Gene-specific DNA Methylation Changes Research Articles

DNA Methylation in Cancer: Epigenetic View of Dietary and Lifestyle Factors.

Alterations in DNA methylation play an important role in cancer development and progression. Dietary nutrients and lifestyle behaviors can influence DNA methylation patterns and thereby modulate cancer risk. To comprehensively review available evidence on how dietary and lifestyle factors impact DNA methylation and contribute to carcinogenesis through epigenetic mechanisms. A literature search was conducted using PubMed to identify relevant studies published between 2005 and 2022 that examined relationships between dietary/lifestyle factors and DNA methylation in cancer. Studies investigating the effects of dietary components (eg, micronutrients, phytochemicals), physical activity, smoking, and obesity on global and gene-specific DNA methylation changes in animal and human cancer models were included. Data on specific dietary/lifestyle exposures, cancer types, DNA methylation targets and underlying mechanisms were extracted. Multiple dietary and lifestyle factors were found to influence DNA methylation patterns through effects on DNA methyltransferase activity, methyl donor availability, and generation of oxidative stress. Altered methylation of specific genes regulating cell proliferation, apoptosis, and inflammation were linked to cancer development and progression. Dietary and lifestyle interventions aimed at modulating DNA methylation have potential for both cancer prevention and treatment through epigenetic mechanisms. Further research is needed to identify actionable targets for nutrition and lifestyle-based epigenetic therapies.

Traffic-related air pollution and genome-wide DNA methylation: A randomized, crossover trial

BackgroundTraffic-related air pollution (TRAP) has been associated with changes in gene-specific DNA methylation. However, few studies have investigated impact of TRAP exposure on genome-wide DNA methylation in circulating blood of human. ObjectiveTo explore the association between TRAP exposure and genome-wide DNA methylation. MethodsWe conducted a randomized, crossover exposure trial among 35 healthy adults in Shanghai, China. All subjects were randomly allocated to a traffic-free park or a main road for consecutive 4 h, respectively. Blood genome-wide DNA methylation after each exposure session was measured by the Infinium Methylation EPIC BeadChip (850K). The differentially methylated CpGs loci associated with TRAP exposure were identified using linear mixed-effect model. ResultsThe average concentrations of traffic-related air pollutants including black carbon, ultrafine particles, carbon dioxide, and nitrogen dioxide were 2–3 times higher in the road compared to those in the park. Methylation levels of 68 CpG loci were significantly changed (false discovery rate < 0.05) following TRAP exposure, among which 49 were hypermethylated and 19 were hypomethylated. The annotated genes based on the differential CpGs loci were related to pathways in cardiovascular signaling, cytokine signaling, immune response, nervous system signaling, and metabolism. ConclusionsWe found that TRAP exposure was associated with DNA methylation in dozens of genes concerning cardiometabolic health. This trial for the first-time profiled genome-wide methylation changes induced by TRAP exposure using the 850K assay, providing epigenetic insights in understanding the cardiometabolic effects of TRAP exposure.

DNA methylation changes in promoter region of CDKN2A gene in workers exposed in construction environment

Purpose Construction workers are exposed to a mixture of substances in the workplace considered carcinogenic. This study aimed to characterise gene-specific changes in DNA methylation over the workweek in this population as this type of environmental exposure has not been studied extensively. Materials and methods We evaluated their DNA methylation in 4 gene-promoter regions (CDKN2A, RASSF1A, MLH1 and APC) and 2 repeat elements (ALU and LINE-1) in blood samples obtained on the first and fifth day of the same workweek of a group of 39 male construction workers. DNA methylation was measured by bisulphite-PCR-Pyrosequencing. We also measured the levels of trace elements in the whole blood by ICP-MS. Results Only the CDKN2A gene had significant differences in the average methylation level between the first and fifth day of the workweek. We also observed that the levels of Cu, Pb, Se, Mn, and Ti decreased during the fifth day of exposure, and only lead, titanium and copper showed a low significant correlation with the methylation level mean for three specific CpG sites of the CDKN2A. Conclusions In summary, the data suggest that altered levels of CDKN2A methylation in construction workers may be a potential biomarker of recent exposure in this environment.

