Differentially Methylated Probes Research Articles

Blood-based DNA methylation in advanced Nasopharyngeal Carcinoma exhibited distinct CpG methylation signature

The TNM staging system is currently used to detect cancer stages. Regardless, a small proportion of cancer patients recur even after therapy, suggesting more specific molecular tools are required to justify the stage-specific detection and prompt cancer diagnosis. Thus, we aimed to explore the blood-based DNA methylation signature of metastatic nasopharyngeal carcinoma (NPC) to establish a holistic methylation biomarker panel. For the identification of methylation signature, the EPIC BeadChip-based array was performed. Comparative analysis for identifying unique probes, validation, and functional studies was investigated by analyzing GEO and TCGA datasets. We observed 4093 differentially methylated probes (DMPs), 1232 hydroxymethylated probes, and 25 CpG islands. Gene expression study revealed both upregulated and downregulated genes. Correlation analysis suggested a positive (with a positive r, p ≤ 0.05) and negative (with a negative r, p ≤ 0.05) association with different cancers. TFBS analysis exhibited the binding site for many TFs. Furthermore, gene enrichment analysis indicated the involvement of those identified genes in biological pathways. However, blood-based DNA methylation data uncovered a distinct DNA methylation pattern, which might have an additive role in NPC progression by altering the TFs binding. Moreover, based on tissue-specificity, a variation of correlation between methylation and gene expression was noted in different cancers.

Deciphering the DNA methylome in women with PCOS diagnosed using the new international evidence-based guidelines.

Is there any methylome alteration in women with PCOS who were diagnosed using the new international evidence-based guidelines? A total of 264 differentially methylated probes (DMPs) and 53 differentially methylated regions (DMRs) were identified in patients with PCOS and healthy controls. PCOS is a common endocrine disorder among women of reproductive age and polycystic ovarian morphology (PCOM) is one of the main features of the disease. Owing to the availability of more sensitive ultrasound machines, the traditional diagnosis of PCOM according to the Rotterdam criteria (≥12 antral follicles per ovary) is currently debated as there is a risk of overdiagnosis. The new international evidence-based guidelines set the threshold for PCOM as ≥20 antral follicles per ovary when using endovaginal ultrasound transducers with a frequency bandwidth that includes 8 MHz. However, current DNA methylation studies in PCOS are still based on the Rotterdam criteria. This study aimed to explore aberrant DNA methylation in patients diagnosed with PCOS according to the new evidence-based guidelines. This cross-sectional case-control study included 34 PCOS cases diagnosed using new international evidence-based guidelines and 36 controls. A total of 70 women, including 34 PCOS cases and 36 controls, were recruited. DNA extracted from whole blood samples of participants were profiled using array technology. Data quality control, preprocessing, annotation, and statistical analyses were performed. Least absolute shrinkage and selection operator (LASSO) regression were used to build a PCOS diagnosis model with DNA methylation sites. We identified 264 DMPs between PCOS cases and controls, which were mainly located in intergenic regions or gene bodies of the genome, CpG open sea sites, and heterochromatin of functional elements. Pathway enrichment analysis showed that DMPs were significantly enriched in biological processes involved in triglyceride regulation. Three of these DMPs overlapped with the PCOS susceptibility genes thyroid adenoma-associated protein (THADA), aminopeptidase O (AOPEP), and tripartite motif family-like protein 2 (TRIML2). Fifty-three DMRs were identified and their annotated genes were largely enriched in allograft rejection, thyroid hormone production, and peripheral downstream signaling effects. Two DMRs were closely related to the PCOS susceptibility genes, potassium voltage-gated channel subfamily A member 4 (KCNA4) and farnesyl-diphosphate farnesyltransferase 1 (FDFT1). Finally, based on LASSO regression, we built a methylation marker model with high accuracy for PCOS diagnosis (AUC=0.952). The study cohort was single-center and the sample size was relatively limited. Further analyses with a larger number of participants are required. This is the first study to identify DNA methylation alterations in women with PCOS diagnosed using the new international evidence-based guideline, and it provided new molecular insight into the application of the new guidelines. This study was supported by the National Key Research and Development Program of China (2021YFC2700400), Basic Science Center Program of NSFC (31988101), CAMS Innovation Fund for Medical Sciences (2021-I2M-5-001), National Natural Science Foundation of China (32370916, 82071606, 82101707, 82192874, and 31871509), Shandong Provincial Key Research and Development Program (2020ZLYS02), Taishan Scholars Program of Shandong Province (ts20190988), and Fundamental Research Funds of Shandong University. The authors declare no conflicts of interest. N/A.

