Aberrant Gene Methylation Research Articles

Advances in Nutritional Epigenetics-A Fresh Perspective for an Old Idea. Lessons Learned, Limitations, and Future Directions.

Nutritional epigenetics is a rapidly expanding field of research, and the natural modulation of the genome is a non-invasive, sustainable, and personalized alternative to gene-editing for chronic disease management. Genetic differences and epigenetic inflexibility resulting in abnormal gene expression, differential or aberrant methylation patterns account for the vast majority of diseases. The expanding understanding of biological evolution and the environmental influence on epigenetics and natural selection requires relearning of once thought to be well-understood concepts. This research explores the potential for natural modulation by the less understood epigenetic modifications such as ubiquitination, nitrosylation, glycosylation, phosphorylation, and serotonylation concluding that the under-appreciated acetylation and mitochondrial dependant downstream epigenetic post-translational modifications may be the pinnacle of the epigenomic hierarchy, essential for optimal health, including sustainable cellular energy production. With an emphasis on lessons learned, this conceptional exploration provides a fresh perspective on methylation, demonstrating how increases in environmental methane drive an evolutionary down regulation of endogenous methyl groups synthesis and demonstrates how epigenetic mechanisms are cell-specific, making supplementation with methyl cofactors throughout differentiation unpredictable. Interference with the epigenomic hierarchy may result in epigenetic inflexibility, symptom relief and disease concomitantly and may be responsible for the increased incidence of neurological disease such as autism spectrum disorder.

Integrative analysis of DNA methylation-driven genes for the prognosis of lung squamous cell carcinoma using MethylMix.

Background: DNA methylation acts as a key component in epigenetic modifications of genomic function and functions as disease-specific prognostic biomarkers for lung squamous cell carcinoma (LUSC). This present study aimed to identify methylation-driven genes as prognostic biomarkers for LUSC using bioinformatics analysis.Materials and Methods: Differentially expressed RNAs were obtained using the edge R package from 502 LUSC tissues and 49 adjacent non-LUSC tissues. Differentially methylated genes were obtained using the limma R package from 504 LUSC tissues and 69 adjacent non-LUSC tissues. The methylation-driven genes were obtained using the MethylMix R package from 500 LUSC tissues with matched DNA methylation data and gene expression data and 69 non-LUSC tissues with DNA methylation data. Gene ontology and ConsensusPathDB pathway analysis were performed to analyze the functional enrichment of methylation-driven genes. Univariate and multivariate Cox regression analyses were performed to identify the independent effect of differentially methylated genes for predicting the prognosis of LUSC.Results: A total of 44 methylation-driven genes were obtained. Univariate and multivariate Cox regression analyses showed that twelve aberrant methylated genes (ATP6V0CP3, AGGF1P3, RP11-264L1.4, HIST1H4K, LINC01158, CH17-140K24.1, CTC-523E23.14, ADCYAP1, COX11P1, TRIM58, FOXD4L6, CBLN1) were entered into a Cox predictive model associated with overall survival in LUSC patients. Methylation and gene expression combined survival analysis showed that the survival rate of hypermethylation and low-expression of DQX1 and WDR61 were low. The expression of DQX1 had a significantly negatively correlated with the methylation site cg02034222.Conclusion: Methylation-driven genes DQX1 and WDR61 might be potential biomarkers for predicting the prognosis of LUSC.

Combined Analysis of the Aberrant Epigenetic Alteration of Pancreatic Ductal Adenocarcinoma.

