Genome-wide DNA Methylation Studies Research Articles

Epigenetic Landscape of DNA Methylation in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, characterized by its aggressive progression and dismal prognosis. Advances in epigenetic profiling, specifically DNA methylation analysis, have significantly deepened our understanding of PDAC pathogenesis. This review synthesizes findings from recent genome-wide DNA methylation studies, which have delineated a complex DNA methylation landscape differentiating between normal and cancerous pancreatic tissues, as well as across various stages and molecular subtypes of PDAC. These studies identified specific differentially methylated regions (DMRs) that not only enhance our grasp of the epigenetic drivers of PDAC but also offer potential biomarkers for early diagnosis and prognosis, enabling the customization of therapeutic approaches. The review further explores how DNA methylation profiling could facilitate the development of subtype-tailored therapies, potentially improving treatment outcomes based on precise molecular characterizations. Overall, leveraging DNA methylation alterations as functional biomarkers holds promise for advancing our understanding of disease progression and refining PDAC management strategies, which could lead to improved patient outcomes and a deeper comprehension of the disease’s underlying biological mechanisms.

Epigenetic patterns, accelerated biological aging, and enhanced epigenetic drift detected 6 months following COVID-19 infection: insights from a genome-wide DNA methylation study

BackgroundThe epigenetic status of patients 6-month post-COVID-19 infection remains largely unexplored. The existence of long-COVID, or post-acute sequelae of SARS-CoV-2 infection (PASC), suggests potential long-term changes. Long-COVID includes symptoms like fatigue, neurological issues, and organ-related problems, regardless of initial infection severity. The mechanisms behind long-COVID are unclear, but virus-induced epigenetic changes could play a role.Methods and resultsOur study explores the lasting epigenetic impacts of SARS-CoV-2 infection. We analyzed genome-wide DNA methylation patterns in an Italian cohort of 96 patients 6 months after COVID-19 exposure, comparing them to 191 healthy controls. We identified 42 CpG sites with significant methylation differences (FDR < 0.05), primarily within CpG islands and gene promoters. Dysregulated genes highlighted potential links to glutamate/glutamine metabolism, which may be relevant to PASC symptoms. Key genes with potential significance to COVID-19 infection and long-term effects include GLUD1, ATP1A3, and ARRB2. Furthermore, Horvath's epigenetic clock showed a slight but significant age acceleration in post-COVID-19 patients. We also observed a substantial increase in stochastic epigenetic mutations (SEMs) in the post-COVID-19 group, implying potential epigenetic drift. SEM analysis identified 790 affected genes, indicating dysregulation in pathways related to insulin resistance, VEGF signaling, apoptosis, hypoxia response, T-cell activation, and endothelin signaling.ConclusionsOur study provides valuable insights into the epigenetic consequences of COVID-19. Results suggest possible associations with accelerated aging, epigenetic drift, and the disruption of critical biological pathways linked to insulin resistance, immune response, and vascular health. Understanding these epigenetic changes could be crucial for elucidating the complex mechanisms behind long-COVID and developing targeted therapeutic interventions.

Age- and sex- divergent translatomic responses of the mouse retinal pigmented epithelium

Aging is the main risk factor for age-related macular degeneration (AMD), a retinal neurodegenerative disease that leads to irreversible blindness, particularly in people over 60 years old. Retinal pigmented epithelium (RPE) atrophy is an AMD hallmark. Genome-wide chromatin accessibility, DNA methylation, and gene expression studies of AMD and control RPE demonstrate epigenomic/transcriptomic changes occur during AMD onset and progression. However, mechanisms by which molecular alterations of normal aging impair RPE function and contribute to AMD pathogenesis are unclear.Here, we specifically interrogate the RPE translatome with advanced age and across sexes in a novel RPE reporter mouse model. We find differential age- and sex- associated transcript expression with overrepresentation of pathways related to inflammation in the RPE. Concordant with impaired RPE function, the phenotypic changes in the aged translatome suggest that aged RPE becomes immunologically active, in both males and females, with some sex-specific signatures, which supports the need for sex representation for in vivo studies.

Sphingolipids in Childhood Asthma and Obesity (SOAP Study): A Protocol of a Cross-Sectional Study.

