Differentially Methylated Probes Research Articles

Genome-Wide DNA methylation profile analysis identifies differentially methylated loci associated with personal PM2.5 exposure in adults with asthma

Particulate matter with aerodynamic diameters ≤2.5 µm (PM2.5) is a major environmental risk factor for acute asthma exacerbation, and the underlying mechanism is not completely understood. Studies have indicated that DNA methylation is a potential mechanism linking PM2.5 to its health effects. We conducted a panel study involving 24 adult patients with asthma in Beijing,China between 2017 and 2019. PM2.5 and other atmospheric pollutant exposure data were repeatedly measured. Blood samples were collected for genome-wide DNA methylation analysis. A linear mixed-effects (LME) model was conducted to identify differentially methylated probes (DMPs) associated with PM2.5 exposure. After filtering out probes that did not meet the criteria through quality control, 811,001 CpG sites were included in the LME model, and 36 DMPs were strongly associated with personal PM2.5 exposure at false discovery rate (FDR) < 0.05, of which 22 and 14 DMPs negatively and positively correlated with personal PM2.5 exposure, respectively. Functional analysis revealed that DMPs affected smooth muscle cell contraction and development, extracellular matrix synthesis and secretion, T cell activation and differentiation, and inflammatory factor production. This study provides evidence linking personal PM2.5 exposure to genome-wide DNA methylation in adult patients with asthma. Identifying enrichment pathways can provide biological insights into the acute health effects of PM2.5.

Unraveling epigenomic signatures and effectiveness of electroconvulsive therapy in treatment-resistant depression patients: a prospective longitudinal study

BackgroundElectroconvulsive therapy (ECT) benefits patients with treatment-resistant depression (TRD), but the underlying biological processes are unclear. We conducted an epigenome-wide association study in 32 TRD patients undergoing ECT to depict ECT-associated methylation changes. Illness severity and ECT outcomes were assessed with the Montgomery–Åsberg Depression Rating Scale at baseline (T0) and 1 month after its end (T1). Methylation was profiled at T0 and T1 with the Illumina Infinium Methylation EPIC BeadChip array.ResultsLongitudinal T0–T1 analyses showed 3 differentially methylated probes (DMPs) with nominal p values ≤ 10−5, with 2 annotated in the genes CYB5B and PVRL4. Including covariates, we found 4 DMPs for symptoms variation, annotated in FAM20C, EPB41, OTUB1 and ADARB1, and 3 DMPs for response status, with 2 annotated in IQCE and FAM20C. Regional analysis revealed 54 differentially methylated regions (DMRs) with nominal p value area ≤ 0.05, with 9 presenting adjusted p-value area ≤ 0.10, annotated in MCF2L, SLC25A24, RUNX3, MIR637, FOXK2, FAM180B, POU6F1, ALS2CL and CCRL2. Considering covariates, we found 21 DMRs for symptoms variation and 26 DMRs for response (nominal p value area ≤ 0.05), with 4 presenting adjusted p-value area ≤ 0.10 for response, annotated in SNORD34, NLRP6, GALNT2 and SFT2D3. None remained significant after false discovery rate correction. Notably, ADARB1 variants are associated with suicide attempt in patients with psychiatric disorders, and SLC25A24 relates to conduct disorder. Several DMPs and DMRs are annotated in genes associated with inflammatory/immune processes. Longitudinal analyses on females (n = 22) revealed statistically significant DMRs (adjusted p value area ≤ 0.05) and trend-significant DMRs (adjusted p value area ≤ 0.07) for symptoms variation and response status, annotated in genes related to psychiatric disorders (ZFP57, POLD4, TRIM10, GAS7, ADORA2A, TOLLIP), trauma exposure (RIPOR2) and inflammatory/immune responses (LAT, DLX4, POLD4, FAM30A, H19). Pathway analysis on females revealed enrichment for transcriptional activity, growth factors, DNA maintenance, and immune pathways including IRF7 and IRF2.ConclusionAlthough no significant results were found for the whole cohort, the study provides insights into ECT-associated methylation changes, highlighting DMPs and DMRs related to ECT outcomes. Analyses on females revealed significant DMRs and pathways related to psychiatric disorders and inflammatory/immune processes.

