Search

Background: Cancer is the leading cause of disease-related mortality in children. Causes of leukemia, the most common form, are largely unknown. Growing evidence points to an origin in-utero, when global redistribution of DNA methylation occurs driving tissue differentiation., Methods: Epigenome-wide DNA methylation was profiled in surrogate (blood) and target (bone marrow) tissues at birth, diagnosis, remission and relapse of pediatric pre-B acute lymphoblastic leukemia (pre-B ALL) patients. Double-blinded analyses was performed between prospective cohorts extending from birth to diagnosis and retrospective studies backtracking from clinical disease to birth. Validation was carried out using independent technologies and populations., Results: The imprinted and immuno-modulating VTRNA2-1 was hypermethylated (FDR<0.05) at birth in nested cases relative to controls in all tested populations (totaling 317 cases and 483 controls), including European and Hispanic ancestries. VTRNA2-1 methylation was stable over follow-up years after birth and across surrogate, target and other tissues (n=5,023 tissues; 30 types). When profiled in leukemic tissues from two clinical cohorts (totaling 644 cases), VTRNA2-1 methylation exhibited higher levels at diagnosis relative to controls, it reset back to normal levels at remission, and then re-increased to above control levels at relapse. Hypermethylation was significantly associated with worse pre-B ALL patient survival and with reduced VTRNA2-1 expression (n=2,294 tissues; 26 types), supporting a functional and translational role for VTRNA2-1 methylation., Conclusion: This study provides proof-of-concept to detect at birth epigenetic precursors of pediatric pre-B ALL. These alterations were reproducible with different technologies, in three continents and in two ethnicities, and can offer biomarkers for early detection and prognosis as well as actionable targets for therapy., (© 2024. The Author(s).)

Maternal educational attainment (MEA) shapes offspring health through multiple potential pathways. Differential DNA methylation may provide a mechanistic understanding of these long-term associations. We aimed to quantify the associations of MEA with offspring DNA methylation levels at birth, in childhood and in adolescence. Using 37 studies from high-income countries, we performed meta-analysis of epigenome-wide association studies (EWAS) to quantify the associations of completed years of MEA at the time of pregnancy with offspring DNA methylation levels at birth (n = 9 881), in childhood (n = 2 017), and adolescence (n = 2 740), adjusting for relevant covariates. MEA was found to be associated with DNA methylation at 473 cytosine-phosphate-guanine sites at birth, one in childhood, and four in adolescence. We observed enrichment for findings from previous EWAS on maternal folate, vitamin-B 12 concentrations, maternal smoking, and pre-pregnancy BMI. The associations were directionally consistent with MEA being inversely associated with behaviours including smoking and BMI. Our findings form a bridge between socio-economic factors and biology and highlight potential pathways underlying effects of maternal education. The results broaden our understanding of bio-social associations linked to differential DNA methylation in multiple early stages of life. The data generated also offers an important resource to help a more precise understanding of the social determinants of health., (© 2023. The Author(s).)

Higher birth order is associated with altered risk of many disease states. Changes in placentation and exposures to in utero growth factors with successive pregnancies may impact later life disease risk via persistent DNA methylation alterations. We investigated birth order with Illumina DNA methylation array data in each of 16 birth cohorts (8164 newborns) with European, African, and Latino ancestries from the Pregnancy and Childhood Epigenetics Consortium. Meta-analyzed data demonstrated systematic DNA methylation variation in 341 CpGs (FDR adjusted P < 0.05) and 1107 regions. Forty CpGs were located within known quantitative trait loci for gene expression traits in blood, and trait enrichment analysis suggested a strong association with immune-related, transcriptional control, and blood pressure regulation phenotypes. Decreasing fertility rates worldwide with the concomitant increased proportion of first-born children highlights a potential reflection of birth order-related epigenomic states on changing disease incidence trends., (© 2024. The Author(s).)

Background: Seasonal variations in environmental exposures at birth or during gestation are associated with numerous adult traits and health outcomes later in life. Whether DNA methylation (DNAm) plays a role in the molecular mechanisms underlying the associations between birth season and lifelong phenotypes remains unclear., Methods: We carried out epigenome-wide meta-analyses within the Pregnancy And Childhood Epigenetic Consortium to identify associations of DNAm with birth season, both at differentially methylated probes (DMPs) and regions (DMRs). Associations were examined at two time points: at birth (21 cohorts, N = 9358) and in children aged 1-11 years (12 cohorts, N = 3610). We conducted meta-analyses to assess the impact of latitude on birth season-specific associations at both time points., Results: We identified associations between birth season and DNAm (False Discovery Rate-adjusted p values < 0.05) at two CpGs at birth (winter-born) and four in the childhood (summer-born) analyses when compared to children born in autumn. Furthermore, we identified twenty-six differentially methylated regions (DMR) at birth (winter-born: 8, spring-born: 15, summer-born: 3) and thirty-two in childhood (winter-born: 12, spring and summer: 10 each) meta-analyses with few overlapping DMRs between the birth seasons or the two time points. The DMRs were associated with genes of known functions in tumorigenesis, psychiatric/neurological disorders, inflammation, or immunity, amongst others. Latitude-stratified meta-analyses [higher (≥ 50°N), lower (< 50°N, northern hemisphere only)] revealed differences in associations between birth season and DNAm by birth latitude. DMR analysis implicated genes with previously reported links to schizophrenia (LAX1), skin disorders (PSORS1C, LTB4R), and airway inflammation including asthma (LTB4R), present only at birth in the higher latitudes (≥ 50°N)., Conclusions: In this large epigenome-wide meta-analysis study, we provide evidence for (i) associations between DNAm and season of birth that are unique for the seasons of the year (temporal effect) and (ii) latitude-dependent variations in the seasonal associations (spatial effect). DNAm could play a role in the molecular mechanisms underlying the effect of birth season on adult health outcomes., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Increasing evidence supports the concept of prenatal programming as an early factor in the aging process. DNA methylation age (DNAm age), global genome-wide DNA methylation (global methylation), telomere length (TL), and mitochondrial DNA content (mtDNA content) have independently been shown to be markers of aging, but their interrelationship and determinants at birth remain uncertain., Methods: We assessed the inter-correlation between the aging biomarkers DNAm age, global methylation, TL and mtDNA content using Pearson's correlation in 190 cord blood samples of the ENVIRONAGE birth cohort. TL and mtDNA content was measured via qPCR, while the DNA methylome was determined using the human 450K methylation Illumina microarray. Subsequently, DNAm age was calculated according to Horvath's epigenetic clock, and mean global, promoter, gene-body, and intergenic DNA methylation were determined. Path analysis, a form of structural equation modeling, was performed to disentangle the complex causal relationships among the aging biomarkers and their potential determinants., Results: DNAm age was inversely correlated with global methylation (r = -0.64, p < 0.001) and mtDNA content (r = - 0.16, p = 0.027). Cord blood TL was correlated with mtDNA content (r = 0.26, p < 0.001) but not with global methylation or DNAm age. Path analysis showed the strongest effect for global methylation on DNAm age with a decrease of 0.64 standard deviations (SD) in DNAm age for each SD (0.01%) increase in global methylation (p < 0.001). Among the applied covariates, newborn sex and season of delivery were the strongest determinants of aging biomarkers., Conclusions: We provide insight into molecular aging signatures at the start of life, including their interrelations and determinants, showing that cord blood DNAm age is inversely associated with global methylation and mtDNA content but not with newborn telomere length. Our findings demonstrate that cord blood TL and DNAm age relate to different pathways/mechanisms of biological aging and can be influenced by environmental factors already at the start of life. These findings are relevant for understanding fetal programming and for the early prevention of noncommunicable diseases., (© 2022. The Author(s).)

In humans, DNA methylation marks inherited from gametes are largely erased following fertilisation, prior to construction of the embryonic methylome. Exploiting a natural experiment of seasonal variation including changes in diet and nutritional status in rural Gambia, we analysed three datasets covering two independent child cohorts and identified 259 CpGs showing consistent associations between season of conception (SoC) and DNA methylation. SoC effects were most apparent in early infancy, with evidence of attenuation by mid-childhood. SoC-associated CpGs were enriched for metastable epialleles, parent-of-origin-specific methylation and germline differentially methylated regions, supporting a periconceptional environmental influence. Many SoC-associated CpGs overlapped enhancers or sites of active transcription in H1 embryonic stem cells and fetal tissues. Half were influenced but not determined by measured genetic variants that were independent of SoC. Environmental 'hotspots' providing a record of environmental influence at periconception constitute a valuable resource for investigating epigenetic mechanisms linking early exposures to lifelong health and disease., Competing Interests: MS, AS, NK, GC, CF, PI, AD, MB, SM, MR, ZH, CC, MD, PJ, DM, AP No competing interests declared, (© 2022, Silver et al.)