Background Pancreatic ductal adenocarcinoma (PDAC) remains one of the most fatal malignancies due to its high morbidity and mortality. DNA methylation exerts a vital part in the development of PDAC. However, a mechanistic role of mutual interactions between DNA methylation and mRNA as epigenetic regulators on transcriptomic alterations and its correlation with clinical outcomes such as survival have remained largely uncovered in cancer. Therefore, elucidation of aberrant epigenetic alteration in the development of PDAC is an urgent problem to be solved. In this work, we conduct an integrative epigenetic analysis of PDAC to identify aberrant DNA methylation-driven cancer genes during the occurrence of cancer. Methods DNA methylation matrix and mRNA profile were obtained from the TCGA database. The integration of methylation and gene expression datasets was analyzed using an R package MethylMix. The genes with hypomethylation/hypermethylation were further validated in the Kaplan–Meier analysis. The correlation analysis of gene expression and aberrant DNA methylation was also conducted. We performed a pathway analysis on aberrant DNG methylation genes identified by MethylMix criteria using ConsensusPathDB. Results 188 patients with both methylation data and mRNA data were considered eligible. A mixture model was constructed, and differential methylation genes in normal and tumor groups using the Wilcoxon rank test was performed. With the inclusion criteria, 95 differential methylation genes were detected. Among these genes, 74 hypermethylation and 21 hypomethylation genes were found. The pathway analysis revealed an increase in hypermethylation of genes involved in ATP-sensitive potassium channels, Robo4, and VEGF signaling pathways crosstalk, and generic transcription pathway. Conclusion Integrated analysis of the aberrant epigenetic alteration in pancreatic ductal adenocarcinoma indicated that differentially methylated genes could play a vital role in the occurrence of PDAC by bioinformatics analysis. The present work can help clinicians to elaborate on the function of differentially methylated expressed genes and pathways in PDAC. CDO1, GJD2, ID4, NOL4, PAX6, TRIM58, and ZNF382 might act as aberrantly DNA-methylated biomarkers for early screening and therapy of PDAC in the future.

Resveratrol alleviates non-alcoholic fatty liver disease through epigenetic modification of the Nrf2 signaling pathway.

Recent findings have demonstrated the aberrant DNA methylation of the Nrf2-Keap1 genes in human cancers; however, the epigenetic control of this pathway in non-alcoholic fatty liver disease (NAFLD) is unknown. Resveratrol can modify epigenetic mechanisms. Our objectives in this study were to explore the correlation between promoter methylation of the Nrf2-Keap1 genes and NAFLD, and that investigate the effect of resveratrol on the epigenetic regulation Nrf2-Keap1 in vitro and in vivo models of NAFLD. Resveratrol attenuated high fat-diet (HFD)-induced methylation of the Nrf2 promoter in the liver of mice, and this effect was correlated with reduction in triglyceride level and decrease in the expression of lipogenesis-related genes such as FAS and SREBP-1c. In addition, treatment of HepG2 cells with high glucose (HG) enhanced methylation level of the Nrf2 promoter, whereas resveratrol reversed this effect. Treatment of the cells with resveratrol or 5-aza, a demethylating agent, could prevent HG-induced reactive oxygen species production and expression of Nrf2-controlled antioxidant genes. Moreover, resveratrol or 5-aza could significantly attenuate HG-induced triglyceride accumulation in HepG2 cells. These findings indicate that resveratrol attenuates NAFLD through the epigenetic modification the Nrf2 signaling.

Methylation of tumour suppressor genes RUNX3, RASSF1A and E-Cadherin in HCV-related liver cirrhosis and hepatocellular carcinoma

ABSTRACTBackground: HCV infection is related to aberrant methylation of several genes. RASSF1A, E-Cadherin and RUNX3 are tumour suppressor genes that may be inactivated by hypermethylation in many tumours including hepatocellular carcinoma (HCC). We hypothesized that methylation is a diagnostic biomarker for HCC in patients with HCV-related liver cirrhosis.Methods: We recruited 207 cases of HCV-related liver cirrhosis, 193 HCC patients and 53 healthy controls. Methylation-specific polymerase chain reaction for detection of circulating hypermethylated RASSF1A, E-Cadherinand RUNX3. Alpha fetoprotein (AFP) was measured by commercial immunoassay.Results: Significant hypermethylation of the three genes was found in the HCC group compared to both cirrhosis and healthy groups (P < 0.001), whereas no significant difference in hypermethylation was found between cirrhosis and healthy groups (P = 0.17, 0.50 and 0.14, respectively). No significant links were found between hypermethylated RASSF1A, E-Cadherin and RUNX3 and stages of Barcelona Clinic of Liver Cancer score (P =0.21, 0.63 and 0.98, respectively). No significant associations were found between AFP value and hypermethylated genes in cirrhosis and HCC groups (P = 0.82) except with E-Cadherin in HCC (P = 0.02). In multiple regression analysis, RASSF1A and E-Cadherin were predictors of HCC within cirrhosis cases, but only E-Cadherin was an independent risk factor for prediction of HCC in cases with low AFP (P = 0.01).Conclusions: The presence of hypermethylated serum RASSF1A, E-Cadherin and RUNX3 is linked to HCC in patients with HCV-related cirrhosis. Only E-Cadherin is an independent risk factor for prediction of HCC with low AFP. These findings may be of diagnostic value.

