Significant CpG Research Articles

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

Exercise intervention during pregnancy induces DNA methylation alterations in maternal blood and cord blood.

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

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

Dietary and supplemental intake of vitamins C and E is associated with altered DNA methylation in an epigenome-wide association study meta-analysis

ABSTRACT Background: Dietary intake of antioxidants such as vitamins C and E protect against oxidative stress, and may also be associated with altered DNA methylation patterns. Methods: We meta-analysed epigenome-wide association study (EWAS) results from 11,866 participants across eight population-based cohorts to evaluate the association between self-reported dietary and supplemental intake of vitamins C and E with DNA methylation. EWAS were adjusted for age, sex, BMI, caloric intake, blood cell type proportion, smoking status, alcohol consumption, and technical covariates. Significant results of the meta-analysis were subsequently evaluated in gene set enrichment analysis (GSEA) and expression quantitative trait methylation (eQTM) analysis. Results: In meta-analysis, methylation at 4,656 CpG sites was significantly associated with vitamin C intake at FDR ≤ 0.05. The most significant CpG sites associated with vitamin C (at FDR ≤ 0.01) were enriched for pathways associated with systems development and cell signalling in GSEA, and were associated with downstream expression of genes enriched in the immune response in eQTM analysis. Furthermore, methylation at 160 CpG sites was significantly associated with vitamin E intake at FDR ≤ 0.05, but GSEA and eQTM analysis of the top most significant CpG sites associated with vitamin E did not identify significant enrichment of any biological pathways investigated. Conclusions: We identified significant associations of many CpG sites with vitamin C and E intake, and our results suggest that vitamin C intake may be associated with systems development and the immune response.

In utero exposure to diethylstilbestrol and blood DNA methylation in adult women: Results from a meta-analysis of two cohort studies

BackgroundPrenatal exposure to diethylstilbestrol (DES) is associated with several adverse health outcomes. Animal studies have shown associations between prenatal DES exposure and DNA methylation. ObjectiveThe aim of this study was to explore blood DNA methylation in women exposed and unexposed to DES in utero. MethodsSixty women (40 exposed and 20 unexposed) in the National Cancer Institute's Combined DES Cohort Study and 199 women (99 exposed and 100 unexposed women) in the Sister Study Cohort were included in this analysis. Within each study, robust linear regression models were used to assess associations between DES exposure and blood DNA methylation. Study-specific associations were combined using fixed-effect meta-analysis with inverse variance weights. Our analysis focused on CpG sites located within nine candidate genes identified in animal models. We further explored whether in utero DES exposure was associated with age acceleration. ResultsBlood DNA methylation levels at 10 CpG sites in six of the nine candidate genes were statistically significantly associated with prenatal DES exposure (P < 0.05) in this meta-analysis. Genes included EGF, EMB, EGFR, WNT11, FOS, and TGFB1, which are related to cell proliferation and differentiation. The most statistically significant CpG site was cg19830739 in gene EGF, and it was associated with lower methylation levels in women prenatally exposed to DES compared with those not exposed (P < 0.0001; false discovery rate<0.05). The association between prenatal DES exposure in utero and age acceleration was not statistically significant (P = 0.07 for meta-analyzed results). ConclusionsThere are few opportunities to investigate the effects of prenatal DES exposure. These findings suggest that in utero DES exposure may be associated with differential blood DNA methylation levels, which could mediate the increased risk of several adverse health outcomes observed in exposed women. Our findings need further evaluation using larger data sets.

The X-factor in ART: does the use of assisted reproductive technologies influence DNA methylation on the X chromosome?

