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Transcriptional regulation is commonly governed by alternative promoters. However, the regulatory architecture in alternative and reference promoters, and how they differ, remains elusive. In 100 CAGE-seq libraries from hepatocellular carcinoma patients, here we annotate 4083 alternative promoters in 2926 multi-promoter genes, which are largely undetected in normal livers. These genes are enriched in oncogenic processes and predominantly show association with overall survival. Alternative promoters are narrow nucleosome depleted regions, CpG island depleted, and enriched for tissue-specific transcription factors. Globally tumors lose DNA methylation. We show hierarchical retention of intragenic DNA methylation with CG-poor regions rapidly losing methylation, while CG-rich regions retain it, a process mediated by differential SETD2, H3K36me3, DNMT3B, and TET1 binding. This mechanism is validated in SETD2 knockdown cells and SETD2-mutated patients. Selective DNA methylation loss in CG-poor regions makes the chromatin accessible for alternative transcription. We show alternative promoters can control tumor transcriptomes and their regulatory architecture.

Childhood allergic diseases, including asthma, rhinitis and eczema, are prevalent conditions that share strong genetic and environmental components. Diagnosis relies on clinical history and measurements of allergen-specific IgE. We hypothesize that a multi-omics model could accurately diagnose childhood allergic disease. We show that nasal DNA methylation has the strongest predictive power to diagnose childhood allergy, surpassing blood DNA methylation, genetic risk scores, and environmental factors. DNA methylation at only three nasal CpG sites classifies allergic disease in Dutch children aged 16 years well, with an area under the curve (AUC) of 0.86. This is replicated in Puerto Rican children aged 9–20 years (AUC 0.82). DNA methylation at these CpGs additionally detects allergic multimorbidity and symptomatic IgE sensitization. Using nasal single-cell RNA-sequencing data, these three CpGs associate with influx of T cells and macrophages that contribute to allergic inflammation. Our study suggests the potential of methylation-based allergy diagnosis.

Cannabidiol (CBD) is a non-psychotomimetic compound present in cannabis sativa. Many recent studies have indicated that CBD has a promising therapeutic profile for stress-related psychiatric disorders, such as anxiety, schizophrenia and depression. Such a diverse profile has been associated with its complex pharmacology, since CBD can target different neurotransmitter receptors, enzymes, transporters and ion channels. However, the precise contribution of each of those mechanisms for CBD effects is still not yet completely understood. Considering that epigenetic changes make the bridge between gene expression and environment interactions, we review and discuss herein how CBD affects one of the main epigenetic mechanisms associated with the development of stress-related psychiatric disorders: DNA methylation (DNAm). Evidence from in vivo and in silico studies indicate that CBD can regulate the activity of the enzymes responsible for DNAm, due to directly binding to the enzymes and/or by indirectly regulating their activities as a consequence of neurotransmitter-mediated signaling. The implications of this new potential pharmacological target for CBD are discussed in light of its therapeutic and neurodevelopmental effects.

Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by the progressive accumulation of amyloid-beta and neurofibrillary tangles of tau in the neocortex. Utilizing extensive neuropathology data from the Brains for Dementia Research (BDR) cohort we performed the most systematic epigenome-wide association study (EWAS) of multiple measures of AD neuropathology yet undertaken, profiling DNA methylation in two cortical regions from 631 donors. We meta-analyzed our results with those from previous studies of DNA methylation in AD cortex (total n = 2,013 donors), identifying 334 cortical differentially methylated positions (DMPs) associated with AD pathology including methylomic variation at novel loci not previously implicated in dementia. We subsequently characterized DNA methylation in purified nuclei populations - enriched for neurons, oligodendrocytes and microglia - exploring the extent to which cortex AD-associated DMPs reflect differences manifest in specific cell populations. We find that the majority of DMPs identified in ‘bulk’ cortex tissue actually reflect DNA methylation differences occurring in non-neuronal cells, with dramatically increased effect sizes observed in microglia-enriched nuclei populations. Our study highlights the power of utilizing multiple measures of neuropathology to identify epigenetic signatures of AD and the importance of characterizing disease-associated variation in purified neural cell-types.

Bromodomain and extra-terminal tail (BET) proteins have been identified as potential epigenetic targets in cancer, including glioblastoma. These epigenetic modifiers link the histone code to gene transcription that can be disrupted with small molecule BET inhibitors (BETi). With the aim of developing rational combination treatments for glioblastoma, we analyzed BETi-induced differential gene expression in glioblastoma derived-spheres, and identified 6 distinct response patterns. To uncover emerging actionable vulnerabilities that can be targeted with a second drug, we extracted the 169 significantly disturbed DNA Damage Response genes and inspected their response pattern. The most prominent candidate with consistent downregulation, was the O-6-methylguanine-DNA methyltransferase (MGMT) gene, a known resistance factor for alkylating agent therapy in glioblastoma. BETi not only reduced MGMT expression in GBM cells, but also inhibited its induction, typically observed upon temozolomide treatment. To determine the potential clinical relevance, we evaluated the specificity of the effect on MGMT expression and MGMT mediated treatment resistance to temozolomide. BETi-mediated attenuation of MGMT expression was associated with reduction of BRD4- and Pol II-binding at the MGMT promoter. On the functional level, we demonstrated that ectopic expression of MGMT under an unrelated promoter was not affected by BETi, while under the same conditions, pharmacologic inhibition of MGMT restored the sensitivity to temozolomide, reflected in an increased level of g-H2AX, a proxy for DNA double-strand breaks. Importantly, expression of MSH6 and MSH2, which are required for sensitivity to unrepaired O6-methylGuanin-lesions, was only briefly affected by BETi. Taken together, the addition of BET-inhibitors to the current standard of care, comprising temozolomide treatment, may sensitize the 50% of patients whose glioblastoma exert an unmethylated MGMT promoter.