Aberrant p16 methylation as an early diagnostic marker in blood of hepatocellular carcinoma patients

BackgroundAlpha-fetoprotein (AFP) is currently used for serologic screening in hepatocellular carcinoma (HCC) but with low sensitivity ranging 41–65% with a high rate of false-negative and false-positive results. For the hypermethylation of the p16 inhibitor of cyclin-dependent kinase 4 (p16INK4A), a tumor suppressor gene results in the uncontrolled division of cells. This suggests that the loss of p16INK4A function due to promoter methylation may be an early event in HCC pathogenesis so the study aimed to assess aberrant p16INK4A gene methylation as an early diagnostic marker in HCC patients.ResultsOur study revealed a highly significant increase of p16INK4A methylation in patients versus controls (Fisher, 36.11; p < 0.01). P16INK4A methylation was detected in 86.6% (26/30) and none of the controls were methylated (100% specificity) compared to the low sensitivity of AFP 65.38% at a cutoff value of 28 ng/mL. Data revealed non-significant difference of p16INK4A methylation status between different HCC Barcelona stages (Fisher, 0.055; p > 0.05). While, AFP levels were statistically significantly higher in stages B and C (median = 243,400 ng/mL, respectively, when compared to stage A (median = 10 ng/mL) (H:16.667, p < 0.01)).ConclusionEarly diagnosis of HCC can be achieved through the detection of p16INK4A gene methylation in chronic liver disease (CLD) patients with normal serum AFP especially in known cirrhotic patients that deteriorate clinically without apparent etiology.

MTHFR C677T genetic polymorphism in combination with serum vitamin B2, B12 and aberrant DNA methylation of P16 and P53 genes in esophageal squamous cell carcinoma and esophageal precancerous lesions: a case\u2013control study

BackgroundThe study aimed to explore the associations between the interactions of serum vitamin B2 or B12 levels, aberrant DNA methylation of p16 or p53 and MTHFR C677T polymorphism and the risks of esophageal squamous cell carcinoma (ESCC) and esophageal precancerous lesion (EPL).Methods200 ESCC cases, 200 EPL cases and 200 normal controls were matched by age (± 2 years) and gender. Serum vitamin B2 and B12 levels, MTHFR C677T genetic polymorphisms and the methylation status of genes were assessed. Chi square test, one-way analysis of variance and binary logistic regression were performed.ResultsThe lowest quartile of both serum vitamin B2 and B12 with TT genotype showed significant increased EPL risk (OR = 4.91, 95% CI 1.31–18.35; OR = 6.88, 95% CI 1.10–42.80). The highest quartile of both serum vitamin B2 and B12 with CC genotype showed significant decreased ESCC risk (OR = 0.16, 95% CI 0.04–0.60; OR = 0.10, 95% CI 0.02–0.46). The ORs of p16 methylation for genotype CT and TT were 1.98 (95% CI 1.01–3.89) and 17.79 (95% CI 2.26–140.22) in EPL, 4.86 (95% CI 2.48–9.50) and 20.40 (95% CI 2.53–164.81) in ESCC, respectively. Similarly, p53 methylation with genotype TT was associated with increased EPL and ESCC risks (OR = 13.28, 95% CI 1.67–105.70; OR = 15.24, 95% CI 1.90–122.62).ConclusionsThe MTHFR C677T genotype and serum vitamin B2 or B12 levels may interact in ways which associated with the EPL and ESCC risks. The gene–gene interaction suggested that aberrant DNA methyaltion of either p16 or p53 combined with T alleles of MTHFR was associated with increased risks of both EPL and ESCC.