Cord blood DNA methylation of immune and lipid metabolism genes is associated with maternal triglycerides and child adiposity.

Fetal exposures may impact offspring epigenetic signatures and adiposity. The authors hypothesized that maternal metabolic traits associate with cord blood DNA methylation, which, in turn, associates with child adiposity. Fasting serum was obtained in 588 pregnant women (27-34 weeks' gestation), and insulin, glucose, high-density lipoprotein cholesterol, triglycerides, and free fatty acids were measured. Cord blood DNA methylation and child adiposity were measured at birth, 4-6 months, and 4-6 years. The association of maternal metabolic traits with DNA methylation (429,246 CpGs) for differentially methylated probes (DMPs) and regions (DMRs) was tested. The association of the first principal component of each DMR with child adiposity was tested, and mediation analysis was performed. Maternal triglycerides were associated with the most DMPs and DMRs of all traits tested (261 and 198, respectively, false discovery rate < 0.05). DMRs were near genes involved in immune function and lipid metabolism. Triglyceride-associated CpGs were associated with child adiposity at 4-6 months (32 CpGs) and 4-6 years (2 CpGs). One, near CD226, was observed at both timepoints, mediating 10% and 22% of the relationship between maternal triglycerides and child adiposity at 4-6 months and 4-6 years, respectively. DNA methylation may play a role in the association of maternal triglycerides and child adiposity.

Comprehensive Analyses Revealed Eight Immune Related Signatures Correlated With Aberrant Methylations as Prognosis and Diagnosis Biomarkers for Kidney Renal Papillary Cell Carcinoma.

Kidney renal papillary cell carcinoma (KIRP) is a common type of renal cell carcinoma. DNA methylation plays an important role in the development of several cancers. The aim of our study was to identify differentially expressed genes associated with abnormal DNA methylation as biomarkers for predicting the outcome of KIRP. We downloaded KIRP methylation data, RNA sequencing (RNAseq) data, and their corresponding clinical information from the Cancer Genome Atlas (TCGA) database. ChAMP and DEGseq2 packages in R software were used to screen differentially methylated probes (DMPs) and differentially expressed genes (DEGs). Univariate and multivariate Cox regression analyses were used to identify suitable immune related genes correlated with aberrant methylations as prognosis biomarkers. We identified 8 DEGs (Cysteine And Glycine Rich Protein 1 [CSRP1], major histocompatibility complex, Class II, DM Beta [HLA-DMB], LIF Receptor Subunit Alpha [LIFR], Leukotriene B4 receptor 2 [LTB4R2], Mitogen-Activated Protein Kinase Kinase Kinase 14 [MAP3K14], Nuclear Receptor Subfamily 2 Group F Member 1 [NR2F1], Secreted And Transmembrane 1 [SECTM1], and Vimentin [VIM]) that were independently associated with the overall survival (months) (OS) of KIRP. The time dependent area under the curve (AUC) for each receiver operating characteristic (ROC) of the risk assessment model at 1, 3, 5, and 10-years reached 0.8415, 0.8131, 0.7873, and 0.7667. The risk assessment model was correlated with several immune cells and factors. The AUC value of the diagnosis model using those 8 DEGs reached 0.99. The risk assessment model constructed by those 8 DEGs was well able to predict the prognosis and diagnose of KIRP. However, whether the prognosis and diagnosis model could be applied in clinical practice requires further study.

Analysis of DNA methylation at birth and in childhood reveals changes associated with season of birth and latitude

BackgroundSeasonal variations in environmental exposures at birth or during gestation are associated with numerous adult traits and health outcomes later in life. Whether DNA methylation (DNAm) plays a role in the molecular mechanisms underlying the associations between birth season and lifelong phenotypes remains unclear.MethodsWe carried out epigenome-wide meta-analyses within the Pregnancy And Childhood Epigenetic Consortium to identify associations of DNAm with birth season, both at differentially methylated probes (DMPs) and regions (DMRs). Associations were examined at two time points: at birth (21 cohorts, N = 9358) and in children aged 1–11 years (12 cohorts, N = 3610). We conducted meta-analyses to assess the impact of latitude on birth season-specific associations at both time points.ResultsWe identified associations between birth season and DNAm (False Discovery Rate-adjusted p values < 0.05) at two CpGs at birth (winter-born) and four in the childhood (summer-born) analyses when compared to children born in autumn. Furthermore, we identified twenty-six differentially methylated regions (DMR) at birth (winter-born: 8, spring-born: 15, summer-born: 3) and thirty-two in childhood (winter-born: 12, spring and summer: 10 each) meta-analyses with few overlapping DMRs between the birth seasons or the two time points. The DMRs were associated with genes of known functions in tumorigenesis, psychiatric/neurological disorders, inflammation, or immunity, amongst others. Latitude-stratified meta-analyses [higher (≥ 50°N), lower (< 50°N, northern hemisphere only)] revealed differences in associations between birth season and DNAm by birth latitude. DMR analysis implicated genes with previously reported links to schizophrenia (LAX1), skin disorders (PSORS1C, LTB4R), and airway inflammation including asthma (LTB4R), present only at birth in the higher latitudes (≥ 50°N).ConclusionsIn this large epigenome-wide meta-analysis study, we provide evidence for (i) associations between DNAm and season of birth that are unique for the seasons of the year (temporal effect) and (ii) latitude-dependent variations in the seasonal associations (spatial effect). DNAm could play a role in the molecular mechanisms underlying the effect of birth season on adult health outcomes.