Asthma and obesity are two of the most common chronic conditions in children and adolescents. There is increasing evidence that sphingolipid metabolism is altered in childhood asthma and is linked to airway hyperreactivity. Dysregulated sphingolipid metabolism is also reported in obesity. However, the functional link between sphingolipid metabolism, asthma, and obesity is not completely understood. This paper describes the protocol of an ongoing study on sphingolipids that aims to examine the pathophysiology of sphingolipids in childhood asthma and obesity. In addition, this study aims to explore the novel biomarkers through a comprehensive multi-omics approach including genomics, genome-wide DNA methylation, RNA-Seq, microRNA (miRNA) profiling, lipidomics, metabolomics, and cytokine profiling. This is a cross-sectional study aiming to recruit 440 children from different groups: children with asthma and normal weight (n = 100), asthma with overweight or obesity (n = 100), overweight or obesity (n = 100), normal weight (n = 70), and siblings of asthmatic children with normal weight, overweight, or obesity (n = 70). These participants will be recruited from the pediatric pulmonology, pediatric endocrinology, and general pediatric outpatient clinics at Sidra Medicine, Doha, Qatar. Information will be obtained from self-reported questionnaires on asthma, quality of life, food frequency (FFQ), and a 3-day food diary that are completed by the children and their parents. Clinical measurements will include anthropometry, blood pressure, biochemistry, bioelectrical impedance, and pulmonary function tests. Blood samples will be obtained for sphingolipid analysis, serine palmitoyltransferase (SPT) assay, whole-genome sequencing (WGS), genome-wide DNA methylation study, RNA-Seq, miRNA profiling, metabolomics, lipidomics, and cytokine analysis. Group comparisons of continuous outcome variables will be carried out by a one-way analysis of variance or the Kruskal-Wallis test using an appropriate pairwise multiple comparison test. The chi-squared test or a Fisher's exact test will be used to test the associations between categorical variables. Finally, multivariate analysis will be carried out to integrate the clinical data with multi-omics data. This study will help us to understand the role of dysregulated sphingolipid metabolism in obesity and asthma. In addition, the multi-omics data from the study will help to identify novel genetic and epigenetic signatures, inflammatory markers, and mechanistic pathways that link asthma and obesity in children. Furthermore, the integration of clinical and multi-omics data will help us to uncover the potential interactions between these diseases and to offer a new paradigm for the treatment of pediatric obesity-associated asthma.

Pilot study of genome-wide DNA methylation and gene expression for treatment response to escitalopram in panic disorder.

Antidepressants, particularly selective serotonin reuptake inhibitors, are currently considered the first-line treatment for panic disorder (PD). However, little is known about the relationship between the biomarkers that may predict better treatment. To compare genome-wide methylation and gene expression patterns between responsive and non-responsive patients with PD after 4 wk of escitalopram treatment. Thirty patients with PD were enrolled in this study (responders = 13; non-responders = 17). All patients were assessed using the PD Severity Scale-Chinese version before and after treatment. The Illumina Infinium MethylationEPIC (850k) BeadChip for genome-wide methylation screening and mRNA sequencing was used in all patients with PD. A total of 701 differentially methylated positions (DMPs) were found between responders and non-responders (|Δβ| ≥ 0.06, q < 0.05), and the hyper- and hypomethylated CpG sites were 511 (72.9%) and 190 (27.1%), respectively. Relative to non-responders, there were 59 differential transcripts, of which 20 were downregulated and 39 were upregulated (q < 0.05). However, no differentially expressed genes were identified by mRNA sequencing after correcting for multiple testing (|log2(FC)| > 1, q > 0.05). This preliminary study showed that DMPs might be associated with the treatment response to escitalopram in PD; however, these DMPs need to be verified in large samples.

