Identification of methylation driver genes for predicting the prognosis of pancreatic cancer patients based on whole-genome DNA methylation sequencing technology.

This study was based on the use of whole-genome DNA methylation sequencing technology to identify DNA methylation biomarkers in tumor tissue that can predict the prognosis of patients with pancreatic cancer (PCa). TCGA database was used to download PCa-related DNA methylation and transcriptome atlas data. Methylation driver genes (MDGs) were obtained using the MethylMix package. Candidate genes in the MDGs were screened for prognostic relevance to PCa patients by univariate Cox analysis, and a prognostic risk score model was constructed based on the key MDGs. ROC curve analysis was performed to assess the accuracy of the prognostic risk score model. The effects of PIK3C2B knockdown on malignant phenotypes of PCa cells were investigated in vitro. A total of 2737 differentially expressed genes were identified, with 649 upregulated and 2088 downregulated, using 178 PCa samples and 171 normal samples. MethylMix was employed to identify 71 methylation-driven genes (47 hypermethylated and 24 hypomethylated) from 185 TCGA PCa samples. Cox regression analyses identified eight key MDGs (LEF1, ZIC3, VAV3, TBC1D4, FABP4, MAP3K5, PIK3C2B, IGF1R) associated with prognosis in PCa. Seven of them were hypermethylated, while PIK3C2B was hypomethylated. A prognostic risk prediction model was constructed based on the eight key MDGs, which was found to accurately predict the prognosis of PCa patients. In addition, the malignant phenotypes of PANC-1 cells were decreased after the knockdown of PIK3C2B. Therefore, the prognostic risk prediction model based on the eight key MDGs could accurately predict the prognosis of PCa patients.

Ganglioglioma with anaplastic/high-grade transformation: Histopathologic, molecular, and epigenetic characterization of 3 cases.

Ganglioglioma (GG) with anaplasia (anaplastic ganglioglioma) is a rare and controversial diagnosis. When present, anaplasia involves the glial component of the tumor, either at presentation or at recurrence. To date, most published cases lack molecular characterization. We describe the histologic and molecular features of 3 patients presenting with BRAF p. V600E-mutant GG (CNS WHO grade 1) with high-grade glial transformation at recurrence. The tumors occurred in pediatric patients (age 9-16 years) with time to recurrence from 20 months to 7 years. At presentation, each tumor was low-grade, with a BRAFV600E-positive ganglion cell component and a glial component resembling pleomorphic xanthoastrocytoma (PXA) or fibrillary astrocytoma. At recurrence, tumors resembled anaplastic PXA or high-grade astrocytomas without neuronal differentiation. CDKN2A homozygous deletion (HD) was absent in all primary tumors. At recurrence, 2 cases acquired CDKN2A HD; the third case showed loss of p16 and MTAP immunoexpression, but no CDKN2A/B HD or mutation was identified. By DNA methylation profiling, all primary and recurrent tumors either grouped or definitely matched to different methylation classes. Our findings indicate that malignant progression of the glial component can occur in GG and suggest that CDKN2A/B inactivation plays a significant role in this process.

Methylome and Transcriptome-Based Integration Analysis Identified Molecular Signatures Associated With Meningitis Induced by Glaesserella parasuis.

Glaesserella parasuis (G. parasuis) can elicit serious inflammatory responses and cause meningitis in piglets. Previous epigenetic studies have indicated that alterations in host DNA methylation may modify the inflammatory response to bacterial infection. However, to date, genome-wide analysis of the DNA methylome during meningitis caused by G. parasuis infection is still lacking. In this study, we employed an unbiased approach using deep sequencing to profile the DNA methylome and transcriptome from G. parasuis infected porcine brain (cerebrum) and integrated the data to identify key differential methylation regions/sites involved in the regulation of the inflammatory response. Results showed that DNA methylation patterns and gene expression profiles from porcine brain were changed after G. parasuis infection. The majority of the altered DNA methylation regions were found in the intergenic regions and introns and not associated with CpG islands, with only a low percentage occurring at promoter or exon regions. Integrated analysis of the DNA methylome and transcriptome identified a number of inversely and positively correlated genes between DNA methylation and gene expression, following the criteria of |log2FC| > 0.5, |diffMethy| > 0.1, and P < 0.05. Differential expression and methylation of two significant genes, semaphoring 4D (SEMA4D) and von Willebrand factor A domain containing 1 (VWA1), were validated by qRT-PCR and bisulfite sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that DNA methylation inversely correlated genes in G. parasuis infected porcine brains were mainly involved with cell adhesion molecules (CAMs), bacterial invasion of epithelial cells, RIG-1-like receptor signaling pathways, and hematopoietic cell lineage signaling pathways. In addition, a protein-protein interaction network of differentially methylated genes found potential candidate molecular interactions relevant to the pathology of G. parasuis infection. To the best of our knowledge, this is the first attempt to integrate the DNA methylome and transcriptome data from G. parasuis infected porcine brains. Our findings will help understanding the contribution of genome-wide DNA methylation to the pathogenesis of meningitis in pigs and developing epigenetic biomarkers and therapeutic targets for the treatment of G. parasuis induced meningitis.