Low prosocial behavior in childhood has been consistently linked to later psychopathology, with evidence supporting the influence of both genetic and environmental factors on its development. Although neonatal DNA methylation (DNAm) has been found to prospectively associate with a range of psychological traits in childhood, its potential role in prosocial development has yet to be investigated. This study investigated prospective associations between cord blood DNAm at birth and low prosocial behavior within and across four longitudinal birth cohorts from the Pregnancy And Childhood Epigenetics (PACE) Consortium. We examined (a) developmental trajectories of "chronic-low" versus "typical" prosocial behavior across childhood in a case-control design (N = 2,095), and (b) continuous "low prosocial" scores at comparable cross-cohort time-points (N = 2,121). Meta-analyses were performed to examine differentially methylated positions and regions. At the cohort-specific level, three CpGs were found to associate with chronic low prosocial behavior; however, none of these associations was replicated in another cohort. Meta-analysis revealed no epigenome-wide significant CpGs or regions. Overall, we found no evidence for associations between DNAm patterns at birth and low prosocial behavior across childhood. Findings highlight the importance of employing multi-cohort approaches to replicate epigenetic associations and reduce the risk of false positive discoveries. The epigenetic studies in INMA were mainly funded by grants from Instituto de Salud Carlos III (Red INMA G03/176, CB06/02/0041, CP18/00018), Spanish Ministry of Health (FIS-PI04/1436, FIS-PI08/1151 including FEDER funds, FIS-PI11/00610, FIS-FEDER-PI06/0867, FIS-FEDER-PI03-1615), Generalitat de Catalunya-CIRIT 1999SGR 00241, Fundació La marató de TV3 (090430), EU Commission (261357-MeDALL: Mechanisms of the Development of ALLergy), and European Research Council (268479-BREATHE: BRain dEvelopment and Air polluTion ultrafine particles in scHool childrEn). MCT is funded by a Juan de la Cierva—Formación Postdoctoral Contract awarded by Ministry of Science and Innovation (FJC2018-036335-I). The 4, 7, and 11 year follow-up visits was funded by grants from Instituto de Salud Carlos III (Red INMA G03/176; CB06/02/0041; PI041436; PI081151 incl. FEDER funds; PI12/01890 incl. FEDER funds; CP13/00054 incl. FEDER funds; PI15/00118 incl. FEDER funds; CP16/00128 incl. FEDER funds; PI16/00118 incl. FEDER funds; PI16/00261 incl. FEDER funds; PI18/00547 incl. FEDER funds), CIBERESP, Generalitat de Catalunya-CIRIT 1999SGR 00241, Generalitat de Catalunya-AGAUR (2009 SGR 501, 2014 SGR 822), Fundació La marató de TV3 (090430), Spanish Ministry of Economy and Competitiveness (SAF2012-32991 incl. FEDER funds), Agence Nationale de Securite Sanitaire de l'Alimentation de l'Environnement et du Travail (1262C0010; EST-2016 RF-21), EU Commission (261357, 308333, 603794 and 634453). We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.

Chronic inflammation, marked by C-reactive protein, has been associated with changes in methylation, but the causal relationship is unclear. Here, the authors perform a Epigenome-wide association meta-analysis for C-reactive protein levels and find that these methylation changes are likely the consequence of inflammation and could contribute to disease.We performed a multi-ethnic Epigenome Wide Association study on 22,774 individuals to describe the DNA methylation signature of chronic low-grade inflammation as measured by C-Reactive protein (CRP). We find 1,511 independent differentially methylated loci associated with CRP. These CpG sites show correlation structures across chromosomes, and are primarily situated in euchromatin, depleted in CpG islands. These genomic loci are predominantly situated in transcription factor binding sites and genomic enhancer regions. Mendelian randomization analysis suggests altered CpG methylation is a consequence of increased blood CRP levels. Mediation analysis reveals obesity and smoking as important underlying driving factors for changed CpG methylation. Finally, we find that an activated CpG signature significantly increases the risk for cardiometabolic diseases and COPD.

Purpose: Beckwith-Wiedemann syndrome (BWS) is a developmental disorder caused by dysregulation of the imprinted gene cluster of chromosome 11p15.5 and often associated with loss of methylation (LOM) of the imprinting center 2 (IC2) located in KCNQ1 intron 10. To unravel the etiological mechanisms underlying these epimutations, we searched for genetic variants associated with IC2 LOM.Methods: We looked for cases showing the clinical features of both BWS and long QT syndrome (LQTS), which is often associated with KCNQ1 variants. Pathogenic variants were identified by genomic analysis and targeted sequencing. Functional experiments were performed to link these pathogenic variants to the imprinting defect.Results: We found three rare cases in which complete IC2 LOM is associated with maternal transmission of KCNQ1 variants, two of which were demonstrated to affect KCNQ1 transcription upstream of IC2. As a consequence of KCNQ1 haploinsufficiency, these variants also cause LQTS on both maternal and paternal transmission.Conclusion: These results are consistent with the hypothesis that, similar to what has been demonstrated in mouse, lack of transcription across IC2 results in failure of methylation establishment in the female germline and BWS later in development, and also suggest a new link between LQTS and BWS that is important for genetic counseling.

DNA methylation (5mC) is central to cellular identity. The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells., Erasure of DNA methylation from the parental genomes is critical to reset the methylome of differentiated gametes to pluripotent cells in the blastocyst. Here, the authors present a high-throughput single-cell method that enables strand-specific quantification of DNA methylation and identify distinct modes of DNA demethylation dynamics during early mammalian development.