Liver saturated fat content associates with hepatic DNA methylation in obese individuals

BackgroundAccumulation of saturated fatty acids (SFAs) in the liver is known to induce hepatic steatosis and inflammation causing non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Although SFAs have been shown to affect the epigenome in whole blood, pancreatic islets, and adipose tissue in humans, and genome-wide DNA methylation studies have linked epigenetic changes to NAFLD and NASH, studies focusing on the association of SFAs and DNA methylation in human liver are missing. We, therefore, investigated whether human liver SFA content associates with DNA methylation and tested if SFA-linked alterations in DNA methylation associate with NAFLD-related clinical phenotypes in obese individuals.ResultsWe identified DNA methylation (Infinium HumanMethylation450 BeadChip) of 3169 CpGs to be associated with liver total SFA content (q-value < 0.05) measured using proton NMR spectroscopy in participants of the Kuopio Obesity Surgery Study (n = 51; mean ± SD:49.3 ± 8.5 years old; BMI:43.7 ± 6.2 kg/m2). Of these 3169 sites, 797 overlapped with previously published NASH-associated CpGs (NASH-SFA), while 2372 CpGs were exclusively associated with SFA (Only-SFA). The corresponding annotated genes of these only-SFA CpGs were found to be enriched in pathways linked to satiety and hunger. Among the 54 genes mapping to these enriched pathways, DNA methylation of CpGs mapping to PRKCA and TSPO correlated with their own mRNA expression (HumanHT-12 Expression BeadChip). In addition, DNA methylation of another ten of these CpGs correlated with the mRNA expression of their neighboring genes (p value < 0.05). The proportion of CpGs demonstrating a correlation of DNA methylation with plasma glucose was higher in NASH-SFA and only-SFA groups, while the proportion of significant correlations with plasma insulin was higher in only-NASH and NASH-SFA groups as compared to all CpGs on the Illumina 450 K array (Illumina, San Diego, CA, USA).ConclusionsOur results suggest that one of the mechanisms how SFA could contribute to metabolic dysregulation in NAFLD is at the level of DNA methylation. We further propose that liver SFA-related DNA methylation profile may contribute more to hyperglycemia, while insulin-related methylation profile is more linked to NAFLD or NASH. Further research is needed to elucidate the molecular mechanisms behind these observations.

Differential CpG DNA methylation of peripheral B cells, CD4+ T cells, and salivary gland tissues in IgG4-related disease

ObjectivesImmunoglobulin-G4-related disease (IgG4-RD) is a distinct systemic autoimmune-mediated disease manifesting as chronic inflammation and tissue fibrosis. Since the role of DNA methylation in the pathogenesis of IgG4-RD is still unclear, we conduct this study to investigate epigenetic modifications in IgG4-RD.MethodsA genome-wide DNA methylation study was conducted with B cells, CD4+ T cells, and salivary gland tissues from IgG4-RD patients and matched controls by using the Illumina HumanMethylation 850K BeadChip. We further performed pyrosequencing and immunohistochemistry assays to validate the methylation status of some targets of interest.ResultsWe identified differentially methylated CpG sites including 44 hypomethylated and 166 hypermethylated differentially methylated probes (DMPs) in B cells and 260 hypomethylated and 112 hypermethylated DMPs in CD4+ T cells from 10 IgG4-RD patients compared with 10 healthy controls. We also identified 36945 hypomethylated and 78380 hypermethylated DMPs in salivary gland tissues of 4 IgG4-RD patients compared with 4 controls. DPM2 (cg21181453), IQCK (cg10266221), and ABCC13 (cg05699681, cg04985582) were hypermethylated and MBP (cg18455083) was hypomethylated in B cells, CD4+ T cells, and salivary gland tissues of IgG4-RD patients. We also observed the hypomethylated HLA-DQB2 in CD4+ T cells from IgG4-RD patients. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DMPs in salivary gland tissues of IgG4-RD patients revealed enrichment of pathways involved in the regulation of immune cell responses and fibrosis.ConclusionThis is the first DNA methylation study in peripheral B cells, CD4+ T cells, and salivary gland tissues from IgG4-RD patients. Our findings highlighted the role of epigenetic modification of DNA methylation and identified several genes and pathways possibly involved in IgG4-RD pathogenesis.

Genome-wide DNA methylation profiling in tongue squamous cell carcinoma.