Background: Among children, sex-specific differences in disease prevalence, age of onset, and susceptibility have been observed in health conditions including asthma, immune response, metabolic health, some pediatric and adult cancers, and psychiatric disorders. Epigenetic modifications such as DNA methylation may play a role in the sexual differences observed in diseases and other physiological traits., Methods: We performed a meta-analysis of the association of sex and cord blood DNA methylation at over 450,000 CpG sites in 8438 newborns from 17 cohorts participating in the Pregnancy And Childhood Epigenetics (PACE) Consortium. We also examined associations of child sex with DNA methylation in older children ages 5.5-10 years from 8 cohorts (n = 4268)., Results: In newborn blood, sex was associated at Bonferroni level significance with differences in DNA methylation at 46,979 autosomal CpG sites (p < 1.3 × 10 -7 ) after adjusting for white blood cell proportions and batch. Most of those sites had lower methylation levels in males than in females. Of the differentially methylated CpG sites identified in newborn blood, 68% (31,727) met look-up level significance (p < 1.1 × 10 -6 ) in older children and had methylation differences in the same direction., Conclusions: This is a large-scale meta-analysis examining sex differences in DNA methylation in newborns and older children. Expanding upon previous studies, we replicated previous findings and identified additional autosomal sites with sex-specific differences in DNA methylation. Differentially methylated sites were enriched in genes involved in cancer, psychiatric disorders, and cardiovascular phenotypes., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background : DNA methylation is an epigenetic control mechanism that may be altered by environmental exposures. We have previously reported that in utero exposure to the mycotoxin and liver carcinogen aflatoxin B1 from the maternal diet, as measured using biomarkers in the mothers' blood, was associated with differential DNA methylation in white blood cells of 6-month-old infants from The Gambia. Methods : Here we examined aflatoxin B1-associated differential DNA methylation in white blood cells of 24-month-old children from the same population ( n = 244), in relation to the child's dietary exposure assessed using aflatoxin albumin biomarkers in blood samples collected at 6, 12 and 18 months of age. HM450 BeadChip arrays were used to assess DNA methylation, with data compared to aflatoxin albumin adduct levels using two approaches; a continuous model comparing aflatoxin adducts measured in samples collected at 18 months to DNA methylation at 24 months, and a categorical time-dose model that took into account aflatoxin adduct levels at 6, 12 and 18 months, for comparison to DNA methylation at 24 months. Results : Geometric mean (95% confidence intervals) for aflatoxin albumin levels were 3.78 (3.29, 4.34) at 6 months, 25.1 (21.67, 29.13) at 12 months and 49.48 (43.34, 56.49) at 18 months of age. A number of differentially methylated CpG positions and regions were associated with aflatoxin exposure, some of which affected gene expression. Pathway analysis highlighted effects on genes involved with with inflammatory, signalling and growth pathways. Conclusions : This study provides further evidence that exposure to aflatoxin in early childhood may impact on DNA methylation.

Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here, we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissues adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritized by weighting methylation results using RNA-sequencing data from The Cancer Genome Atlas and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR), with a 30% methylation (Δβ) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2 , and THSD4 , had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high-incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization. SIGNIFICANCE: This largest genome-wide DNA methylation study on ESCC from high-incidence populations of the world identifies functionally relevant and robust DNAme events that could serve as potential tumor-specific markers. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2612/F1.large.jpg., (©2021 American Association for Cancer Research.)

Coffee and tea are extensively consumed beverages worldwide which have received considerable attention regarding health. Intake of these beverages is consistently linked to, among others, reduced risk of diabetes and liver diseases; however, the mechanisms of action remain elusive. Epigenetics is suggested as a mechanism mediating the effects of dietary and lifestyle factors on disease onset. Here we report the results from epigenome-wide association studies (EWAS) on coffee and tea consumption in 15,789 participants of European and African-American ancestries from 15 cohorts. EWAS meta-analysis of coffee consumption reveals 11 CpGs surpassing the epigenome-wide significance threshold (P-value <1.1×10 -7 ), which annotated to the AHRR, F2RL3, FLJ43663, HDAC4, GFI1 and PHGDH genes. Among them, cg14476101 is significantly associated with expression of the PHGDH and risk of fatty liver disease. Knockdown of PHGDH expression in liver cells shows a correlation with expression levels of genes associated with circulating lipids, suggesting a role of PHGDH in hepatic-lipid metabolism. EWAS meta-analysis on tea consumption reveals no significant association, only two CpGs annotated to CACNA1A and PRDM16 genes show suggestive association (P-value <5.0×10 -6 ). These findings indicate that coffee-associated changes in DNA methylation levels may explain the mechanism of action of coffee consumption in conferring risk of diseases.

The protocol described here for methylome profiling consists of two parts. One is the experimental part for a genome-wide analysis of methylation level, and the other is the bioinformatics analysis of the methylome data. DNA methylation measurement is conducted using the commercially available array-based "Infinium Human Methylation 450K BeadChip" kit (or its updated version, Infinium MethylationEPICBeadChip). This BeadChip allows the high-throughput DNA methylation analysis suitable for genome-wide studies with large sample size. The results give intensities of the beads providing information on the unmethylated and methylated CpG sites. Bioinformatics data analysis involves reading the intensities as methylation values using R packages. Here, we provide a detailed analysis tool for each of the data analysis steps.

Background: Preterm birth and shorter duration of pregnancy are associated with increased morbidity in neonatal and later life. As the epigenome is known to have an important role during fetal development, we investigated associations between gestational age and blood DNA methylation in children., Methods: We performed meta-analysis of Illumina's HumanMethylation450-array associations between gestational age and cord blood DNA methylation in 3648 newborns from 17 cohorts without common pregnancy complications, induced delivery or caesarean section. We also explored associations of gestational age with DNA methylation measured at 4-18 years in additional pediatric cohorts. Follow-up analyses of DNA methylation and gene expression correlations were performed in cord blood. DNA methylation profiles were also explored in tissues relevant for gestational age health effects: fetal brain and lung., Results: We identified 8899 CpGs in cord blood that were associated with gestational age (range 27-42 weeks), at Bonferroni significance, P < 1.06 × 10 - 7 , of which 3343 were novel. These were annotated to 4966 genes. After restricting findings to at least three significant adjacent CpGs, we identified 1276 CpGs annotated to 325 genes. Results were generally consistent when analyses were restricted to term births. Cord blood findings tended not to persist into childhood and adolescence. Pathway analyses identified enrichment for biological processes critical to embryonic development. Follow-up of identified genes showed correlations between gestational age and DNA methylation levels in fetal brain and lung tissue, as well as correlation with expression levels., Conclusions: We identified numerous CpGs differentially methylated in relation to gestational age at birth that appear to reflect fetal developmental processes across tissues. These findings may contribute to understanding mechanisms linking gestational age to health effects.

Objective: Maternal gestational diabetes mellitus (GDM) has been associated with adverse outcomes in the offspring. Growing evidence suggests that the epigenome may play a role, but most previous studies have been small and adjusted for few covariates. The current study meta-analyzed the association between maternal GDM and cord blood DNA methylation in the Pregnancy and Childhood Epigenetics (PACE) consortium., Research Design and Methods: Seven pregnancy cohorts (3,677 mother-newborn pairs [317 with GDM]) contributed results from epigenome-wide association studies, using DNA methylation data acquired by the Infinium HumanMethylation450 BeadChip array. Associations between GDM and DNA methylation were examined using robust linear regression, with adjustment for potential confounders. Fixed-effects meta-analyses were performed using METAL. Differentially methylated regions (DMRs) were identified by taking the intersection of results obtained using two regional approaches: comb-p and DMRcate., Results: Two DMRs were identified by both comb-p and DMRcate. Both regions were hypomethylated in newborns exposed to GDM in utero compared with control subjects. One DMR (chr 1: 248100345-248100614) was located in the OR2L13 promoter, and the other (chr 10: 135341870-135342620) was located in the gene body of CYP2E1 . Individual CpG analyses did not reveal any differentially methylated loci based on a false discovery rate-adjusted P value threshold of 0.05., Conclusions: Maternal GDM was associated with lower cord blood methylation levels within two regions, including the promoter of OR2L13 , a gene associated with autism spectrum disorder, and the gene body of CYP2E1 , which is upregulated in type 1 and type 2 diabetes. Future studies are needed to understand whether these associations are causal and possible health consequences., (© 2019 by the American Diabetes Association.)