Genome-wide DNA methylation profiling of leukocytes identifies CpG methylation signatures of aggressive prostate cancer.

Most of screening-detected prostate cancer (PCa) are indolent and not lethal. Biomarkers that can predict aggressive diseases independently of clinical features are needed to improve risk stratification of localized PCa patients and reduce overtreatment. We aimed to identify leukocyte DNA methylation differences between clinically defined aggressive and non-aggressive PCa. We performed whole genome DNA methylation profiling in leukocyte DNA from 287 PCa patients with Gleason Score (GS) 6 and ≥8 using Illumina 450k methylation arrays. We observed a global hypomethylation in GS≥8 patients compared to GS=6 PCa patients; in contrast, the methylation level in core promoter and exon 1 region was significantly higher in GS≥8 patients than GS=6 PCa. We then performed 5-fold cross validated random forest model training on 1,459 differentially methylated CpG Probes (DMPs) with false discovery rate (FDR) <0.01 between GS=6 and GS≥8 groups. The power of the predictive model was further reinforced by ranking the DMPs with Decreased Gini and re-train the model with the top 97 DMPs (Testing AUC=0.920, predict accuracy =0.847). In conclusion, we identified a CpG methylation signature in leukocyte DNA that is associated with aggressive clinical features of PCa at diagnosis.

The human aortic endothelium undergoes dose-dependent DNA methylation in response to transient hyperglycemia.

BACKGROUND: Glycemic control is a strong predictor of long-term cardiovascular risk in patients with diabetes mellitus, and poor glycemic control influences long-term risk of cardiovascular disease even decades after optimal medical management. This phenomenon, termed glycemic memory, has been proposed to occur due to stable programs of cardiac and endothelial cell gene expression. This transcriptional remodeling has been shown to occur in the vascular endothelium through a yet undefined mechanism of cellular reprogramming. METHODS: In the current study, we quantified genome-wide DNA methylation of cultured human endothelial aortic cells (HAECs) via reduced-representation bisulfite sequencing (RRBS) following exposure to diabetic (250 mg/dL), pre-diabetic (125 mg/dL), or euglycemic (100 mg/dL) glucose concentrations for 72 h (n = 2). RESULTS: We discovered glucose-dependent methylation of genomic regions (DMRs) encompassing 2199 genes, with a disproportionate number found among genes associated with angiogenesis and nitric oxide (NO) signaling-related pathways. Multi-omics analysis revealed differential methylation and gene expression of VEGF (↑5.6% DMR, ↑3.6-fold expression), and NOS3 (↓20.3% DMR, ↓1.6-fold expression), nodal regulators of angiogenesis and NO signaling, respectively. CONCLUSION: In the current exploratory study, we examine glucose-dependent and dose-responsive alterations in endothelial DNA methylation to examine a putative epigenetic mechanism underlying diabetic vasculopathy. Specifically, we uncover the disproportionate glucose-dependent methylation and gene expression of VEGF and NO signaling cascades, a physiologic imbalance known to cause endothelial dysfunction in diabetes. We therefore hypothesize that epigenetic mechanisms encode a glycemic memory within endothelial cells.

Genome-wide methylation profiles in monozygotic twins with discordance for ovarian carcinoma.