To provide a comprehensive characterization of DNA methylome of oral tongue squamous cell carcinoma (OTSCC) and identify novel tumor-specific DNA methylation markers for early detection using saliva. Genome-wide DNA methylation analysis including six OTSCC matched adjacent non-tumoral tissue and saliva was performed using Infinium MethylationEPIC array. Differentially methylated levels of selected genes in our OTSCC cohort were further validated using OTSCC methylation data from The Cancer Genome Atlas database (TCGA). The methylation levels of a set of tumor-specific hypermethylated genes associated with a downregulated expression were evaluated in saliva. Receiver operating characteristic (ROC) curves were performed to assess the diagnostic value of DNA methylation markers. A total of 25,890 CpGs (20,505 hypomethylated and 5385 hypermethylated) were differentially methylated (DMCpGs) between OTSCC and adjacent non-tumoral tissue. Hypermethylation of 11 tumor-specific genes was validated in OTSCC TCGA cohort. Of these 11 genes, A2BP1, ANK1, ALDH1A2, GFRA1, TTYH1, and PDE4B were also hypermethylated in saliva. These six salivary methylated genes showed high diagnostic accuracy (≥0.800) for discriminating patients from controls. This is the first largest genome-wide DNA methylation study on OTSCC that identifies a group of novel tumor-specific DNA methylation markers with diagnostic potential in saliva.

Genome-wide DNA methylation study identifies significant epigenomic changes associated with internalized stigma in adults with non-specific chronic low back pain.

Non-specific chronic low back pain (cLBP) represents a common musculoskeletal condition with no identifiable cause. It cannot be diagnosed with conventional neuroimaging techniques such as computerized tomography (CT). The diagnostic uncertainty that characterizes non-specific cLBP can lead to stigmatizing responses from others that can become internalized Among individuals with non-specific cLBP, internalized stigma is associated with greater pain intensity and disability. Yet, no study has examined the biological mechanism linking high internalized stigma to worse outcomes in individuals with non-specific cLBP. We aimed to identify differentially methylated loci (DML), enrichment pathways, and associated network interactions among individuals with non-specific cLBP experiencing low vs. high internalized stigma. We examined DNA methylation in whole blood samples from 48 adults, ages 19-85, using reduced representation bisulfite sequencing (RRBS). After controlling for age, sex, race, and multiple testing, differentially methylated loci (DML) differed in adults with low vs. high internalized stigma by at least 10% and q < 0.01 in 3,665 CpG sites: 2,280 hypomethylated and 1,385 hypermethylated. Gene ontology (GO) analyses of the annotated genes from these sites revealed significant enrichment of 274 biological processes, 29 cellular components, and 24 molecular functions (adjusted p < 0.05). The top enriched molecular functions regulate protein binding and DNA binding of transcription factor activity. Pathway analyses indicated that many functional genomic pathways, including Hippo Signaling, Melanogenesis, and Pathways in Cancer, were enriched with differentially methylated genes. Also, there was a significant interaction between relevance pathways such as P53, mTOR, PI3K-Akt, and Wnt signaling pathways. These pathways have previously been associated with neuroinflammation, neurodegeneration, and stress-related conditions. Thus, findings point to possible stress-induced DNAm changes as the link between high levels of internalized stigma and worse outcomes in adults with non-specific cLBP.

Artificial Intelligence and Circulating Cell-Free DNA Methylation Profiling: Mechanism and Detection of Alzheimer's Disease.

Background: Despite extensive efforts, significant gaps remain in our understanding of Alzheimer’s disease (AD) pathophysiology. Novel approaches using circulating cell-free DNA (cfDNA) have the potential to revolutionize our understanding of neurodegenerative disorders. Methods: We performed DNA methylation profiling of cfDNA from AD patients and compared them to cognitively normal controls. Six Artificial Intelligence (AI) platforms were utilized for the diagnosis of AD while enrichment analysis was used to elucidate the pathogenesis of AD. Results: A total of 3684 CpGs were significantly (adj. p-value < 0.05) differentially methylated in AD versus controls. All six AI algorithms achieved high predictive accuracy (AUC = 0.949–0.998) in an independent test group. As an example, Deep Learning (DL) achieved an AUC (95% CI) = 0.99 (0.95–1.0), with 94.5% sensitivity and specificity. Conclusion: We describe numerous epigenetically altered genes which were previously reported to be differentially expressed in the brain of AD sufferers. Genes identified by AI to be the best predictors of AD were either known to be expressed in the brain or have been previously linked to AD. We highlight enrichment in the Calcium signaling pathway, Glutamatergic synapse, Hedgehog signaling pathway, Axon guidance and Olfactory transduction in AD sufferers. To the best of our knowledge, this is the first reported genome-wide DNA methylation study using cfDNA to detect AD.