BackgroundAssisted reproductive technologies (ART) may perturb DNA methylation (DNAm) in early embryonic development. Although a handful of epigenome-wide association studies of ART have been published, none have investigated CpGs on the X chromosome. To bridge this knowledge gap, we leveraged one of the largest collections of mother–father–newborn trios of ART and non-ART (natural) conceptions to date to investigate sex-specific DNAm differences on the X chromosome. The discovery cohort consisted of 982 ART and 963 non-ART trios from the Norwegian Mother, Father, and Child Cohort Study (MoBa). To verify our results from the MoBa cohort, we used an external cohort of 149 ART and 58 non-ART neonates from the Australian ‘Clinical review of the Health of adults conceived following Assisted Reproductive Technologies’ (CHART) study. The Illumina EPIC array was used to measure DNAm in both datasets. In the MoBa cohort, we performed a set of X-chromosome-wide association studies (‘XWASs’ hereafter) to search for sex-specific DNAm differences between ART and non-ART newborns. We tested several models to investigate the influence of various confounders, including parental DNAm. We also searched for differentially methylated regions (DMRs) and regions of co-methylation flanking the most significant CpGs. Additionally, we ran an analogous model to our main model on the external CHART dataset.ResultsIn the MoBa cohort, we found more differentially methylated CpGs and DMRs in girls than boys. Most of the associations persisted after controlling for parental DNAm and other confounders. Many of the significant CpGs and DMRs were in gene-promoter regions, and several of the genes linked to these CpGs are expressed in tissues relevant for both ART and sex (testis, placenta, and fallopian tube). We found no support for parental DNAm-dependent features as an explanation for the observed associations in the newborns. The most significant CpG in the boys-only analysis was in UBE2DNL, which is expressed in testes but with unknown function. The most significant CpGs in the girls-only analysis were in EIF2S3 and AMOT. These three loci also displayed differential DNAm in the CHART cohort.ConclusionsGenes that co-localized with the significant CpGs and DMRs associated with ART are implicated in several key biological processes (e.g., neurodevelopment) and disorders (e.g., intellectual disability and autism). These connections are particularly compelling in light of previous findings indicating that neurodevelopmental outcomes differ in ART-conceived children compared to those naturally conceived.

Impact of intrauterine exposure to maternal diabetes on preterm birth: fetal DNA methylation alteration is an important mediator

BackgroundIn utero exposure to diabetes has been shown to contribute to preterm birth, though the underlying biological mechanisms are yet to be fully elucidated. Fetal epigenetic variations established in utero may be a possible pathway. This study aimed to investigate whether in utero exposure to diabetes was associated with a change in newborn DNA methylation, and whether the identified CpG sites mediate the association between diabetes and preterm birth in a racially diverse birth cohort population.MethodsThis study included 954 mother–newborn pairs. Methylation levels in the cord blood were determined using the Illumina Infinium MethylationEPIC BeadChip 850 K array platform. In utero exposure to diabetes was defined by the presence of maternal pregestational or gestational diabetes. Preterm birth was defined as gestational age at birth less than 37 weeks. Linear regression analysis was employed to identify differentially methylated CpG sites. Differentially methylated regions were identified using the DMRcate Package.Results126 (13%) newborns were born to mothers with diabetes in pregnancy and 173 (18%) newborns were born preterm, while 41 newborns were born both preterm and to mothers with diabetes in pregnancy. Genomic-wide CpG analysis found that eighteen CpG sites in cord blood were differentially methylated by maternal diabetes status at an FDR threshold of 5%. These significant CpG sites were mapped to 12 known genes, one of which was annotated to gene Major Histocompatibility Complex, Class II, DM Beta (HLA-DMB). Consistently, one of the two identified significant methylated regions overlapped with HLA-DMB. The identified differentially methylated CpG sites mediated the association between diabetes in pregnancy and preterm birth by 61%.ConclusionsIn this US birth cohort, we found that maternal diabetes was associated with altered fetal DNA methylation patterns, which substantially explained the link between diabetes and preterm birth.