The Differential Effect of Carbon Dots on Gene Expression and DNA Methylation of Human Embryonic Lung Fibroblasts as a Function of Surface Charge and Dose.

This study presents a toxicological evaluation of two types of carbon dots (CD), similar in size (<10 nm) but differing in surface charge. Whole-genome mRNA and miRNA expression (RNAseq), as well as gene-specific DNA methylation changes, were analyzed in human embryonic lung fibroblasts (HEL 12469) after 4 h and 24 h exposure to concentrations of 10 and 50 µg/mL (for positive charged CD; pCD) or 10 and 100 µg/mL (for negative charged CD, nCD). The results showed a distinct response for the tested nanomaterials (NMs). The exposure to pCD induced the expression of a substantially lower number of mRNAs than those to nCD, with few commonly differentially expressed genes between the two CDs. For both CDs, the number of deregulated mRNAs increased with the dose and exposure time. The pathway analysis revealed a deregulation of processes associated with immune response, tumorigenesis and cell cycle regulation, after exposure to pCD. For nCD treatment, pathways relating to cell proliferation, apoptosis, oxidative stress, gene expression, and cycle regulation were detected. The expression of miRNAs followed a similar pattern: more pronounced changes after nCD exposure and few commonly differentially expressed miRNAs between the two CDs. For both CDs the pathway analysis based on miRNA-mRNA interactions, showed a deregulation of cancer-related pathways, immune processes and processes involved in extracellular matrix interactions. DNA methylation was not affected by exposure to any of the two CDs. In summary, although the tested CDs induced distinct responses on the level of mRNA and miRNA expression, pathway analyses revealed a potential common biological impact of both NMs independent of their surface charge.

Global DNA Hypomethylation and Rassf1a and c-myc Promoter Hypermethylation in Rat Kidney Cells after Bisphenol A Exposure.

Bisphenol A (BPA) is a synthetic monomer used in the production of polycarbonate and an environmental contaminant with endocrine disrupting properties. BPA release from plastic carriers is thought to cause high amounts of exposure, which result in high risk to human and environment health. The study examined the possible changes in global DNA methylation, CpG promoter DNA methylation, and gene expressions of Rassf1a and c-myc after BPA exposure in rat kidney epithelial cells (NRK-52E). The IC50 values of BPA in NRK-52E cells were 133.42 and 101.74 μM in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake tests, respectively. The cells were treated with BPA at 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM concentrations for 24 h and at 100 nM concentration for 24, 48, 72, 96 h, and 6 days. Decreased global 5-methylcytosine levels were observed after 48, 72, 96 h, and 6 days at the concentration of 100 nM BPA. Changes in CpG promoter DNA methylation were detected in the genes of Rassf1a and c-myc in BPA-treated NRK-52E cells. Expression levels of Rassf1a and c-myc changed in response to BPA at the high concentrations after 24 h treatment, whereas 100 nM exposure to BPA altered gene expression after 48, 72, and 96 h. These results indicate that changes in global and gene-specific DNA methylation may play an important role in the mechanism of BPA toxicity in kidney cells.

Telomere length correlates with subtelomeric DNA methylation in long-term mindfulness practitioners

Mindfulness and meditation techniques have proven successful for the reduction of stress and improvement in general health. In addition, meditation is linked to longevity and longer telomere length, a proposed biomarker of human aging. Interestingly, DNA methylation changes have been described at specific subtelomeric regions in long-term meditators compared to controls. However, the molecular basis underlying these beneficial effects of meditation on human health still remains unclear. Here we show that DNA methylation levels, measured by the Infinium HumanMethylation450 BeadChip (Illumina) array, at specific subtelomeric regions containing GPR31 and SERPINB9 genes were associated with telomere length in long-term meditators with a strong statistical trend when correcting for multiple testing. Notably, age showed no association with telomere length in the group of long-term meditators. These results may suggest that long-term meditation could be related to epigenetic mechanisms, in particular gene-specific DNA methylation changes at distinct subtelomeric regions.