DNA methylation profiles after ART during human lifespan: a systematic review and meta-analysis.

The many manipulations and processes used in ART coincide with the timing of epigenetic reprogramming and imprinting during female gametogenesis and pre-implantation embryo development, leading to concerns that the actual ART could negatively affect epigenetic reprogramming and imprinting in gametes and early embryos. A growing body of literature suggests that ART may affect epigenetic marks, such as DNA methylation, in the fetus and placenta. Potentially, this may be responsible later in life for the increased risk of adverse outcomes associated with ART. Unfortunately, the conclusions are inconsistent and, despite the increasing usage of ART, its safety at the epigenetic level is still not established. To examine whether ART is associated with DNA methylation modifications and if these modifications persist throughout life, we provide an update on the current understanding of epigenetic reprogramming in human gametes and embryos, and then focus on the assessment of fetal and postnatal DNA methylation modifications that may remain until adulthood following the use of ART in humans. We reviewed studies using targeted or epigenome-wide techniques to assess the DNA methylation patterns of the conceptus after ART compared with natural conceptions. A search for relevant studies was performed in the PubMed and EMBASE databases on 15 July 2021 with an extensive search equation. Studies on animals, gametes and embryos were subsequently excluded. After an in-depth review of full-text articles, studies on specific populations with imprinting disorders were removed and not further discussed. Before comprehensive analysis, the risk of bias of each included study was assessed with the Newcastle-Ottawa scale and quality of evidence was graded using the Grading of Recommendations, Assessment, Development and Evaluations criteria. In total, 928 records were initially identified, and 51 were finally included in the systematic review. Given the variability in the genomic scale at which DNA methylation was measured in the different studies, they were separated into two categories: targeted DNA methylation or genome-wide DNA methylation study. The present systematic review has made it possible to assess a substantial number of children since more than 4000 DNA methylation profiles of ART concepti were compared to more than 7000 controls. There is evidence that ART conception is associated with aberrant DNA methylation in imprinted loci and other genes in various tissues. One isolated modification notably occur in the paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) region, and we cannot rule out other studied sequences owing to the heterogeneity of the evidence base. Differences in DNA methylation after ART conceptions are modest, and the functional relevance in adult tissues is unknown. Functional effects in terms of gene expression as well as the roles of other epigenetic marks need to be further explored. Moreover, there is little overlap of findings obtained in targeted and genome-scale analyses owing to the lack of comparability of CpGs analyzed between both techniques. This issue also stems from small sample sizes and marked differences in methodology and cohort characteristics. Standardization of methodologies and large collaborative efforts are required to reduce the inconsistency of results and increase the robustness of findings. Finally, further studies are required to determine the contribution of parental infertility per se from the ART treatment.

Genome-wide study of DNA methylation shows alterations in metabolic, inflammatory, and cholesterol pathways in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an estimated heritability between 40 and 50%. DNA methylation patterns can serve as proxies of (past) exposures and disease progression, as well as providing a potential mechanism that mediates genetic or environmental risk. Here, we present a blood-based epigenome-wide association study meta-analysis in 9706 samples passing stringent quality control (6763 patients, 2943 controls). We identified a total of 45 differentially methylated positions (DMPs) annotated to 42 genes, which are enriched for pathways and traits related to metabolism, cholesterol biosynthesis, and immunity. We then tested 39 DNA methylation-based proxies of putative ALS risk factors and found that high-density lipoprotein cholesterol, body mass index, white blood cell proportions, and alcohol intake were independently associated with ALS. Integration of these results with our latest genome-wide association study showed that cholesterol biosynthesis was potentially causally related to ALS. Last, DNA methylation at several DMPs and blood cell proportion estimates derived from DNA methylation data were associated with survival rate in patients, suggesting that they might represent indicators of underlying disease processes potentially amenable to therapeutic interventions.

DNA Methylation Signature in Monozygotic Twins Discordant for Psoriatic Disease.

