg03179312, cg21976560, cg25262698, and cg09433843 showed significant association with serum Hcy levels (FDR-corrected p &lt;0.05). Six additional CpG sites met the threshold for suggestive significance (p&lt;1 × 10-6), among which three DMPs (cg25781123, cg26463106, and cg06679221) were annotated to the THUMPD3 gene. Furthermore, the methylation levels of cg13169662 and cg25262698 (RPRD1A) were significantly associated with Cf-PWV. Conclusions: Our study provides preliminary evidence for an association between DNA methylation and serum Hcy levels in patients with hypertension. This study may give an insight into the role of DNA methylation in cardiovascular injury underlying HHcy in patients with hypertension.

A novel principal component based method for identifying differentially methylated regions in Illumina Infinium MethylationEPIC BeadChip data

ABSTRACT Differentially methylated regions (DMRs) are genomic regions with methylation patterns across multiple CpG sites that are associated with a phenotype. In this study, we proposed a Principal Component (PC) based DMR analysis method for use with data generated using the Illumina Infinium MethylationEPIC BeadChip (EPIC) array. We obtained methylation residuals by regressing the M-values of CpGs within a region on covariates, extracted PCs of the residuals, and then combined association information across PCs to obtain regional significance. Simulation-based genome-wide false positive (GFP) rates and true positive rates were estimated under a variety of conditions before determining the final version of our method, which we have named DMRPC. Then, DMRPC and another DMR method, coMethDMR, were used to perform epigenome-wide analyses of several phenotypes known to have multiple associated methylation loci (age, sex, and smoking) in a discovery and a replication cohort. Among regions that were analysed by both methods, DMRPC identified 50% more genome-wide significant age-associated DMRs than coMethDMR. The replication rate for the loci that were identified by only DMRPC was higher than the rate for those that were identified by only coMethDMR (90% for DMRPC vs. 76% for coMethDMR). Furthermore, DMRPC identified replicable associations in regions of moderate between-CpG correlation which are typically not analysed by coMethDMR. For the analyses of sex and smoking, the advantage of DMRPC was less clear. In conclusion, DMRPC is a new powerful DMR discovery tool that retains power in genomic regions with moderate correlation across CpGs.