Transplacental arsenic exposure produced 5-methylcytosine methylation changes and aberrant microRNA expressions in livers of male fetal mice

Arsenic is a known human carcinogen. Early-life exposure to inorganic arsenic induces tumors in humans and in C3H mice. We hypothesized that arsenic exposure in utero may induce epigenetic changes at the level of DNA methylation and miRNA alterations that could lead to greater postnatal susceptibility to cancer. To test this hypothesis, pregnant C3H mice were given sodium arsenite at doses known to cause liver cancer (42.5 and 85 ppm in the drinking water) from gestation day 8–19, and the livers from male fetal mice were collected for analysis. The antibody against 5-methylcytosine was used to perform chromatin-immunoprecipitation coupled with sequencing (ChIP-Seq) to determine genome-wide methylation alterations. In utero arsenic exposure produced global DNA hypomethylation and an array of gene-specific DNA methylation changes, including hypomethylation of Cyclin D1 and hypermethylation of Tp53. Illumina Correlation Engine analysis revealed 260 methylation alterations that would affect 143 microRNAs. MicroRNA array further revealed 140 aberrantly expressed miRNAs out of the 718 miRNAs. The increased expression of miR-205, miR-203, miR-215, miR-34a, and decreased expression of miR-217 were confirmed by qPCR. Comparison of the methylation changes to those of microarray analyses indicates little if any correspondence between gene methylation and gene expression. The increased expression of Xist, Prrc2, Krit1, Nish, and decreased expression of Prss2, Spp1, Col1a2, and Lox were confirmed by qPCR. In summary, in utero arsenic exposure induced global alterations in DNA methylation and aberrant miRNA expression that might contribute to adult adverse outcomes including liver cancer.

Modified CDKN2B (p15) and CDKN2A (p16) DNA methylation profiles in urban pesticide applicators.

Gene-specific changes in DNA methylation by pesticides in occupationally exposed populations have not been studied extensively. Of particular concern are changes in the methylation profile of tumor-suppressor, such as CDKN2B and CDKN2A, genes involved in oncogenesis. The aim of this study was to evaluate the methylation profiles of CDKN2B and CDKN2A genes in urban pesticide applicators and their relationship with occupational exposure to pesticides. A cross-sectional study was conducted in 186 urban pesticide applicators (categorized as high or moderate exposures) and 102 participants without documented occupational exposures to pesticides. Acute and chronic pesticide exposures were evaluated by direct measurement of urinary dialkylphosphates, organophosphate metabolites, and a structured questionnaire, respectively. Anthropometric characteristics, diet, clinical histories, and other variables were estimated through a validated self-reported survey. DNA methylation was determined by pyrosequencing of bisulfite-treated DNA. Decreased DNA methylation of the CDKN2B gene was observed in pesticide-exposed groups compared to the non-exposed group. In addition, increased methylation of the CDKN2A promoter was observed in the moderate-exposure group compared to the non-exposed group. Bivariate analysis showed an association between CDKN2B methylation and pesticide exposure, general characteristics, smoking status, and micronutrients, while changes in CDKN2A methylation were associated with pesticide exposure, sex, educational level, body mass index, smoking status, supplement intake, clinical parameters, and caffeine consumption. These data suggest that pesticide exposure modifies the methylation pattern of CDKN2B and CDKN2A genes and raise important questions about the role that these changes may play in the regulation of cell cycle activities, senescence, and aging.

