A pilot examination of the genome-wide DNA methylation signatures of subjects entering and exiting short-term alcohol dependence treatment programs

Alcoholism has a profound impact on millions of people throughout the world. However, the ability to determine if a patient needs treatment is hindered by reliance on self-reporting and the clinician’s capability to monitor the patient’s response to treatment is challenged by the lack of reliable biomarkers. Using a genome-wide approach, we have previously shown that chronic alcohol use is associated with methylation changes in DNA from human cell lines. In this pilot study, we now examine DNA methylation in peripheral mononuclear cell DNA gathered from subjects as they enter and leave short-term alcohol treatment. When compared with abstinent controls, subjects with heavy alcohol use show widespread changes in DNA methylation that have a tendency to reverse with abstinence. Pathway analysis demonstrates that these changes map to gene networks involved in apoptosis. There is no significant overlap of the alcohol signature with the methylation signature previously derived for smoking. We conclude that DNA methylation may have future clinical utility in assessing acute alcohol use status and monitoring treatment response.     

Identification of DNA methylation signature to predict prognosis in gastric adenocarcinoma

Gastric adenocarcinoma is an important death-related cancer. To find factors related to survival and prognosis, and thus improve recovery prospects, a powerful signature is needed. DNA methylation plays an important role in gastric adenocarcinoma processes and development, and here we report on the search for a significant DNA methylation gene to aid with the earlier diagnosis of gastric adenocarcinoma patients. A Cox proportional risk regression analysis and random survival forest algorithm were used to analyze gastric adenocarcinoma patients’ DNA methylation data from The Cancer Genome Atlas, a public database. DNA methylation gene signature consisting of five genes (SERPINA3, AP000357.4, GZMA, AC004702.2, and GREB1L) were selected. As the most accurate predictor, the area under the curve in the training and test group were 0.72 and 0.61, respectively. The signature was able to sort patients into high- and low-risk groups with meaningful overall survival rates (median: 18.36 vs 72.23 months, log-rank test, P < 0.001) in the training group, which predictive ability was validated in a test data set (median: 25.56 vs 58.80 months, log-rank test, P < 0.016). A multivariate Cox regression analysis showed the significant DNA methylation was an independent prediction prognostic factor for gastric adenocarcinoma patients. Functional analysis suggests that these signature genes may be related to pathways and biological processes associated with tumorigenesis. The significant DNA methylation gene could be a novel prediction and prognostic biomarker that both aids in the treatment and predicts the overall survival likelihoods of gastric adenocarcinoma patients.     

LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors

Multiple clinical trials are investigating the use of the DNA methylation inhibitors azacitidine and decitabine for the treatment of solid tumors. Clinical trials in hematological malignancies have shown that optimal activity does not occur at their maximum tolerated doses but selection of an optimal biological dose and schedule for use in solid tumor patients is hampered by the difficulty of obtaining tumor tissue to measure their activity. Here we investigate the feasibility of using plasma DNA to measure the demethylating activity of the DNA methylation inhibitors in patients with solid tumors. We compared four methods to measure LINE-1 and MAGE-A1 promoter methylation in T24 and HCT116 cancer cells treated with decitabine treatment and selected Pyrosequencing for its greater reproducibility and higher signal to noise ratio. We then obtained DNA from plasma, peripheral blood mononuclear cells, buccal mucosa cells and saliva from ten patients with metastatic solid tumors at two different time points, without any intervening treatment. DNA methylation measurements were not significantly different between time point 1 and time point 2 in patient samples. We conclude that measurement of LINE-1 methylation in DNA extracted from the plasma of patients with advanced solid tumors, using Pyrosequencing, is feasible and has low within patient variability. Ongoing studies will determine whether changes in LINE-1 methylation in plasma DNA occur as a result of treatment with DNA methylation inhibitors and parallel changes in tumor tissue DNA.     

Effects of epigenetic-based anti-cancer drugs in leukaemia and multiple myeloma cells

Here, we focus on epigenetic changes in leukaemia and MM (multiple myeloma) cells. We show how the histone signature, DNA methylation and levels of select tumour-suppressor proteins can be affected by inhibitors of HDACs (histone deacetylases) and Dnmts (DNA methyltransferases). Both inhibitors, TSA (trichostatin A) and 5-AZA (5-azacytidine), have the ability to change the histone signature in a tumour-specific manner. In MM cells, we observed changes in H3K4 methylation, while in leukaemia cells, H3K9 methylation was especially affected by select inhibitors. Compared with normal peripheral blood lymphocytes, tumour cell samples were characterized by increased H3K9 acetylation, increased H3K4me2, H3K9me2 and HP1α (heterochromatin protein 1α) levels and specific changes were also observed for DNA methylation. Additionally, we showed that the tumour suppressor pRb1 (retinoblastoma protein) is more sensitive to epigenetic-based anti-cancer stimuli than p53. We have found significant decrease in the levels of pRb1 and p53 in both myeloma and leukaemia cells after HDAC inhibition.     

