Search Results (54)

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors primarily involving the adrenal medulla and its associated paraganglia, with heterogeneous clinical behavior and complex molecular drivers. This study aimed to characterize DNA methylation and gene expression patterns in PPGLs to understand the molecular differences between tumor subtypes and malignancy. We performed an integrative analysis of DNA methylation (Illumina EPIC 850K) and gene expression profiles (Affymetrix microarrays) in 24 PPGLs, comparing these with The Cancer Genome Atlas (TCGA) data, to delineate cluster- and malignancy-specific epigenetic patterns. Comparison between pseudohypoxic Cluster I and kinase-signaling Cluster II tumors revealed 13 differentially methylated CpG sites, with a specific CpG within DSCAML1 showing hypermethylation in Cluster II accompanied by increased expression, suggesting context-dependent gene body methylation effects. Benign versus malignant comparisons identified 101 differentially methylated CpGs, including hypermethylated CpG in BAIAP2L1 and hypomethylated CpG in SHANK1 in malignant tumors. Pathway enrichment of differentially methylated genes revealed alterations in Notch signaling, adherens junctions, cytoskeletal regulation, and intracellular transport. Gene expression analysis demonstrated partial overlap between clusters, with malignant tumors exhibiting distinct transcriptional profiles involving RNA processing, metabolism, and adhesion pathways. Correlation between methylation and expression was generally limited, emphasizing that methylation-dependent gene regulation is a locus-specific and context-dependent regulation. These findings illustrate a complex interplay between epigenetic modifications and transcriptional programs in PPGLs, enhancing our understanding of molecular heterogeneity and tumor classification, and identifying candidate biomarkers and therapeutic targets for malignant progression. Full article

Background/Objectives: Parkinson’s disease (PD) is an adult-onset neurodegenerative disorder whose pathogenesis is still not completely understood. Several lines of evidence suggest that alterations in epigenetic architecture may contribute to the development of this condition. Here, we present a pilot DNA methylation study from peripheral blood in a cohort of Sicilian PD patients and matched controls. Peripheral tissue analysis has previously been shown to reflect molecular and functional profiles relevant to neurological diseases, supporting their validity as a proxy for studying brain-related epigenetic mechanisms. Methods: We analyzed 20 PD patients and 20 healthy controls (19 males and 21 females overall), matched for sex, with an age range of 60–87 years (mean 72.3 years). Peripheral blood DNA was extracted and processed using the Illumina Infinium MethylationEPIC v2.0 BeadChip, which interrogates over 935,000 CpG sites across the genome, including promoters, enhancers, CpG islands, and other regulatory elements. The assay relies on sodium bisulfite conversion of DNA to detect methylation status at single-base resolution. Results: Epigenome-wide association study (EWAS) data allowed for multiple levels of analysis, including immune cell-type deconvolution, estimation of biological age (epigenetic clocks), quantification of stochastic epigenetic mutations (SEMs) as a measure of epigenomic stability, and differential methylation profiling. Immune cell-type inference revealed an increased but not significant proportion of monocytes in PD patients, consistent with previous reports. In contrast, epigenetic clock analysis did not reveal significant differences in biological age acceleration between cases and controls, partially at odds with earlier studies—likely due to the limited sample size. SEMs burden did not differ significantly between groups. Epivariations reveal genes involved in pathways known to be altered in dopaminergic neuron dysfunction and α-synuclein toxicity. Differential methylation analysis, however, yielded 167 CpG sites, of which 55 were located within genes, corresponding to 54 unique loci. Gene Ontology enrichment analysis highlighted significant overrepresentation of pathways with neurological relevance, including regulation of synapse structure and activity, axonogenesis, neuron migration, and synapse organization. Notably, alterations in KIAA0319, a gene involved in neuronal migration, synaptic formation, and cortical development, have previously been associated with Parkinson’s disease at the gene expression level, while methylation changes in FAM50B have been reported in neurotoxic and cognitive contexts; our data suggest, for the first time, a potential epigenetic involvement of both genes in Parkinson’s disease. Conclusions: This pilot study on a Sicilian population provides further evidence that DNA methylation profiling can yield valuable molecular insights into PD. Despite the small sample size, our results confirm previously reported findings and highlight biological pathways relevant to neuronal structure and function that may contribute to disease pathogenesis. These data support the potential of epigenetic profiling of peripheral blood as a tool to advance the understanding of PD and generate hypotheses for future large-scale studies. Full article