Maternal obesity and high leptin levels prime pro-inflammatory pathways in human cord blood leukocytes

IntroductionMaternal obesity alters the immune function in the offspring. We hypothesize that maternal obesity and pro-inflammatory pathways induce leptin-related genes in neonatal monocytes, whereby high leptin levels enhance their inflammatory response. MethodsTranscriptional profiles of cord blood leukocytes (CBL) in basal and pro-inflammatory conditions were studied to determine differentially expressed genes (DEG). The DNA methylation profile of CB monocytes (CBM) of neonates born to control BMI mothers and women with obesity was assayed to identify differentially methylated probes (DMP). CBM-derived macrophages were cultured with or without leptin (10–100 ng/ml) and then stimulated with lipopolysaccharide (LPS, 100 ng/ml) and interferon-gamma (20 ng/ml) to assess the induction of TNF-α and IL-10 transcripts. ResultsCBL from pregnancies with obesity (CBL-Ob) showed 12,183 DEG, affecting 49 out of 78 from the leptin pathway. Control CBM exposed to LPS showed 45 leptin-related DEG, an effect prevented by the co-exposure to LPS and IL-10. Conversely, CBM-Ob showed 5279 DMP enriched in insulin- and leptin-related genes, and Lasso regression of leptin-related DMP showed high predictive value for plasma leptin levels (r2 = 0.9897) and maternal BMI categories (AUC = 1). Chronic exposure to leptin increased TNF-α and decreased IL-10 levels in control BMI samples but not in Ob-CBM. Enhanced TNF-α induction after proinflammatory stimulation was observed in leptin-treated control BMI samples. DiscussionObesity in pregnancy is associated with a distinctive expression and DNA methylation profile of leptin-related genes in cord blood monocytes, meanwhile, leptin enhances the expression of pro-inflammatory cytokines upon stimulation with M1-skewing agents.

DNA methylation mediated genetic risk in severe acne in a young men population.

Acne is a chronic inflammatory skin disease that affects the pilosebaceous follicle and is influenced by heredity, hormones, inflammation, and the environment. At present, the recognized pathogenesis mainly includes four categories: excessive sebum secretion, excessive Cutibacterium acnes proliferation, excessive keratinization of sebaceous glands in hair follicles, and inflammatory mechanisms. Previous studies have found that DNA methylation is closely related to some chronic inflammatory skin diseases, and there is evidence that DNA methylation is controlled by genetic factors, making us want to know the relationship between DNA methylation, genetic variation and acne. In our previous study, we performed genome-wide DNA methylation analysis in peripheral blood samples from 44 patients with severe acne and 44 unaffected normal subjects, and identified 23 differentially methylated probes (DMPs). In this study, we identified single nucleotide polymorphisms (SNPs) associated with severe acne by genome-wide association analysis in these 88 samples. To test the association between SNPs and DMPs, we conducted DNA methylation quantitative trait loci (methQTL) analysis. Next, causal inference testing (CIT) was used to determine whether genetic variation influences DNA methylation, which impacts disease phenotypes. We found 38,269 SNPs associated with severe acne. By methQTL analysis, we obtained 24 SNP-CpG pairs that reached the threshold (FDR < 0.05), which included 7 unique CpGs and 22 unique methQTL SNPs. After CIT analysis, we found that 11 out of 24 pairs of SNP-CpG showed a weakened SNP effect after adjustment for methylation, indicating a methylation-mediated relationship between SNPs and severe acne. These 11 SNP-CpG pairs consist of four unique CpG sites and 11 SNPs, of which three CpG sites, cg03020863, cg20652636, and cg19964325, are located on the gene body of PDGFD, the intron of SH2D6, and the 5'UTR of the IL1R1 gene, respectively. During this study, the DNA methylation of certain genes was found to be influenced by genetic factors and mediated the risk of severe acne in a young Chinese male population, providing a new perspective on the pathogenesis of severe acne.