Aberrant DNA methylation of M1-macrophage genes in coronary artery disease

M1 and M2 macrophage balance in atherosclerosis has attracted much interest. Though, it remains unknown how macrophage heterogeneity is regulated. Moreover, the regulation of macrophage polarization and activation also involve DNA methylation. However, it remains ambiguous which genes are under direct regulation by DNA methylation. Our aim was to evaluate the gene-specific promoter DNA methylation status of M1/M2 polarization markers in PBMCs of CAD patients. A case-control study was performed with 25 CAD patients and 25 controls to study the promoter DNA methylation status of STAT1, STAT6, MHC2, IL12b, iNOS, JAK1, JAK2 and SOCS5 using MS-HRM analysis. Our data indicates that there was a clear-cut difference in the pattern of gene-specific promoter DNA methylation of CAD patients in comparison to controls. A significant difference was observed between the percentage methylation of STAT1, IL12b, MHC2, iNOS, JAK1 and JAK2 in CAD patients and control subjects. In conclusion, our data show that MS-HRM assay is a rapid and inexpensive method for qualitatively identifying aberrant gene-specific promoter DNA methylation changes in CAD. Furthermore, we propose that gene-specific promoter DNA methylation based on monocyte/macrophage might aid as diagnostic marker for clinical application or DNA methylation-related drug interventions may offer novel possibilities for atherosclerotic disease management.

Epigenetic regulation in AKI and kidney repair: mechanisms and therapeutic implications.

Acute kidney injury (AKI) is a major public health concern associated with high morbidity and mortality. Despite decades of research, the pathogenesis of AKI remains incompletely understood and effective therapies are lacking. An increasing body of evidence suggests a role for epigenetic regulation in the process of AKI and kidney repair, involving remarkable changes in histone modifications, DNA methylation and the expression of various non-coding RNAs. For instance, increases in levels of histone acetylation seem to protect kidneys from AKI and promote kidney repair. AKI is also associated with changes in genome-wide and gene-specific DNA methylation; however, the role and regulation of DNA methylation in kidney injury and repair remains largely elusive. MicroRNAs have been studied quite extensively in AKI, and a plethora of specific microRNAs have been implicated in the pathogenesis of AKI. Emerging research suggests potential for microRNAs as novel diagnostic biomarkers of AKI. Further investigation into these epigenetic mechanisms will not only generate novel insights into the mechanisms of AKI and kidney repair but also might lead to new strategies for the diagnosis and therapy of this disease.

Effects of Major Epigenetic Factors on Systemic Lupus Erythematosus

The pathogenesis of systemic lupus erythematosus (SLE) is influenced by both genetic factors and epigenetic modifications; the latter is a result of exposure to various environmental factors. Epigenetic modifications affect gene expression and alter cellular functions without modifying the genomic sequences. CpG-DNA methylation, histone modifications, and miRNAs are the main epigenetic factors of gene regulation. In SLE, global and gene-specific DNA methylation changes have been demonstrated to occur in CD4+ T-cells. Moreover, histone acetylation and deacetylation inhibitors reverse the expression of multiple genes involved in SLE, indicating histone modification in SLE. Autoreactive T-cells and B-cells have been shown to alter the patterns of epigenetic changes in SLE patients. Understanding the molecular mechanisms involved in the pathogenesis of SLE is critical for the introduction of effective, target-directed and tolerated therapies. In this review, we summarize the recent findings that highlight the importance of epigenetic modifications and their mechanisms in SLE.

Effect of aflatoxin B1, benzo[a]pyrene, and methapyrilene on transcriptomic and epigenetic alterations in human liver HepaRG cells