Tumor cell invasion is one of the key processes during cancer progression, leading to life-threatening metastatic lesions in melanoma. As methylation of cancer-related genes plays a fundamental role during tumorigenesis and may lead to cellular plasticity which promotes invasion, our aim was to identify novel epigenetic markers on selected invasive melanoma cells. Using Illumina BeadChip assays and Affymetrix Human Gene 1.0 microarrays, we explored the DNA methylation landscape of selected invasive melanoma cells and examined the impact of DNA methylation on gene expression patterns. Our data revealed predominantly hypermethylated genes in the invasive cells affecting the neural crest differentiation pathway and regulation of the actin cytoskeleton. Integrative analysis of the methylation and gene expression profiles resulted in a cohort of hypermethylated genes (IL12RB2, LYPD6B, CHL1, SLC9A3, BAALC, FAM213A, SORCS1, GPR158, FBN1 and ADORA2B) with decreased expression. On the other hand, hypermethylation in the gene body of the EGFR and RBP4 genes was positively correlated with overexpression of the genes. We identified several methylation changes that can have role during melanoma progression, including hypermethylation of the promoter regions of the ARHGAP22 and NAV2 genes that are commonly altered in locally invasive primary melanomas as well as during metastasis. Interestingly, the down-regulation of the methylcytosine dioxygenase TET2 gene, which regulates DNA methylation, was associated with hypermethylated promoter region of the gene. This can probably lead to the observed global hypermethylation pattern of invasive cells and might be one of the key changes during the development of malignant melanoma cells., (© 2019 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Bisphenol A (BPA), an estrogen-like endocrine disruptor used in plastics, has been associated with development and promotion of breast cancer, so plastic manufacturers shifted towards less-studied analogs, BPF and BPS. Studying the associated DNA methylome-wide mechanisms of these derivatives is timely, particularly in comparison with BPA., Methods: We assessed proliferation, cell cycle, and migration of breast cancer cells (estrogen receptor (ER)-positive: MCF-7 and ER-negative: MDA-MB-231) treated with BPF and BPS ± estrogen receptor inhibitor (ERI) in comparison to BPA ± ERI. RNA expression and activity of DNA (de)methylation enzymes and LINE-1 methylation were quantified. DNA methylome-wide analysis was evaluated in bisphenol-exposed cells and compared to clinical breast cancer data., Results: The three bisphenols caused ER-dependent increased proliferation and migration of MCF-7 but not MDA-MB-231 cells, with BPS being 10 times less potent than BPA and BPF. Although they have similar chemical structures, the three bisphenols induced differential DNA methylation alterations at several genomic clusters of or single CpG sites, with the majority of these being ER-dependent. At equipotent doses, BPA had the strongest effect on the methylome, followed by BPS then BPF. No pathways were enriched for BPF while BPA- and BPS-induced methylome alterations were enriched in focal adhesion, cGMP-PKG, and cancer pathways, which were also dysregulated in methylome-wide alterations comparing ER-positive breast cancer samples to adjacent normal tissues., Conclusions: The three bisphenols have important epigenetic effects in breast cell lines, with those of BPA and BPS overlapping with cancer-related pathways in clinical breast cancer models. Hence, further investigation of their safety is warranted.

Aim: Cigarette smoking influences DNA methylation genome wide, in newborns from pregnancy exposure and in adults from personal smoking. Whether a unique methylation signature exists for in utero exposure in newborns is unknown. Materials & methods: We separately meta-analyzed newborn blood DNA methylation (assessed using Illumina450k Beadchip), in relation to sustained maternal smoking during pregnancy (9 cohorts, 5648 newborns, 897 exposed) and adult blood methylation and personal smoking (16 cohorts, 15907 participants, 2433 current smokers). Results & conclusion: Comparing meta-analyses, we identified numerous signatures specific to newborns along with many shared between newborns and adults. Unique smoking-associated genes in newborns were enriched in xenobiotic metabolism pathways. Our findings may provide insights into specific health impacts of prenatal exposure on offspring.

Background: Environmental and genetic factors play an important role in the etiology of breast cancer. Several small blood-based DNA methylation studies have reported risk associations with methylation at individual CpGs and average methylation levels; however, these findings require validation in larger prospective cohort studies. To investigate the role of blood DNA methylation on breast cancer risk, we conducted a meta-analysis of four prospective cohort studies, including a total of 1663 incident cases and 1885 controls, the largest study of blood DNA methylation and breast cancer risk to date., Methods: We assessed associations with methylation at 365,145 CpGs present in the HumanMethylation450 (HM450K) Beadchip, after excluding CpGs that did not pass quality controls in all studies. Each of the four cohorts estimated odds ratios (ORs) and 95% confidence intervals (CI) for the association between each individual CpG and breast cancer risk. In addition, each study assessed the association between average methylation measures and breast cancer risk, adjusted and unadjusted for cell-type composition. Study-specific ORs were combined using fixed-effect meta-analysis with inverse variance weights. Stratified analyses were conducted by age at diagnosis (< 50, ≥ 50), estrogen receptor (ER) status (+/-), and time since blood collection (< 5, 5-10, > 10 years). The false discovery rate (q value) was used to account for multiple testing., Results: The average age at blood draw ranged from 52.2 to 62.2 years across the four cohorts. Median follow-up time ranged from 6.6 to 8.4 years. The methylation measured at individual CpGs was not associated with breast cancer risk (q value > 0.59). In addition, higher average methylation level was not associated with risk of breast cancer (OR = 0.94, 95% CI = 0.85, 1.05; P = 0.26; P for study heterogeneity = 0.86). We found no evidence of modification of this association by age at diagnosis (P = 0.17), ER status (P = 0.88), time since blood collection (P = 0.98), or CpG location (P = 0.98)., Conclusions: Our data indicate that DNA methylation measured in the blood prior to breast cancer diagnosis in predominantly postmenopausal women is unlikely to be associated with substantial breast cancer risk on the HM450K array. Larger studies or with greater methylation coverage are needed to determine if associations exist between blood DNA methylation and breast cancer risk.

Background: Prenatal exposure to air pollution has been associated with childhood respiratory disease and other adverse outcomes. Epigenetics is a suggested link between exposures and health outcomes., Objectives: We aimed to investigate associations between prenatal exposure to particulate matter (PM) with diameter [Formula: see text] ([Formula: see text]) or [Formula: see text] ([Formula: see text]) and DNA methylation in newborns and children., Methods: We meta-analyzed associations between exposure to [Formula: see text] ([Formula: see text]) and [Formula: see text] ([Formula: see text]) at maternal home addresses during pregnancy and newborn DNA methylation assessed by Illumina Infinium HumanMethylation450K BeadChip in nine European and American studies, with replication in 688 independent newborns and look-up analyses in 2,118 older children. We used two approaches, one focusing on single cytosine-phosphate-guanine (CpG) sites and another on differentially methylated regions (DMRs). We also related PM exposures to blood mRNA expression., Results: Six CpGs were significantly associated [false discovery rate (FDR) [Formula: see text]] with prenatal [Formula: see text] and 14 with [Formula: see text] exposure. Two of the [Formula: see text] CpGs mapped to FAM13A (cg00905156) and NOTCH4 (cg06849931) previously associated with lung function and asthma. Although these associations did not replicate in the smaller newborn sample, both CpGs were significant ([Formula: see text]) in 7- to 9-y-olds. For cg06849931, however, the direction of the association was inconsistent. Concurrent [Formula: see text] exposure was associated with a significantly higher NOTCH4 expression at age 16 y. We also identified several DMRs associated with either prenatal [Formula: see text] and or [Formula: see text] exposure, of which two [Formula: see text] DMRs, including H19 and MARCH11, replicated in newborns., Conclusions: Several differentially methylated CpGs and DMRs associated with prenatal PM exposure were identified in newborns, with annotation to genes previously implicated in lung-related outcomes. https://doi.org/10.1289/EHP4522.