DNA methylation, combined with RNA sequencing, provide novel insight into molecular classification of chordomas and their microenvironment

Chordomas are rare tumors of notochord remnants, occurring mainly in the sacrum and skull base. Despite of their unusually slow growth, chordomas are highly invasive and the involvement of adjacent critical structures causes treatment challenges. Due to the low incidence, the molecular pathogenesis of this entity remains largely unknown. This study aimed to investigate DNA methylation abnormalities and their impact on gene expression profiles in skull base chordomas. 32 tumor and 4 normal nucleus pulposus samples were subjected to DNA methylation and gene expression profiling with methylation microarrays and RNA sequencing. Genome-wide DNA methylation analysis revealed two distinct clusters for chordoma (termed subtypes C and I) with different patterns of aberrant DNA methylation. C Chordomas were characterized by general hypomethylation with hypermethylation of CpG islands, while I chordomas were generally hypermethylated. These differences were reflected by distinct distribution of differentially methylated probes (DMPs). Differentially methylated regions (DMRs) were identified, indicating aberrant methylation in known tumor-related genes in booth chordoma subtypes and regions encoding small RNAs in subtype C chordomas. Correlation between methylation and expression was observed in a minority of genes. Upregulation of TBXT in chordomas appeared to be related to lower methylation of tumor-specific DMR in gene promoter. Gene expression-based clusters of tumor samples did not overlap with DNA methylation-based subtypes. Nevertheless, they differ in transcriptomic profile that shows immune infiltration in I chordomas and up-regulation of cell cycle in C chordomas. Immune enrichment in chordomas I was confirmed with 3 independent deconvolution methods and immunohistochemistry. Copy number analysis showed higher chromosomal instability in C chordomas. Nine out of eight had deletion of CDKN2A/B loci and downregulation of genes encoded in related chromosomal band. No significant difference in patients’ survival was observed between tumor subtypes, however, shorter survival was observed in patients with higher number of copy number alterations.

A Glimpse into the Genome-wide DNA Methylation Changes in 6-hydroxydopamine-induced In Vitro Model of Parkinson's Disease.

A cell-based model of Parkinson's disease (PD) is a well-established in vitro experimental prototype to investigate the disease mechanism and therapeutic approach for a potential anti-PD drug. The SH-SY5Y human neuroblastoma cells and 6-OHDA combo is one of the many neurotoxininduced neuronal cell models employed in numerous neuroscience-related research for discovering neuroprotective drug compounds. Emerging studies have reported a significant correlation between PD and epigenetic alterations, particularly DNA methylation. However, the DNA methylation changes of PD-related CpG sites on the 6-OHDA-induced toxicity on human neuronal cells have not yet been reported. We performed a genome-wide association study (GWAS) using Infinium Epic beadchip array surveying 850000 CpG sites in differentiated human neuroblastoma cells exposed to 6-OHDA. We identified 236 differentially methylated probes (DMPs) or 163 differentially methylated regions (DMRs) in 6-OHDA treated differentiated neuroblastoma cells than the untreated reference group with p<0.01, Δbeta cut-off of 0.1. Among 236 DMPs, hypermethylated DMPs are 110 (47%), whereas 126 (53%) are hypomethylated. Our bioinformatic analysis revealed 3 DMRs that are significantly hypermethylated and associated with neurological disorders, namely AKT1, ITPR1 and GNG7. This preliminary study demonstrates the methylation status of PD-related CpGs in the 6-OHDA-induced toxicity in the differentiated neuroblastoma cells model.

Investigation of changes in DNA methylation associated with alterations in gene expression resulting in differences between lean and obese adipogenesis

DNA methylation (DNAm) is an important epigenetic regulator controlling various cellular activities, including cell proliferation and differentiation. In the present work, we examined alterations in DNAm associated with obesity using methylome and transcriptome data from 27 purified adipocytes (ACs) and 7 preadipocytes (preACs) in human visceral adipose tissue (VAT) samples. We identified differentially methylated probes (DMPs) using two methods: (i) DMPs that were obtained from a comparison of the DNAm levels between AC and preAC samples (AGDMPs) and (ii) DMPs that were obtained from a comparison of the DNAm levels between obese and lean AC samples (ACDMPs). These two classes of DMPs were obtained to identify a relationship between adipogenesis and obesity. We also investigated how hyper and hypomethylation of the promoter and/or gene body differentially affected changes in gene expression by estimating the odds ratios (ORs) of changes in gene expression without DMPs in the background. Interestingly, the magnitude of DNAm alterations during AC differentiation was greater under lean conditions than under obese conditions. In conclusion, several adipogenesis-related genes were affected by complicated methylation changes and ultimately cause differences in gene expression in ACs under lean and obese conditions.