GENE-50. SWI/SNF COMPLEX HETEROGENEITY RELATES WITH POLYPHENOTYPIC DIFFERENTIATION, PROGNOSIS AND IMMUNE RESPONSE IN RHABDOID TUMORS

Abstract PURPOSE Rhabdoid tumors (RTs) arise within (Atypical Teratoid/Rhabdoid Tumor-AT/RT) or outside the brain (extraCNS-RT-eCNS-RT) and are driven mainly by inactivation of the SWI/SNF complex subunit SMARCB1. A pathognomonic hallmark of RTs is heterogeneity characterized by multilineage differentiation of tumor cells including anomalous neuronal differentiation in a subset of eCNS-RT. The mechanisms that regulate heterogeneity in RTs are unknown. Because remodeling of the SWI/SNF complex regulates differentiation, we hypothesized that SWI/SNF-BAF and PBAF complex heterogeneity correlates with both multilineage differentiation and clinical outcome. EXPERIMENTAL DESIGN: We performed an integrated analysis of SWI/SNF complex subunit alterations in the developing kidney and cerebellum (most common regions of origin for RT) in comparison to eCNS-RT (n=14) and AT/RT (n=25) tumors. RT samples were interrogated using immunohistochemistry, DNA methylation and gene expression analyses. RESULTS (A) The SWI/SNF-BAF paralogs ACTL6A/ACTL6B were expressed in a mutually exclusive manner in the developing cerebellum and kidney. In contrast, a subset of eCNS-RTs lost mutual exclusivity and co-expressed both subunits. These tumors showed aberrant DNA methylation of genes that regulate neuronal and renal development and demonstrated immunohistochemical evidence of neuronal differentiation. (B) Low expression of the PBAF subunit-PBRM1 identified a group of AT/RTs in younger children with better overall prognosis. PBRM1-low AT/RT and eCNS-RTs showed altered DNA methylation and gene expression in immune-related genes accompanied by increased CD8 cytotoxic T-cell infiltration. CONCLUSIONS Heterogeneity in SWI/SNF BAF (ACTL6A/ACTL6B) and PBAF (PBRM1) subunits correlates with histogenesis, contributes to the immune microenvironment and prognosis in RTs and may inform opportunities to develop immunotherapies.

Aberrant DNA methylation profiling affecting the endometrial receptivity in recurrent implantation failure patients undergoing in vitro fertilization.

DNA methylation profile in mid-secretory phase of endometrium is reported to be varied from other phases in natural menstrual cycle. Therefore, we intended to study the impairment in endometrial receptivity by performing whole-genome methylation and gene expression profiling in endometrium of recurrent implantation failure patients (RIF) during IVF under controlled ovarian stimulation (COS). Endometrial biopsies were collected from IVF-RIF patients (cases, n=6) and healthy fertile oocyte donors (controls, n=6) undergoing COS after 6/7th day of human chorionic gonadotropin administration. The whole-genome methylation and gene expression microarray were performed and analysed by GenomeStudio software (P<.05 by Illumina Custom Model), whereas the enrichment analysis was performed using "Database for Annotation, Visualization and Integrated Discovery" (DAVID, V6.8). Significant differentially methylated genes were correlated with dys-regulated genes using Pearson's correlation. Differential methylation in RIF patients revealed 448 CpG sites. The enrichment analysis showed aberrant methylation in genes involved in immunological response and G protein activity. Methylation in NLRP2 gene in inflammatory pathway had significant negative correlation with gene expression (P=.008), whereas SERPINA5 gene that is already known to be involved in endometrial receptivity was observed to be hypomethylated in promoter region with highest delta beta value and up-regulated in gene expression analysis. The aberrant methylation of genes involved in immunological functions and G protein activation was found to be prevalent which might suggest a role in endometrial receptivity. However, the findings need to be further validated on a larger cohort of IVF-RIF patients.