Machine Learning-Based Identification of Diagnostic Biomarkers for Korean Male Sarcopenia Through Integrative DNA Methylation and Methylation Risk Score: From the Korean Genomic Epidemiology Study (KoGES).

Sarcopenia, characterized by a progressive decline in muscle mass, strength, and function, is primarily attributable to aging. DNA methylation, influenced by both genetic predispositions and environmental exposures, plays a significant role in sarcopenia occurrence. This study employed machine learning (ML) methods to identify differentially methylated probes (DMPs) capable of diagnosing sarcopenia in middle-aged individuals. We also investigated the relationship between muscle strength, muscle mass, age, and sarcopenia risk as reflected in methylation profiles. Data from 509 male participants in the urban cohort of the Korean Genome Epidemiology Study_Health Examinee study were categorized into quartile groups based on the sarcopenia criteria for appendicular skeletal muscle index (ASMI) and handgrip strength (HG). To identify diagnostic biomarkers for sarcopenia, we used recursive feature elimination with cross validation (RFECV), to pinpoint DMPs significantly associated with sarcopenia. An ensemble model, leveraging majority voting, was utilized for evaluation. Furthermore, a methylation risk score (MRS) was calculated, and its correlation with muscle strength, function, and age was assessed using likelihood ratio analysis and multinomial logistic regression. Participants were classified into two groups based on quartile thresholds: sarcopenia (n = 37) with ASMI and HG in the lowest quartile, and normal ranges (n = 48) in the highest. In total, 238 DMPs were identified and eight probes were selected using RFECV. These DMPs were used to build an ensemble model with robust diagnostic capabilities for sarcopenia, as evidenced by an area under the receiver operating characteristic curve of 0.94. Based on eight probes, the MRS was calculated and then validated by analyzing age, HG, and ASMI among the control group (n = 424). Age was positively correlated with high MRS (coefficient, 1.2494; odds ratio [OR], 3.4882), whereas ASMI and HG were negatively correlated with high MRS (ASMI coefficient, -0.4275; OR, 0.6521; HG coefficient, -0.3116; OR, 0.7323). Overall, this study identified key epigenetic markers of sarcopenia in Korean males and developed a ML model with high diagnostic accuracy for sarcopenia. The MRS also revealed significant correlations between these markers and age, HG, and ASMI. These findings suggest that both diagnostic models and the MRS can play an important role in managing sarcopenia in middle-aged populations.

Neuroimaging and epigenetic analysis reveal novel epigenetic loci in major depressive disorder.

Epigenetic modifications, such as DNA methylation, contribute to the pathophysiology of major depressive disorder (MDD). This study aimed to identify novel MDD-associated epigenetic loci using DNA methylation profiles and explore the correlations between epigenetic loci and cortical thickness changes in patients with MDD. A total of 350 patients with MDD and 161 healthy controls (HCs) were included in the epigenome-wide association studies (EWAS). We analyzed methylation, copy number alteration (CNA), and gene network profiles in the MDD group. A total of 234 patients with MDD and 135 HCs were included in neuroimaging methylation analysis. Pearson's partial correlation analysis was used to estimate the correlation between cortical thickness of brain regions and DNA methylation levels of the loci. In total, 2018 differentially methylated probes (DMPs) and 351 differentially methylated regions (DMRs) were identified. DMP-related genes were enriched in two networks involved in the central nervous system. In neuroimaging analysis, patients with MDD showed cortical thinning in the prefrontal regions and cortical thickening in several occipital regions. Cortical thickness of the left ventrolateral prefrontal cortex (VLPFC, i.e. pars triangularis) was negatively correlated with eight DMPs associated with six genes (EML6, ZFP64, CLSTN3, KCNMA1, TAOK2, and NT5E). Through combining DNA methylation and neuroimaging analyses, negative correlations were identified between the cortical thickness of the left VLPFC and DNA methylation levels of eight DMPs. Our findings could improve our understanding of the pathophysiology of MDD.