Cardiometabolic phenotypes such as obesity and impaired insulin action are key determinants of type 2 diabetes (T2D). Growing evidence highlights the postprandial state as a critical window in metabolic regulation, where epigenetic mechanisms, particularly DNA methylation in insulin-sensitive tissues, may play pivotal roles. However, their dynamics across prandial states in subcutaneous adipose tissue (SAT) remain unclear. We analyzed genome-wide DNA methylation in paired fasting and postprandial SAT biopsies from 29 asymptomatic, drug-naïve individuals classified as controls (n = 8), prediabetes n = 9), or T2D (n = 12). Postprandial samples followed a standardized mixed-meal test. DNA methylation was quantified using the Illumina MethylationEPIC array and analyzed through the Chip Analysis Methylation Pipeline (ChAMP) pipeline. Differential methylation was more pronounced postprandially, especially in the T2D group. After adjusting for age and sex, 4599 differentially methylated CpG sites (DMCs) were identified, with increased hypermethylation in T2D. A total of 130 DMCs across 99 genes, including LCLAT1, HLA-C, ZNF714, and HOOK2, were shared by prediabetes and T2D groups. Over-representation analysis revealed 202 enriched pathways related to insulin resistance, AMPK signaling, and immune responses. Additionally, 110 Differentially Methylated Regions (DMRs), including ZNF577 and AGPAT1, were detected. These findings reveal early, prandial-dependent epigenetic alterations in SAT that precede overt dysglycemia, offering insights into personalized prevention in T2D. Full article

Pediatric obesity is rising globally, and emerging evidence suggests that sleep timing may influence metabolic health through epigenetic mechanisms. This study investigated epigenome-wide DNA methylation patterns associated with bedtime in children and explored their biological relevance. Children aged 6–10 years were classified as early (≤8:30 PM) or late (>8:30 PM) bedtime groups. Saliva-derived DNA was analyzed using the Illumina Infinium MethylationEPIC BeadChip Array, and the Sparse Wrapper Algorithm (SWAG) was applied to identify differentially methylated loci. A total of 1006 CpG sites, representing 571 unique genes, were significantly associated with bedtime (p < 0.001). Significant methylation differences were observed between early and late bedtime groups, with ABCG2, ABHD4, MOBKL1A, AK3, SDE2, PRAMEF4, CREM, CDH4, BRAT1, and SDK1 showing the most consistent variation. Functional enrichment analyses (Gene Ontology, KEGG, and DisGeNET) conducted on the SWAG-identified gene set revealed enrichment in biological processes including peptidyl-lysin demethylation, regulation of sodium ion transport, DNA repair, and lipo-protein particle assembly. Key KEGG pathways included circadian entrainment, neurotransmission (GABAergic, dopaminergic, and glutamatergic), growth hormone synthesis, and insulin secretion. DisGeNET analysis identified associations with neurodevelopmental disorders and cognitive impairment. Cross-comparison with established sleep and obesity gene sets identified ten overlapping genes(CDH4, NR3C2, ACTG1, COG5, CAT, HDAC4, FTO, DOK7, OCLN, and ATXN1). These findings suggest that variations in bedtime during childhood may epigenetically modify genes regulating circadian rhythm, metabolism, neuronal connectivity, and stress response, potentially predisposing to later-life developmental, and metabolic challenges. Full article

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. In Mexico, its higher incidence and lower survival suggest a role for epigenetic factors like DNA methylation (DNAme). We conducted an epigenome-wide association study (EWAS) to define the methylation landscape and identify the profiles associated with ALL and relapse. Bone marrow or peripheral blood samples from pediatric ALL patients at diagnosis and controls without ALL were analyzed using an Infinium MethylationEPIC v2.0 array. Differential methylation was assessed using the ChAMP package. We identified a significant hypermethylated profile in ALL patients compared to controls. Probes in MAD1L1 and RPTOR contained the most differentially methylated CpG sites. Key affected pathways included proliferation, neurotransmission, and neuronal signaling. Survival analysis revealed that hypomethylation of four specific CpGs—cg01052776 (RNH1), cg20747787, cg05001671, and cg01767116 (FBXL22)—was significantly associated with an increased risk of relapse, highlighting their potential as prognostic biomarkers. This study underscores the importance of epigenetic mechanisms in pediatric ALL. Full article