Abstract 5516: Identifying DNA methylation biomarkers in Brazilian women of African descent

Abstract Breast cancer is the most common cancer, and the leading cause of cancer-related deaths in Brazilian women. Studies have shown that in Brazil while breast cancer mortality has increased in the last decade for all women, mortality in women of African descent has doubled. A recent study showed that Afro-Brazilian women suffer from higher excess breast cancer mortality even after adjusting for extent of disease, year of diagnosis, age and socioeconomic status, which suggests biological factors also contribute to the differences in the clinicopathological make-up of the disease in women in this group. DNA methylation biomarkers, representing a combination of genetic and non-genetic factors, have the potential to be especially valuable when considering the combined contribution of the environment and genetics in breast cancer etiology. Several groups have found differences in DNA methylation levels associated with race and ethnicity in breast cancer. In addition, research has shown that women of different races/ethnicities with similar breast cancer tumor subtypes have distinct DNA methylation patterns. To explore further the association between race, ancestry and DNA methylation, we conducted a study in paired tumor and non-tumor tissue samples of forty-eight Brazilian women who self-identified as Black or Brown and were treated at National Cancer Institute Breast Cancer Hospital in Rio de Janeiro, Brazil. We carried out a DNA methylation profiling analysis using Illumina EPIC (850k) arrays in bisulfite converted DNA originally extracted from frozen tissue samples. A total of 739,883 differentially methylated probes (DMP) were identified, of which a large proportion 6,241 (78.4%) were hypomethylated. Lower DNA methylation was more common on intragenic regions and gene bodies, while hypermethylation was more commonly found on the promoter regions. DNA methylation was often higher in non-tumor than tumor samples. Among our top hits we found genes previously associated with estrogen receptor negative tumor types, CDH4, CERK, PROX1 and ADHFE1, and with high risk of breast cancer mortality (ST6GAL1, LYN, TFF1 and BMP3). A DPM enrichment pathway analysis predicted effects in known signaling pathways and transcriptional misregulation. We are currently analyzing the associations between the top differentially methylated regions with ancestry informative markers, and the clinicopathological characteristics of disease, to gain a better understanding of the role of DNA methylation marks in this population. This study adds to our current knowledge on epigenetics marks that will ultimately help us address mortality due to this disease in this population. Citation Format: Lissette Delgado-Cruzata, Diego J. Gomes de Paula, Jennifer Vieira Gomes, Tatiana A. Simão, Leonor Gusmão, Luis Felipe Ribeiro Pinto, Sheila C. Soares-Lima. Identifying DNA methylation biomarkers in Brazilian women of African descent. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5516.

DNA Methylation of Window of Implantation Genes in Cervical Secretions Predicts Ongoing Pregnancy in Infertility Treatment

Window of implantation (WOI) genes have been comprehensively identified at the single cell level. DNA methylation changes in cervical secretions are associated with in vitro fertilization embryo transfer (IVF-ET) outcomes. Using a machine learning (ML) approach, we aimed to determine which methylation changes in WOI genes from cervical secretions best predict ongoing pregnancy during embryo transfer. A total of 2708 promoter probes were extracted from mid-secretory phase cervical secretion methylomic profiles for 158 WOI genes, and 152 differentially methylated probes (DMPs) were selected. Fifteen DMPs in 14 genes (BMP2, CTSA, DEFB1, GRN, MTF1, SERPINE1, SERPINE2, SFRP1, STAT3, TAGLN2, TCF4, THBS1, ZBTB20, ZNF292) were identified as the most relevant to ongoing pregnancy status. These 15 DMPs yielded accuracy rates of 83.53%, 85.26%, 85.78%, and 76.44%, and areas under the receiver operating characteristic curves (AUCs) of 0.90, 0.91, 0.89, and 0.86 for prediction by random forest (RF), naïve Bayes (NB), support vector machine (SVM), and k-nearest neighbors (KNN), respectively. SERPINE1, SERPINE2, and TAGLN2 maintained their methylation difference trends in an independent set of cervical secretion samples, resulting in accuracy rates of 71.46%, 80.06%, 80.72%, and 80.68%, and AUCs of 0.79, 0.84, 0.83, and 0.82 for prediction by RF, NB, SVM, and KNN, respectively. Our findings demonstrate that methylation changes in WOI genes detected noninvasively from cervical secretions are potential markers for predicting IVF-ET outcomes. Further studies of cervical secretion of DNA methylation markers may provide a novel approach for precision embryo transfer.