Pruning and thresholding approach for methylation risk scores in multi-ancestry populations

ABSTRACT Recent efforts have focused on developing methylation risk scores (MRS), a weighted sum of the individual’s DNA methylation (DNAm) values of pre-selected CpG sites. Most of the current MRS approaches that utilize Epigenome-wide association studies (EWAS) summary statistics only include genome-wide significant CpG sites and do not consider co-methylation. New methods that relax the p-value threshold to include more CpG sites and account for the inter-correlation of DNAm might improve the predictive performance of MRS. We paired informed co-methylation pruning with P-value thresholding to generate pruning and thresholding (P+T) MRS and evaluated its performance among multi-ancestry populations. Through simulation studies and real data analyses, we demonstrated that pruning provides an improvement over simple thresholding methods for prediction of phenotypes. We demonstrated that European-derived summary statistics can be used to develop P+T MRS among other populations such as African populations. However, the prediction accuracy of P+T MRS may differ across multi-ancestry population due to environmental/cultural/social differences.

Epigenome-Wide Association Study Reveals CpG Sites Associated with Thyroid Function and Regulatory Effects on KLF9.

Background: Thyroid hormones play a key role in differentiation and metabolism and are known regulators of gene expression through both genomic and epigenetic processes including DNA methylation. The aim of this study was to examine associations between thyroid hormones and DNA methylation. Methods: We carried out a fixed-effect meta-analysis of epigenome-wide association study (EWAS) of blood DNA methylation sites from 8 cohorts from the ThyroidOmics Consortium, incorporating up to 7073 participants of both European and African ancestry, implementing a discovery and replication stage. Statistical analyses were conducted using normalized beta CpG values as dependent and log-transformed thyrotropin (TSH), free thyroxine, and free triiodothyronine levels, respectively, as independent variable in a linear model. The replicated findings were correlated with gene expression levels in whole blood and tested for causal influence of TSH and free thyroxine by two-sample Mendelian randomization (MR). Results: Epigenome-wide significant associations (p-value <1.1E-7) of three CpGs for free thyroxine, five for free triiodothyronine, and two for TSH concentrations were discovered and replicated (combined p-values = 1.5E-9 to 4.3E-28). The associations included CpG sites annotated to KLF9 (cg00049440) and DOT1L (cg04173586) that overlap with all three traits, consistent with hypothalamic-pituitary-thyroid axis physiology. Significant associations were also found for CpGs in FKBP5 for free thyroxine, and at CSNK1D/LINCO1970 and LRRC8D for free triiodothyronine. MR analyses supported a causal effect of thyroid status on DNA methylation of KLF9. DNA methylation of cg00049440 in KLF9 was inversely correlated with KLF9 gene expression in blood. The CpG at CSNK1D/LINC01970 overlapped with thyroid hormone receptor alpha binding peaks in liver cells. The total additive heritability of the methylation levels of the six significant CpG sites was between 25% and 57%. Significant methylation QTLs were identified for CpGs at KLF9, FKBP5, LRRC8D, and CSNK1D/LINC01970. Conclusions: We report novel associations between TSH, thyroid hormones, and blood-based DNA methylation. This study advances our understanding of thyroid hormone action particularly related to KLF9 and serves as a proof-of-concept that integrations of EWAS with other -omics data can provide a valuable tool for unraveling thyroid hormone signaling in humans by complementing and feeding classical in vitro and animal studies.

Association of DNA methylation from the temporal cortex and cerebellum with AD‐related neuropathology and biochemistry