The increasing number of man-made chemicals in the environment that may pose a carcinogenic risk highlights the need for developing reliable time- and cost-effective approaches for carcinogen detection and identification. To address this issue, we investigated the utility of high-throughput microarray gene expression and next-generation genome-wide DNA methylation sequencing for the in vitro identification of genotoxic and non-genotoxic carcinogens. Terminally differentiated and metabolically competent human liver HepaRG cells were treated at minimally cytotoxic concentrations of (i) the genotoxic human liver carcinogen aflatoxin B1 (AFB1) and its structural non-carcinogenic analog aflatoxin B2 (AFB2); (ii) the genotoxic human lung carcinogen benzo[a]pyrene (B[a]P) and its non-carcinogenic isomer benzo[e]pyrene (B[e]P); and (iii) the non-genotoxic liver carcinogen methapyrilene for 72 h and transcriptomic and DNA methylation profiles were examined. Treatment of HepaRG cells with the liver carcinogens AFB1 and methapyrilene generated distinct gene-expression profiles, whereas B[a]P had only a slight effect on gene expression. In contrast to transcriptomic alterations, treatment of HepaRG cells with the carcinogenic and non-carcinogenic chemicals resulted in profound changes in the DNA methylation footprint; however, the correlation between gene-specific DNA methylation and gene expression changes was minimal. Among the carcinogen-altered genes, transferrin (TF) emerged as sensitive marker for an initial screening of chemicals for their potential liver carcinogenicity. Potential liver carcinogens (i.e., chemicals causing altered TF gene expression) could then be subjected to gene-expression analyses to differentiate genotoxic from non-genotoxic liver carcinogens. This approach may substantially enhance the identification and assessment of potential liver carcinogens.

Short-term exposure to fine particulate air pollution and genome-wide DNA methylation: A randomized, double-blind, crossover trial

BackgroundPrevious studies have associated fine particulate (PM2.5) exposure with changes in gene-specific DNA methylation. However, the evidence was still limited and inconsistent in genome-wide DNA methylation. ObjectiveTo examine the impact of short-term PM2.5 exposure on genome-wide DNA methylation. MethodsWe designed a randomized, double-blind, crossover trial among 36 healthy young adults in Shanghai, China. A two-stage intervention with alternative use of real and sham air purifiers in dormitory rooms for consecutive 9 days were conducted to create natural low and high exposure scenarios of PM2.5. Blood genome-wide DNA methylation was analyzed using the Illumina Infinium Human Methylation EPIC BeadChip (850k). Mixed-effect models were used to evaluate the impacts of changes in PM2.5 levels on genome-wide DNA methylation. ResultsThere was a drastic contrast for PM2.5 exposure levels in the two scenarios (24-h averages: 53.1 and 24.3 μg/m3). Between the high and low exposure groups, methylation levels were changed significantly with a false discovery rate < 0.01 at 49 CpG loci, of which 31 CpG sites were annotated to the specific genes. DNA methylation of these annotated genes were elevated in response to increased PM2.5 exposure, which were implicated in insulin resistance, glucose and lipid metabolism, inflammation, oxidative stress, platelet activation, and cell survival and apoptosis. ConclusionsOur results provided novel biological pathways linking ambient PM2.5 exposure to systemic adverse response through variations in DNA methylation and reinforced the hypothesized role of epigenetics in the development of cardiometabolic diseases induced by PM2.5 exposure.

Changes in first trimester fetal CYP1A1 and AHRR DNA methylation and mRNA expression in response to exposure to maternal cigarette smoking

Prenatal exposure to maternal cigarette smoking increases the risk of intrauterine growth retardation, adverse pregnancy outcomes, and diseases later in life. Exposure can result in postnatal global and gene-specific DNA methylation changes, with the latter well documented for the CYP1A1 and AHRR genes involved in the detoxification of xenobiotic substances. This study assessed the impact of exposure to maternal smoking on first trimester fetal CYP1A1 and AHRR mRNA expression and DNA methylation for CpG-sites displaying maternal smoking during pregnancy-mediated methylation changes at birth. The analyses included first trimester (6–12 weeks) placentas (N=39) and livers (N=43). For AHRR, exposure to maternal smoking was associated with increased DNA methylation in the placentas of female fetuses; mRNA expression, however, was unchanged. While exposure to maternal smoking was not associated with AHRR DNA methylation changes in fetal livers; mRNA expression was increased. For CYP1A1, exposure to maternal smoking was not associated with fetal DNA methylation changes whereas mRNA expression increased in placentas and male fetal livers. These results show that first trimester exposure to maternal smoking is associated with CYP1A1 and AHRR DNA methylation and mRNA expression changes. However, the results also indicate that maternal smoking during pregnancy-mediated postnatal CYP1A1 and AHRR DNA methylation changes are not imprinted during the first trimester.