Glucocorticoid-induced loss of DNA methylation in non-neuronal cells and potential involvement of DNMT1 in epigenetic regulation of Fkbp5

Glucocorticoids may play a significant role in the etiology of neuropsychiatric illnesses. Abnormalities in plasma cortisol levels, glucocorticoid sensitivity, and HPA-axis function often accompany clinical symptoms of stress-related illnesses such as PTSD and depression. Of particular interest are genetic association studies that link single nucleotide polymorphisms of HPA-axis genes with illnesses only in the context of an early-life trauma exposure such as child abuse. These studies suggest that dysregulation of HPA-axis function can have lasting repercussions in shaping mood and anxiety, long after termination of the traumatic experience. As persistent glucocorticoid-induced loss of DNA methylation in FK506 binding protein 5 (Fkbp5) was previously observed in the hippocampus and blood and in the neuronal cell line HT-22, we asked whether these epigenetic alterations occur in non-neuronal, HPA-axis relevant cells. We used the pituitary adenoma cell line AtT-20 to demonstrate that the intronic enhancer region of Fkbp5 undergoes loss of DNA methylation in response to dexamethasone treatment in a dose-dependent manner. We also focused on the mouse hippocampal dentate gyrus to test whether these changes would be enriched in a region implicated in the HPA-axis stress response, neurogenesis, and synaptic plasticity. We observed an increase in enrichment of DNA methylation loss in the dentate gyrus, as compared to whole hippocampal tissues that were similarly treated with glucocorticoids. We then asked whether DNA methyltransferase 1 (Dnmt1), a methyltransferase enzyme involved in maintaining DNA methylation following cell division, is involved in the observed epigenetic alterations. We found a dose-dependent decrease of Dnmt1 expression in the AtT-20 cells following dexamethasone treatment, and a similar decrease in corticosterone-treated mouse hippocampus. Taken together, we provide evidence that these glucocorticoid-induced epigenetic alterations have a broader validity in non-neuronal cells and that they may involve the DNA methylation machinery.     

The dynamics and prognostic potential of DNA methylation changes at stem cell gene loci in women's cancer

Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs) occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs) and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers.     

The defining DNA methylation signature of Kabuki syndrome enables functional assessment of genetic variants of unknown clinical significance

Kabuki syndrome (KS) is caused by mutations in KMT2D, which is a histone methyltransferase involved in methylation of H3K4, a histone marker associated with DNA methylation. Analysis of >450,000 CpGs in 24 KS patients with pathogenic mutations in KMT2D and 216 controls, identified 24 genomic regions, along with 1,504 CpG sites with significant DNA methylation changes including a number of Hox genes and the MYO1F gene. Using the most differentiating and significant probes and regions we developed a “methylation variant pathogenicity (MVP) score,” which enables 100% sensitive and specific identification of individuals with KS, which was confirmed using multiple public and internal patient DNA methylation databases. We also demonstrated the ability of the MVP score to accurately reclassify variants of unknown significance in subjects with apparent clinical features of KS, enabling its potential use in molecular diagnostics. These findings provide novel insights into the molecular etiology of KS and illustrate that DNA methylation patterns can be interpreted as ‘epigenetic echoes’ in certain clinical disorders.     

DNA Methylation Signature of Childhood Chronic Physical Aggression in T Cells of Both Men and Women

Background

High frequency of physical aggression is the central feature of severe conduct disorder and is associated with a wide range of social, mental and physical health problems. We have previously tested the hypothesis that differential DNA methylation signatures in peripheral T cells are associated with a chronic aggression trajectory in males. Despite the fact that sex differences appear to play a pivotal role in determining the development, magnitude and frequency of aggression, most of previous studies focused on males, so little is known about female chronic physical aggression. We therefore tested here whether or not there is a signature of physical aggression in female DNA methylation and, if there is, how it relates to the signature observed in males.

Methodology/Principal Findings

Methylation profiles were created using the method of methylated DNA immunoprecipitation (MeDIP) followed by microarray hybridization and statistical and bioinformatic analyses on T cell DNA obtained from adult women who were found to be on a chronic physical aggression trajectory (CPA) between 6 and 12 years of age compared to women who followed a normal physical aggression trajectory. We confirmed the existence of a well-defined, genome-wide signature of DNA methylation associated with chronic physical aggression in the peripheral T cells of adult females that includes many of the genes similarly associated with physical aggression in the same cell types of adult males.