Abstract PO2-01-08: Differential HOXB13 gene expression and promoter methylation analysis in breast cancer

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature comprising two functional biomarker panels: the Molecular Grade Index (MGI) and BCI (H/I), which is a ratio of the HOXB13 and IL17BR gene expression. Several studies have shown that BCI (H/I) is a predictive biomarker for extended adjuvant endocrine therapy benefit in hormone receptor-positive (HR+) early-stage breast cancer. Molecular mechanisms underlying endocrine responsiveness remain to be fully explored. The objective of this analysis is to evaluate the correlation between gene expression and methylation in breast cancer patients displaying different HOXB13 mRNA expression levels from The Cancer Genome Atlas (TCGA) project. Methods: Data from methylation microarrays and mRNA sequencing were examined in combination with clinical metadata from 1,095 TCGA breast cancer patients. After removing outliers, HR+ samples were ordered based on their HOXB13 expression levels and the top 10% of samples (HOXB13-high; n=95) and bottom 10% (HOXB13-low; n=95) were selected for further analysis. Differentially expressed genes (DEGs) were determined by comparing all protein-coding genes between the HOXB13-high and HOXB13-low sets, based on an absolute log-fold change above 2 and a Benjamini-Hochberg adjusted p-value threshold of 0.01. Among the subset of samples with matching methylation data (n=60 for the HOXB13-high group and n=48 for the HOXB13-low group), hypo-methylated and hyper-methylated differentially methylated probes (DMPs) were identified based on at least 10% change of β (i.e., average promoter methylation) and a Benjamini-Hochberg adjusted p-value threshold of 0.01. Motif and transcription factor (TF) enrichment analyses were identified at a minimum incidence of 10 and a lower boundary of 1.5 for the 95% confidence interval of the odds ratio. Results: Our analysis identified a total of 613 DEGs between the HOXB13-high and HOXB13-low groups. Gene ontology analysis of DEGs revealed a statistically significant enrichment of genes associated with PTK6 regulated cell cycle, oncogene-induced cellular senescence, and regulation of T cell activation. Analysis of protein-protein interactions of differentially expressed genes revealed differences in key biological processes including changes in cell cycle regulation, p53 pathway, and IL-17 signaling. A total of 5,295 hyper-methylated and 580 hypo-methylated DMPs were discovered when comparing HOXB13-high and HOXB13-low samples. Changes in global methylation patterns enabled classification of breast cancer samples with high or low HOXB13 expression with an accuracy of 0.90. Reconstruction of gene regulatory networks from DNA methylation and transcriptome profiles elucidated significant pairs of DMPs and differentially expressed genes. Specifically, a total of 197 differentially expressed genes were attributed to hyper-methylated DMPs, including genes involved in response to estrogen, activation of HOX genes during differentiation, and drug-mediated inhibition of CDK4/6 activity. Additionally, 8 differentially expressed genes were attributed to hypo-methylated DMPs and were associated with proteasomal protein catabolic processes. Lastly, motif analysis of transcriptional factors for the hyper-methylated DMPs revealed enrichment of binding motifs attributed to FOXA1, ESR1, XBR1, and FOXP1. Conclusion: A comprehensive and comparative analysis of mRNA expression levels and methylation patterns between TCGA breast cancer samples with high and low expression of HOXB13 revealed key signaling pathways and biological processes that may provide insights on the molecular mechanism of HOXB13 in the regulation of response to endocrine therapy in HR+ breast cancer. Citation Format: Natalia Siuliukina, Yi Zhang, Kai Treuner, Mark Pegram. Differential HOXB13 gene expression and promoter methylation analysis in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-01-08.

Genome- and epigenome-wide association studies identify susceptibility of CpG sites and regions for metabolic syndrome in a Korean population