DNA methylation (DNAm) is shaped by genetic and environmental factors and modulated by aging. Here, we examine interrelations between epigenetic aging, body weight (BW), and life span in 12 isogenic strains from the BXD family of mice that exhibit over twofold variation in longevity. Genome‐wide DNAm was assayed in 70 liver specimens from predominantly female cases, 6–25 months old, that were maintained on normal chow or high‐fat diet (HFD). We defined subsets of CpG regions associated with age, BW at young adulthood, and strain‐by‐diet‐dependent life span. These age‐associated differentially methylated CpG regions (age‐DMRs) featured distinct genomic characteristics, with DNAm gains over time occurring in sites such as promoters and exons that have high CpG density and low average methylation. CpG regions associated with BW were enriched in introns, tended to have lower methylation in mice with higher BW, and were inversely correlated with gene expression (i.e., higher mRNA levels in mice with higher BW). CpG regions associated with life span were linked to genes involved in life span modulation, including the telomerase reverse transcriptase gene, Tert, which had both lower methylation and higher expression in long‐lived strains. An epigenetic clock defined from age‐DMRs revealed accelerated aging in mice belonging to strains with shorter life spans. Both higher BW and the HFD were associated with accelerated epigenetic aging. Our results highlight the age‐accelerating effect of heavier BW. Furthermore, we demonstrate that the measure of epigenetic aging derived from age‐DMRs can predict genotype and diet‐induced differences in life span among female BXD members., The epigenetic clock, also referred to as DNA methylation age, provides a measure of biological aging. This clock can predict life expectancy in genetically distinct mouse stains, and is accelerated by higher body weight and high‐fat diet.

Epigenetic processes that regulate gene expression, such as DNA methylation (DNAm), have been linked to individual differences in physical aggression. Yet, it is currently unclear whether: (a) DNAm patterns in humans associate with physical aggression independently of other co-occurring psychiatric and behavioral symptoms; (b) whether these patterns are observable across multiple tissues; and (c) whether they may function as a causal versus noncausal biomarker of physical aggression. Here, we used a multisample, cross-tissue design to address these questions. First, we examined genome-wide DNAm patterns (buccal swabs; Illumina 450k) associated with engagement in physical fights in a sample of high-risk youth (n = 119; age = 16-24 years; 53% female). We identified one differentially methylated region in DRD4, which survived genome-wide correction, associated with physical aggression above and beyond co-occurring symptomatology (e.g., ADHD, substance use), and showed strong cross-tissue concordance with both blood and brain. Second, we found that DNAm sites within this region were also differentially methylated in an independent sample of young adults, between individuals with a history of chronic-high versus low physical aggression (peripheral T cells; ages 26-28). Finally, we ran a Mendelian randomization analysis using GWAS data from the EAGLE consortium to test for a causal association of DRD4 methylation with physical aggression. Only one genetic instrument was eligible for the analysis, and results provided no evidence for a causal association. Overall, our findings lend support for peripheral DRD4 methylation as a potential biomarker of physically aggressive behavior, with no evidence yet of a causal relationship.

ObjectiveEpigenetic changes contribute to pancreatic neuroendocrine tumor (PanNET) development. Hypermethylation of promoter DNA as a cause of tumor suppressor gene silencing is a well-established oncogenic mechanism that is potentially reversible and therefore an interesting therapeutic target. Multiple endocrine neoplasia type 1 (MEN1) is the most frequent cause of inherited PanNETs. The aim of this study was to determine promoter methylation profiles in MEN1-related PanNETs.Design and methodsMethylation-specific multiplex ligation-dependent probe amplification was used to assess promoter methylation of 56 tumor suppressor genes in MEN1-related (n = 61) and sporadic (n = 34) PanNETs. Differences in cumulative methylation index (CMI), individual methylation percentages and frequency of promoter hypermethylation between subgroups were analyzed.ResultsWe found promoter methylation of a large number of potential tumor suppressor genes. CMI (median CMI: 912 vs 876,P = 0.207) was the same in MEN1-related and sporadic PanNETs. We found higher methylation percentages ofCASP8in MEN1-related PanNETs (median: 59% vs 16.5%,P = 0.002). In MEN1-related non-functioning PanNETs, the CMI was higher in larger PanNETs (>2 cm) (median: 969.5 vs 838.5;P = 0.021) and in PanNETs with liver metastases (median: 1036 vs 869;P = 0.013). Hypermethylation ofMGMT2was more frequent in non-functioning PanNETs compared to insulinomas (median: 44.7% vs 8.3%;P = 0.022). Hypermethylation of the Von Hippel–Lindau gene promoter was observed in one MEN1-related PanNET and was associated with loss of protein expression.ConclusionPromoter hypermethylation is a frequent event in MEN1-related and sporadic PanNETs. Targeting DNA methylation could be of therapeutic value in MEN1 patients with advanced PanNETs.

// Allyson C. Espinal 1, * , Dan Wang 2, * , Li Yan 2 , Song Liu 2 , Li Tang 3 , Qiang Hu 2 , Carl D. Morrison 4 , Christine B. Ambrosone 4 , Michael J. Higgins 1, # , Lara E. Sucheston-Campbell 5, 6, # 1 Department of Molecular and Cell Biology, Roswell Park Cancer Institute, Buffalo NY, USA 2 Department of Biostatistics, Roswell Park Cancer Institute, Buffalo, NY, USA 3 Department of Cancer Prevention and Control, Roswell Park Cancer Institute, Buffalo, NY, USA 4 Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY, USA 5 College of Pharmacy, The Ohio State University, Columbus, OH, USA 6 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA * These authors contributed jointly as first authors # These authors contributed jointly as senior authors Correspondence to: Lara E. Sucheston-Campbell, email: sucheston-campbell.1@osu.edu Michael J. Higgins, email: Michael.Higgins@roswellpark.org Keywords: DNA methylation/epigenetics, breast cancer, estrogen receptor negative Received: May 27, 2016 Accepted: January 10, 2017 Published: January 19, 2017 ABSTRACT Background: DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. Results: For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER– tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Materials and Methods: Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER−) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. Conclusions: FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.