Backgrounds: Epstein–Barr virus (EBV) is implicated in the pathogenesis of different B-cell lymphomas and lymphoproliferative disorders, including diffuse large B-cell lymphoma (DLBCL) arising in immunodeficiency settings. Despite its clinical significance, the mechanisms of EBV-mediated lymphomagenesis across different disease subtypes remain poorly understood. Global DNA methylation profiling can provide insight into tumor heterogeneity and disease mechanisms. Methods: To further characterize the underlying biology of EBV(+) DLBCL, we performed a global methylome analysis of a cohort of EBV(+)/(−) DLBCL. Illumina MethylationEPIC array data were generated from a curated set of DLBCL tissue samples (n = 43) from a rural patient population with defined EBV status and immunodeficiency background. Differential methylation analyses were conducted using linear mixed models to identify significant methylation changes associated with EBV status. Results: Principle component analysis (PCA) and probe-level comparisons revealed a distinct, globally hypermethylated DNA methylome in EBV(+) DLBCL compared to EBV(−) cases, and an overall hypomethylated profile in all DLBCL relative to control tissues. We identified a total of 117,334 differentially methylated probes mapping to 1557 cancer-associated genes in EBV(+) versus EBV(−) DLBCL, and 330,872 probes mapping to 4230 cancer-associated genes in all DLBCL versus controls. Pathway enrichment analysis highlighted distinct biological processes in EBV(+) DLBCL, including P53 feedback loops (hypermethylated genes) and MAPK signaling (hypomethylated genes). Conclusions: These findings demonstrate that EBV(+) DLBCL is epigenetically distinct from EBV(−) disease, with alterations that may contribute to clinical heterogeneity and potentially serve as biomarkers for disease classification and therapeutic targeting. Full article

Fetal exposure to antiseizure medications (ASMs) can impact organogenesis, resulting in elevated risk of congenital malformations. Despite longstanding clinical awareness of the teratogenic potential of ASMs, the molecular mechanisms remain largely unexplored. To address this multisystem impact of ASMs, an OMIC-based approach was considered to understand the impact of ASMs on methylome and subsequently on proteome and how folic acid (FA) supplementation can counter the teratogenic impact. The study employed an established in vitro embryonic cell line model system, treated with varying concentrations of first-generation ASMs, alone and in combination with FA. Integrated analyses included quantification of global DNA methylation, expression analysis of key epigenetic regulators (DNMTs and TETs), genome-wide methylation profiling using the 935K EPIC array, and LC-MS/MS-based proteomics analysis. The study identified that ASMs can induce global DNA hypomethylation, which was likely to be impacted by dysregulation of DNMT and TET expression. Interestingly, FA co-treatment partially restored DNA methylation as evidenced by global DNA methylation and epigenetic gene expression, and also by compensatory effect via one-carbon metabolism. Genome-wide DNA methylation revealed site-specific hypermethylation at key developmental genes, several of which were reversed with FA. Proteomics analysis identified downregulation of developmentally critical proteins, including those linked to key metabolic processes, while FA co-treatment reversed expression of several such proteins. Integrative methylome–proteome analysis revealed the coordinated regulation of target genes that are linked to congenital abnormalities. Together, these findings offer mechanistic insight into ASM-induced teratogenesis and support FA’s potential to mitigate epigenetic and proteomic disruptions. This integrated OMICs based approach identifies key biomarkers which can be used for therapeutic monitoring and help in optimizing maternal epilepsy management. Full article