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often measured by Thal phase and Braak stage, respectively. Frequently, Aβ also deposits in the brain vasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. Assessment of these and related endophenotypes can reveal biological insights into the phenotypic variability in AD. We hypothesize that, amongst AD patients, severity of neuropathology and brain levels of AD‐related proteins are influenced by variation in DNA methylation (DNAm), the identification of which may uncover biological pathways for AD pathogenesis. To test this, we performed epigenome‐wide association studies (EWAS) using DNAm from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathology measures (Braak, Thal, and CAA) and brain levels of five proteins (apoE, Aβ40, Aβ42, tau, and p‐tau).MethodsDNAm from autopsy‐confirmed AD cases available through the Mayo Clinic Brain Bank was measured by reduced representation bisulfite sequencing (RRBS) from 471 brain samples, 200 of which had both TCX and CER measures. Bisulfite converted reads were sequenced on the Illumina High Seq4000 and quality filtered. Neuropathology scoring was performed by an experienced neuropathologist. TCX levels of five AD‐related proteins from three fractions (buffer‐soluble, detergent‐soluble, and insoluble) were measured by ELISA (Liu et al. 2020). Individual CpG methylation ratios were tested for association with each neuropathological and biochemical measure using the R package CpGassoc.ResultsWe identified multiple epigenome‐wide significant CpGs unique to each AD‐related measure. Comparisons between the TCX and CER revealed region specific CpG associations. The effects of sex, age at death, APOE genotype, and presence of genetic variants for each significant CpG association were also identified.ConclusionsAlthough all neuropathological and biochemical measures tested reflect core AD pathologies, our results demonstrate unique associations of epigenetic factors with these individual phenotypes, suggesting that their variability may be governed by distinct epigenetic processes. Discovery of DNAm changes that underlie specific neuropathological or biochemical outcomes can enhance our understanding of AD pathophysiology and may lead to the identification of novel gene or pathway targets for the different facets of this complex condition.

DNA methylation of the TPMT gene and azathioprine pharmacokinetics in children with very early onset inflammatory bowel disease

BackgroundThiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). AimsThe aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. MethodsThe association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients’ erythrocytes by HPLC and associated with patients’ age group and TPMT DNA methylation. ResultsWhole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10−5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. ConclusionMethylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.

Differential Gene Expression and DNA Methylation in the Risk of Depression in LOAD Patients.

Depression is common among late-onset Alzheimer's Disease (LOAD) patients. Only a few studies investigated the genetic variability underlying the comorbidity of depression in LOAD. Moreover, the epigenetic and transcriptomic factors that may contribute to comorbid depression in LOAD have yet to be studied. Using transcriptomic and DNA-methylomic datasets from the ROSMAP cohorts, we investigated differential gene expression and DNA-methylation in LOAD patients with and without comorbid depression. Differential expression analysis did not reveal significant association between differences in gene expression and the risk of depression in LOAD. Upon sex-stratification, we identified 25 differential expressed genes (DEG) in males, of which CHI3L2 showed the strongest upregulation, and only 3 DEGs in females. Additionally, testing differences in DNA-methylation found significant hypomethylation of CpG (cg20442550) on chromosome 17 (log2FC = -0.500, p = 0.004). Sex-stratified differential DNA-methylation analysis did not identify any significant CpG probes. Integrating the transcriptomic and DNA-methylomic datasets did not discover relationships underlying the comorbidity of depression and LOAD. Overall, our study is the first multi-omics genome-wide exploration of the role of gene expression and epigenome alterations in the risk of comorbid depression in LOAD patients. Furthermore, we discovered sex-specific differences in gene expression underlying the risk of depression symptoms in LOAD.

Infertility and treatments used have minimal effects on first-trimester placental DNA methylation and gene expression