DOP88 Understanding the molecular mechanisms of anti-TNF treatment failure: Whole blood DNA methylation changes associated with primary non-response to anti-TNF treatment in patients with Crohn’s disease

Abstract Background Anti-TNF treatment failure in patients with IBD is common and frequently related to low drug concentrations. DNA methylation sites from whole blood have been identified as effective biomarkers predicting treatment response to methotrexate and etanercept in patients with rheumatoid arthritis. We sought to define differences in DNA methylation associated with primary non-response (PNR) to anti-TNF treatment in patients with Crohn’s disease. Methods The Personalised Anti-TNF Therapy in Crohn’s disease (PANTS) study is a prospective UK-wide study investigating anti-TNF treatment failure in patients with active luminal Crohn’s disease. DNA methylation from whole blood was assessed using the Illumina EPIC Beadchip at baseline, weeks 14, 30 and 52. We compared DNA methylation profiles in a subset of adalimumab- and infliximab-treated patients who experienced PNR at week 14 and were not in remission at week 54 (infliximab = 99, adalimumab = 94) with patients who responded at week 14 and were in remission at week 54 (infliximab = 99, adalimumab = 93). Low anti-TNF drug concentration was defined as an infliximab level &amp;lt;3mg/L and adalimumab level &amp;lt;5mg/L. Epigenome-wide association (EWAS) analysis was performed using linear mixed models, where p values &amp;lt; 9 x 10-8 were considered significant. Because we observed significant changes in CD4 and CD8 T cells, B cells, NK cells, monocytes and granulocytes following anti-TNF treatment we adjusted our models for derived cell proportions (Figure 1). Results Overall, between baseline and week 14, we observed 4999 differentially methylated probes (DMPs) annotated to 2376 genes. Pathway analysis identified 108 significant gene ontology terms enriched in biological processes related to immune system processes and responses (Figure 2). EWAS analysis at baseline identified 48 DMPs annotated to 36 genes associated with PNR (Figure 3). The majority of DMPs (40/48) were hypermethylated (p &amp;lt; 0.001): of those annotated to genes, 25 were located in the gene body, 5 in the 5’ untranslated regions (UTR), 4 in transcription start sites and the remaining in 3’UTR. When EWAS analysis at baseline was stratified by anti-TNF drug concentration, 27 DMPs annotated to 17 genes were associated with PNR and low drug concentration. Of these, 11 DMPs were independently associated with low anti-TNF drug concentration at week 14 (Figure 4). We did not identify any DMPs associated with PNR with adequate drug concentration. Conclusion Our data suggests that baseline DNA methylation profiles may be used as a predictor for anti-TNF drug concentration at week 14.

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.

DNA Methylation Description of Hippocampus, Cortex, Amygdala, and Blood of Drug-Resistant Temporal Lobe Epilepsy.

Epigenetic changes such as DNA methylation were observed in drug-resistant temporal lobe epilepsy (DR-TLE), a disease that affects 25-30% of epilepsy patients. The main objective is to simultaneously describe DNA methylation patterns associated with DR-TLE in hippocampus, amygdala, surrounding cortex to the epileptogenic zone (SCEZ), and peripheral blood. An Illumina Infinium MethylationEPIC BeadChip array was performed in 19 DR-TLE patients and 10 postmortem non-epileptic controls. Overall, 32, 59, and 3210 differentially methylated probes (DMPs) were associated with DR-TLE in the hippocampus, amygdala, and SCEZ, respectively. These DMP-affected genes were involved in neurotrophic and calcium signaling in the hippocampus and voltage-gated channels in SCEZ, among others. One of the hippocampus DMPs (cg26834418 (CHORDC1)) showed a strong blood-brain correlation with BECon and IMAGE-CpG, suggesting that it could be a potential surrogate peripheral biomarker of DR-TLE. Moreover, in three of the top SCEZ's DMPs (SHANK3, SBF1, and MCF2L), methylation status was verified with methylation-specific qPCR. The differentially methylated CpGs were classified in DMRs: 2 in the hippocampus, 12 in the amygdala, and 531 in the SCEZ. We identified genes that had not been associated to DR-TLE so far such as TBX5, EXOC7, and WRHN. The area with more DMPs associated with DR-TLE was the SCEZ, some of them related to voltage-gated channels. The DMPs found in the amygdala were involved in inflammatory processes. We also found a potential surrogate peripheral biomarker of DR-TLE. Thus, these results provide new insights into epigenetic modifications involved in DR-TLE.