BackgroundWhile multiple studies have investigated the relationship between metabolic syndrome (MetS) and its related traits (fasting glucose, triglyceride, HDL cholesterol, blood pressure, waist circumference) and DNA methylation, our understanding of the epigenetic mechanisms in MetS remains limited. Therefore, we performed an epigenome-wide meta-analysis of blood DNA methylation to identify differentially methylated probes (DMPs) and differentially methylated regions (DMRs) associated with MetS and its components using two independent cohorts comprising a total of 2,334 participants. We also investigated the specific genetic effects on DNA methylation, identified methylation quantitative trait loci (meQTLs) through genome-wide association studies and further utilized Mendelian randomization (MR) to assess how these meQTLs subsequently influence MetS status.ResultsWe identified 40 DMPs and 27 DMRs that are significantly associated with MetS. In addition, we identified many novel DMPs and DMRs underlying inflammatory and steroid hormonal processes. The most significant associations were observed in 3 DMPs (cg19693031, cg26974062, cg02988288) and a DMR (chr1:145440444–145441553) at the TXNIP, which are involved in lipid metabolism. These CpG sites were identified as coregulators of DNA methylation in MetS, TG and FAG levels. We identified a total of 144 cis-meQTLs, out of which only 13 were found to be associated with DMPs for MetS. Among these, we confirmed the identified causal mediators of genetic effects at CpG sites cg01881899 at ABCG1 and cg00021659 at the TANK genes for MetS.ConclusionsThis study observed whether specific CpGs and methylated regions act independently or are influenced by genetic effects for MetS and its components in the Korean population. These associations between the identified DNA methylation and MetS, along with its individual components, may serve as promising targets for the development of preventive interventions for MetS.

An epigenetic candidate-gene association study of parental styles in suicide attempters with substance use disorders.

Suicide attempts (SA) are prevalent in substance use disorders (SUD). Epigenetic mechanisms may play a pivotal role in the molecular mechanisms of environmental effects eliciting suicidal behaviour in this population. Hypothalamic-pituitary-adrenal axis (HPA), oxytocin and neurotrophin pathways have been consistently involved in SA, yet , their interplay with childhood adversity remains unclear, particularly in SUD. In 24 outpatients with SUDs, we examined the relation between three parental dysfunctional styles and history of SA with methylation of 32 genes from these pathways, eventually analysing 823 methylation sites. Extensive phenotypic characterization was obtained using a semi-structured interview. Parental style was patient-reported using the Measure of Parental Style (MOPS) questionnaire, analysed with and without imputation of missing items. Linear regressions were performed to adjust for possible confounders, followed by multiple testing correction. We describe both differentially methylated probes (DMPs) and regions (DMRs) for each set of analyses (with and without imputation of MOPS items). Without imputation, five DMRs in OXTR, CRH and NTF3 significantly interacted with MOPS father abuse to increase the risk for lifetime SA, thus covering the three pathways. After imputation of missing MOPS items, two other DMPs from FKBP5 and SOCS3 significantly interactedwith each of the three father styles to increase the risk for SA. Although our findings must be interpreted with caution due to small sample size, they suggest implications of stress reactivity genes in the suicidal risk of SUD patients and highlight the significance offather dysfunction as a potential marker of childhood adversity in SUD patients.

Abstract 4414: Genome-wide DNA methylation classifier diagnoses pancreatic ductal adenocarcinoma in CUP setting