Background: Psoriatic disease is a multifactorial inflammatory condition spanning from skin and nail psoriasis (Pso) to spine and joint involvement characterizing psoriatic arthritis (PsA). Monozygotic twins provide a model to investigate genetic, early life environmental exposure and stochastic influences to complex diseases, mainly mediated by epigenetics. Methods: We performed a genome-wide DNA methylation study on whole blood of monozygotic twins from 7 pairs discordant for Pso/PsA using the Infinium Methylation EPIC array (Illumina). MeDiP—qPCR was used to confirm specific signals. Data were replicated in an independent cohort of seven patients with Pso/PsA and 3 healthy controls. Transcriptomic profiling was performed by RNAsequence on the same 7 monozygotic twin pairs. Results: We identified 2,564 differentially methylated positions between psoriatic disease and controls, corresponding to 1,703 genes, 59% within gene bodies. There were 19 regions with at least two DMPs within 1 kb of distance and significant within-pair Δβ-values (p < 0.005), among them SNX25, BRG1 and SMAD3 genes, all involved in TGF-β signaling pathway, were identified. Co-expression analyses on transcriptome data identified IL-6/JAK/STAT3 and TNF-α pathways as important signaling axes involved in the disease, and they also suggested an altered glucose metabolism in patients’ immune cells, characteristic of pro-inflammatory T lymphocytes. Conclusion: The study suggests the presence of an epigenetic signature in affected individuals, pointing to genes involved in immunological and inflammatory responses. This result is also supported by transcriptome data, that altogether suggest a higher activation state of the immune system, that could promote the disease status.

Invertebrate methylomes provide insight into mechanisms of environmental tolerance and reveal methodological biases.

There is a growing focus on the role of DNA methylation in the ability of marine invertebrates to rapidly respond to changing environmental factors and anthropogenic impacts. However, genome-wide DNA methylation studies in nonmodel organisms are currently hampered by a limited understanding of methodological biases. Here, we compare three methods for quantifying DNA methylation at single base-pair resolution-whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and methyl-CpG binding domain bisulfite sequencing (MBDBS)-using multiple individuals from two reef-building coral species with contrasting environmental sensitivity. All methods reveal substantially greater methylation in Montipora capitata (11.4%) than the more sensitive Pocillopora acuta (2.9%). The majority of CpG methylation in both species occurs in gene bodies and flanking regions. In both species, MBDBS has the greatest capacity for detecting CpGs in coding regions at our sequencing depth, but MBDBS may be influenced by intrasample methylation heterogeneity. RRBS yields robust information for specific loci albeit without enrichment of any particular genome feature and with significantly reduced genome coverage. Relative genome size strongly influences the number and location of CpGs detected by each method when sequencing depth is limited, illuminating nuances in cross-species comparisons. As genome-wide methylation differences, supported by data across bisulfite sequencing methods, may contribute to environmental sensitivity phenotypes in critical marine invertebrate taxa, these data provide a genomic resource for investigating the functional role of DNA methylation in environmental tolerance.

Genome-wide DNA methylation pattern in systemic sclerosis microvascular endothelial cells: Identification of epigenetically affected key genes and pathways.

The etiology of systemic sclerosis is not clear, but there is evidence suggesting a critical role for epigenetic alterations in disease pathogenesis and clinical expression. We sought, in this study, to characterize the genome-wide DNA methylation signature in systemic sclerosis microvascular endothelial cells. We performed a genome-wide DNA methylation study in microvascular endothelial cells derived from seven diffuse cutaneous systemic sclerosis patients compared to seven age-, sex-, and ethnicity-matched healthy controls. We paired matched samples on Illumina HumanMethylation450 (three diffuse cutaneous systemic sclerosis microvascular endothelial cells and three controls), and reproduced the results in an independent set of matched patient and controls using Illumina Infinium MethylationEPIC (four diffuse cutaneous systemic sclerosis patients and four controls) to identify differentially methylated genes. We identified 71,353 differentially methylated CpG sites in systemic sclerosis microvascular endothelial cells using Infinium MethylationEPIC microarray in the first group (0.081% of representative probes) and 33,170 CpG sites in the second group using HumanMethylation450 microarray (0.073% of representative probes) in diffuse cutaneous systemic sclerosis microvascular endothelial cells. Among the two groups of subjects, we identified differential methylation of 2455 CpG sites, representing 1301 genes. Most of the differentially methylated CpG sites were hypermethylated (1625 CpG), corresponding to 910 genes. Common hypermethylated genes in systemic sclerosis microvascular endothelial cells include NOS1, DNMT3A, DNMT3B, HDAC4, and ANGPT2. We also identified hypomethylation of IL17RA, CTNNA3, ICAM2, and SDK1 in systemic sclerosis microvascular endothelial cells. Furthermore, we demonstrate significant inverse correlation between DNA methylation status and gene expression in the majority of genes evaluated. Gene ontology analysis of hypermethylated genes demonstrated enrichment of genes involved in angiogenesis (p = 0.0006). Pathway analysis of hypomethylated genes includes genes involved in vascular smooth muscle contraction (p = 0.014) and adherens junctions (p = 0.013). Our data suggest the presence of significant genome-wide DNA methylation aberrancies in systemic sclerosis microvascular endothelial cells, and identify novel affected genes and pathways in systemic sclerosis microvascular endothelial cells.