To determine if DNA methylation alterations exist in first trimester human placenta between conceptions utilizing fertility treatments and those that do not, and if so, if they are the result of underlying infertility or fertility treatments. We also assessed if significant alterations led to changes in gene expression. We compared DNA methylation of first trimester placenta from singleton pregnancies that resulted in live births from unassisted, non-IVF fertility treatment (NIFT), and IVF conceptions using the Infinium MethylationEPIC BeadChip array. Significant CpG sites were compared to corresponding RNA-seq analysis in similar cohorts to determine if methylation alterations lead to differences in gene expression. 138 singleton pregnancies undergoing chorionic villus sampling resulting in a live birth were recruited for methylation analysis (56 unassisted, 38 NIFT, 44 IVF conceptions). RNA-seq data consisted of 141 subjects (74 unassisted, 33 NIFT, and 34 IVF) of which 116 overlap with the methylation cohort. IVF conceived pregnancy, or pregnancy conceived via non-IVF fertility treatment such as ovulation induction and/or intrauterine insemination. Significant methylation changes at CpG sites after adjustment for multiple comparisons. Secondary outcome is gene expression changes of significant CpG sites. Of the 741,145 probes analyzed in placenta, few were significant at Bonferroni<0.05: 185 CpG sites (0.025%) significant in pregnancies conceived with fertility treatment (NIFT+IVF) vs unassisted conceptions; 28 in NIFT vs unassisted; 195 in IVF vs unassisted; and only 13 (0.0018%) significant in IVF vs NIFT conceptions. Of all significant CpG sites combined, 10% (35 total) were located in genes with suggestive gene expression changes (RNA-seq P<0.05), but none were significant after adjustment for multiple comparisons (RNA-seq FDR<0.05). None of the 13 differentially methylated probes in IVF vs NIFT placenta were located in genes with suggestive IVF vs NIFT gene expression differences. Underlying infertility is the most significant contributor to the minimal differences in first trimester placental methylation, and not the specific fertility treatment utilized, such as IVF.

Epigenetic Mediation of Prenatal Tobacco and Alcohol Exposure and Infant Neurodevelopment in a South African Birth Cohort: A High-Dimensional Mediation Analysis

BACKGROUND AND AIMS Prenatal tobacco exposure (PTE) and prenatal alcohol exposure (PAE) have been associated with an increased risk of delayed neurodevelopment in children as well as differential newborn DNA methylation (DNAm). However, the biological mechanisms connecting PTE and PAE, DNAm, and neurodevelopment are largely unknown. Here we aim to determine whether differential DNAm in cord blood mediates the association between PTE and PAE and neurodevelopment in 113 children of 6 months of age from the South African Drakenstein Child Health Study. METHODS PTE and PAE were assessed antenatally using urine cotinine measurements and the ASSIST questionnaire, respectively. DNAm was measured from cord blood using the EPIC and 450K BeadChips. Neurodevelopment was measured at 6 months across five domains (cognitive, language, motor, adaptive behavior, socioemotional) using the Bayley Scale of Infant and Toddler Development, Third Edition. We conducted a high-dimensional mediation analysis using a novel approach called the divide-aggregate composite null test (DACT), followed by causal mediation analysis to estimate the average causal mediation effects (ACME) and total effect (TE). RESULTS In this population, 35% of mothers actively smoked and 23% of mothers consumed alcohol during pregnancy. We identified 31 CpG sites and 8 CpG sites to have a significant mediating effect (ACME and TE P &amp;#x3c;0.05) between PTE and cognitive and motor development, respectively. For PAE, 16 CpG sites and 1 CpG site had a significant mediating effect on motor and adaptive behavior development, respectively. CONCLUSIONS These findings suggest that changes in the methylome may in part explain the biological mechanisms underlying the relationship between PTE and PAE and infant neurodevelopment. The identification of significant CpG sites could provide novel insights for early detection of disease and potential prevention targets in translational research and community interventions in at-risk populations. KEYWORDS: epigenomics; mediation analysis; neurodevelopment; prenatal smoking; prenatal alcohol exposure

Maternal pre-pregnancy body mass index and offspring with overweight/obesity at preschool age: The possible role of epigenome-wide DNA methylation changes in cord blood.