HSD11B2, RUNX3, and LINE-1 Methylation in Placental DNA of Hypertensive Disorders of Pregnancy Patients.

Hypertensive Disorders of Pregnancy (HDsP) remain leading causes of maternal and perinatal morbidity and mortality. Growing evidence suggests the involvement of epigenetic factors, such as gene-specific and global DNA methylation changes, both in the etiology and as an effect of HDsP. In this study, we investigated the potential association between placental DNA methylation status in selected CpGs of HSD11B2 cortisol level controlling gene, RUNX3 tumor suppressor gene, and long interspersed nucleotide element-1 (LINE-1) repetitive elements and HDsP-preeclampsia (PE), gestational hypertension (GH), and chronic hypertension (CH). Methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ) were used to analyze placental DNA methylation. Plasma and urine cortisol and cortisone levels were measured using high performance liquid chromatography with fluorescence detection (HPLC-FLD), whereas serum progesterone level was determined by electrochemiluminescence immunoassay. The mean percentage of HSD11B2, RUNX3, and LINE-1 methylation was not altered in the placentas of patients with HDsP, as compared to the controls. However, among patients from PE, GH, and CH groups, several significant correlations were observed between the methylation status of HSD11B2, RUNX3, or LINE-1 and children's birth weight, gestational age at delivery, mother's age, and body mass index as well as hormones levels. These results indicate lack of association between methylation status of HSD11B2, RUNX3, or LINE-1 repetitive elements and HDsP. However, association of these parameters with some clinical variables may suggest the role of placental DNA methylation in fetal development and should be further explored.

Distinctions in gene-specific changes in DNA methylation in response to folic acid supplementation between women with normal weight and obesity

Obesity and maternal folate deficiency are associated with increased risk for neural tube defects (NTDs). Limited knowledge exists on the impact of folate status or obesity on DNA methylation of genes related to NTD risk and folate metabolism. Women (18-35y) with normal weight (NW; BMI 18.5-24.9kg/m2; n=12) and obesity (OB; BMI >30kg/m2; n=6) were provided FA (800μg/d) for 8-weeks. Serum folate concentration and changes in DNA methylation across 2098 CpG sites in 91 genes related to NTD risk and folate metabolism were examined. Serum folate concentration increased in both groups following FA supplementation, but OB maintained a relative lower concentration (NW; 38.36±2.50-71.41±3.02nmol/L and OB; 27.12±3.09-56.85±3.90nmol/L). Methylation of 56 and 99 CpG sites changed in response to supplementation in NW and OB, respectively, and majority of these sites decreased in methylation in both groups. Only 4 CpG sites responded to supplementation in both groups. Gene ontology analysis revealed a response to supplementation in 61 biological processes (BPs) from the selected genes. Five of the 61 BPs were identified only in NW, including neural tube closure, while 13 of the 61 BPs were enriched only in OB, including folate metabolism, vitamin B12 metabolism and methylation related processes. Changes in DNA methylation in genes related to NTD risk and folate metabolism in response to FA supplementation were different in NW and OB. Increased NTD risk and abnormal folate metabolism in obesity may be due to a distinctive epigenetic response to folate status in these genes.

Abstract 2407: Inhibition of gene expression during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis is mediated by histone H4 lysine 16 deacetylation