Ovarian cancer is a disease that is generally diagnosed at an advanced stage, and has poor survival. Monozygotic (MZ) twins are considered to be good research models for investigating the epigenetic changes associated with diseases. In the present study, the involvement of epigenetic mechanisms in ovarian cancer etiology were evaluated using the MZ twin model. Whole-genome methylation patterns were investigated in a BRCA1 gene mutation-carrying family comprising MZ twins, only one of whom had ovarian cancer, and other healthy siblings. Whole-genome methylation patterns were assessed in peripheral blood DNA using Infinium MethylationEPIC BeadChips on an Illumina iScan device. The hypermethylated and hypomethylated genes were detected between cases and controls in four different comparison groups in order to evaluate the differences in methylation levels according to cancer diagnosis and BRCA mutation status. The obtained results showed that the differential methylations in 12 different genes, namely PR/SET domain 6, cytochrome B5 reductase 4, ZNF714, OR52M1, SEMA4D, CHD1L, CAPZB, clustered mitochondria homolog, RB-binding protein 7, chromatin repair factor, ankyrin repeat domain 23, RIB43A domain with coiled-coils 1 and C6orf227, were associated with ovarian cancer. Biological functional analysis of the genes detected in the study using the PANTHER classification system revealed that they have roles in biological processes including 'biologic adhesion', 'regulation', 'cellular components organization', 'biogenesis', 'immune system functioning', 'metabolic functioning' and 'localization'. Overall, the present study suggested that epigenetic differences, such as methylation status, could be used as a non-invasive biological markers for the early diagnosis and follow-up of ovarian cancer.

Changes of DNA Methylation Pattern in Metabolic Pathways Induced by High-Carbohydrate Diet Contribute to Hyperglycemia and Fat Deposition in Grass Carp (Ctenopharyngodon idellus).

Although studies have determined that epigenetics plays an essential role in regulating metabolism in mammals, research on nutrition-related DNA methylation remains to be lacking in teleosts. In the present study, we provided a hepatic whole-genome DNA methylation analysis in grass carp fed with moderate- or excessive-carbohydrate-level diet. Although a high-carbohydrate (HC) diet significantly changed the mRNA expression levels of metabolic genes, it did not affect the global genomic DNA methylation levels in grass carp liver. However, compared with the control group, 3,972 genes were hyper-methylated and 2,904 genes were hypo-methylated in the promoter region. Meanwhile, 10,711 genes were hyper-methylated and 6,764 genes were hypo-methylated in the gene body region in the HC group. These differentially methylated genes (DMGs) were enriched in multiple pathways, including carbohydrate metabolism, insulin pathway, lipid metabolism, and adipocytokine signaling pathway. In addition, the variations in DNA methylation significantly regulated the transcription levels of key genes of metabolism, which could affect the glucose concentrations and the lipid deposition of grass carp. Furthermore, we compared the DNA methylation alterations of genes in glucose metabolism and obesity pathways of grass carp with those of mammalian models in different nutritional states. The results showed that most of the DMGs in grass carp were also regulated by DNA methylation in mammals when the nutritional state changed. The findings revealed more differentially methylated regions and candidate genes for glucose metabolism and broken species boundaries.

Differential DNA Methylation Encodes Proliferation and Senescence Programs in Human Adipose-Derived Mesenchymal Stem Cells.

Adult adipose tissue-derived mesenchymal stem cells (ASCs) constitute a vital population of multipotent cells capable of differentiating into numerous end-organ phenotypes. However, scientific and translational endeavors to harness the regenerative potential of ASCs are currently limited by an incomplete understanding of the mechanisms that determine cell-lineage commitment and stemness. In the current study, we used reduced representation bisulfite sequencing (RRBS) analysis to identify epigenetic gene targets and cellular processes that are responsive to 5'-azacitidine (5'-AZA). We describe specific changes to DNA methylation of ASCs, uncovering pathways likely associated with the enhancement of their proliferative capacity. We identified 4,797 differentially methylated regions (FDR < 0.05) associated with 3,625 genes, of which 1,584 DMRs annotated to the promoter region. Gene set enrichment of differentially methylated promoters identified "phagocytosis," "type 2 diabetes," and "metabolic pathways" as disproportionately hypomethylated, whereas "adipocyte differentiation" was the most-enriched pathway among hyper-methylated gene promoters. Weighted coexpression network analysis of DMRs identified clusters associated with cellular proliferation and other developmental programs. Furthermore, the ELK4 binding site was disproportionately hyper-methylated within the promoters of genes associated with AKT signaling. Overall, this study offers numerous preliminary insights into the epigenetic landscape that influences the regenerative capacity of human ASCs.