Background: It is well established that estrogens and other hormonal factors influence breast cancer susceptibility. We hypothesized that a woman's total lifetime estrogen exposure accumulates changes in DNA methylation, detectable in the blood, which could be used in risk assessment for breast cancer., Methods: An estimated lifetime estrogen exposure (ELEE) model was defined using epidemiological data from EPIC-Italy (n = 31,864). An epigenome-wide association study (EWAS) of ELEE was performed using existing Illumina HumanMethylation450K Beadchip (HM450K) methylation data obtained from EPIC-Italy blood DNA samples (n = 216). A methylation index (MI) of ELEE based on 31 CpG sites was developed using HM450K data from EPIC-Italy and the Generations Study and evaluated for association with breast cancer risk in an independent dataset from the Generations Study (n = 440 incident breast cancer cases matched to 440 healthy controls) using targeted bisulfite sequencing. Lastly, a meta-analysis was conducted including three additional cohorts, consisting of 1187 case-control pairs., Results: We observed an estimated 5% increase in breast cancer risk per 1-year longer ELEE (OR = 1.05, 95% CI 1.04-1.07, P = 3 × 10 -12 ) in EPIC-Italy. The EWAS identified 694 CpG sites associated with ELEE (FDR Q < 0.05). We report a DNA methylation index (MI) associated with breast cancer risk that is validated in the Generations Study targeted bisulfite sequencing data (OR Q4&#95;vs&#95;Q1  = 1.77, 95% CI 1.07-2.93, P = 0.027) and in the meta-analysis (OR Q4&#95;vs&#95;Q1  = 1.43, 95% CI 1.05-2.00, P = 0.024); however, the correlation between the MI and ELEE was not validated across study cohorts., Conclusion: We have identified a blood DNA methylation signature associated with breast cancer risk in this study. Further investigation is required to confirm the interaction between estrogen exposure and DNA methylation in the blood.

Background: There is increasing evidence that folate, an important component of one-carbon metabolism, modulates the epigenome. Alcohol, which can disrupt folate absorption, is also known to affect the epigenome. We investigated the association of dietary folate and alcohol intake on leukocyte DNA methylation levels in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Leukocyte genome-wide DNA methylation profiles on approximately 450,000 CpG sites were acquired with Illumina HumanMethylation 450K BeadChip measured among 450 women control participants of a case-control study on breast cancer nested within the EPIC cohort. After data preprocessing using surrogate variable analysis to reduce systematic variation, associations of DNA methylation with dietary folate and alcohol intake, assessed with dietary questionnaires, were investigated using CpG site-specific linear models. Specific regions of the methylome were explored using differentially methylated region (DMR) analysis and fused lasso (FL) regressions. The DMR analysis combined results from the feature-specific analysis for a specific chromosome and using distances between features as weights whereas FL regression combined two penalties to encourage sparsity of single features and the difference between two consecutive features., Results: After correction for multiple testing, intake of dietary folate was not associated with methylation level at any DNA methylation site, while weak associations were observed between alcohol intake and methylation level at CpG sites cg03199996 and cg07382687, with q val  = 0.029 and q val  = 0.048, respectively. Interestingly, the DMR analysis revealed a total of 24 and 90 regions associated with dietary folate and alcohol, respectively. For alcohol intake, 6 of the 15 most significant DMRs were identified through FL., Conclusions: Alcohol intake was associated with methylation levels at two CpG sites. Evidence from DMR and FL analyses indicated that dietary folate and alcohol intake may be associated with genomic regions with tumor suppressor activity such as the GSDMD and HOXA5 genes. These results were in line with the hypothesis that epigenetic mechanisms play a role in the association between folate and alcohol, although further studies are warranted to clarify the importance of these mechanisms in cancer.

Methylation levels in tumor-suppressor genes and repetitive sequences have previously been used to study the relationship between environmental air pollution and epigenetic changes related to cancer. In this study, we measured the methylation profiles of the promoter regions CDKN2A , MLH1 and APC and the repetitive sequence LINE-1 in 59 workers exposed to the construction environment and in 49 unexposed workers. We also evaluated the micronuclei frequency and levels of trace elements in the blood of all workers. We evaluated of levels of particulate matter and polycyclic aromatic hydrocarbons (PAHs) at the construction site to characterize the environmental exposure. Our findings demonstrated that exposed workers exhibited significantly higher average levels of promoter methylation of CDKN2A , APC , and MLH1 genes and increased hypomethylation of the LINE-1 in comparison to unexposed workers (all p < 0.05). A higher frequency of micronuclei was observed in the exposed group (2 ± 2) compared to the unexposed group (1 ± 1) with p < 0.001. High levels of particulate matter (51⁻841 μg/m³) and some PAHs were found in samples from the construction environment. In summary, we provide evidence of increased DNA damage and altered DNA methylation of exposed workers, suggesting that genomic approaches to biomonitoring may be an effective way of estimating future cancer risk for construction workers.

A high body mass (BMI) index has repeatedly been associated with non-atopic asthma, but the biological mechanism linking obesity to asthma is still poorly understood. We aimed to test the hypothesis that inflammation and/or innate immunity plays a role in the obesity-asthma link. DNA methylome was measured in blood samples of 61 non-atopic participants with asthma and 146 non-atopic participants without asthma (non-smokers for at least 10 years) taking part in the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA) study. Modification by DNA methylation of the association of BMI or BMI change over 10 years with adult-onset asthma was examined at each CpG site and differentially methylated region. Pathway enrichment tests were conducted for genes in a priori curated inflammatory pathways and the NLRP3-IL1B-IL17 axis. The latter was chosen on the basis of previous work in mice. Inflammatory pathways including glucocorticoid/PPAR signaling ( p = 0.0023), MAPK signaling ( p = 0.013), NF-κB signaling ( p = 0.031), and PI3K/AKT signaling ( p = 0.031) were enriched for the effect modification of BMI, while NLRP3-IL1B-IL17 axis was enriched for the effect modification of BMI change over 10 years ( p = 0.046). DNA methylation measured in peripheral blood is consistent with inflammation as a link between BMI and adult-onset asthma and with the NLRP3-IL1B-IL17 axis as a link between BMI change over 10 years and adult-onset asthma in non-atopic participants.

The Human Leucocyte Antigen (HLA) locus and other DNA sequence variants identified in Genome-Wide Association (GWA) studies explain around 50% of the heritability of celiac disease (CD). However, the pathogenesis of CD could be driven by other layers of genomic information independent from sequence variation, such as DNA methylation, and it is possible that allele-specific methylation explains part of the SNP associations. Since the DNA methylation landscape is expected to be different among cell types, we analyzed the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and controls separately. We found a cell type-specific methylation signature that includes genes mapping to the HLA region, namely TAP1 and HLA-B. We also performed Immunochip SNP genotyping of the same samples and interrogated the expression of some of the affected genes. Our analysis revealed that the epithelial methylome is characterized by the loss of CpG island (CGI) boundaries, often associated to altered gene expression, and by the increased variability of the methylation across the samples. The overlap between differentially methylated positions (DMPs) and CD-associated SNPs or variants contributing to methylation quantitative trait loci (mQTLs) is minimal. In contrast, there is a notable enrichment of mQTLs among the most significant CD-associated SNPs. Our results support the notion that DNA methylation alterations constitute a genotype-independent event and confirm its role in the HLA region (apart from the well-known, DQ allele-specific effect). Finally, we find that a fraction of the CD-associated variants could exert its phenotypic effect through DNA methylation.