Abstract Introduction: The metastasis of a pancreatic ductal adenocarcinoma (PAAD) is a diagnosis of exclusion and one of the most common causes of cancer of unknown primary (CUP). We have recently developed a genome-wide DNA methylation-based neural network classifier that can accurately differentiate between liver metastasis of a PAAD and intrahepatic cholangiocarcinoma (iCCA) (PAAD-iCCA-Classifier). Therefore, the aim of our study was to test whether our PAAD-iCCA-Classifier can be extended to be used to correctly diagnose PAAD metastases from other sites in CUP setting. Methods: For this purpose, we enhanced the anomaly detection layer of the classifier by incorporating ten mimicker carcinomas to be excluded by this layer. We used a validation set 1 (n=3786) including primary PAAD (n=242), PAAD liver metastases (n=20), iCCA (n=151) and 10 other mimicker carcinomas (n=3373) and a validation set 2 (n=26) including primary PAAD from a real-life clinical cohort from an independent institution to validate the classifier. Next, we tested the classifier on 16 PAAD initially considered CUP samples (test set) from different sites: peritoneum, lung, liver, and lymph node. The clinical history and diagnostic imaging of these samples were used to confirm PAAD as the most probable origin. We further performed differentially methylated probes (DMP) and copy number alterations (CNA) analysis of primary PAAD and metastatic PAAD from different locations. Results: The improved version of the PAAD-iCCA-Classifier achieved an accuracy of 98.43% on the validation set 1 and was able to exclude most of the mimicker carcinomas. On validation set 2, the classifier achieved an accuracy of 88.46%. Medical history, imaging and immunohistochemical analysis of the test set samples confirmed the diagnosis of PAAD. The DNA methylation classifier correctly labeled 15/16 PAAD metastatic samples as PAAD (93.75% accuracy). We observed that the classifier performance was negatively affected by a high CD3+ immune infiltrate and positively affected by high tumor purity and high proliferation rate. CNA revealed that PAAD liver metastases have a distinct CNA profile characterized by chromosome 6, 9 (CDKN2A/B) and 18q (SMAD4) deletions. DMP analysis showed that PAAD liver metastases have global hypomethylation of both promoter- and enhancer-associated CpGs compared to primary PAAD and PAAD peritoneal carcinomatosis. Finally, gene ontology analysis revealed that different epithelial-mesenchymal transition pathways are activated in PAAD liver metastases compared to PAAD peritoneal carcinomatosis. Conclusion: Our tool performs well in classifying metastatic PAAD samples and could be of great clinical use when a PAAD origin is suspected in the case of a CUP. DMP and CNA profiles show that PAAD liver metastases may have a distinct DNA methylation and copy number profile compared to primary and peritoneal carcinomatosis PAAD. Citation Format: Teodor G. Calina, Eilís Perez, Simon Schallenberg, Horst David, Erik Knutsen, David Capper, Mihnea P. Dragomir. Genome-wide DNA methylation classifier diagnoses pancreatic ductal adenocarcinoma in CUP setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4414.

Aberrant PRDM2 methylation as an early event in serrated lesions destined to evolve into microsatellite-instable colorectal cancers.

Up to 30% of colorectal cancers (CRCs) develop from sessile serrated lesions (SSLs). Within the serrated neoplasia pathway, at least two principally distinct oncogenetic routes exist generating microsatellite-stable and microsatellite-instable CRCs, respectively. Aberrant DNA methylation (DNAm) is found early in the serrated pathway and might play a role in both oncogenetic routes. We studied a cohort of 23 SSLs with a small focus (<10 mm) of dysplasia or cancer, 10 of which were MLH1 deficient and 13 MLH1 proficient. By comparing, for each SSL, the methylation status of (1) the region of dysplasia or cancer (SSL-D), (2) the nondysplastic SSL (SSL), and (3) adjacent normal mucosa, differentially methylated probes (DMPs) and regions (DMRs) were assessed both genome-wide as well as in a tumor-suppressor gene-focused approach. By comparing DNAm of MLH1-deficient SSL-Ds with their corresponding SSLs, we identified five DMRs, including those annotating for PRDM2 and, not unexpectedly, MLH1. PRDM2 gene promotor methylation was associated with MLH1 expression status, as it was largely hypermethylated in MLH1-deficient SSL-Ds and hypomethylated in MLH1-proficient SSL-Ds. Significantly increased DNAm levels of PRDM2 and MLH1, in particular at 'critical' MLH1 probe sites, were to some extent already visible in SSLs as compared to normal mucosa (p = 0.02, p = 0.01, p < 0.0001, respectively). No DMRs, nor DMPs, were identified for SSLs destined to evolve into MLH1-proficient SSL-Ds. Our data indicate that, within both arms of the serrated CRC pathway, the majority of the epigenetic alterations are introduced early during SSL formation. Promoter hypermethylation of PRDM2 and MLH1 on the other hand specifically initiates in SSLs destined to transform into MLH1-deficient CRCs suggesting that the fate of SSLs may not necessarily result from a stochastic process but possibly is already imprinted and predisposed.

Genome-wide DNA methylation and transcriptomic patterns of precancerous gastric cardia lesions.