Hypertensive disorders of pregnancy (HDP) are associated with low birth weight, shorter gestational age, and increased risk of maternal and offspring cardiovascular diseases later in life. The mechanisms involved are poorly understood, but epigenetic regulation of gene expression may play a part. We performed meta-analyses in the Pregnancy and Childhood Epigenetics Consortium to test the association between either maternal HDP (10 cohorts; n=5242 [cases=476]) or preeclampsia (3 cohorts; n=2219 [cases=135]) and epigenome-wide DNA methylation in cord blood using the Illumina HumanMethylation450 BeadChip. In models adjusted for confounders, and with Bonferroni correction, HDP and preeclampsia were associated with DNA methylation at 43 and 26 CpG sites, respectively. HDP was associated with higher methylation at 27 (63%) of the 43 sites, and across all 43 sites, the mean absolute difference in methylation was between 0.6% and 2.6%. Epigenome-wide associations of HDP with offspring DNA methylation were modestly consistent with the equivalent epigenome-wide associations of preeclampsia with offspring DNA methylation (R-2=0.26). In longitudinal analyses conducted in 1 study (n=108 HDP cases; 550 controls), there were similar changes in DNA methylation in offspring of those with and without HDP up to adolescence. Pathway analysis suggested that genes located at/near HDP-associated sites may be involved in developmental, embryogenesis, or neurological pathways. HDP is associated with offspring DNA methylation with potential relevance to development.

We use a genome-wide association of 1 million parental lifespans of genotyped subjects and data on mortality risk factors to validate previously unreplicated findings near CDKN2B-AS1, ATXN2/BRAP, FURIN/FES, ZW10, PSORS1C3, and 13q21.31, and identify and replicate novel findings near ABO, ZC3HC1, and IGF2R. We also validate previous findings near 5q33.3/EBF1 and FOXO3, whilst finding contradictory evidence at other loci. Gene set and cell-specific analyses show that expression in foetal brain cells and adult dorsolateral prefrontal cortex is enriched for lifespan variation, as are gene pathways involving lipid proteins and homeostasis, vesicle-mediated transport, and synaptic function. Individual genetic variants that increase dementia, cardiovascular disease, and lung cancer – but not other cancers – explain the most variance. Resulting polygenic scores show a mean lifespan difference of around five years of life across the deciles. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., eLife digest Ageing happens to us all, and as the cabaret singer Maurice Chevalier pointed out, "old age is not that bad when you consider the alternative". Yet, the growing ageing population of most developed countries presents challenges to healthcare systems and government finances. For many older people, long periods of ill health are part of the end of life, and so a better understanding of ageing could offer the opportunity to prolong healthy living into old age. Ageing is complex and takes a long time to study – a lifetime in fact. This makes it difficult to discern its causes, among the countless possibilities based on an individual’s genes, behaviour or environment. While thousands of regions in an individual’s genetic makeup are known to influence their risk of different diseases, those that affect how long they will live have proved harder to disentangle. Timmers et al. sought to pinpoint such regions, and then use this information to predict, based on their DNA, whether someone had a better or worse chance of living longer than average. The DNA of over 500,000 people was read to reveal the specific ‘genetic fingerprints’ of each participant. Then, after asking each of the participants how long both of their parents had lived, Timmers et al. pinpointed 12 DNA regions that affect lifespan. Five of these regions were new and had not been linked to lifespan before. Across the twelve as a whole several were known to be involved in Alzheimer’s disease, smoking-related cancer or heart disease. Looking at the entire genome, Timmers et al. could then predict a lifespan score for each individual, and when they sorted participants into ten groups based on these scores they found that top group lived five years longer than the bottom, on average. Many factors beside genetics influence how long a person will live and our lifespan cannot be read from our DNA alone. Nevertheless, Timmers et al. had hoped to narrow down their search and discover specific genes that directly influence how quickly people age, beyond diseases. If such genes exist, their effects were too small to be detected in this study. The next step will be to expand the study to include more participants, which will hopefully pinpoint further genomic regions and help disentangle the biology of ageing and disease.

Background Lynch syndrome (LS) predisposes to endometrial cancer (EC), colorectal cancer, and other cancers through inherited pathogenic variants affecting mismatch-repair (MMR) genes. Diagnosing LS in women with EC can reduce subsequent cancer mortality through colonoscopic surveillance and aspirin chemoprevention; it also enables cascade testing of relatives. A growing consensus supports LS screening in EC; however, the expected proportion of test positives, and optimal testing strategy is uncertain. Previous studies from insurance-based healthcare systems were limited by narrow selection criteria, failure to apply reference standard tests consistently, and poor conversion to definitive testing. The aim of this study was to establish the prevalence of LS and the diagnostic accuracy of LS testing strategies in an unselected EC population. Methods and findings This was a prospective cross-sectional study carried out at a large United Kingdom gynaecological cancer centre between October 2015 and January 2017. Women diagnosed with EC or atypical hyperplasia (AH) were offered LS testing. Tumours underwent MMR immunohistochemistry (IHC), microsatellite instability (MSI), and targeted MLH1-methylation testing. Women, Emma Crosbie and colleagues investigate the prevalence of and strategies for identifying Lynch syndrome among women with endometrial cancer., Author summary Why was this study done? Endometrial (womb) cancer (EC) is the most common gynaecological cancer in the developed world, and its incidence is rising. A significant minority (around 3%) of EC are caused by an inherited genetic predisposition called Lynch syndrome (LS). EC may be the first sign that a woman has LS. She is likely to survive this cancer but develop other preventable cancers related to LS later in life. Her family members are also at risk. Identifying women with LS can enable them to reduce their risk of new cancers, for example, through bowel (colorectal) cancer surveillance (colonoscopy). We do not know how many women with EC have LS or how best to identify them; current practice is based on experience in bowel cancer and may not be accurate in EC. What did this research do and find? We tested 500 women with EC treated in a large tertiary referral centre in the North West of England for LS. We did not preselect women to test based on clinical or tumour characteristics. We tested tumours for features of LS called mismatch repair (MMR) deficiency and microsatellite instability (MSI). Women with strongly suggestive clinical or tumour characteristics underwent germline LS testing. In total, 16 of 500 women (3%) had LS, and these women could not always be predicted by their age or family history. MMR deficiency was more accurate than MSI at identifying LS-EC, picking up 16/16 (100%) versus 9/16 (56%). What do these findings mean? In our study, we found that 3% of women with endometrial cancer have LS and can benefit from strategies to reduce their future cancer risk. Our results suggest that it may be best to test everyone because preselecting women to test based on clinical or tumour characteristics misses cases of LS. In this population, tumour MMR deficiency was more accurate than MSI at identifying LS in EC. Our results should be interpreted with caution because we did not do germline testing on all women, and the number of women we tested was relatively small.