Response to upfront azacitidine in juvenile myelomonocytic leukemia in the AZA-JMML-001 trial

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for most children with juvenile myelomonocytic leukemia (JMML). Novel therapies controlling the disorder prior to HSCT are needed. We conducted a phase 2, multicenter, open-label study to evaluate the safety and antileukemic activity of azacitidine monotherapy prior to HSCT in newly diagnosed JMML patients. Eighteen patients enrolled from September 2015 to November 2017 were treated with azacitidine (75 mg/m2) administered IV once daily on days 1 to 7 of a 28-day cycle. The primary end point was the number of patients with clinical complete remission (cCR) or clinical partial remission (cPR) after 3 cycles of therapy. Pharmacokinetics, genome-wide DNA-methylation levels, and variant allele frequencies of leukemia-specific index mutations were also analyzed. Sixteen patients completed 3 cycles and 5 patients completed 6 cycles. After 3 cycles, 11 patients (61%) were in cPR and 7 (39%) had progressive disease. Six of 16 patients (38%) who needed platelet transfusions were transfusion-free after 3 cycles. All 7 patients with intermediate- or low-methylation signatures in genome-wide DNA-methylation studies achieved cPR. Seventeen patients received HSCT; 14 (82%) were leukemia-free at a median follow-up of 23.8 months (range, 7.0-39.3 months) after HSCT. Azacitidine was well tolerated and plasma concentration-–time profiles were similar to observed profiles in adults. In conclusion, azacitidine monotherapy is a suitable option for children with newly diagnosed JMML. Although long-term safety and efficacy remain to be fully elucidated in this population, these data demonstrate that azacitidine provides valuable clinical benefit to JMML patients prior to HSCT. This trial was registered at www.clinicaltrials.gov as #NCT02447666.

DNA methylome analysis identifies BMI-related epigenetic changes associated with non-small cell lung cancer susceptibility.

BackgroundBody mass index (BMI) has been reported to be inversely associated with incident risk of non‐small cell lung cancer (NSCLC). However, the underlying mechanism is still unclear. This study aimed to investigate the role of DNA methylation in the relationship between BMI and NSCLC.MethodsWe carried out a genome‐wide DNA methylation study of BMI in peripheral blood among 2266 Chinese participants by using Illumina Methylation arrays. For the BMI‐related DNA methylation changes, their associations with NSCLC risk were further analyzed and their mediation effects on BMI‐NSCLC association were also evaluated.ResultsThe methylation levels of four CpGs (cg12593793, cg17061862, cg11024682, and cg06500161, annotated to LMNA, ZNF143, SREBF1, and ABCG1, respectively) were found to be significantly associated with BMI. Methylation levels of cg12593793, cg11024682, and cg06500161 were observed to be inversely associated with NSCLC risk [OR (95%CI) =0.22 (0.16, 0.31), 0.39 (0.30, 0.50), and 0.66 (0.53, 0.82), respectively]. Additionally, cg11024682 in SREBF1 and cg06500161 in ABCG1 mediated 45.3% and 19.5% of the association between BMI and decreased NSCLC risk, respectively.ConclusionsIn this study, we identified four DNA methylation sites associated with BMI in the Chinese populations at the genome‐wide significant level. We also found that the BMI‐related methylations of SREBF1 and ABCG1 could mediate about a quintile‐to‐half of the effect of BMI on reduced NSCLC risk, which adds a potential mechanism underlying this association.