Epigenome-wide association studies have identified some DNA methylation sites associated with body mass index (BMI) or obesity. Studies in the Asian population are lacking. To examine the association of cord blood genome-wide DNA methylation (GWDm) changes with maternal pre-pregnancy BMI and children's BMI-z score at preschool age. Additionally, we also explored the genome-wide differentially methylated regions and differentially methylated probes between preschoolers with overweight/obesity and normal-weight counterparts. This two-stage study design included (1) a GWDm analysis of 30 mother-child pairs from 633 participants of the Zhuhai birth cohort with data on newborn cord blood, maternal pre-pregnancy BMI, and children's BMI at 3 years of age; and (2) a targeted validation analysis of the cord blood of ten children with overweight/obesity and ten matched controls to validate the CpG sites. In the first stage, no significant CpG sites were found to be associated with children's BMI-z score at preschool age after FDR correction with the p-values of the CpG sites in FOXN3 (cg23501836) and ZNF264 (cg27437574) being close to 1 × 10-6 . In the second stage, a significant difference of CpG sites in AHRR (chr5:355067-355068) and FOXN3 (chr14: 89630264-89630272 and chr14: 89630387-89630388) was found between the ten children with overweight/obesity and ten controls (p < 0.05). The CpG sites in FOXN3 (chr14:89630264-89630272 and chr14:89630295-89630296) and ZNF264 (chr19: 57703104-57703107 and chr19: 57703301-57703307) were associated with children's BMI-z score; and the CpG sites in FOXN3 (chr14: 89630264-89630272 and chr14: 89630387-89630388) were associated with maternal pre-pregnancy BMI. DNA methylation in FOXN3 and AHRR is associated with overweight/obesity in preschool-aged children, and the methylation in FOXN3 and ZNF264 might be associated with children's BMI-z score. FOXN3 methylation may be associated with maternal pre-pregnancy BMI, suggesting its potential role in the children's BMI-z score or overweight/obesity. Our results provide novel insights into the mechanisms of children's obesity.

Pre-diagnostic DNA methylation in blood leucocytes in cutaneous melanoma; a nested case–control study within the Norwegian Women and Cancer cohort

The prognosis of cutaneous melanoma depends on early detection, and good biomarkers for melanoma risk may provide a valuable tool to detect melanoma development at a pre-clinical stage. By studying the epigenetic profile in pre-diagnostic blood samples of melanoma cases and cancer free controls, we aimed to identify DNA methylation sites conferring melanoma risk. DNA methylation was measured at 775,528 CpG sites using the Illumina EPIC array in whole blood in incident melanoma cases (n = 183) and matched cancer-free controls (n = 183) in the Norwegian Women and Cancer cohort. Phenotypic information and ultraviolet radiation exposure were obtained from questionnaires. Epigenome wide association (EWAS) was analyzed in future melanoma cases and controls with conditional logistic regression, with correction for multiple testing using the false discovery rate (FDR). We extended the analysis by including a public data set on melanoma (GSE120878), and combining these different data sets using a version of covariate modulated FDR (AdaPT). The analysis on future melanoma cases and controls did not identify any genome wide significant CpG sites (0.85 ≤ padj ≤ 0.99). In the restricted AdaPT analysis, 7 CpG sites were suggestive at the FDR level of 0.15. These CpG sites may potentially be used as pre-diagnostic biomarkers of melanoma risk.

Docetaxel targets aggressive methylation profiles and serves as a radiosensitizer in high-risk meningiomas.

Meningioma is the most common primary intracranial tumor in adults. A subset of these tumors recur and invade the brain, even after surgery and radiation, resulting in significant disability. There is currently no standard-of-care chemotherapy for meningiomas. As genomic DNA methylation profiling can prognostically stratify these lesions, we sought to determine whether any existing chemotherapies might be effective against meningiomas with high-risk methylation profiles. A previously published dataset of meningioma methylation profiles was used to screen for clinically significant CpG methylation events and associated cellular pathways. Based on these results, patient-derived meningioma cell lines were used to test candidate drugs in vitro and in vivo, including efficacy in conjunction with radiotherapy. We identified 981 genes for which methylation of mapped CpG sites was related to progression-free survival in meningiomas. Associated molecular pathways were cross-referenced with FDA-approved cancer drugs, which nominated Docetaxel as a promising candidate for further preclinical analyses. Docetaxel arrested growth in 17 meningioma cell sources, representing all tumor grades, with a clinically favorable IC50 values ranging from 0.3 nM to 10.7 mM. The inhibitory effects of this medication scaled with tumor doubling time, with maximal benefit in fast-growing lesions. The combination of Docetaxel and radiation therapy increased markers of apoptosis and double-stranded DNA breaks, and extended the survival of mice engrafted with meningioma cells relative to either modality alone. Global patterns of DNA methylation may be informative for the selection of chemotherapies against meningiomas, and existing drugs may enhance radiation sensitivity in high-risk cases.