Intestinal metaplasia (IM) and intraepithelial neoplasia (IEN) are considered precursors of gastric cardia cancer (GCC). Here, we investigated the histopathologic and molecular profiles of precancerous gastric cardia lesions (PGCLs) and biomarkers for risk stratification of gastric cardia IM. We conducted a hospital-based evaluation (n = 4578) for PGCL profiles in high-incidence and non-high-incidence regions for GCC in China. We next performed 850K methylation arrays (n = 42) and RNA-seq (n = 44) in tissues with PGCLs. We then examined the protein expression of candidate biomarker using immunohistochemistry. Of the 4578 participants, 791 were diagnosed with PGCLs (600 IM, 62 IM with IEN, and 129 IEN). We found that individuals from high-incidence regions (26.7%) were more likely to develop PGCLs than those from non-high-incidence areas (13.5%). DNA methylation and gene expression alterations, indicated by differentially methylated probes (DMPs) and differentially expressed genes (DEGs), exhibited a progressive increase from type I IM (DMP = 210, DEG = 24), type II IM (DMP = 3402, DEG = 129), to type III IM (DMP = 3735, DEG = 328), peaking in IEN (DMP = 47 373, DEG = 2278). Three DEGs with aberrant promoter methylation were identified, shared exclusively by type III IM and IEN. Of these DEGs, we found that OLFM4 expression appears in IMs and increases remarkably in IENs (P < .001). We highlight that type III IM and IEN share similar epigenetic and transcriptional features in gastric cardia and propose biomarkers with potential utility in risk prediction.

Epigenome-wide DNA methylation analysis of late-stage mild cognitive impairment.

Background: Patients with late-stage mild cognitive impairment (LMCI) have a higher risk of progression to Alzheimer's disease (AD) than those with early-stage mild cognitive impairment (EMCI). However, previous studies have often pooled EMCI and LMCI patients into a single MCI group, with limited independent investigation into the pathogenesis of LMCI. Methods: In this study, we employed whole-genome methylation association analysis to determine the differences in peripheral blood methylation profiles between 663 cognitive aging (CN) and 554 LMCI patients. Results: Our results revealed 2,333 differentially methylated probes (DMPs) and 85 differentially methylated regions (DMRs) specific to LMCI. The top hit methylation sites or regions were associated with genes such as SNED1, histone deacetylases coding gene HDACs, and HOX and ZNF gene family. The DNA methylations upregulated the expression of HDAC4, HDAC8, and HOX family genes HOXC5 and HOXC9, but they downregulated the expression of SNED1, ADCYAP1, and ZNF family genes ZNF415 and ZNF502. Gene Ontology (GO) and KEGG analysis showed that the genes associated with these methylation sites were predominantly related to the processes of addiction disorders, neurotransmission, and neurogenesis. Out of the 554 LMCI patients included in this study, 358 subjects (65%) had progressed to AD. Further association analysis between the LMCI subjects with a stable course (sLMCI) and those who progressed to AD (pLMCI) indicated that the methylation signal intensities of HDAC6, ZNF502, HOXC5, HOXC6, and HOXD8 were associated with increased susceptibility to AD. Protective effects against progression to AD were noticed when the methylation of SNED1 and ZNF727 appeared in LMCI patients. Conclusion: Our findings highlight a substantial number of LMCI-specific methylated biomarkers that differ from those identified in previous MCI case-control studies. These biomarkers have the potential to contribute to a better understanding of the pathogenesis of LMCI.

The intrinsic defects of T cells impact the efficacy of CAR-T therapy in patients with diffuse large B-cell lymphoma

CAR-T cell therapy did not achieve the desired efficacy in some patients with diffuse large B-cell lymphoma (DLBCL). We conducted single-cell RNA and TCR sequencing as well as methylation chip profiling of peripheral blood samples in DLBCL patients. Patients who achieved complete remission (CR) showed an upward trend in T-cell levels, especially CD8-effector T cells. The responders exhibited T-cell clone expansion, more active T-cell transformation, and frequent cell communication. Highly expressed genes in the CR group were enriched in functions like leukocyte-mediated cytotoxicity and activation of immune response, while the non-CR group was enriched in pathways related to DNA damage and P53-mediated intrinsic apoptotic. More differentially methylated probes (DMPs) were identified in the baseline of the non-CR group (779 vs 350). GSEA analysis revealed that the genes annotated by DMPs were associated with cellular immune functions in T cells, including the generation of chemokines, leukocyte-mediated cytotoxicity, and cell-killing functions. The genes with low expression in the non-CR group exhibited a high methylation status. There is heterogeneity in the cellular, molecular, and epigenetic characteristics of host T cells in patients with different clinical outcomes. Intrinsic defects in T cells are important factors leading to poor efficacy of CAR-T therapy.