Aberrant DNA methylation of PAX1, SOX1 and ZNF582 genes as potential biomarkers for esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is a highly invasive malignant tumor and the majority of patients have advanced stage of ESCC at diagnosis with poor outcome. Identification of sensitive and specific biomarkers for early screening of ESCC is critical for improving patient overall survival. MethodsPyrosequencing was used to determine the magnitude of DNA methylation on the selected regions PAX1 (paired box gene1), SOX1(sex determining region Y-box-1), and ZNF582 (zinc finger protein 582) in ESCC. ResultsThe methylation levels ofPAX1, SOX1, and ZNF582 genes were significantly higher in the cancerous tissues compared to those in the non-cancerous (all P < 0.0001). The accuracy, sensitivity and specificity of PAX1 methylation for the detection of cancer were respectively 0.754, 96.0% and 51.4%; for SOX1 which were 0.781, 89.2% and 59.5%; for ZNF582 which were 0.898, 93.2% and 75.7%. Most importantly, both the sensitivity and specificity of ZNF582 methylation testing achieved 100% in female ESCC patients. Hypermethylation of PAX1/SOX1/ZNF582 exhibited as an independent risk factor for ESCC development. In addition, ZNF582 methylation level in tumor tissue from the female patients was higher than that from male patients, and it was higher in the moderate and poor differentiated tumors compared to that in well-differentiated tumors. SOX1 methylation level was significantly higher in tumors located in the upper region than those located in the middle or lower regions. ConclusionThe methylation levels ofPAX1, SOX1 and ZNF582 genes were all higher in cancer tissues than in paired-adjacent and normal tissues, suggesting that detection of PAX1/SOX1/ZNF582 methylation status may serve as a promising biomarker for ESCC screening and diagnosis.

Chromate exposure induces DNA hypermethylation of the mismatch repair gene MLH1 in lung cancer.

Hexavalent chromium is recognized as a human carcinogen. Our previous studies revealed that lung cancer (LC) in chromate-exposed workers (chromate LC) had molecular features of frequent microsatellite instability (MSI), repression of MLH1 level, and aberrant DNA methylation of several tumor-suppressor genes, including MLH1. In the present study, we quantitatively investigated MLH1-promoter methylation status using bisulfite pyrosequencing of paired tumorous/nontumorous tissues from chromate and nonchromate LCs to determine the effect of chromate exposure on MLH1-promoter methylation. The methylation level of MLH1 promoter was significantly higher in chromate LC tumors (P < .001) than nonchromate LC tumors and, among chromate LC, significantly higher in tumorous tissue than nontumorous tissue (P = .004). Moreover, the methylation level of MLH1 promoter in normal lung tissue tended to be higher in chromate LC than nonchromate LC (P = .062). In addition, LC with reduced levels of MLH1 showed significantly higher methylation levels of MLH1 promoter than LC exhibiting normal MLH1 levels (P = .019). Moreover, immunohistochemical analyses determined that levels of SUV39H1, an H3K9me2-related methyltransferase, were higher in chromate LC than nonchromate LC (P = .076). Furthermore, we evaluated three DNA double-strand break-repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment-length polymorphism analysis, revealing the mutation frequency of RAD50 as significantly higher in chromate LC than nonchromate LC (P = .047). These results suggest that chromate exposure might induce MLH1 hypermethylation in LC as a mechanism of chromate-induced carcinogenesis.

Abstract P2042: Alteration of Mitochondrial Deoxyribonucleic Acid Methylation and Nuclear Transcription Factors Reduces Mitochondrial Biogenesis in Aged Hypertensive Kidney