Nasal Transcriptome and Epigenome Analysis Identifies the Pathogenic Features of Aspirin-Exacerbated Respiratory Disease.

Dysregulation of the arachidonic acid metabolic pathway is the most widely known pathomechanism of aspirin-exacerbated respiratory disease (AERD). This study aimed to perform integrative analysis of transcriptomic and epigenomic profiling with network analysis to determine the novel pathogenic features of AERD. Ten patients with asthma including 5 patients with AERD and another 5 patients with aspirin tolerant asthma (ATA) were enrolled. Nasal scraping was performed and nasal mucosa was used in omics profiling. Peripheral eosinophil counts, sputum eosinophil counts, fractional exhaled nitric oxide levels, and pulmonary function test results were evaluated. Differentially expressed genes (DEGs), differentially methylated probes (DMPs) and differentially correlated genes (DCGs) between patients with AERD and those with ATA were analyzed. Network analysis using ingenuity pathway analysis (IPA) was performed to determine the gene connection network and signaling pathways. In total, 1,736 DEGs, 1,401 DMPs, and 19 pairs for DCGs were identified. Among DCGs, genes related to vesicle transport (e.g., RAB3B and STX2) and sphingolipid dysregulation (e.g., SMPD3) were found to be hypo-methylated and up-regulated in AERD. Using the canonical pathway analysis of IPA with 78 asthma-related DEGs, signaling pathways of T helper cell differentiation/activation and Fcε receptor I were generated. Up-regulation of RORγt and FcER1A were noted in AERD. Gene expression levels of RAB3B, SYNE1, STX2, SMPD3 and RORγt were significantly associated with sputum eosinophil counts. Quantitative real-time polymerase chain reaction was performed and mRNA expression levels of STX2, SMPD3, RORγt, and FcER1A were significantly higher in AERD compared to ATA. Distinct pathogenic features were identified by using integrative multi-omics data analysis in patients with AERD.

Periconceptional folate intake influences DNA methylation at birth based on dietary source in an analysis of pediatric acute lymphoblastic leukemia cases and controls

Periconceptional folate intake is associated with the establishment of DNA methylation in offspring; however, variations in this relation by food sources compared with folic acid supplements are not described. Also, maternal folate intake is associated with decreased risk of pediatric acute lymphoblastic leukemia (ALL), but the mechanism is not known. We evaluated the relation between periconceptional folate intake by source and DNA methylation at birth in a cohort of pediatric ALL cases and controls in an epigenome-wide association study. Genome-wide DNA methylation status obtained from archived neonatal blood spots from pediatric ALL cases (n=189) and controls (n=205) in the California Childhood Leukemia Study (CCLS) from 1995-2008 was compared with periconceptional folate from total, food, and supplemental sources using multivariable linear regression. Further stratification was performed by income, education, ethnicity, and total folate intake. We evaluated variable DNA methylation response to periconceptional folate by ALL case status through an interaction term. Two significant differentially methylated probes (DMPs) were associated with food and supplemental periconceptional folate intake in all subjects (n=394). The top differentially methylated region at the promoter region of DUSP22(dual specificity phosphatase 22) demonstrated DNA hypermethylation in ALL cases but not in controls in response to total and food folate intake. We further identified 8 interaction term DMPs with variable DNA methylation response to folate intake by ALL case status. Further stratification of the cohort by education and ethnicity revealed a substantially higher number of DMPs associated with supplemental folic acid intake in Hispanic subjects with lower income and educational level. We identified modest associations between periconceptional folate intake and DNA methylation differing by source, including variation by ALL case status. Hispanic subjects of lower income and education appear uniquely responsive to periconceptional folate supplementation.

Genome-wide DNA methylation profiling of gastric cardia cancer.