DNA methylation analysis for smoking status prediction in the Chinese population based on the methylation-sensitive single-nucleotide primer extension method

In some criminal cases, the identity of suspect is unknown and there is no matching DNA profile in the DNA database. Forensic DNA Phenotyping can provide useful investigative information for these cases. Most forensic studies focus on visible characteristics rather than behavioral characteristics. However, smoking is prevalent in the Chinese population, and DNA methylation is the most promising biomarker for smoking. We collected 204 whole blood samples from the Chinese population and measured methylation levels of 9 smoking-related CpG loci using the methylation-sensitive single-nucleotide primer extension method (Ms-SnuPE). But the single-base extension primers of loci cg12803068 and cg21566642 contained other CpG sites, which may introduce bias, and only the other 7 CpG loci were included in subsequent statistical analysis. The methylation level of locus cg05575921 near the aromatic hydrocarbon receptor repressor (AHRR) gene was much lower in the current smoker group than in the never smoker group. To evaluate the ability of each of 7 CpG loci to predict smoking status, the logistic regression (LR) models were established separately, and locus cg05575921 had the best ability to predict smoking status compared with the other 6 loci. Then, combined (including loci cg19572487, cg05575921, cg23480021, cg23576855, cg21161138, cg01940273, and cg09935388) and stepwise (including loci cg05575921 and cg01940273) multinomial logistic regression (MLR) models were also established. Both combined and stepwise MLR models had good efficiencies in predicting smoking status, and outperformed the above 7 LR models. However, the accuracy, specificity and area under the curve (AUC) of stepwise MLR model in the testing dataset were slightly higher than those of combined MLR model, and the stepwise MLR model required less loci information. Therefore, the stepwise MLR model based on 2 significant CpG loci was more recommended model for predicting smoking status in the Chinese population, and the formula was as follow: P = 1/(1 +e-(10.621–10.005*cg05575921–8.770*cg01940273)). Mainly 2 CpG loci (cg05575921 and cg01940273) played a major role in the prediction of smoking status, and the other 5 CpG loci contributed less. Moreover, for evaluating the ability of each of 7 CpG loci to predict cigarette consumption, the polynomial regression formulas were established separately. As the adjusted R2 was between 0.00 and 0.20, the methylation levels of these 7 loci were not closely associated with the cigarette consumption. Our methylation assay is simple, economical, and available in conventional forensic laboratories, and may be useful in assessing the smoking status of unknown suspects.

A multiomic ferroptosis-associated prognostic signature incorporating epigenetic and transcriptional biomarkers for hepatocellular carcinoma.

