Methylated CpG Sites Research Articles

Changes in Gut Microbial Composition and DNA Methylation in Obese Patients with NAFLD After Bariatric Surgery

This study investigates the effects of bariatric surgery on non-alcoholic fatty liver disease (NAFLD) by examining the interplay between gut microbiota, epigenetics, and metabolic health. A cohort of 22 patients undergoing sleeve gastrectomy (SG) was analyzed for changes in gut microbial composition and DNA methylation profiles before and six months after surgery. Correlations between gut microbial abundance and clinical markers at baseline revealed that certain genera were associated with worse metabolic health and liver markers. Following SG, significant improvements were observed in the clinical, anthropometric, and biochemical parameters of the NAFLD patients. Although alpha-diversity indices (i.e., Chao1, Simpson, Shannon) did not show significant changes, beta-diversity analysis revealed a slight shift in microbial composition (PERMANOVA, p = 0.036). Differential abundance analysis identified significant changes in specific bacterial taxa, including an increase in beneficial Lactobacillus species such as Lactobacillus crispatus and Lactobacillus iners and a decrease in harmful taxa like Erysipelotrichia. Additionally, DNA methylation analysis revealed 609 significant differentially methylated CpG sites between the baseline values and six months post-surgery, with notable enrichment in genes related to the autophagy pathway, such as IRS4 and ATG4B. The results highlight the individualized responses to bariatric surgery and underscore the potential for personalized treatment strategies. In conclusion, integrating gut microbiota and epigenetic factors into NAFLD management could enhance treatment outcomes, suggesting that future research should explore microbiome-targeted therapies and long-term follow-ups on liver health post-surgery.

A DNA Polymerase Variant Senses the Epigenetic Marker 5-Methylcytosine by Increased Misincorporation.

Dysregulation of DNA methylation is associated with human disease, particularly cancer, and the assessment of aberrant methylation patterns holds great promise for clinical diagnostics. However, DNA polymerases do not effectively discriminate between processing 5-methylcytosine (5 mC) and unmethylated cytosine, resulting in the silencing of methylation information during amplification or sequencing. As a result, current detection methods require multi-step DNA conversion treatments or careful analysis of sequencing data to decipher individual 5 mC bases. To overcome these challenges, we propose a novel DNA polymerase-mediated 5 mC detection approach. Here, we describe the engineering of a thermostable DNA polymerase variant derived from Thermus aquaticus with altered fidelity towards 5 mC. Using a screening-based evolutionary approach, we have identified a DNA polymerase that exhibits increased misincorporation towards 5 mC during DNA synthesis. This DNA polymerase generates mutation signatures at methylated CpG sites, allowing direct detection of 5 mC by reading an increased error rate after sequencing without prior treatment of the sample DNA.

Decoding the Methylation Patterns of ABC Transporters in Colorectal Cancer

Colorectal cancer (CRC) is a significant global health concern, posing a major threat to morbidity and mortality rates. The molecular mechanisms that drive CRC, particularly the DNA methylation patterns in ATP-binding cassette (ABC) genes, have attracted considerable attention because of their potential roles in CRC drug resistance, initiation, and progression. This study aimed to investigate the methylation patterns of ABC genes in CRC using a high-throughput microarray technology. Microarray methylation data from CRC and adjacent normal tissues were subjected to preprocessing, differential methylation analysis, and correlation analysis using the MethSurv tool for a detailed study of methylation patterns. The study revealed global hypomethylation of 43 ABC transporters and two pseudogenes in CRC compared to normal tissues. Receiver operating characteristic curve analysis identified 369 CpG sites as potential biomarkers for CRC diagnosis, with area under the curve values ranging from acceptable to outstanding. Survival analysis demonstrated the correlation between DNA methylation of individual CpG sites and patient survival probability in colon and rectal adenocarcinoma datasets from The Cancer Genome Atlas. The study provides insights into the epigenetic landscape of ABC genes in CRC, highlighting their potential roles in drug resistance, disease initiation, and progression. The findings offer opportunities for developing innovative therapeutic approaches and improving patient outcomes in the fight against CRC. Further research is needed to validate these results and investigate the functional implications of ABC gene methylation in CRC pathogenesis and treatment response.