Background: Socioeconomic experiences are recognized determinants of health, and recent work has shown that social disadvantages in early life may induce sustained biological changes at molecular level that are detectable later in life. However, the dynamics and persistence of biological embedding of socioeconomic position (SEP) remains vastly unexplored., Methods: Using the data from the ALSPAC birth cohort, we performed epigenome-wide association studies of DNA methylation changes at three life stages (birth, n = 914; childhood at mean age 7.5 years, n = 973; and adolescence at mean age 15.5 years, n = 974), measured using the Illumina HumanMethylation450 Beadchip, in relation to pregnancy SEP indicators (maternal and paternal education and occupation)., Results: Across the four early life SEP metrics investigated, only maternal education was associated with methylation levels at birth, and four CpGs mapped to SULF1, GLB1L2 and RPUSD1 genes were identified [false discovery rate (FDR)-corrected P-value <0.05]. No epigenetic signature was found associated with maternal education in child samples, but methylation levels at 20 CpG loci were found significantly associated with maternal education in adolescence. Although no overlap was found between the differentially methylated CpG sites at different ages, we identified two CpG sites at birth and during adolescence which are 219 bp apart in the SULF1 gene that encodes an heparan sulphatase involved in modulation of signalling pathways. Using data from an independent birth cohort, the ENVIRONAGE cohort, we were not able to replicate these findings., Conclusions: Taken together, our results suggest that parental SEP, and particularly maternal education, may influence the offspring's methylome at birth and adolescence., (© The Author(s) 2018; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Background: One of the potential mechanisms linking air pollution to health effects is through changes in DNA-methylation, which so far has mainly been analyzed globally or at candidate sites., Objective: We investigated the association of personal and ambient air pollution exposure measures with genome-wide DNA-methylation changes., Methods: We collected repeated 24-hour personal and ambient exposure measurements of particulate matter (PM 2.5 ), PM 2.5 absorbance, and ultrafine particles (UFP) and peripheral blood samples from a panel of 157 healthy non-smoking adults living in four European countries. We applied univariate mixed-effects models to investigate the association between air pollution and genome-wide DNA-methylation perturbations at single CpG (cytosine-guanine dinucleotide) sites and in Differentially Methylated Regions (DMRs). Subsequently, we explored the association of air pollution-induced methylation alterations with gene expression and serum immune marker levels measured in the same subjects., Results: Personal exposure to PM 2.5 was associated with methylation changes at 13 CpG sites and 69 DMRs. Two of the 13 identified CpG sites (mapped to genes KNDC1 and FAM50B) were located within these DMRs. In addition, 42 DMRs were associated with personal PM 2.5 absorbance exposure, 16 DMRs with personal exposure to UFP, 4 DMRs with ambient exposure to PM 2.5 , 16 DMRs with ambient PM 2.5 absorbance exposure, and 15 DMRs with ambient UFP exposure. Correlation between methylation levels at identified CpG sites and gene expression and immune markers was generally moderate., Conclusion: This study provides evidence for an association between 24-hour exposure to air pollution and DNA-methylation at single sites and regional clusters of CpGs. Analysis of differentially methylated regions provides a promising avenue to further explore the subtle impact of environmental exposures on DNA-methylation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The large geographic variations in the incidence of gastric cancer (GC) are likely due to differential environmental exposures, in particular to Helicobacter pylori (H. pylori) infection. We aimed to investigate the impact of H. pylori on the epigenome in normal gastric mucosa and methylation changes associated with cancer risk independent of H. pylori. A discovery set of normal gastric mucosa from GC cases (n = 42) and controls (n = 42), nested in a large case-control study and stratified by H. pylori status, were subjected to genome-wide methylation profiling. Single-nucleotide polymorphism arrays from peripheral blood leukocytes were used to conduct methylation quantitative trait loci (mQTL) analysis. A validation set of gastric mucosa samples (n = 180) was used in the replication phase. We found 1,924 differentially methylated positions (DMPs) and 438 differentially methylated regions (DMRs) associated with H. pylori infection, most of which were hypermethylated. Significant methylation alterations identified in the initial set were successfully replicated. Furthermore, the H. pylori-associated DMP/Rs showed marked stability ('epigenetic memory') after H. pylori clearance. Interestingly, we found 152 DMRs associated with cancer risk independent of the H. pylori status in normal gastric mucosa. The methylation score derived from three biomarkers was a strong predictor of GC. Finally, the mQTL analysis indicated that the H. pylori- and cancer-specific methylation signatures were minimally affected by genetic variation. The comprehensively characterized methylome changes associated with H. pylori infection and GC risk in our study might serve as potential biomarkers for early cancer progression in tumour-free gastric mucosa., (© 2018 International Agency for Research on Cancer (IARC/WHO); licensed by UICC.)

Introduction and Aim: Epigenetic alterations play an essential role in cancer onset and progression, thus studies of drugs targeting the epigenetic machinery are a principal concern for cancer treatment. Here, we evaluated the potential of the combination of the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5aza-dC) and the pan-deacetylase inhibitor Trichostatin A (TSA), at low cytotoxic concentrations, to modulate the canonical Wnt/β-catenin pathway in liver cancer cells., Material and Methods: Pyrosequencing was used for DNA methylation analyses of LINE-1 sequences and the Wnt/β-catenin pathway antagonist DKK3, SFRP1, WIF1 and CDH1. qRT-PCR was employed to verify the expression of the antagonist. Pathway regulation were evaluated looking at the expression of β-catenin and E-cadherin by confocal microscopy and the antitumoral effects of the drugs was studied by wound healing and clonogenic assays., Results: Our result suggest that 5aza-dC and TSA treatments were enough to induce a significant expression of the pathway antagonists, decrease of β-catenin protein levels, re-localization of the protein to the plasma membrane, and pathway transcriptional activity reduction. These important effects exerted an antitumoral outcome shown by the reduction of the migration and clonogenic capabilities of the cells., Conclusion: We were able to demonstrate Wnt/ β-catenin pathway modulation through E-cadherin up-regulation induced by 5aza-dC and TSA treatments, under an activation-pathway background, like CTNNB1 and TP53 mutations. These findings provide evidences of the potential effect of epigenetic modifier drugs for liver cancer treatment. However, further research needs to be conducted, to determine the in vivo potential of this treatment regimen for the management of liver cancer.