Abstract Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers and the incidence is steadily increasing worldwide. The molecular mechanisms leading to the development of HCC consist of genetic and epigenetic aberrations, including changes in global and gene-specific DNA methylation and altered expression of several classes of non-coding RNAs. While changes in DNA methylation and microRNA expression during the development of HCC are well-studied, the role of aberrant post-translational histone modifications remains unexplored. Using a mouse Stelic Animal Model (STAM) of NASH-associated liver carcinogenesis, we have investigated alterations in hepatic histone modifications at steatotic (6 weeks), fibrotic (12 weeks), and full-fledged HCC (20 weeks) stages of the HCC development. NASH-related liver carcinogenesis was characterized by a progressive decrease in the levels of histone H4 lysine 20 trimethylation (H4K20me3) and histone H4 lysine 16 acetylation (H4K16ac), with the greatest decrease occurring in full-fledged tumors. Mechanistically, the deacetylation of H4K16ac was associated with nuclear protein 1 (Nupr1)-mediated inhibition of histone lysine acetyltransferase KAT8. In addition to a global loss of hepatic H4K16ac, there was a significant decrease of gene-specific H4K16ac, as indicated by a reduced level of H4K16ac in 16 out of 25 critical cancer-related genes. More importantly, the changes in the extent of gene-specific H4K16 deacetylation were positively correlated with the level of gene transcription (r = 0.965, p &amp;lt; 0.01). These results indicate that a reduction of global and gene-specific of H4K16ac is a key pathophysiological mechanism contributing to the development of NAFLD-derived HCC. Citation Format: Kostiantyn Dreval, Aline de Conti, Orish Ebere Orisakwe, Frederick A. Beland, Igor P. Pogribny. Inhibition of gene expression during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis is mediated by histone H4 lysine 16 deacetylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2407. doi:10.1158/1538-7445.AM2017-2407

DNA methylation alterations in Alzheimer's disease.

The observation that Alzheimer’s disease (AD) patients with similar and even identical genetic backgrounds often present with heterogeneous pathologies has prompted the hypothesis that epigenetics may contribute to AD. While the study of epigenetics encompasses a variety of modifications including histone modifications and non-coding RNAs, much of the research on how epigenetics might impact AD pathology has been focused on DNA methylation. To this end, several studies have characterized DNA methylation alterations in various brain regions of individuals with AD, with conflicting results. This review examines the results of studies analyzing both global and gene-specific DNA methylation changes in AD and also assesses the results of studies analyzing DNA hydroxymethylation in patients with AD.

Hypermethylation of CDKN2A exon 2 in tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients

BackgroundBreast carcinogenesis is a multistep process involving genetic and epigenetic changes. Tumor tissues are frequently characterized by gene-specific hypermethylation and global DNA hypomethylation. Aberrant DNA methylation levels have, however, not only been found in tumors, but also in tumor-surrounding tissue appearing histologically normal. This phenomenon is called field cancerization. Knowledge of the existence of a cancer field and its spread are of clinical relevance. If the tissue showing pre-neoplastic lesions is not removed by surgery, it may develop into invasive carcinoma.MethodsWe investigated the prevalence of gene-specific and global DNA methylation changes in tumor-adjacent and tumor-distant tissues in comparison to tumor tissues from the same breast cancer patients (n = 18) and normal breast tissues from healthy women (n = 4). Methylation-sensitive high resolution melting (MS-HRM) analysis was applied to determine methylation levels in the promoters of APC, BRCA1, CDKN2A (p16), ESR1, HER2/neu and PTEN, in CDKN2A exon 2 and in LINE-1, as indicator for the global DNA methylation extent. The methylation status of the ESR2 promoter was determined by pyrosequencing.ResultsTumor-adjacent and tumor-distant tissues frequently showed pre-neoplastic gene-specific and global DNA methylation changes. The APC promoter (p = 0.003) and exon 2 of CDKN2A (p < 0.001) were significantly higher methylated in tumors than in normal breast tissues from healthy women. For both regions, significant differences were also found between tumor and tumor-adjacent tissues (p = 0.001 and p < 0.001, respectively) and tumor and tumor-distant tissues (p = 0.001 and p < 0.001, respectively) from breast cancer patients. In addition, tumor-adjacent (p = 0.002) and tumor-distant tissues (p = 0.005) showed significantly higher methylation levels of CDKN2A exon 2 than normal breast tissues serving as control. Significant correlations were found between the proliferative activity and the methylation status of CDKN2A exon 2 in tumor (r = −0.485, p = 0.041) and tumor-distant tissues (r = −0.498, p = 0.036).ConclusionsFrom our results we can conclude that methylation changes in CDKN2A exon 2 are associated with breast carcinogenesis. Further investigations are, however, necessary to confirm that hypermethylation of CDKN2A exon 2 is associated with tumor proliferative activity.