Aberrant DNA methylation has been found in various cancer types including gastric cancer, yet the genome-wide DNA methylation profile of gastric cardia cancer (GCC) remains unclear. Therefore, we aimed to profile the DNA methylation pattern of GCC and identify promising diagnostic epigenetic biomarkers. We investigated the genome-wide DNA methylation pattern in eight pairs of GCC and adjacent normal tissues using Illumina 850K microarrays. Subsequently, bisulfite-pyrosequencing and quantitative real-time PCR were performed on eight pairs of GCC-adjacent normal tissues for validation. Finally, we performed immunohistochemistry to examine ADHFE1 expression on 126 pairs of GCC-adjacent normal samples. DNAmethylome analysis showed global hypomethylation and local hypermethylation of promoter cytosine-phosphate-guanine (CpG) islands (CGIs) in GCC tissues compared with gastric cardia normal mucosa (P<2.2×10-16 ). Differential methylation analysis identified a total of 91 723 differentially-methylated probes (DMPs), and the candidate gene with the largest average DNA methylation difference mapped to ADHFE1 (mean Δβ=0.53). Subsequently, three DMPs in the ADHFE1 promoter were validated by pyrosequencing. Notably,the mean methylation level of the three candidate DMPs (ADHFE1_cg08090772, ADHFE1_cg19283840, and ADHFE1_cg20295442) was negatively associated with ADHFE1 mRNA expression level (Spearman rho=-0.64, P=0.01). Moreover, both mRNA (P=0.0213) and protein (P<0.0001) expression of ADHFE1 were significantly decreased in GCCs compared with the adjacent normal tissues. Our results reveal DNA methylation aberrations in GCC and that ADHFE1 gene DNA methylation contributes to the risk of GCC, thus providing novel mechanistic insights into gastric cardia cancer carcinogenesis.

Identification of DNA methylation-regulated differentially expressed genes in RA by integrated analysis of DNA methylation and RNA-Seq data

ObjectiveTo identify novel DNA methylation-regulated differentially expressed genes (MeDEGs) in RA by integrated analysis of DNA methylation and RNA-Seq data.MethodsThe transcription and DNA methylation profiles of 9 RA and 15 OA synovial tissue were generated by RNA-Seq and Illumina 850K DNA methylation BeadChip. Gene set enrichment analysis (GSEA) and Weighted gene co-expression network analysis (WGCNA) were used to analyze methylation-regulated expressed genes by R software. The differentially expressed genes (DEGs), differentially methylated probes (DMPs), differentially methylated genes (DMGs) were analyzed by DESeq and ChAMP R package. The functional correlation of MeDEGs was analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein–protein interaction (PPI) network of MeDEGs was constructed by STRING and Reactome FI Cytoscape Plugin. Correlation analysis between methylation level and mRNA expression was conducted with R software.ResultsA total of 17,736 genes, 25,578 methylated genes and 755,852 methylation probes were detected. A total of 16,421 methylation-regulated expressed genes were obtained. The GSEA showed that these genes are associated with activation of immune response, adaptive immune response, Inflammatory response in C5 (ontology gene sets). For KEGG analysis, these genes are associated with rheumatoid arthritis, NF-kappa B signaling pathway, T cell receptor signaling pathway. The WGCNA showed that the turquoise module exhibited the strongest correlation with RA (R = 0.78, P = 1.27 × 10− 05), 660 genes were screened in the turquoise module. A total of 707 MeDEGs were obtained. GO analysis showed that MeDEGs were enriched in signal transduction, cell adhesion for BP, enriched in plasma membrane, integral component of membrane for CC, and enriched in identical protein binding, calcium ion binding for MF. The KEGG pathway analysis showed that the MeDEGs were enriched in calcium signaling pathway, T cell receptor signaling pathway, NF-kappa B signaling pathway, Rheumatoid arthritis. The PPI network containing 706 nodes and 882 edges, and the enrichment p value < 1.0 × 10− 16. With Cytoscape, based on the range of more than 10 genes, a total of 8 modules were screened out. Spearman correlation analysis showed RGS1(cg10718027), RGS1(cg02586212), RGS1(cg10861751) were significantly correlated with RA.ConclusionsRGS1 can be used as novel methylated biomarkers for RA.

DNA METHYLATION CHANGES IN EARLY PLACENTA FROM ASSISTED REPRODUCTIVE TECHNOLOGIES ARE NOT ASSOCIATED WITH SIGNIFICANT GENE EXPRESSION CHANGES

Studies suggest assisted reproductive technologies (ART) cause term placenta methylation differences when compared to spontaneous conceptions. However, 1st trimester placenta, closer to the utilization of fertility treatments has not been well studied. We aimed to determine whether methylation differences affecting gene expression exist in 1st trimester placenta between ART and spontaneous pregnancies and whether ART or infertility are causal. We compared differentially methylated probes (DMPs) in 1st trimester placenta from spontaneous, non in vitro fertilization treatment (NIFT) , and in vitro fertilization (IVF) conceptions to corresponding differences in gene expression in these cohorts.