Accumulating evidence suggests that epigenetic mechanisms contribute to aging and target organ injury during hypertension. Aberrant DNA methylation of mitochondrial genes can affect mitochondrial copy numbers, replication and metabolism. Recently, the presence of mitochondrial DNA (mtDNA) methylation was reported; however, its functional significance in aging and hypertension-induced chronic kidney disease is unknown. We hypothesized that alteration in mtDNA methylation aggravates hypertension injury in aged kidney by disrupting mitochondrial homeostasis leading to reduced mitochondrial biogenesis and dysfunction. Hydrogen sulfide (H 2 S) was shown to modulate chromatin recently, we therefore investigated whether H 2 S can act as an epigenetic modifying agent to protect renal mitochondria and improve renal function. Aged (72-75 weeks) and young (10-12 weeks) wild type (WT, C57BL/6J) mice were treated without or with angiotensin-II (Ang II, 1000 ng/Kg/d) and H 2 S donor, GYY4137 (0.5 mg/Kg/d, i.p.), for 28 d. Ang-II increased mean blood pressure in aged mice compared to young (136.54±3.55 vs. 108.68±2.86 mmHg) and reduced glomerular filtration rate (688.3±24.83 vs. 1029.3±30.8 μL/min/100 g b.w.). H 2 S treatment reduced mean BP (122.34±3.47) and increased GFR (962.7±21.5) in aged mice receiving Ang II. In aged mice, querying renal mitochondrial genes for methylation status revealed increased methylation of mt-ND1 and reduction of mt-CO1 and mt-CO2 and H 2 S treatment reversed the changes. Methylation of mt-Cytb was undetected and mt-Dloop was low in Ang II treated aged mice and increased with H 2 S treatment. Ang II treated aged mice exhibited decreased PGC-1α and nuclear transcription factors, NRFI, NRF2, ERRα and TFAM, indicating reduced mitochondrial biogenesis compared to young mice and H 2 S restored their levels. ATP synthase activity, ATP generation was reduced in aged mice receiving Ang II compared to young and increased with H 2 S treatment. Together, our results suggest that alteration in mtDNA methylation and impaired nuclear DNA genes is associated with reduced mitochondrial replication and disrupted metabolism and H 2 S therapy protects the aged kidney from hypertension-induced damage by restoring mitochondrial homeostasis.

Identification of Methylation Markers and Differentially Expressed Genes with Prognostic Value in Breast Cancer.

Breast cancer is one of the most common cancers causing a high mortality worldwide. This study aimed to identify differential methylation and expression genes with prognostic value in breast cancer. DNA methylation and gene expression profiles (GSE60185, GSE42568, GSE21653, GSE58812, and GSE52865) were downloaded from TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) databases. The differentially expressed genes (DEGs) and differential methylation genes were identified between breast cancer samples and normal samples. Functional analysis was performed using DAVID (Database for Annotation, Visualization, and Integrated Discovery) tool. Furthermore, functional epigenetic modules (FEM) were analyzed to identify critical genes with prognostic values. A large amount of DEGs and aberrant methylation genes were identified between breast cancer samples and normal samples. These genes were mainly associated with several GO (Gene Ontology) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, such as neuroactive ligand-receptor interaction, alcoholism, gamma-aminobutyric acid signaling pathway, and G-protein-coupled receptor signaling pathway. Additionally, 10 DEGs with differential methylation levels were significantly correlated with survival outcomes in breast cancer patients. FEM analysis revealed that several DEGs (e.g., GABRA4, GABRG1, and GABRA1) in module GABRA4 were identified as potential biomarkers in breast cancer patients. Several DEGs identified were associated with breast cancer prognosis. These DEGs might act as prognostic and diagnostic markers in breast cancer.

Abstract 907: Bioinformatic method to define epigenetically regulated enhancer elements associated with cancer