Whole Genome DNA Methylation Analysis of Active Pulmonary Tuberculosis Disease Identifies Novel Epigenotypes: PARP9/miR-505/RASGRP4/GNG12 Gene Methylation and Clinical Phenotypes.

We hypothesized that DNA methylation patterns may contribute to the development of active pulmonary tuberculosis (TB). Illumina's DNA methylation 450 K assay was used to identify differentially methylated loci (DML) in a discovery cohort of 12 active pulmonary TB patients and 6 healthy subjects (HS). DNA methylation levels were validated in an independent cohort of 64 TB patients and 24 HS. Microarray analysis identified 1028 DMLs in TB patients versus HS, and 3747 DMLs in TB patients after versus before anti-TB treatment, while autophagy was the most enriched signaling pathway. In the validation cohort, PARP9 and miR505 genes were hypomethylated in the TB patients versus HS, while RASGRP4 and GNG12 genes were hypermethylated, with the former two further hypomethylated in those with delayed sputum conversion, systemic symptoms, or far advanced lesions. MRPS18B and RPTOR genes were hypomethylated in TB patients with pleural involvement. RASGRP4 gene hypermethylation and RPTOR gene down-regulation were associated with high mycobacterial burden. TB patients with WIPI2/GNG12 hypermethylation or MRPS18B/FOXO3 hypomethylation had lower one-year survival. In vitro ESAT6 and CFP10 stimuli of THP-1 cells resulted in DNA de-methylation changes of the PARP9, RASGRP4, WIPI2, and FOXO3 genes. In conclusions, aberrant DNA methylation over the PARP9/miR505/RASGRP4/GNG12 genes may contribute to the development of active pulmonary TB disease and its clinical phenotypes, while aberrant DNA methylation over the WIPI2/GNG12/MARPS18B/FOXO3 genes may constitute a determinant of long-term outcomes.

Linking endotypes to omics profiles in difficult-to-control asthma using the diagnostic Chinese medicine syndrome differentiation algorithm.

Objective: Patients with difficult-to-control asthma have difficulty breathing almost all of the time, even leading to life-threatening asthma attacks. However, only few diagnostic markers for this disease have been identified. We aimed to take advantage of unique Chinese medicine theories for phenotypic classification and to explore molecular signatures in difficult-to-control asthma. Methods: The Chinese medicine syndrome differentiation algorithm (CMSDA) is a syndrome-scoring classification method based on the Chinese medicine overall observation theory. Patients with difficult-to-control asthma were classified into Cold- and Hot-pattern groups according to the CMSDA. DNA methylation and metabolomic profiles were obtained using Infinium Human Methylation 450 BeadChip and gas chromatography-mass spectrometer. Subsequently, an integrated bioinformatics analysis was performed to compare those two patterns and identify Cold/Hot-associated candidates, followed by functional validation studies. Results: A total of 20 patients with difficult-to-control asthma were enrolled in the study. Ten were grouped as Cold and 10 as Hot according to the CMSDA. We identified distinct whole-genome DNA methylation and metabolomic profiles between Cold- and Hot-pattern groups. ALDH3A1 gene exhibited variations in the DNA methylation probe cg10791966, while two metabolic pathways were associated with those two patterns. Conclusions: Our study introduced a novel diagnostic classification approach, the CMSDA, for difficult-to-control asthma. This is an alternative way to categorize diverse syndromes and link endotypes with omics profiles of this disease. ALDH3A1 might be a potential biomarker for precision diagnosis of difficult-to-control asthma.

DNA methylation reprograms cardiac metabolic gene expression in end-stage human heart failure.