Epigenetic Profiling of Type 2 Diabetes Mellitus: An Epigenome-Wide Association Study of DNA Methylation in the Korean Genome and Epidemiology Study.

Diabetes is characterized by persistently high blood glucose levels and severe complications and affects millions of people worldwide. In this study, we explored the epigenetic landscape of diabetes using data from the Korean Genome and Epidemiology Study (KoGES), specifically the Ansung-Ansan (AS-AS) cohort. Using epigenome-wide association studies, we investigated DNA methylation patterns in patients with type 2 diabetes mellitus (T2DM) and those with normal glucose regulation. Differential methylation analysis revealed 106 differentially methylated probes (DMPs), with the 10 top DMPs prominently associated with TXNIP, PDK4, NBPF20, ARRDC4, UFM1, PFKFB2, C7orf50, and ABCG1, indicating significant changes in methylation. Correlation analysis highlighted the association between the leading DMPs (e.g., cg19693031 and cg26974062 for TXNIP and cg26823705 for NBPF20) and key glycemic markers (fasting plasma glucose and hemoglobin A1c), confirming their relevance in T2DM. Moreover, we identified 62 significantly differentially methylated regions (DMRs) spanning 61 genes. A DMR associated with PDE1C showed hypermethylation, whereas DMRs associated with DIP2C, FLJ90757, PRSS50, and TDRD9 showed hypomethylation. PDE1C and TDRD9 showed a strong positive correlation between the CpG sites included in each DMR, which have previously been implicated in T2DM-related processes. This study contributes to the understanding of epigenetic modifications in T2DM. These valuable insights can be utilized in identifying potential biomarkers and therapeutic targets for effective management and prevention of diabetes.

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.

Whole blood DNA methylation changes are associated with anti-TNF drug concentration in patients with Crohn's disease.

Anti-TNF treatment failure in patients with inflammatory bowel disease (IBD) is common and frequently related to low drug concentrations. In order to identify patients who may benefit from dose optimisation at the outset of anti-TNF therapy, we sought to define epigenetic biomarkers in whole blood at baseline associated with anti-TNF drug concentrations at week 14. DNA methylation from 1,104 whole blood samples from 385 patients in the Personalised Anti-TNF Therapy in Crohn's disease (PANTS) study were assessed using the Illumina EPIC Beadchip (v1.0) at baseline, weeks 14, 30 and 54. We compared DNA methylation profiles in anti-TNF-treated patients who experienced primary non-response at week 14 and if they were assessed at subsequent time points, were not in remission at week 30 or 54 (infliximab n = 99, adalimumab n = 94), with patients who responded at week 14 and when assessed at subsequent time points, were in remission at week 30 or 54 (infliximab n = 99, adalimumab n = 93). Overall, between baseline and week 14, we observed 4,999 differentially methylated probes (DMPs) annotated to 2376 genes following anti-TNF treatment. Pathway analysis identified 108 significant gene ontology terms enriched in biological processes related to immune system processes and responses.Epigenome-wide association (EWAS) analysis identified 323 DMPs annotated to 210 genes at baseline associated with higher anti-TNF drug concentrations at week 14. Of these, 125 DMPs demonstrated shared associations with other common traits (proportion of shared CpGs compared to DMPs) including body mass index (23.2%), followed by CRP (11.5%), smoking (7.4%), alcohol consumption per day (7.1%) and IBD type (6.8%). EWAS of primary non-response to anti-TNF identified 20 DMPs that were associated with both anti-TNF drug concentration and primary non-response to anti-TNF with a strong correlation of the coefficients (Spearman's rho = -0.94, p < 0.001). Baseline DNA methylation profiles may be used as a predictor for anti-TNF drug concentration at week 14 to identify patients who may benefit from dose optimisation at the outset of anti-TNF therapy.