Background: Methylation measures quantified by microarray techniques can be affected by systematic variation due to the technical processing of samples, which may compromise the accuracy of the measurement process and contribute to bias the estimate of the association under investigation. The quantification of the contribution of the systematic source of variation is challenging in datasets characterized by hundreds of thousands of features.In this study, we introduce a method previously developed for the analysis of metabolomics data to evaluate the performance of existing normalizing techniques to correct for unwanted variation. Illumina Infinium HumanMethylation450K was used to acquire methylation levels in over 421,000 CpG sites for 902 study participants of a case-control study on breast cancer nested within the EPIC cohort. The principal component partial R-square (PC-PR2) analysis was used to identify and quantify the variability attributable to potential systematic sources of variation. Three correcting techniques, namely ComBat, surrogate variables analysis (SVA) and a linear regression model to compute residuals were applied. The impact of each correcting method on the association between smoking status and DNA methylation levels was evaluated, and results were compared with findings from a large meta-analysis., Results: A sizeable proportion of systematic variability due to variables expressing 'batch' and 'sample position' within 'chip' was identified, with values of the partial R 2 statistics equal to 9.5 and 11.4% of total variation, respectively. After application of ComBat or the residuals' methods, the contribution was 1.3 and 0.2%, respectively. The SVA technique resulted in a reduced variability due to 'batch' (1.3%) and 'sample position' (0.6%), and in a diminished variability attributable to 'chip' within a batch (0.9%). After ComBat or the residuals' corrections, a larger number of significant sites ( k  = 600 and k  = 427, respectively) were associated to smoking status than the SVA correction ( k  = 96)., Conclusions: The three correction methods removed systematic variation in DNA methylation data, as assessed by the PC-PR2, which lent itself as a useful tool to explore variability in large dimension data. SVA produced more conservative findings than ComBat in the association between smoking and DNA methylation., Competing Interests: The study was approved by the Ethical Review Board of the International Agency for Research on Cancer, and by the local Ethics Committees in the participating centres. This study was also conducted in accordance with the IARC Ethic Committee (Project No 10-22).Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Blood represents an easily accessible human tissue for numerous research and clinical applications, including surrogate roles for biomarkers of other tissues. Dried blood spots (DBS) are space- and cost-efficient storage forms of blood while stably retaining many of its chemical constituents. Consequently, neonatal DBS are routinely collected in many countries, and their biobanks represent gold mines for research. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome-wide profiling. In particular, DNA methylome analysis in DBS has proven to be technically challenging, mainly due to the requirement for stringent preprocessing, such as bisulfite conversion. Moreover, DNA amplification, required to increase its yield, often leads to a bias in the analysis, particularly in methylome profiles. Thus, it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses. Using a combination of in-house-derived and modified commercial extraction methods, we developed two robust protocols that produced increased DNA yield and quality from DBS. Though both protocols are more efficient relative to other published methods, one protocol yields less DNA compared to the other, but shows improved 260/280 spectrophotometric ratios, which are useful for sample quality assessment. Both protocols consist of two sequential phases, each involving several critical steps. Phase I comprises blood extraction off the filter papers, cell lysis, and protein digestion. Phase II involves DNA precipitation, purification, and elution. Results from subsequent locus-specific and genome-wide DNA methylation analyses demonstrate the high quality, reproducibility, and consistency of the data. This work may prove useful to meet the increased demand for research on DBS, particularly with a focus on the epigenetic origins of human diseases and newborn screening programs.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the 10 most incident cancer types in the world, and it is mainly associated with tobacco and alcohol consumption. ESCC mortality rates stand very close to its incidence, which is a direct consequence of a late diagnosis and an inefficient treatment. Although this scenery is quite alarming, the major molecular alterations that drive this carcinogenesis process remain unclear. We have previously shown through the first ESCC methylome analysis that TFF1 promoter is frequently hypermethylated in ESCC. Here, to evaluate TFF1 methylation as a potential biomarker of early ESCC diagnosis, we investigated the status of TFF1 promoter methylation and its expression in ESSC and histologically normal tumor surrounding tissue of ESCC patients in comparison to healthy esophagus of non-cancer individuals., Results: Analysis of TFF1 promoter methylation, and gene and protein expression in 65 ESCC patients and 88 controls revealed that TFF1 methylation levels were already increased in histologically normal tumor surrounding tissue of ESCC patients when compared to healthy esophagus of non-cancer individuals. This increase in DNA methylation was followed by the reduction of TFF1 mRNA expression. Interestingly, TFF1 expression was capable of distinguishing tumor surrounding normal tissue from normal mucosa of healthy individuals with 92% accuracy. In addition, TFF1 protein was undetectable both in tumor and surrounding mucosa by immunohistochemistry, while submucosa glands of the healthy esophagus showed positive staining. Furthermore, treatment of TE-1 and TE-13 ESCC cell lines with decitabine led to a reduction of promoter methylation and consequent upregulation of TFF1 gene and protein expression. Finally, using TCGA data we showed that TFF1 loss is observed in ESCC, but not in esophageal adenocarcinoma, highlighting the different molecular mechanisms involved in the development of each histological subtype of esophageal cancer., Conclusions: This study shows that TFF1 expression is silenced in early phases of ESCC development, which seems to be mediated at least in part by promoter hypermethylation, and provides the basis for the use of TFF1 expression as a potential biomarker for early ESCC detection., Competing Interests: The project was approved by the local ethic committees (Comitê de Ética em Pesquisa-Instituto Nacional de Câncer #116/11 and Comitê de Ética em Pesquisa-Hospital Universitário Pedro Ernesto #416).Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Previous studies have revealed a robust association between exposure to asbestos and human lung cancer. Accumulating evidence has highlighted the role of epigenome deregulation in the mechanism of carcinogen-induced malignancies. We examined the impact of asbestos on DNA methylation. Our genome-wide studies (using Illumina HumanMethylation450K BeadChip) of lung cancer tissue and paired normal lung from 28 asbestos-exposed or non-exposed patients, mostly smokers, revealed distinctive DNA methylation changes. We identified a number of differentially methylated regions (DMR) and differentially variable, differentially methylated CpGs (DVMC), with individual CpGs further validated by pyrosequencing in an independent series of 91 non-small cell lung cancer and paired normal lung. We discovered and validated BEND4, ZSCAN31 and GPR135 as significantly hypermethylated in lung cancer. DMRs in genes such as RARB (FDR 1.1 × 10 -19 , mean change in beta [Δ] -0.09), GPR135 (FDR 1.87 × 10 -8 , mean Δ -0.09) and TPO (FDR 8.58 × 10 -5 , mean Δ -0.11), and DVMCs in NPTN, NRG2, GLT25D2 and TRPC3 (all with p <0.05, t-test) were significantly associated with asbestos exposure status in exposed versus non-exposed lung tumors. Hypomethylation was characteristic to DVMCs in lung cancer tissue from asbestos-exposed subjects. When DVMCs related to asbestos or smoking were analyzed, 96% of the elements were unique to either of the exposures, consistent with the concept that the methylation changes in tumors may be specific for risk factors. In conclusion, we identified novel DNA methylation changes associated with lung tumors and asbestos exposure, suggesting that changes may be present in causal pathway from asbestos exposure to lung cancer., (© 2017 UICC.)

Understanding the processes that govern liver progenitor cell differentiation has important implications for the design of strategies targeting chronic liver diseases, whereby regeneration of liver tissue is critical. Although DNA methylation (5mC) and hydroxymethylation (5hmC) are highly dynamic during early embryonic development, less is known about their roles at later stages of differentiation. Using an in vitro model of hepatocyte differentiation, we show here that 5hmC precedes the expression of promoter 1 (P1)-dependent isoforms of HNF4A, a master transcription factor of hepatocyte identity. 5hmC and HNF4A expression from P1 are dependent on ten-eleven translocation (TET) dioxygenases. In turn, the liver pioneer factor FOXA2 is necessary for TET1 binding to the P1 locus. Both FOXA2 and TETs are required for the 5hmC-related switch in HNF4A expression. The epigenetic event identified here may be a key step for the establishment of the hepatocyte program by HNF4A., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Papillary thyroid carcinoma (PTC) is a common endocrine neoplasm with a recent increase in incidence in many countries. Although PTC has been explored by gene expression and DNA methylation studies, the regulatory mechanisms of the methylation on the gene expression was poorly clarified. In this study, DNA methylation profile (Illumina HumanMethylation 450K) of 41 PTC paired with non-neoplastic adjacent tissues (NT) was carried out to identify and contribute to the elucidation of the role of novel genic and intergenic regions beyond those described in the promoter and CpG islands (CGI). An integrative and cross-validation analysis were performed aiming to identify molecular drivers and pathways that are PTC-related., Results: The comparisons between PTC and NT revealed 4995 methylated probes (88% hypomethylated in PTC) and 1446 differentially expressed transcripts cross-validated by the The Cancer Genome Atlas data. The majority of these probes was found in non-promoters regions, distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes (mainly in the promoter and body regions, respectively) with negative and positive correlation, respectively. Genes showing negative correlation underlined FGF and retinoic acid signaling as critical canonical pathways disrupted by DNA methylation in PTC. BRAF mutation was detected in 68% (28 of 41) of the tumors, which presented a higher level of demethylation (95% hypomethylated probes) compared with BRAF wild-type tumors. A similar integrative analysis uncovered 40 of 254 differentially expressed genes, which are potentially regulated by DNA methylation in BRAF V600E-positive tumors. The methylation and expression pattern of six selected genes ( ERBB3 , FGF1 , FGFR2 , GABRB2 , HMGA2 , and RDH5 ) were confirmed as altered by pyrosequencing and RT-qPCR., Conclusions: DNA methylation loss in non-promoter, poor CGI and enhancer-enriched regions was a significant event in PTC, especially in tumors harboring BRAF V600E. In addition to the promoter region, gene body and 3'UTR methylation have also the potential to influence the gene expression levels (both, repressing and inducing). The integrative analysis revealed genes potentially regulated by DNA methylation pointing out potential drivers and biomarkers related to PTC development.

Background: Head and neck squamous cell carcinomas (HNSCCs) represent a heterogeneous group of cancers for which human papilloma virus (HPV) infection is an emerging risk factor. Previous studies showed promoter hypermethylation in HPV(+) oropharyngeal cancers, but only few consistent target genes have been so far described, and the evidence of a functional impact on gene expression is still limited., Methods: We performed global and stratified pooled analyses of epigenome-wide data in HNSCCs based on the Illumina HumanMethylation450 bead-array data in order to identify tissue-specific components and common viral epigenetic targets in HPV-associated tumours., Results: We identified novel differentially methylated CpGs and regions associated with viral infection that are independent of the anatomic site. In particular, most hypomethylated regions were characterized by a marked loss of CpG island boundaries, which showed significant correlations with expression of neighbouring genes. Moreover, a subset of only five CpGs in a few hypomethylated regions predicted HPV status with a high level of specificity in different cohorts. Finally, this signature was a better predictor of survival compared with HPV status determined by viral gene expression by RNA sequencing in The Cancer Genome Atlas cohort., Conclusions: We identified a novel epigenetic signature of HPV infection in HNSCCs which is independent of the anatomic site, is functionally correlated with gene expression and may be leveraged for improved stratification of prognosis in HNSCCs.