Background/Objectives: This study investigated variations in DNA methylation patterns associated with chronic pain and propensity for recurrent pressure injuries (PrI) in persons with spinal cord injury (SCI). Methods: Whole blood was collected from 81 individuals with SCI. DNA methylation was quantified using Illumina genome-wide arrays (EPIC and EPICv2). Comprehensive clinical profiles collected included secondary health complications, in particular current PrI and chronic pain. Relationships between recurrent PrI and chronic pain and whether the co-occurrence of both traits was mediated by changes in DNA methylation were investigated using R packages limma, DMRcate and mCSEA. Results: Three differentially methylated positions (DMPs) (cg09867095, cg26559694, cg24890286) and one region in the micro-imprinted locus for BLCAP/NNAT are associated with chronic pain in persons with SCI. The study cohort was stratified by PrI status to identify any sites associated with chronic pain and while the same three sites and region were replicated in the group with no recurrent PrI, two novel, hypermethylated (cg21756558, cg26217441) sites and one region in the protein-coding gene FDFT1 were identified in the group with recurrent PrI. Gene enrichment and genes associated with specific promoters using MetaScape identified several shared disorders and ontology terms between independent phenotypes of pain and recurrent PrI and interactive sub-groups. Conclusions: DMR analysis using mCSEA identified several shared genes, promoter-associated regions and CGI associated with overall pain and PrI history, as well as sub-groups based on recurrent PrI history. These findings suggest that a much larger gene regulatory network is associated with each phenotype. These findings require further validation. Full article

Recent studies show that patients with Alzheimer’s disease (AD) harbor specific methylation marks in the brain that, if accessible, could be used as epigenetic biomarkers. Liquid biopsy enables the study of circulating cell-free DNA (cfDNA) fragments originated from dead cells, including neurons affected by neurodegenerative processes. Here, we isolated and epigenetically characterized plasma cfDNA from 35 patients with AD and 35 cognitively healthy controls by using the Infinium^® MethylationEPIC BeadChip array. Bioinformatics analysis was performed to identify differential methylation positions (DMPs) and regions (DMRs), including APOE ε4 genotype stratified analysis. Plasma pTau181 (Simoa) and cerebrospinal fluid (CSF) core biomarkers (Fujirebio) were also measured and correlated with differential methylation marks. Validation was performed with bisulfite pyrosequencing and bisulfite cloning sequencing. Epigenome-wide cfDNA analysis identified 102 DMPs associated with AD status. Most DMPs correlated with clinical cognitive and functional tests including 60% for Mini-Mental State Examination (MMSE) and 80% for Global Deterioration Scale (GDS), and with AD blood and CSF biomarkers. In silico functional analysis connected 30 DMPs to neurological processes, identifying key regulators such as SPTBN4 and APOE genes. Several DMRs were annotated to genes previously reported to harbor epigenetic brain changes in AD (HKR1, ZNF154, HOXA5, TRIM40, ATG16L2, ADAMST2) and were linked to APOE ε4 genotypes. Notably, a DMR in the HKR1 gene, previously shown to be hypermethylated in the AD hippocampus, was validated in cfDNA from an orthogonal perspective. These results support the feasibility of studying cfDNA to identify potential epigenetic biomarkers in AD. Thus, liquid biopsy could improve non-invasive AD diagnosis and aid personalized medicine by detecting epigenetic brain markers in blood. Full article

Objective: This in vitro study aimed to investigate the impact of folic acid on DNA methylation and gene expression in adipocytes from subcutaneous adipose tissue of patients with systemic lupus erythematosus (SLE), with a focus on the influence of obesity on these epigenetic changes. Methods: Tissue biopsies were collected from patients with normal weight (NW) and obesity (OBS). Adipocytes were isolated via enzymatic digestion and density separation. Each group was divided into control (standard medium) and folic acid treatment (2 mg/24 h for 48 h) conditions. After treatment, DNA methylation levels were analyzed using the Infinium Methylation EPIC v2.0 Kit, and gene expression analyses were performed by RT-qPCR. A pathway enrichment analysis was conducted using the KEGG database for functional insight. Results: Folic acid induced differential methylation at 755 CpG sites in NW adipocytes, which were associated with immune regulation, including MAPK signaling. Also, OBS adipocytes showed methylation changes at 92 CpG sites, affecting pathways related to metabolic regulation, such as cAMP signaling. LEP gene expression was upregulated (5.2-fold) in OBS adipocytes, while CREM2 expression was increased (2.8-fold) in NW adipocytes after treatment. These gene expression differences underscore weight-dependent responses to folic acid, with LEP upregulation in OBS cells suggesting links to metabolic dysregulation and CREM2 upregulation in NW cells potentially contributing to immune modulation. Conclusions: Folic acid treatment exerts distinct epigenetic and gene expression effects in adipocytes of SLE patients, modulated by obesity status. This weight-dependent response, marked by changes in pathways relevant to immune and metabolic function, highlights the need for further investigation into how nutrient-based interventions might support SLE management. From a clinical perspective, this study underscores the potential of targeted nutrient-based interventions to address immunometabolic dysfunctions in SLE patients. Further research could explore folic acid supplementation as a complementary approach to personalized treatment strategies, particularly for patients with obesity. Full article