Abstract BACKGROUND: Several mechanisms involved in gene regulation are altered in cancer. Cataloging these alterations can lead to a better understanding of tumorigenesis. In addition, the alterations can be used to classify patients with similar clinical features and thereby lead to better targeted treatment. Epigenetics (e.g. DNA methylation) is the process by which cells define gene regulation and aberrant DNA methylation patterns have been observed in many cancer types. Alterations in non-promoter (intergenic regions) have been shown to be tightly associated with functional genomic elements such as enhancers or transcription factor binding. In order to identify altered candidate functional elements associated with specific gene or pathways, we developed a method to integrate enhancer, DNA methylation and gene expression data, using tumor and non-tumor data. METHOD: Using epigenome-wide platform (Illumina 850K), CpG probes were separated into promoters and intergenic regions. The intragenic CpGs were further filtered by overlapping with known functional enhancer database from multiple studies. The nearest genes to each CpG enhancer is further stratified based on differential gene expression. Each CpG/gene pair is classified as methylated or unmethylated by sample, using a 50% methylation cutoff. The mean expression of the methylated samples is calculated and pairs with lower than the bottom 10% (1.28 standard deviation) of the mean expression in the unmethylated group of samples are selected. Finally, CpG/gene pairs with at least 75% of the methylated samples have expression values lower than the mean expression in the unmethylated group of samples are classified as epigenetically silenced. CpG/gene pairs unmethylated and upregulated are called as epigenetically active. By separating samples into different epigenetically deregulated states (silenced or active), we can further characterize each sample by evaluating the association or enrichment for specific clinical features such as outcome, treatment, age at diagnosis, etc. RESULTS: As a proof of concept, we applied our method across the TCGA PanCan cohort to identify potential enhancers regulating genes encoding subunits of the SWI/SNF protein complex. Our method was able to detect several deregulated enhancers associated with SWI/SNF genes specifically altered in each tumor type, independent of mutation. We validated the results using Hi-C data from primary cancer cell lines. Citation Format: Thais S. Sabedot, Seth H. Cassel, Galen F. Gao, Caleb A. Lareau, Andrew Cherniack, Alexander Lazar, Cigall Kadoch, Houtan Noushmehr. Bioinformatic method to define epigenetically regulated enhancer elements associated with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 907.

Gene Expression and DNA Methylation Alterations During Non-alcoholic Steatohepatitis-Associated Liver Carcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers. HCC is characterized by an acquisition of multiple abnormal phenotypes driven by genetic and epigenetic alterations, especially abnormal DNA methylation. Most of the existing clinical and experimental reports provide only a snapshot of abnormal DNA methylation patterns in HCC rather than their dynamic changes. This makes it difficult to elucidate the significance of these changes in the development of HCC. In the present study, we investigated hepatic gene expression and gene-specific DNA methylation alterations in mice using the Stelic Animal Model (STAM) of non-alcoholic steatohepatitis (NASH)-derived liver carcinogenesis. Analysis of the DNA methylation status in aberrantly expressed epigenetically regulated genes showed the accumulation of DNA methylation abnormalities during the development of HCC, with the greatest number of aberrantly methylated genes being found in full-fledged HCC. Among these genes, only one gene, tubulin, beta 2B class IIB (Tubb2b), was increasingly hypomethylated and over-expressed during the progression of the carcinogenic process. Furthermore, the TUBB2B gene was also over-expressed and hypomethylated in poorly differentiated human HepG2 cells as compared to well-differentiated HepaRG cells. The results of this study indicate that unique gene-expression alterations mediated by aberrant DNA methylation of selective genes may contribute to the development of HCC and may have diagnostic value as the disease-specific indicator.

Autofluorescence Imaging Reflects the Nuclear Enlargement of Tumor Cells as well as the Cell Proliferation Ability and Aberrant Status of the p53, Ki-67, and p16 Genes in Colon Neoplasms.

Background: Autofluorescence imaging (AFI) is useful for diagnosing colon neoplasms, but what affects the AFI intensity remains unclear. This study investigated the association between AFI and the histological characteristics, aberrant methylation status, and aberrant expression in colon neoplasms. Methods: Fifty-three patients with colorectal neoplasms who underwent AFI were enrolled. The AFI intensity (F index) was compared with the pathological findings and gene alterations. The F index was calculated using an image analysis software program. The pathological findings were assessed by the tumor crypt density, cell densities, and N/C ratio. The aberrant methylation of p16, E-cadherin, Apc, Runx3, and hMLH1 genes was determined by a methylation-specific polymerase chain reaction. The aberrant expression of p53 and Ki-67 was evaluated by immunohistochemical staining. Results: An increased N/C ratio, the aberrant expression of p53, Ki-67, and the altered methylation of p16 went together with a lower F index. The other pathological findings and the methylation status showed no association with the F index. Conclusions: AFI reflects the nuclear enlargement of tumor cells, the cell proliferation ability, and the altered status of cell proliferation-related genes, indicating that AFI is a useful and practical method for predicting the dysplastic grade of tumor cells and cell proliferation.