Heart failure (HF) is a leading cause of morbidity and mortality in the United States and worldwide. As a multifactorial syndrome with unpredictable clinical outcomes, identifying the common molecular underpinnings that drive HF pathogenesis remains a major focus of investigation. Disruption of cardiac gene expression has been shown to mediate a common final cascade of pathological hallmarks wherein the heart reactivates numerous developmental pathways. Although the central regulatory mechanisms that drive this cardiac transcriptional reprogramming remain unknown, epigenetic contributions are likely. In the current study, we examined whether the epigenome, specifically DNA methylation, is reprogrammed in HF to potentiate a pathological shift in cardiac gene expression. To accomplish this, we used paired-end whole genome bisulfite sequencing and next-generation RNA sequencing of left ventricle tissue obtained from seven patients with end-stage HF and three nonfailing donor hearts. We found that differential methylation was localized to promoter-associated cytosine-phosphate-guanine islands, which are established regulatory regions of downstream genes. Hypermethylated promoters were associated with genes involved in oxidative metabolism, whereas promoter hypomethylation enriched glycolytic pathways. Overexpression of plasmid-derived DNA methyltransferase 3A in vitro was sufficient to lower the expression of numerous oxidative metabolic genes in H9c2 rat cardiomyoblasts, further supporting the importance of epigenetic factors in the regulation of cardiac metabolism. Last, we identified binding-site competition via hypermethylation of the nuclear respiratory factor 1 (NRF1) motif, an established upstream regulator of mitochondrial biogenesis. These preliminary observations are the first to uncover an etiology-independent shift in cardiac DNA methylation that corresponds with altered metabolic gene expression in HF.NEW & NOTEWORTHY The failing heart undergoes profound metabolic changes because of alterations in cardiac gene expression, reactivating glycolytic genes and suppressing oxidative metabolic genes. In the current study, we discover that alterations to cardiac DNA methylation encode this fetal-like metabolic gene reprogramming. We also identify novel epigenetic interference of nuclear respiratory factor 1 via hypermethylation of its downstream promoter targets, further supporting a novel contribution of DNA methylation in the metabolic remodeling of heart failure.

Prenatal Alcohol Exposure Is Associated With Adverse Cognitive Effects and Distinct Whole-Genome DNA Methylation Patterns in Primary School Children.

Prenatal alcohol exposure (PAE) is known to elicit a broad range of systemic effects, including neurophysiological alterations that result in adverse behavioral and cognitive outcomes. However, molecular pathways underlying these long-term intrauterine effects remain to be investigated. Here, we tested a hypothesis that PAE may lead to epigenetic alterations to the DNA resulting in attentional and cognitive alterations of the children. We report the results of the study that included 156 primary school children of the Franconian Cognition and Emotion Studies (FRANCES) cohort which were tested for an objective marker of PAE, ethyl glucuronide (EtG) in meconium at birth. Thirty-two newborns were found to be exposed to alcohol with EtG values above 30 ng/g (EtG+). Previously we described PAE being associated with lower IQ and smaller amplitude of the event-related potential component P3 in go trials (Go-P3), which indicates a reduced capacity of attentional resources. Whole-genome methylation analysis of the buccal cell DNA revealed 193 differentially methylated genes in children with positive meconium EtG, that were clustered into groups involved in epigenetic modifications, neurodegeneration, neurodevelopment, axon guidance and neuronal excitability. Furthermore, we detected mediation effects of the methylation changes in DPP10 and SLC16A9 genes on the EtG related cognitive and attention-related deficits. Our results suggest that system-wide epigenetic changes are involved in long-term effects of PAE. In particular, we show an epigenetic mediation of PAE effects on cognition and attention-related processes.

Skin score correlates with global DNA methylation and GSTO1 A140D polymorphism in arsenic-affected population of Eastern India.

Arsenic is a potent environmental toxicant causing serious public health concerns in India, Bangladesh and other parts of the world. Gene- and promoter-specific hypermethylation has been reported in different arsenic-exposed cell lines, whereas whole genome DNA methylation study suggested genomic hypo- and hypermethylation after arsenic exposure in in vitro and in vivo studies. Along with other characteristic biomarkers, arsenic toxicity leads to typical skin lesions. The present study demonstrates significant correlation between severities of skin manifestations with their whole genome DNA methylation status as well as with a particular polymorphism (Ala 140 Asp) status in arsenic metabolizing enzyme Glutathione S-transferase Omega-1 (GSTO1) in arsenic-exposed population of the district of Nadia, West Bengal, India.