Aim of the Study: A vast majority of human malignancies are associated with ageing, and age is a strong predictor of cancer risk. Recently, DNA methylation-based marker of ageing, known as 'epigenetic clock', has been linked with cancer risk factors. This study aimed to evaluate whether the epigenetic clock is associated with breast cancer risk susceptibility and to identify potential epigenetics-based biomarkers for risk stratification., Methods: Here, we profiled DNA methylation changes in a nested case-control study embedded in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (n = 960) using the Illumina HumanMethylation 450K BeadChip arrays and used the Horvath age estimation method to calculate epigenetic age for these samples. Intrinsic epigenetic age acceleration (IEAA) was estimated as the residuals by regressing epigenetic age on chronological age., Results: We observed an association between IEAA and breast cancer risk (OR, 1.04; 95% CI, 1.007-1.076, P = 0.016). One unit increase in IEAA was associated with a 4% increased odds of developing breast cancer (OR, 1.04; 95% CI, 1.007-1.076). Stratified analysis based on menopausal status revealed that IEAA was associated with development of postmenopausal breast cancers (OR, 1.07; 95% CI, 1.020-1.11, P = 0.003). In addition, methylome-wide analyses revealed that a higher mean DNA methylation at cytosine-phosphate-guanine (CpG) islands was associated with increased risk of breast cancer development (OR per 1 SD = 1.20; 95 %CI: 1.03-1.40, P = 0.02) whereas mean methylation levels at non-island CpGs were indistinguishable between cancer cases and controls., Conclusion: Epigenetic age acceleration and CpG island methylation have a weak, but statistically significant, association with breast cancer susceptibility., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Breast cancer (BC) encompasses heterogeneous pathologies with different subtypes exhibiting distinct molecular changes, including those related to DNA methylation. However, the role of these changes in mediating BC heterogeneity is poorly understood. Lowly methylated regions (LMRs), non-CpG island loci that usually contain transcription factor (TF) binding sites, have been suggested to act as regulatory elements that define cellular identity. In this study, we aimed to identify the key subtype-specific TFs that may lead to LMR generation and shape the BC methylome and transcription program. We initially used whole-genome bisulfite sequencing (WGBS) data available at The Cancer Genome Atlas (TCGA) portal to identify subtype-specific LMRs. Differentially methylated regions (DMRs) within the BC PAM50 subtype-specific LMRs were selected by comparing tumors and normal tissues in a larger TCGA cohort assessed by HumanMethylation450 BeadChip (450K) arrays and TF enrichment analyses were performed. To assess the impact of LMRs on gene expression, TCGA RNA sequencing data were downloaded and Pearson correlations between methylation levels of loci presenting subtype-specific TF motifs and expression of the nearest genes were calculated. WGBS methylome data revealed a large number of LMRs for each of the BC subtypes. Analysis of these LMRs in the 450K datasets available for a larger sample set identified 7,765, 5,657, and 19 differentially methylated positions (DMPs) between normal adjacent tissues and tumor tissues from basal, luminal, and HER2-enriched subtypes, respectively. Unsupervised clustering showed that the discriminatory power of the top DMPs was remarkably strong for basal BC. Interestingly, in this particular subtype, we found 4,409 differentially hypomethylated positions grouped into 1,185 DMRs with a strong enrichment for the early B-cell factor 1 (EBF1) motifs. The methylation levels of the DMRs containing EBF1 motifs showed a strong negative correlation with the expression of 719 nearby genes, including BTS2 and CD74, two oncogenes known to be specific for basal BC subtype and for poor outcome. This study identifies LMRs specific to the three main BC subtypes and reveals EBF1 as a potentially important regulator of BC subtype-specific methylation and gene expression program.

Background: DNA methylation leaves a long-term signature of smoking exposure and is one potential mechanism by which tobacco exposure predisposes to adverse health outcomes, such as cancers, osteoporosis, lung, and cardiovascular disorders., Methods and Results: To comprehensively determine the association between cigarette smoking and DNA methylation, we conducted a meta-analysis of genome-wide DNA methylation assessed using the Illumina BeadChip 450K array on 15 907 blood-derived DNA samples from participants in 16 cohorts (including 2433 current, 6518 former, and 6956 never smokers). Comparing current versus never smokers, 2623 cytosine-phosphate-guanine sites (CpGs), annotated to 1405 genes, were statistically significantly differentially methylated at Bonferroni threshold of P<1×10 -7 (18 760 CpGs at false discovery rate <0.05). Genes annotated to these CpGs were enriched for associations with several smoking-related traits in genome-wide studies including pulmonary function, cancers, inflammatory diseases, and heart disease. Comparing former versus never smokers, 185 of the CpGs that differed between current and never smokers were significant P<1×10 -7 (2623 CpGs at false discovery rate <0.05), indicating a pattern of persistent altered methylation, with attenuation, after smoking cessation. Transcriptomic integration identified effects on gene expression at many differentially methylated CpGs., Conclusions: Cigarette smoking has a broad impact on genome-wide methylation that, at many loci, persists many years after smoking cessation. Many of the differentially methylated genes were novel genes with respect to biological effects of smoking and might represent therapeutic targets for prevention or treatment of tobacco-related diseases. Methylation at these sites could also serve as sensitive and stable biomarkers of lifetime exposure to tobacco smoke., Competing Interests: B. M. P. serves on Data Safety Monitoring Board (DSMB) of a clinical trial of a device funded by the manufacturer (Zoll LifeCor) and on the Steering Committee of the Yale Open Data Access Project funded by Johnson & Johnson. C.E.E. is currently employed by Astra Zeneca, although the work was completed prior to the employment. All other authors declare no conflicts of interest., (© 2016 American Heart Association, Inc.)

In a number of human cancers, NTN1 upregulation inhibits apoptosis induced by its so-called dependence receptors DCC and UNC5H, thus promoting tumor progression. In other cancers however, the selective inhibition of this dependence receptor death pathway relies on the silencing of pro-apoptotic effector proteins. We show here that a substantial fraction of human breast tumors exhibits simultaneous DNA methylation-dependent loss of expression of NTN1 and of DAPK1, a serine threonine kinase known to transduce the netrin-1 dependence receptor pro-apoptotic pathway. The inhibition of DNA methylation by drugs such as decitabine restores the expression of both NTN1 and DAPK1 in netrin-1-low cancer cells. Furthermore, a combination of decitabine with NTN1 silencing strategies or with an anti-netrin-1 neutralizing antibody potentiates tumor cell death and efficiently blocks tumor growth in different animal models. Thus, combining DNA methylation inhibitors with netrin-1 neutralizing agents may be a valuable strategy for combating cancer., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Aim: Epigenetic changes may occur in response to environmental stressors, and an altered epigenome pattern may represent a stable signature of environmental exposure., Materials & Methods: Here, we examined the potential of DNA methylation changes in 910 prediagnostic peripheral blood samples as a marker of exposure to tobacco smoke in a large multinational cohort., Results: We identified 748 CpG sites that were differentially methylated between smokers and nonsmokers, among which we identified novel regionally clustered CpGs associated with active smoking. Importantly, we found a marked reversibility of methylation changes after smoking cessation, although specific genes remained differentially methylated up to 22 years after cessation., Conclusion: Our study has comprehensively cataloged the smoking-associated DNA methylation alterations and showed that these alterations are reversible after smoking cessation.