Conclusions

This study in a small number of women presents preliminary evidence for a genome-wide variation in promoter DNA methylation that associates with CPA in women that warrant larger studies for further verification. A significant proportion of these associations were previously observed in men with CPA supporting the hypothesis that the epigenetic signature of early life aggression in females is composed of a component specific to females and another common to both males and females.     

Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10⁻⁷ and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.     

Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10^?7 and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.     

A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies.     

The landscape of DNA methylation in hepatocellular carcinoma

Better understanding of the relationship between changes in the overall methylation status of hepatocellular carcinoma (HCC) and disease progression will help us find good strategies for the early detection and treatment of HCC patients. The purpose of the study was to study the relations between the methylation status changes in HCC patients and progression of the disease to enable early detection and treatment of HCC patients. First, the DNA methylation data of 50 HCC samples and the surrounding normal samples were extracted and the change pattern of methylation status in the DNA promoter region of HCC samples against that of normal samples was studied. Then, some DNA methylation genes that could accurately identify cancer and cancer-adjacent tissues were identified using the k-top scoring pair method. Also, a prognostic signature that could predict the survival of HCC patients was constructed based on the overall survival time and death information of the early HCC patients. Finally, the obtained prognostic signature was verified. In conclusion, this study described the changes in the methylation spectrum during the development of HCC and identified genes associated with HCC progression and prognosis, which may offer new opportunities for the diagnosis and treatment of HCC.     

Methylome sequencing for fibrolamellar hepatocellular carcinoma depicts distinctive features

With the goal of studying epigenetic alterations in fibrolamellar hepatocellular carcinoma (FLC) and establish an associated DNA methylation signature, we analyzed LINE-1 methylation in a cohort of FLC and performed next-generation sequencing of DNA methylation in a training set of pure-FLCs and non-cirrhotic hepatocellular carcinomas (nc-HCC). DNA methylation was correlated with gene expression. Furthermore, we established and validated an epigenetic signature differentiating pure-FLC from other HCCs. LINE-1 methylation correlated with shorter recurrence-free survival and overall survival in resected pure-FLC patients. Unsupervised clustering using CG sites located in islands distinguished pure-FLC from nc-HCC. Major DNA methylation changes occurred outside promoters, mainly in gene bodies and intergenic regions located in the vicinity of liver developmental genes (i.e., SMARCA4 and RXRA). Partially methylated domains were more prone to DNA methylation changes. Furthermore, we identified several putative tumor suppressor genes (e.g., DLEU7) and oncogenes (e.g., DUSP4). While ∼70% of identified gene promoters gaining methylation were marked by bivalent histone marks (H3K4me3/H3K27me3) in embryonic stem cells, ∼70% of those losing methylation were marked by H3K4me3. Finally, we established a pure FLC DNA methylation signature and validated it in an independent dataset. Our analysis reveals a distinct epigenetic signature of pure FLC as compared to nc-HCC, with DNA methylation changes occurring in the vicinity of liver developmental genes. These data suggest new options for targeting FLC based on cancer epigenome aberrations.     

DNA methylation-based diagnostic,prognostic, and predictive biomarkers in colorectal cancer

DNA methylation is an epigenetic mechanism regulating gene expression. Changes in DNA methylation were suggested to be useful biomarkers for diagnosis, and for the determination of prognosis and treatment response. Here, we provide an overview of methylation-based biomarkers in colorectal cancer.First, we start with the two methylation-based diagnostic biomarkers already approved for colorectal cancer, SEPT9 and the combination of NDRG4 and BMP3. Then, we provide a list-based overview of new biomarker candidates depending on the sample source including plasma, stool, urine, and surgically removed tumor tissues. The most often identified markers like SDC2, VIM, APC, MGMT, SFRP1, SFRP2, and NDRG4 have distinct functions previously linked to tumor progression.Although numerous studies have identified tumor-specific methylation changes, most of these alterations were observed in a single study only. The lack of validation in independent samples means low reproducibility and is a major limitation. The genome-wide determination of methylation status (methylome) can provide data to solve these issues. In the third section of the review, methylome studies focusing on different aspects related to CRC, including precancerous lesions, CRC-specific changes, molecular subtypes, aging, and chemotherapy response are summarized. Notably, techniques simultaneously analyzing a large set of regions can also uncover epigenetic regulation of genes which have not yet been associated with tumorigenesis previously.A remaining constraint of studies published to date is the low patient number utilized in these preventing the identification of clinically valuable biomarker candidates. Either future large-scale studies or the integration of already available methylome-level data will be necessary to uncover biomarkers sufficiently robust for clinical application.