BackgroundFerroptosis was reported to have tremendous promise in the treatment and prognosis of hepatocellular carcinoma (HCC). Here, we identified a novel ferroptosis-related prognostic signature incorporating epigenetic and transcriptional biomarkers could help predicting survival of patients with HCC.MethodsWe employed multi-omics and clinical data from The Cancer Genome Atlas (TCGA) database to identify the ferroptosis-associated methylation CpG sites associated with HCC survival using sure independence screening (SIS). Then we utilized Kaplan-Meier curves to evaluate the prognostic significance of gene expression and DNA methylation. Receiver operating characteristic (ROC) curve was used predicting the 3- and 5-year survival. Mediation analysis of ferroptosis-related methylation and transcriptional score was performed.ResultsWe firstly identified 114 significant CpG sites under the criteria of false discovery rate (FDR) <0.05 in training set. Then we screened out 5 candidate CpG sites in validation set for multivariate screening and stepwise regression. We found that the high-risk group had significantly shorter survival time than the low-risk group in the prognostic signature combined with epigenetic and transcriptional scores (HR =2.72 95% CI: 2.01–3.68, P=8.75E-11). And the predictive model involving clinical information, gene expression, and methylation data performed best for 3-year survival prediction (AUC =0.672) and 5-year survival prediction (AUC =0.742).ConclusionsOur results suggested a signature combining clinical information, ferroptosis-related gene expression, and methylation presented a superior ability for prognostic prediction in HCC, which may bring us novel tool and targets in the treatment of HCC.

Diagnostic classification of cancers using DNA methylation of paracancerous tissues

The potential role of DNA methylation from paracancerous tissues in cancer diagnosis has not been explored until now. In this study, we built classification models using well-known machine learning models based on DNA methylation profiles of paracancerous tissues. We evaluated our methods on nine cancer datasets collected from The Cancer Genome Atlas (TCGA) and utilized fivefold cross-validation to assess the performance of models. Additionally, we performed gene ontology (GO) enrichment analysis on the basis of the significant CpG sites selected by feature importance scores of XGBoost model, aiming to identify biological pathways involved in cancer progression. We also exploited the XGBoost algorithm to classify cancer types using DNA methylation profiles of paracancerous tissues in external validation datasets. Comparative experiments suggested that XGBoost achieved better predictive performance than the other four machine learning methods in predicting cancer stage. GO enrichment analysis revealed key pathways involved, highlighting the importance of paracancerous tissues in cancer progression. Furthermore, XGBoost model can accurately classify nine different cancers from TCGA, and the feature sets selected by XGBoost can also effectively predict seven cancer types on independent GEO datasets. This study provided new insights into cancer diagnosis from an epigenetic perspective and may facilitate the development of personalized diagnosis and treatment strategies.

Associations of DNA Methylation With Behavioral Problems, Gray Matter Volumes, and Negative Life Events Across Adolescence: Evidence From the Longitudinal IMAGEN Study

BackgroundNegative life events (NLEs) increase the risk for externalizing behaviors (EBs) and internalizing behaviors (IBs) in adolescence and adult psychopathology. DNA methylation associated with behavioral problems may reflect this risk and long-lasting effects of NLEs. MethodsTo identify consistent associations between blood DNA methylation and EBs or IBs across adolescence, we conducted longitudinal epigenome-wide association studies (EWASs) using data from the IMAGEN cohort, collected at ages 14 and 19 years (n = 506). Significant findings were validated in a separate subsample (n = 823). Methylation risk scores were generated by 10-fold cross-validation and further tested for their associations with gray matter volumes and NLEs. ResultsNo significant findings were obtained for the IB-EWAS. The EB-EWAS identified a genome-wide significant locus in a gene linked to attention-deficit/hyperactivity disorder (ADHD) (IQSEC1, cg01460382; p = 1.26 × 10−8). Other most significant CpG sites were near ADHD-related genes and enriched for genes regulating tumor necrosis factor and interferon-γ signaling, highlighting the relevance of EB-EWAS findings for ADHD. Analyses with the EB methylation risk scores suggested that it partly reflected comorbidity with IBs in late adolescence. Specific to EBs, EB methylation risk scores correlated with smaller gray matter volumes in medial orbitofrontal and anterior/middle cingulate cortices, brain regions known to associate with ADHD and conduct problems. Longitudinal mediation analyses indicated that EB-related DNA methylation were more likely the outcomes of problematic behaviors accentuated by NLEs, and less likely the epigenetic bases of such behaviors. ConclusionsOur findings suggest that novel epigenetic mechanisms through which NLEs exert short and longer-term effects on behavior may contribute to ADHD.