Aberrant methylation of multiple promoter CpG islands could be related to the biology of ovarian tumors and its determination could help to improve treatment strategies. DNA methylation profiling was performed using the Methylation Ligation-dependent Macroarray (MLM), an array-based analysis. Promoter regions of 41 genes were analyzed in 102 ovarian tumors and 17 normal ovarian samples. An average of 29% of hypermethylated promoter genes was observed in normal ovarian tissues. This percentage increased slightly in serous, endometrioid, and mucinous carcinomas (32%, 34%, and 45%, respectively), but decreased in germ cell tumors (20%). Ovarian tumors had methylation profiles that were more heterogeneous than other epithelial cancers. Unsupervised hierarchical clustering identified four groups that are very close to the histological subtypes of ovarian tumors. Aberrant methylation of three genes ( BRCA1 , MGMT , and MLH1 ), playing important roles in the different DNA repair mechanisms, were dependent on the tumor subtype and represent powerful biomarkers for precision therapy. Furthermore, a promising relationship between hypermethylation of MGMT , OSMR , ESR1 , and FOXL2 and overall survival was observed. Our study of DNA methylation profiling indicates that the different histotypes of ovarian cancer should be treated as separate diseases both clinically and in research for the development of targeted therapies.

The global rise in metabolic diseases can be attributed to a complex interplay between biology, behavior and environmental factors. This article reviews the current literature concerning DNA methylation-based epigenetic inheritance (intergenerational and transgenerational) of metabolic diseases through the male germ line. Included are a presentation of the basic principles for DNA methylation in developmental programming, and a description of windows of susceptibility for the inheritance of environmentally induced aberrations in DNA methylation and their associated metabolic disease phenotypes. To this end, escapees, genomic regions with the intrinsic potential to transmit acquired paternal epigenetic information across generations by escaping the extensive programmed DNA demethylation that occurs during gametogenesis and in the zygote, are described. The ongoing descriptive and functional examinations of DNA methylation in the relevant biological samples, in conjugation with analyses of non-coding RNA and histone modifications, hold promise for improved delineation of the effect size and mechanistic background for epigenetic inheritance of metabolic diseases.

X-chromosome inactivation (XCI), i.e., the inactivation of one of the female X chromosomes, restores equal expression of X-chromosomal genes between females and males. However, ~10% of genes show variable degrees of escape from XCI between females, although little is known about the causes of variable XCI. Using a discovery data-set of 1867 females and 1398 males and a replication sample of 3351 females, we show that genetic variation at three autosomal loci is associated with female-specific changes in X-chromosome methylation. Through cis-eQTL expression analysis, we map these loci to the genes SMCHD1/METTL4, TRIM6/HBG2, and ZSCAN9. Low-expression alleles of the loci are predominantly associated with mild hypomethylation of CpG islands near genes known to variably escape XCI, implicating the autosomal genes in variable XCI. Together, these results suggest a genetic basis for variable escape from XCI and highlight the potential of a population genomics approach to identify genes involved in XCI.

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.

An adequate intake of vitamin B12 during pregnancy plays an important role in offspring neurodevelopment, potentially via epigenetic processes. We used a two-step Mendelian randomization approach to assess whether DNA methylation plays a mediating and causal role in associations between maternal vitamin B12 status and offspring's cognition. Firstly, we estimated the causal effect of maternal vitamin B12 levels on cord blood DNA methylation using the maternal FUT2 genotypes rs492602:A > G and rs1047781:A > T as proxies for circulating vitamin B12 levels in the Avon Longitudinal Study of Parents and Children (ALSPAC) and we tested the observed associations in a replication cohort. Secondly, we estimated the causal effect of DNA methylation on IQ using the offspring genotype at sites close to the methylated CpG site as a proxy for DNA methylation in ALSPAC and in a replication sample. The first step Mendelian randomization estimated that maternal vitamin B12 had a small causal effect on DNA methylation in offspring at three CpG sites, which was replicated for one of the sites. The second step Mendelian randomization found weak evidence of a causal effect of DNA methylation at two of these sites on childhood performance IQ which was replicated for one of the sites. The findings support a causal effect of maternal vitamin B12 levels on cord blood DNA methylation, and a causal effect of vitamin B12-responsive DNA methylation changes on children's cognition. Some limitations were identified and future studies using a similar approach should aim to overcome such issues.