Aberrant DNA methylation of mTOR pathway genes promotes inflammatory activation of immune cells in diabetic kidney disease

DNA methylation has been implicated in the pathogenesis of diabetic kidney disease (DKD), but the underlying mechanisms remain unclear. In this study, we tested the hypothesis that aberrant DNA methylation in peripheral immune cells contributes to DKD progression. We showed that levels of DNA methyltransferase 1 (DNMT1), a key enzyme for DNA methylation, were increased along with inflammatory activity of peripheral blood mononuclear cells in DKD patients. Inhibition of DNMT1 with 5-aza-2'-deoxycytidine (5-Aza) markedly increased the proportion of CD4+CD25+ regulatory T cells in peripheral blood mononuclear cells in culture and in diabetic animals. Adoptive transfer of immune cells from 5-Aza-treated animals showed beneficial effects on the host immune system, resulting in a significant improvement of DKD. Using genome-wide DNA methylation assays, we identified the differentially methylated cytosines in the promoter regions of mammalian target of rapamycin (mTOR) regulators in peripheral blood mononuclear cells of diabetic patients. Further, mRNA arrays confirmed the consistent induction of genes expressed in the mTOR pathway. Importantly, down-regulation of DNMT1 expression via RNA interference resulted in prominent cytosine demethylation of mTOR negative regulators and subsequent decrease of mTOR activity. Lastly, modulation of mTOR resulted in changes in the effect of 5-aza on diabetic immune cells. Thus, up-regulation of DNMT1 in diabetic immune cells induces aberrant cytosine methylation of the upstream regulators of mTOR, leading to pathogenic activation of the mTOR pathway and consequent inflammation in diabetic kidneys. Hence, this study highlights therapeutic potential of targeting epigenetic events in immune system for treating DKD.

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.

Methylation levels of IGF2 and KCNQ1 in spermatozoa from infertile men are associated with sperm DNA damage.

Abnormal imprinted genes methylation in spermatozoa has been shown to be associated with subfertility. However, the relationship between sperm DNA damage and specific imprinted genes methylation remains unclear. In this study, DNA methylation levels were determined at seven imprinted genes loci (H19, INS-IGF2, KCNQ1, MEG3, MEST, PEG3 and SNRPN) in 66 semen samples using the MSRE-qPCR method. The semen samples were divided into two groups according to the threshold value (25%) of DNA fragmentation index (DFI). We found that the mean methylation level at IGF2 (cg17037101) in the group with DFI≥25% was lower than that in the group with DFI<25% (13.7±3% vs. 31.5±5.3%, p=0.0053). However, the methylation levels of other CpGs did not differ from the imprinted genes. Correlation analysis of DFI with the methylation levels of imprinted genes demonstrated that the IGF2 (cg17037101) methylation level was negatively correlated with sperm DFI (r=-0.448, p=0.0038), and the KCNQ1 (cg24932449) methylation level was positively correlated with sperm DFI (r=0.354, p=0.0273). Our results suggest that the aberrant methylation of IGF2 and KCNQ1 genes may be associated with sperm DNA damage.

DNA methylation biomarkers in stool for early screening of colorectal cancer.

Objective: Detection of aberrant methylated genes in feces has been developed as an early screening method for colorectal cancer. The aim of this study was to probe the methylation status of SEPT9, BMP3, NDRG4, and SDC2 in stool and study whether methylation of these genes is associated with colorectal cancer.Materials and Methods: DNAs were isolated and purified from cancerous and non-cancerous stool samples and colorectal cancer tissue. Gene methylation levels were quantified by methylation-specific PCR on SEPT9, BMP3, NDRG4, and SDC2 and analyzed by a diagnostic model.Results: DNA methylation of SEPT9, NDRG4 and SDC2, but not BMP3, had diagnostic potential for detecting colorectal cancer. Moreover, integration of SEPT9, NDRG4, and SDC2 methylation demonstrated high feasibility for detecting colorectal cancer and adenoma, with better performance on colorectal cancer than adenoma.Conclusion: The methylation of SEPT9, NDRG4, and SDC2 in stool may be a potential biomarker for early screening of colorectal cancer.