Epigenetic modifications, including DNA methylation, represent a potential mechanism for environmental impacts on human disease. Maternal smoking in pregnancy remains an important public health problem that impacts child health in a myriad of ways and has potential lifelong consequences. The mechanisms are largely unknown, but epigenetics most likely plays a role. We formed the Pregnancy And Childhood Epigenetics (PACE) consortium and meta-analyzed, across 13 cohorts (n = 6,685), the association between maternal smoking in pregnancy and newborn blood DNA methylation at over 450,000 CpG sites (CpGs) by using the Illumina 450K BeadChip. Over 6,000 CpGs were differentially methylated in relation to maternal smoking at genome-wide statistical significance (false discovery rate, 5%), including 2,965 CpGs corresponding to 2,017 genes not previously related to smoking and methylation in either newborns or adults. Several genes are relevant to diseases that can be caused by maternal smoking (e.g., orofacial clefts and asthma) or adult smoking (e.g., certain cancers). A number of differentially methylated CpGs were associated with gene expression. We observed enrichment in pathways and processes critical to development. In older children (5 cohorts, n = 3,187), 100% of CpGs gave at least nominal levels of significance, far more than expected by chance (p value < 2.2 × 10(-16)). Results were robust to different normalization methods used across studies and cell type adjustment. In this large scale meta-analysis of methylation data, we identified numerous loci involved in response to maternal smoking in pregnancy with persistence into later childhood and provide insights into mechanisms underlying effects of this important exposure., (Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Viral infections are able to modify the host's cellular programs, with DNA methylation being a biological intermediate in this process. The extent to which viral infections deregulate gene expression and DNA methylation is not fully understood. In the case of Hepatitis B virus (HBV), there is evidence for an interaction between viral proteins and the host DNA methylation machinery. We studied the ability of HBV to modify the host transcriptome and methylome, using naturally infected primary human hepatocytes to better mimic the clinical setting.Gene expression was especially sensitive to culture conditions, independently of HBV infection. However, we identified non-random changes in gene expression and DNA methylation occurring specifically upon HBV infection. There was little correlation between expression and methylation changes, with transcriptome being a more sensitive marker of time-dependent changes induced by HBV. In contrast, a set of differentially methylated sites appeared early and were stable across the time course experiment. Finally, HBV-induced DNA methylation changes were defined by a specific chromatin context characterized by CpG-poor regions outside of gene promoters.These data support the ability of HBV to modulate host cell expression and methylation programs. In addition, it may serve as a reference for studies addressing the genome-wide consequences of HBV infection in human hepatocytes.

Background: Exposure to environmental toxins during embryonic development may lead to epigenetic changes that influence disease risk in later life. Aflatoxin is a contaminant of staple foods in sub-Saharan Africa, is a known human liver carcinogen and has been associated with stunting in infants., Methods: We have measured aflatoxin exposure in 115 pregnant women in The Gambia and examined the DNA methylation status of white blood cells from their infants at 2-8 months old (mean 3.6 ± 0.9). Aflatoxin exposure in women was assessed using an ELISA method to measure aflatoxin albumin (AF-alb) adducts in plasma taken at 1-16 weeks of pregnancy. Genome-wide DNA methylation of infant white blood cells was measured using the Illumina Infinium HumanMethylation450beadchip., Results: AF-alb levels ranged from 3.9 to 458.4 pg/mg albumin. We found that aflatoxin exposure in the mothers was associated to DNA methylation in their infants for 71 CpG sites (false discovery rate < 0.05), with an average effect size of 1.7% change in methylation. Aflatoxin-associated differential methylation was observed in growth factor genes such as FGF12 and IGF1, and immune-related genes such as CCL28, TLR2 and TGFBI. Moreover, one aflatoxin-associated methylation region (corresponding to the miR-4520b locus) was identified., Conclusions: This study shows that maternal exposure to aflatoxin during the early stages of pregnancy is associated with differential DNA methylation patterns of infants, including in genes related to growth and immune function. This reinforces the need for interventions to reduce aflatoxin exposure, especially during critical periods of fetal and infant development., (© The Author 2015; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Background: Interindividual epigenetic variation that occurs systemically must be established prior to gastrulation in the very early embryo and, because it is systemic, can be assessed in easily biopsiable tissues. We employ two independent genome-wide approaches to search for such variants., Results: First, we screen for metastable epialleles by performing genomewide bisulfite sequencing in peripheral blood lymphocyte (PBL) and hair follicle DNA from two Caucasian adults. Second, we conduct a genomewide screen for genomic regions at which PBL DNA methylation is affected by season of conception in rural Gambia. Remarkably, both approaches identify the genomically imprinted VTRNA2-1 as a top environmentally responsive epiallele. We demonstrate systemic and stochastic interindividual variation in DNA methylation at the VTRNA2-1 differentially methylated region in healthy Caucasian and Asian adults and show, in rural Gambians, that periconceptional environment affects offspring VTRNA2-1 epigenotype, which is stable over at least 10 years. This unbiased screen also identifies over 100 additional candidate metastable epialleles, and shows that these are associated with cis genomic features including transposable elements., Conclusions: The non-coding VTRNA2-1 transcript (also called nc886) is a putative tumor suppressor and modulator of innate immunity. Thus, these data indicating environmentally induced loss of imprinting at VTRNA2-1 constitute a plausible causal pathway linking early embryonic environment, epigenetic alteration, and human disease. More broadly, the list of candidate metastable epialleles provides a resource for future studies of epigenetic variation and human disease.

Aim: To set up a targeted methylation analysis using semiconductor sequencing and evaluate the potential for studying methylation in circulating cell-free DNA (cfDNA)., Materials & Methods: Methylation of VIM, FBLN1, LTBP2, HINT2, h19 and IGF2 was analyzed in plasma cfDNA and white blood cell DNA obtained from eight hepatocellular carcinoma patients and eight controls using Ion Torrent™ PGM sequencer., Results: h19 and IGF2 showed consistent methylation levels and methylation was detected for VIM and FBLN1, whereas LTBP2 and HINT2 did not show methylation for target regions. VIM gene promoter methylation was higher in HCC cfDNA than in cfDNA of controls or white blood cell DNA., Conclusion: Semiconductor sequencing is a suitable method for analyzing methylation profiles in cfDNA. Furthermore, differences in cfDNA methylation can be detected between controls and hepatocellular carcinoma cases, even though due to the small sample set these results need further validation.

Epigenetic regulation of imprinted genes is regarded as a highly plausible explanation for linking dietary exposures in early life with the onset of diseases during childhood and adulthood. We sought to test whether prenatal dietary supplementation with docosahexaenoic acid (DHA) during pregnancy may modulate epigenetic states at birth. This study was based on a randomized intervention trial conducted in Mexican pregnant women supplemented daily with 400 mg of DHA or a placebo from gestation week 18-22 to parturition. We applied quantitative profiling of DNA methylation states at IGF2 promoter 3 (IGF2 P3), IGF2 differentially methylated region (DMR), and H19 DMR in cord blood mononuclear cells of the DHA-supplemented group (n = 131) and the control group (n = 130). In stratified analyses, DNA methylation levels in IGF2 P3 were significantly higher in the DHA group than the control group in preterm infants (P = 0.04). We also observed a positive association between DNA methylation levels and maternal body mass index; IGF2 DMR methylation was higher in the DHA group than the control group in infants of overweight mothers (P = 0.03). In addition, at H19 DMR, methylation levels were significantly lower in the DHA group than the control group in infants of normal weight mothers (P = 0.01). Finally, methylation levels at IGF2/H19 imprinted regions were associated with maternal BMI. These findings suggest that epigenetic mechanisms may be modulated by DHA, with potential impacts on child growth and development., (Copyright © 2014 the American Physiological Society.)

Background: Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses., Results: Using combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent., Conclusions: This study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS.

Background: Distinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display pronounced ability to initiate new tumors and induce metastasis. Recent evidence suggests that signals from transforming growth factor beta (TGF-β) may increase the survival of these so called tumor initiating cells leading to poor HCC prognosis. However, how TGF-β establishes and modifies the key features of these cell subpopulations is not fully understood., Results: In the present report we describe the differential DNA methylome of CD133-negative and CD133-expressing liver cancer cells. Next, we show that TGF-β is able to increase the proportion of CD133+ cells in liver cancer cell lines in a way that is stable and persistent across cell division. This process is associated with stable genome-wide changes in DNA methylation that persist through cell division. Differential methylation in response to TGF-β is under-represented at promoter CpG islands and enriched at gene bodies, including a locus in the body of the de novo DNA methyl-transferase DNMT3B gene. Moreover, phenotypic changes induced by TGF-β, including the induction of CD133, are impaired by siRNA silencing of de novo DNA methyl-transferases., Conclusions: Our study reveals a self-perpetuating crosstalk between TGF-β signaling and the DNA methylation machinery, which can be relevant in the establishment of cellular phenotypes. This is the first indication of the ability of TGF-β to induce genome-wide changes in DNA methylation, resulting in a stable change in the proportion of liver cancer cell subpopulations.