// Francesca Pirini 1, * , Sassan Noazin 2, * , Martha H. Jahuira-Arias 3, 4, * , Sebastian Rodriguez-Torres 3 , Leah Friess 3 , Christina Michailidi 3 , Jaime Cok 5 , Juan Combe 6 , Gloria Vargas 7 , William Prado 8 , Ethan Soudry 3 , Jimena Perez 3 , Tikki Yudin 3 , Andrea Mancinelli 3 , Helen Unger 3 , Carmen Ili-Gangas 9, 10 , Priscilla Brebi-Mieville 9, 10 , Douglas E. Berg 11, 12 , Masamichi Hayashi 3, 13 , David Sidransky 3 , Robert H. Gilman 2, 4 , Rafael Guerrero-Preston 3, 14 1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 2 The Johns Hopkins University, Bloomberg School of Public Health, Department of International Health, Baltimore, MD, USA 3 The Johns Hopkins University, School of Medicine, Otolaryngology Department, Head and Neck Cancer Research Division, Baltimore, MD, USA 4 Universidad Peruana Cayetano Heredia, Lima, Peru 5 Hospital Nacional Cayetano Heredia, Pathology Department, Lima, Peru 6 Instituto Nacional de Enfermedades Neoplasicas, Gastroenterology Department, Lima, Peru 7 Hospital Nacional Arzobispo Loayza, Gastroenterology Department, Lima, Peru 8 Hospital Nacional Dos de Mayo, Gastroenterology Department, Lima, Peru 9 Laboratory of Molecular Pathology, Department of Pathological Anatomy, School of Medicine, Universidad de La Frontera, Temuco, Chile 10 Center of Excellence in Translational Medicine - Scientific and Technological Bioresource Nucleus (CEMT-BIOREN), Universidad de La Frontera, Temuco, Chile 11 Washington University Medical School, Department of Molecular Microbiology, St Louis, MO, USA 12 University of California San Diego, Department of Medicine, La Jolla, CA, USA 13 Department of Gastroenterological Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan 14 University of Puerto Rico School of Medicine, Department of Obstetrics and Gynecology, San Juan, Puerto Rico * These authors equally contributed to this work Correspondence to: Rafael Guerrero-Preston, email: rafael.guerrero@upr.edu Robert H. Gilman, email: rgilman1@jhmi.edu Keywords: translational epigenomics, global DNA methylation index, epigenome-wide DNA methylation analysis, IRF4, ELMO1 Received: January 09, 2017 Accepted: February 24, 2017 Published: March 16, 2017 ABSTRACT Clinically useful molecular tools to triage gastric cancer patients are not currently available. We aimed to develop a molecular tool to predict gastric cancer risk in endoscopy-driven biopsies obtained from high-risk gastric cancer clinics in low resource settings. We discovered and validated a DNA methylation biomarker panel in endoscopic samples obtained from 362 patients seen between 2004 and 2009 in three high-risk gastric cancer clinics in Lima, Peru, and validated it in 306 samples from the Cancer Genome Atlas project (“TCGA”). Global, epigenome wide and gene-specific DNA methylation analyses were used in a Phase I Biomarker Development Trial to identify a continuous biomarker panel that combines a Global DNA Methylation Index (GDMI) and promoter DNA methylation levels of IRF4, ELMO1, CLIP4 and MSC . We observed an inverse association between the GDMI and histological progression to gastric cancer, when comparing gastritis patients without metaplasia (mean = 5.74, 95% CI, 4.97−6.50), gastritis patients with metaplasia (mean = 4.81, 95% CI, 3.77−5.84), and gastric cancer cases (mean = 3.38, 95% CI, 2.82−3.94), respectively ( p 4 are useful molecular tools for gastric cancer risk stratification in endoscopic biopsies.

Deletion of the long arm of chromosome 11 (11q deletion) is one of the most frequent events that occur during the development of aggressive neuroblastoma. Clinically, 11q deletion is associated with higher disease stage and decreased survival probability. During the last 25 years, extensive efforts have been invested to identify the precise frequency of 11q aberrations in neuroblastoma, the recurrently involved genes, and to understand the molecular mechanisms of 11q deletion, but definitive answers are still unclear. In this review, it is our intent to compile and review the evidence acquired to date on 11q deletion in neuroblastoma.

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species.

Vertebrate DNA can be chemically modified by methylation of the 5 position of the cytosine base in the context of CpG dinucleotides. This modification creates a binding site for MBD (methyl-CpG-binding domain) proteins which target chromatin-modifying activities that are thought to contribute to transcriptional repression and maintain heterochromatic regions of the genome. In contrast with DNA methylation, which is found broadly across vertebrate genomes, non-methylated DNA is concentrated in regions known as CGIs (CpG islands). Recently, a family of proteins which encode a ZF-CxxC (zinc finger-CxxC) domain have been shown to specifically recognize non-methylated DNA and recruit chromatin-modifying activities to CGI elements. For example, CFP1 (CxxC finger protein 1), MLL (mixed lineage leukaemia protein), KDM (lysine demethylase) 2A and KDM2B regulate lysine methylation on histone tails, whereas TET (ten-eleven translocation) 1 and TET3 hydroxylate methylated cytosine bases. In the present review, we discuss the most recent advances in our understanding of how ZF-CxxC domain-containing proteins recognize non-methylated DNA and describe their role in chromatin modification at CGIs. © 2013 The Author(s).

The etiology of respiratory allergies (RA) can be partly explained by DNA methylation changes caused by adverse environmental and lifestyle factors experienced early in life. Longitudinal, prospective studies can aid in the unravelment of the epigenetic mechanisms involved in the disease development. High compliance rates can be expected in these studies when data is collected using non-invasive and convenient procedures. Saliva is an attractive biofluid to analyze changes in DNA methylation patterns. We investigated in a pilot study the differential methylation in saliva of RA (n = 5) compared to healthy controls (n = 5) using the Illumina Methylation 450K BeadChip platform. We evaluated the results against the results obtained in mononuclear blood cells from the same individuals. Differences in methylation patterns from saliva and mononuclear blood cells were clearly distinguishable (P-Adj0.1), respectively, of which 216 were in common. The methylation levels of these sites were significantly correlated between blood and saliva. The absolute levels of methylation in blood and saliva were confirmed for 3 selected DMS in the PM20D1, STK32C, and FGFR2 genes using pyrosequencing analysis. The differential methylation could only be confirmed for DMS in PM20D1 and STK32C genes in saliva. We show that saliva can be used for genome-wide methylation analysis and that it is possible to identify DMS when comparing RA cases and healthy controls. The results were replicated in blood cells of the same individuals and confirmed by pyrosequencing analysis. This study provides proof-of-concept for the applicability of saliva-based whole-genome methylation analysis in the field of respiratory allergy. SL is the beneficiary of a post-doctoral grant from the AXA Research Fund and the Cefic-LRI Innovative Science Award 2013.