Background: Epigenetic biomarkers, particularly CpG methylation, are increasingly employed in clinical and forensic settings. However, we still lack a cost-effective, sensitive, medium-scale method for the analysis of hundreds to thousands of user-defined CpGs suitable for minute DNA input amounts (<10 ng). In this study, motivated by promising results in the genetics field, we investigated single-molecule molecular inversion probes (smMIPs) for simultaneous analysis of hundreds of CpGs by using an example set of 514 age-associated CpGs (Zhang model). Methods: First, we developed a novel smMIP design tool to suit bisulfite-converted DNA (Locksmith). Then, to optimize the capture process, we performed single-probe capture for ten selected, representative smMIPs. Based on this pilot, the full smMIP panel was tested under varying capture conditions, including hybridization and elongation temperature, smMIP and template DNA amounts, dNTP concentration and elongation time. Results: Overall, we found that the capture efficiency was highly probe-(and hence, sequence-) dependent, with a heterogeneous coverage distribution across CpGs higher than the 1000-fold range. Considering CpGs with at least 20X coverage, we yielded robust methylation detection with levels comparable to those obtained from the gold standard EPIC microarray analysis (Pearsons’s r: 0.96). Conclusions: The observed low specificity and uniformity indicate that smMIPs in their current form are not compatible with the lowered complexity of bisulfite-converted DNA. Full article

Asthma, a chronic respiratory condition characterized by airway inflammation, affects millions of individuals worldwide. Challenges remain in asthma prediction and diagnosis from its complex etiology involving genetic and environmental factors. Here, we investigated the relationship between genome-wide DNA methylation and genetic risk for asthma quantified via polygenic scores in two cohorts from the Netherlands Twin Register; one enriched with asthmatic families measured on the Illumina EPIC array (n = 526) and a general population cohort measured on the Illumina HM450K array (n = 2680). We performed epigenome-wide association studies of asthma polygenic scores in each cohort with results combined through meta-analysis (total samples = 3206). The EWAS meta-analysis identified 63 significantly associated CpGs, (following Bonferroni correction, α = 0.05/358,316). An investigation of previous mQTL associations identified 48 mQTL associations between 24 unique CpGs and 48 SNPs, of which two SNPs have previous associations with asthma. Enrichment analysis using the 63 significant CpGs highlighted previous associations with ancestry, smoking, and air pollution. A dizygotic twin within-pair analysis of the 63 CpGs revealed similar directional effects between the two cohorts in 33 of the 63 CpGs. These findings further characterize the intricate relationship between DNA methylation and genetics relative to asthma. Full article

Background/Objectives: The DNA methylation of neonatal cord blood can be used to accurately estimate gestational age. This is known as epigenetic gestational age. The greater the difference between epigenetic and chronological gestational age, the greater the association with an inappropriate perinatal fetal environment and development. Maternal vitamin D deficiency is common in Japan. The aim of this study was to investigate the associations between maternal serum vitamin D levels and epigenetic gestational age acceleration at birth in Japan. Methods: The data were obtained from the hospital-based birth cohort study conducted at the National Center for Child Health and Development in Tokyo, Japan. Maternal blood was collected in the second trimester to measure the serum vitamin D concentration. Cord blood was collected at birth to measure serum vitamin D and to extract DNA. DNA methylation was assessed using an Illumina methylation EPIC array. Epigenetic gestational age was calculated using the “methylclock” R package. Linear regression analysis was performed to see associations. Results: Maternal serum vitamin D levels in the second trimester were negatively associated with epigenetic gestational age acceleration at birth when calculated by Bohlin’s method (regression coefficient [95% CI]: −0.022 [−0.039, −0.005], n = 157), which was still significant after considering infants’ sex (−0.022 [−0.039, −0.005]). Cord blood serum vitamin D levels were not associated with epigenetic age acceleration. Maternal age at delivery and birth height were associated in positive and negative ways with epigenetic gestational age acceleration, respectively (0.048 [0.012, 0.085] and −0.075 [−0.146, −0.003]). Conclusions: Maternal vitamin D deficiency was related to an infant’s epigenetic gestational age acceleration at birth. These findings suggest that the association between fetal development and maternal vitamin D levels may involve the fetal epigenetic regulation of the fetus. Full article