Genome-Wide DNA Methylation Profiling of Esophageal Squamous Cell Carcinoma from Global High-Incidence Regions Identifies Crucial Genes and Potential Cancer Markers.

Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here, we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissues adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritized by weighting methylation results using RNA-sequencing data from The Cancer Genome Atlas and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR), with a 30% methylation (Δβ) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2, and THSD4, had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high-incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization. SIGNIFICANCE: This largest genome-wide DNA methylation study on ESCC from high-incidence populations of the world identifies functionally relevant and robust DNAme events that could serve as potential tumor-specific markers. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2612/F1.large.jpg.

A genome-wide methylation study of body fat traits in the Norfolk Island isolate

Background and aimsNatural variation in body fat is explained by both genetic and environmental effects. Epigenetic mechanisms such as DNA methylation can mediate these effects causing changes in gene expression leading to onset of obesity. Studies of genetic isolates have the potential to provide new epigenetic insights with advantages such as reduced genetic diversity and environmental exposures. Methods and resultsThis was an exploratory study of genome-wide DNA methylation in relation to body fat traits in 47 healthy adults from the genetic isolate of Norfolk Island. Quantitative body fat traits (body fat percentage, body mass index, hip circumference, waist circumference, waist-hip-ratio and weight) were carefully measured. DNA methylation data was obtained from peripheral blood using Illumina 450K arrays. Multi-trait analysis was performed using Principal Component Analysis (PCA). CpG by trait association testing was performed using stepwise linear regressions. Two components were identified that explained approximately 89% of the phenotypic variance. In total, 5 differential methylated positions (DMPs) were identified at genome-wide significance (P≤ 2.4 × 10−7), which mapped to GOT2-CDH8, LYSMD3, HIBADH, ADGRD1 and EBF4 genes. Gene set enrichment analysis of 848 genes containing suggestive DMPs (P≤ 1.0 × 10−4) implicated the Cadherin (28 genes, Padj = 6.76 × 10−7) and Wnt signaling pathways (38 genes, Padj = 7.78 × 10−6). ConclusionThis study provides new insights into the epigenetically influenced genes and pathways underlying body fat variation in a healthy cohort and provides targets for consideration in future studies of obesity risk.

DNA Methylation in Peripheral Blood and Risk of Gastric Cancer: A Prospective Nested Case-control Study.

DNA methylation in peripheral blood is a potential biomarker of gastric cancer risk which could be used for early detection. We conducted a prospective case-control study nested within the Melbourne Collaborative Cohort Study. Genomic DNA was prepared from blood samples collected a median of 12 years before diagnosis for cases (N = 168). Controls (N = 163) were matched to cases on sex, year of birth, country of birth, and blood sample type using incidence density sampling. Genome-wide DNA methylation was measured using the Infinium HumanMethylation450K Beadchip. Global measures of DNA methylation were defined as the median methylation M value, calculated for each of 13 CpG subsets representing genomic function, mean methylation and location, and reliability of measurement. Conditional logistic regression was conducted to assess associations between these global measures of methylation and gastric cancer risk, adjusting for Helicobacter pylori and other potential confounders. We tested nonlinear associations using quintiles of the global measure distribution. A genome-wide association study of DNA methylation and gastric cancer risk was also conducted (N = 484,989 CpGs) using conditional logistic regression, adjusting for potential confounders. Differentially methylated regions (DMR) were investigated using the R package DMRcate We found no evidence of associations with gastric cancer risk for individual CpGs or DMRs (P > 7.6 × 10-6). No evidence of association was observed with global measures of methylation (OR 1.07 per SD of overall median methylation; 95% confidence interval, 0.80-1.44; P = 0.65). We found no evidence that blood DNA methylation is prospectively associated with gastric cancer risk.Prevention Relevance: We studied DNA methylation in blood to try and predict who was at risk of gastric cancer before symptoms developed, by which stage survival is poor. We did not find any such markers, but the importance of early diagnosis in gastric cancer remains, and the search for markers continues.