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans' lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole.

C13ORF18 is frequently hypermethylated in cervical cancer but not in normal cervix and might serve as a biomarker for the early detection of cervical cancer in scrapings. As hypermethylation is often observed for silenced tumor suppressor genes (TSGs), hypermethylated biomarker genes might exhibit tumor suppressive activities upon re-expression. Epigenetic drugs are successfully exploited to reverse TSG silencing, but act genome-wide. Artificial Transcription Factors (ATFs) provide a gene-specific approach for re-expression of silenced genes. Here, we investigated the potential tumor suppressive role of C13ORF18 in cervical cancer by ATF-induced re-expression.Five zinc finger proteins were engineered to bind the C13ORF18 promoter and fused to a strong transcriptional activator. C13ORF18 expression could be induced in cervical cell lines: ranging from >40-fold in positive (C13ORF18-unmethylated) cells to >110-fold in negative (C13ORF18-methylated) cells. Re-activation of C13ORF18 resulted in significant cell growth inhibition and/or induction of apoptosis. Co-treatment of cell lines with ATFs and epigenetic drugs further enhanced the ATF-induced effects. Interestingly, reactivation of C13ORF18 led to partial demethylation of the C13ORF18 promoter and decreased repressive histone methylation. These data demonstrate the potency of ATFs to re-express and potentially demethylate hypermethylated silenced genes. Concluding, we show that C13ORF18 has a TSG function in cervical cancer and may serve as a therapeutic anti-cancer target. As the amount of epimutations in cancer exceeds the number of gene mutations, ATFs provide promising tools to validate hypermethylated marker genes as therapeutic targets. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Human cancers nearly ubiquitously harbor epigenetic alterations. While such alterations in epigenetic marks, including DNA methylation, are potentially heritable, they can also be dynamically altered. Given this potential for plasticity, the degree to which epigenetic changes can be subject to selection and act as drivers of neoplasia has been questioned. Here, we carried out genome-scale analyses of DNA methylation alterations in lethal metastatic prostate cancer and created DNA methylation “cityscape” plots to visualize these complex data. We show that somatic DNA methylation alterations, despite showing marked inter-individual heterogeneity among men with lethal metastatic prostate cancer, were maintained across all metastases within the same individual. The overall extent of maintenance in DNA methylation changes was comparable to that of genetic copy number alterations. Regions that were frequently hypermethylated across individuals were markedly enriched for cancer and development/differentiation related genes. Additionally, regions exhibiting high consistency of hypermethylation across metastases within individuals, even if variably hypermethylated across individuals, showed enrichment of cancer-related genes. Interestingly, whereas some regions showed intra-individual metastatic tumor heterogeneity in promoter methylation, such methylation alterations were generally not correlated with gene expression. This was despite a general tendency for promoter methylation patterns to be strongly correlated with gene expression, particularly at regions that were variably methylated across individuals. These findings suggest that DNA methylation alterations have the potential for producing selectable driver events in carcinogenesis and disease progression and highlight the possibility of targeting such epigenome alterations for development of longitudinal markers and therapeutic strategies.

Age-related changes in DNA methylation have been implicated in cellular senescence and longevity, yet the causes and functional consequences of these variants remain unclear. To elucidate the role of age-related epigenetic changes in healthy ageing and potential longevity, we tested for association between whole-blood DNA methylation patterns in 172 female twins aged 32 to 80 with age and age-related phenotypes. Twin-based DNA methylation levels at 26,690 CpG-sites showed evidence for mean genome-wide heritability of 18%, which was supported by the identification of 1,537 CpG-sites with methylation QTLs in cis at FDR 5%. We performed genome-wide analyses to discover differentially methylated regions (DMRs) for sixteen age-related phenotypes (ap-DMRs) and chronological age (a-DMRs). Epigenome-wide association scans (EWAS) identified age-related phenotype DMRs (ap-DMRs) associated with LDL (STAT5A), lung function (WT1), and maternal longevity (ARL4A, TBX20). In contrast, EWAS for chronological age identified hundreds of predominantly hyper-methylated age DMRs (490 a-DMRs at FDR 5%), of which only one (TBX20) was also associated with an age-related phenotype. Therefore, the majority of age-related changes in DNA methylation are not associated with phenotypic measures of healthy ageing in later life. We replicated a large proportion of a-DMRs in a sample of 44 younger adult MZ twins aged 20 to 61, suggesting that a-DMRs may initiate at an earlier age. We next explored potential genetic and environmental mechanisms underlying a-DMRs and ap-DMRs. Genome-wide overlap across cis-meQTLs, genotype-phenotype associations, and EWAS ap-DMRs identified CpG-sites that had cis-meQTLs with evidence for genotype–phenotype association, where the CpG-site was also an ap-DMR for the same phenotype. Monozygotic twin methylation difference analyses identified one potential environmentally-mediated ap-DMR associated with total cholesterol and LDL (CSMD1). Our results suggest that in a small set of genes DNA methylation may be a candidate mechanism of mediating not only environmental, but also genetic effects on age-related phenotypes., Author Summary Epigenetic patterns vary during healthy ageing and development. Age-related DNA methylation changes have been implicated in cellular senescence and longevity, yet the causes and functional consequences of these variants remain unclear. To understand the biological mechanisms involved in potential longevity and rate of healthy ageing, we performed genome-wide association of epigenetic and genetic variation with both chronological age and age-related phenotypes. We identified hundreds of DNA methylation variants significantly associated with age and replicated these in an independent sample of young adult twins. Only a small proportion of these variants were also associated with age-related phenotypes. Therefore, the majority of age-related epigenetic changes do not contribute to rate of healthy ageing at later stages in life. Our results suggest that age-related changes in methylation occur throughout an individual's lifespan and that a proportion of these may be initiated from an early age. Intriguingly, a fraction of the age differentially methylated regions also associated with genetic variants in our sample, suggesting that DNA methylation may be a candidate mechanism of mediating not only environmental but also genetic effects on age-related phenotypes.