Background/Objectives: Oral cancers in patients with proliferative verrucous leukoplakia (PVL-OSCC) exhibit different clinical and prognostic outcomes from those seen in conventional oral squamous cell carcinomas (cOSSCs). The aim of the present study is to compare the genome-wide DNA methylation signatures in fresh frozen tissues between oral squamous cell carcinomas in patients with PVL and cOSCC using the Illumina Infinium MethylationEPIC BeadChip. Methods: This case–control study was carried out at the Stomatology and Maxillofacial Surgery Department of the General University Hospital of Valencia. For the epigenomic study, unsupervised exploratory bioinformatic analyses were performed using principal component and heatmap analysis. Supervised differential methylation analyses were conducted using a rank-based regression model and a penalized logistic regression model to identify potential prognostic biomarkers. Results: The unsupervised analyses of the global methylation profiles did not allow us to differentiate between the distinct oral cancer groups. However, the two supervised analyses confirmed the existence of two oral carcinoma phenotypes. We identified 21 differentially methylated CpGs corresponding to 14 genes. Among them, three CpGs had not been previously assigned to any known gene, and the remaining were associated with genes unrelated to oral cancer. The AGL, WRB, and ARL15 genes were identified as potential prognostic biomarkers. Conclusions: This study emphasizes the significant role of epigenetic dysregulation in OSCC, particularly in cases preceded by PVL. We have provided data on differential methylation genes that could be involved in the molecular carcinogenesis of PVL-OSCC. Full article

Background/Objectives: The pathophysiology of cancer anorexia is multifactorial and unclear. Transcriptomic analysis from PBMCs RNA showed diverse patterns of gene expression pathways in anorexic cancer patients. We assessed whether the different transcriptomic signatures are modulated by DNA methylation in lung cancer patients presenting with poor appetite. Methods: Lung cancer patients and controls were enrolled, and anorexia was assessed by the FAACT-score questionnaire. Genome-wide DNA methylation was determined by Human Infinium MethylationEPIC BeadChip Kit. Data from genome-wide methylation analysis were merged with those from gene expression analysis, previously obtained by RNA sequencing (NGS). Four groups of genes were identified for each comparison: hypermethylated repressed, hypermethylated induced, hypomethylated repressed, and hypomethylated induced. Results: Cancer patients (n = 16) showed 382 differentially methylated genes when compared with controls (n = 8). Anorexic patients (n = 8) presented 586 hypomethylated and 174 hypermethylated genes compared with controls. In anorexic patients vs. non-anorexic (n = 8), 211 genes were identified as hypomethylated and 90 hypermethylated. When microarray methylation data were merged with transcriptomic data by RNA sequencing, we observed significant differences in anorexic patients vs. controls; a total of 42 genes resulted as hypomethylated and induced, 5 hypermethylated repressed, 10 hypermethylated induced, and 15 hypomethylated repressed. The CG sites analyzed by targeted bisulfite NGS in four genes of interest (FLNA, PGRMC1, GNL3L, and FHL1) resulting as hypomethylated in anorexic vs. controls allowed the validation of the data obtained from DNA methylation. Interestingly, the four genes resulted as hypomethylated in anorexic patients vs. non-anorexic patients and vs. controls (p < 0.0001). Conclusions: Our data support that methylation is implicated in cancer-associated anorexia and nutritional derangements among lung cancer patients. Full article