DNA Methylation Profiling of Myeloma Trial Patients Reveals Specific Epigenetic Changes Associated with Outcome

IntroductionEpigenetic dysregulation is a hallmark of cancer and has significant impact on disease biology. The epigenetic structure of myeloma is heterogeneous and we previously demonstrated that gene specific DNA methylation changes are associated with outcome, using low-resolution arrays. We now performed a high-resolution genome wide DNA methylation analysis of a larger group of patients from a UK national phase III study to further define the role of epigenetic modifications in disease behaviour and outcome.Patients and MethodsHighly purified (>95%) CD138+ myeloma bone marrow cells from 465 newly diagnosed patients enrolled in the UK NCRI Myeloma XI study were analysed. The extracted DNA was bisulfite-converted using the EZ DNA methylation kit (Zymo) and hybridized to Infinium HumanMethylation450 BeadChip arrays. Raw data was processed using the R Bioconductor package "minfi". SNP containing probes and probes on the sex chromosomes were removed. 464 samples and 441293 probes were retained following inspection of quality control metrics. Beta values were summarized across functional genomic units or differentially methylated regions (DMRs) that included: gene bodies, promoters, insulators, CpG-islands and enhancers. K-means was applied to each DMR to cluster patients into 2 groups (high or low methylation) per region. Filters were applied to define a clinically meaningful minimum group size and methylation differences between the groups. Overall survival (OS) and progression free survival (PFS) were assessed by a Cox proportional hazards regression model fitted to each DMR with a time-dependent covariate of the trial pathway. Pathway analyses were performed using GREAT (Stanford University) and GSEA (Broad Institute).ResultsWe identified 589 differentially methylated regions that were significantly associated with PFS and OS when using a cut-off of P<0.01 (log-rank). Of these, 114 DMRs were located within 10kb of a gene transcription start site (TSS). Among these, several genes implicated in myeloma disease biology, such as immune cell-cell interaction genes (e.g. CD226) or stemness-associated transcription factors (e.g. PAX4) were identified to be differentially methylated. Using pathway analysis on all 589 DMRs, Gene Ontology biologic groups were enriched for positive regulation of proliferation, cell migration and cytoskeleton organisation (FDR P<0.05). This was further supported by enrichment of proliferative E2F1 transcription factor target structures (FDR P<0.05).Matched gene expression profiles have been generated and integrated analyses correlating epigenetic with GEP and genetic risk data and individual gene level methylation-expression associations will be presented at the meeting. This data is also being integrated with drug resistance profiles from the Cancer Cell Line Encyclopedia (CCLE; Barretina, et al, 2016).ConclusionEpigenetic mechanisms play a significant role in influencing tumour cell behaviour. We have identified here differentially methylated regions that are significantly associated with patient outcome. Pathway analyses suggest an epigenetic regulation of biologic mechanisms involved in high risk disease, such as proliferation and migration. Integration of epigenetic data with matched gene expression profiles is currently ongoing to delineate independent epigenetic biomarkers associated with high risk disease behaviour. DisclosuresJones:Celgene: Honoraria, Research Funding. Pawlyn:Takeda Oncology: Consultancy; Celgene: Consultancy, Honoraria, Other: Travel Support. Jenner:Janssen: Consultancy, Honoraria, Other: Travel support, Research Funding; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel support; Takeda: Consultancy, Honoraria, Other: Travel support; Celgene: Consultancy, Honoraria, Research Funding. Cook:Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Glycomimetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Drayson:Abingdon Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Davies:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria. Jackson:MSD: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau. Kaiser:Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Other: Travel Support; BMS: Consultancy, Other: Travel Support; Chugai: Consultancy.