The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research. The Consortium is funded by grants from the NHGRI as follows: production grants: U54HG004570 (B. E. Bernstein); U01HG004695 (E. Birney); U54HG004563 (G. E. Crawford); U54HG004557 (T. R. Gingeras); U54HG004555 (T. J. Hubbard); U41HG004568 /n(W. J. Kent); U54HG004576 (R. M. Myers); U54HG004558 (M. Snyder);/nU54HG004592 (J. A. Stamatoyannopoulos). Pilot grants: R01HG003143 (J. Dekker); RC2HG005591 and R01HG003700 (M. C. Giddings); R01HG004456-03 (Y. Ruan); U01HG004571 (S. A. Tenenbaum); U01HG004561 (Z. Weng); RC2HG005679 (K. P. White). This project was supported in part by American Recovery and/nReinvestment Act (ARRA) funds from the NHGRI through grants U54HG004570, U54HG004563, U41HG004568, U54HG004592, R01HG003143, RC2HG005591,R01HG003541, U01HG004561, RC2HG005679andR01HG003988(L. Pennacchio). In addition, work from NHGRI Groups was supported by the Intramural Research/nProgram of the NHGRI (L. Elnitski, ZIAHG200323; E. H. Margulies, ZIAHG200341). Research in the Pennachio laboratory was performed at Lawrence Berkeley National Laboratory and at the United States Department of Energy Joint Genome Institute, Department of Energy Contract DE-AC02-05CH11231, University of California.

Aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of the tumor-related genes including DNA repair genes. The objective of this study was to determine the frequency of promoter methylation of the O⁶-methylguanine DNA methyltransferase (MGMT) gene as a DNA repair gene in nonsmall cell lung cancer (NSCLC) and to analyze the correlation with clinicopathological parameters including age, gender, smoking status, histological subtype, and clinical stage. Eighty patients with NSCLC were included in this study. The analysis of DNA methylation was performed on formalin-fixed, paraffin-embedded lung cancer tissues. Following DNA isolation and bisulfite treatment, DNA methylation was analyzed by methylation-specific real-time polymerase chain reaction. MGMT promoter methylation was detected in 51 of 80 (64%) NSCLC patients. There was a significant correlation between MGMT methylation and tumor stage (p = 0.01). The frequencies of the promoter methylation of MGMT gene in smokers and older patients were higher than in their counterparts. In conclusion, the present study provides strong evidence for a higher frequency of promoter methylation of the MGMT gene in NSCLC, indicating that it is a common event during the carcinogenesis of NSCLC.

BackgroundDespite their well-established functional roles, histone modifications have received less attention than DNA methylation in the cancer field. In order to evaluate their importance in colorectal cancer (CRC), we generated the first genome-wide histone modification profiles in paired normal colon mucosa and tumor samples.MethodsChromatin immunoprecipitation and microarray hybridization (ChIP-chip) was used to identify promoters enriched for histone H3 trimethylated on lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in paired normal colon mucosa and tumor samples from two CRC patients and for the CRC cell line HT29.ResultsBy comparing histone modification patterns in normal mucosa and tumors, we found that alterations predicted to have major functional consequences were quite rare. Furthermore, when normal or tumor tissue samples were compared to HT29, high similarities were observed for H3K4me3. However, the differences found for H3K27me3, which is important in determining cellular identity, indicates that cell lines do not represent optimal tissue models. Finally, using public expression data, we uncovered previously unknown changes in CRC expression patterns. Genes positive for H3K4me3 in normal and/or tumor samples, which are typically already active in normal mucosa, became hyperactivated in tumors, while genes with H3K27me3 in normal and/or tumor samples and which are expressed at low levels in normal mucosa, became hypersilenced in tumors.ConclusionsGenome wide histone modification profiles can be used to find epigenetic aberrations in genes associated with cancer. This strategy gives further insights into the epigenetic contribution to the oncogenic process and may identify new biomarkers.

Low socioeconomic status (SES) is associated with earlier onset of age-related chronic conditions and reduced life-expectancy, but the underlying biomolecular mechanisms remain unclear. Evidence of DNA-methylation differences by SES suggests a possible association of SES with epigenetic age acceleration (AA). We investigated the association of SES with AA in more than 5,000 individuals belonging to three independent prospective cohorts from Italy, Australia, and Ireland. Low SES was associated with greater AA (β = 0.99 years; 95% CI 0.39,1.59; p = 0.002; comparing extreme categories). The results were consistent across different SES indicators. The associations were only partially modulated by the unhealthy lifestyle habits of individuals with lower SES. Individuals who experienced life-course SES improvement had intermediate AA compared to extreme SES categories, suggesting reversibility of the effect and supporting the relative importance of the early childhood social environment. Socioeconomic adversity is associated with accelerated epigenetic aging, implicating biomolecular mechanisms that may link SES to age-related diseases and longevity.

The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM-450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg1243587 and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p