Effects of Differentially Methylated CpG Sites in Enhancer and Promoter Regions on the Chromatin Structures of Target LncRNAs in Breast Cancer.

Aberrant DNA methylation plays a crucial role in breast cancer progression by regulating gene expression. However, the regulatory pattern of DNA methylation in long noncoding RNAs (lncRNAs) for breast cancer remains unclear. In this study, we integrated gene expression, DNA methylation, and clinical data from breast cancer patients included in The Cancer Genome Atlas (TCGA) database. We examined DNA methylation distribution across various lncRNA categories, revealing distinct methylation characteristics. Through genome-wide correlation analysis, we identified the CpG sites located in lncRNAs and the distally associated CpG sites of lncRNAs. Functional genome enrichment analysis, conducted through the integration of ENCODE ChIP-seq data, revealed that differentially methylated CpG sites (DMCs) in lncRNAs were mostly located in promoter regions, while distally associated DMCs primarily acted on enhancer regions. By integrating Hi-C data, we found that DMCs in enhancer and promoter regions were closely associated with the changes in three-dimensional chromatin structures by affecting the formation of enhancer-promoter loops. Furthermore, through Cox regression analysis and three machine learning models, we identified 11 key methylation-driven lncRNAs (DIO3OS, ELOVL2-AS1, MIAT, LINC00536, C9orf163, AC105398.1, LINC02178, MILIP, HID1-AS1, KCNH1-IT1, and TMEM220-AS1) that were associated with the survival of breast cancer patients and constructed a prognostic risk scoring model, which demonstrated strong prognostic performance. These findings enhance our understanding of DNA methylation's role in lncRNA regulation in breast cancer and provide potential biomarkers for diagnosis.

Epigenetic Age Estimation by Detecting DNA Methylation Status in Buccal Swabs.

The analysis of DNA methylation (DNAm) levels at specific CpG sites represents one of the most promising molecular techniques for estimating an individual's age. To date, a considerable number of studies have reported the development of age prediction models on the basis of DNAm in body fluids, with only a few utilizing buccal swabs. The objective of this study was to identify age-dependent methylation CpG sites in three different genes (HOXC4, TRIM59, and ELOVL2) in buccal swab samples from the Chinese Han population. A total of 461 buccal swabs, with an age range of 0.4-80.8 years, were divided into a training set (n=325) and a validation set (n=136). Samples were analyzed by pyrosequencing in order to identify age-related genes with correlation coefficient. A random forest regression model was ultimately proposed, including eight CpGs in three genes, with a mean absolute error (MAE) of 2.119 years. The model performs independent validation set with an MAE of 4.391 years. Our findings illustrate that buccal swabs present a suitable alternative to biological traces for age prediction based on DNAm pattern using pyrosequencing and random forest regression, offering the additional advantage of being collected noninvasively.

Methylation of Selected CpG-Sites of the Gene CSF1 as a Factor in Regulation of Its Expression and a Marker of Human Biological Aging

—Age-associated transformation of methylation patterns is considered to be an important predictor of human biological age. Changes in the level of CpG-dinucleotide methylation contribute to a shift in the function of a number of genes, including those associated with the functioning of the immune system. One such gene is CSF1. The protein product of this gene is associated with inflammatory aging, making it an important biomarker of age-related diseases. We studied the methylation profile of the promoter-associated CpG island of the CSF1 gene by MALDI-TOF mass spectrometry. Dependences between the character of CpG-site methylation within the investigated regions and the relative level of the gene mRNA and its protein product in people of different age groups were sought. For two CpG sites, a high level of correlation with the studied parameters is shown. A search for the landing sites of transcription factor binding sites associated with gene transcription showed that these CpG dinucleotides are part of motifs for the NFI family transcription factors and the EGR1 factor. We hypothesize that these CpG sites play an important role in the regulation of CSF1 gene expression.

Mechanistic model for epigenetic maintenance by methyl-CpG-binding domain proteins.

DNA methylation is a fundamental element of epigenetic regulation that is governed by the MBD protein superfamily, a group of "readers" that share a highly conserved methyl-CpG-binding domain (MBD) and mediate chromatin remodeler recruitment, transcription regulation, and coordination of DNA and histone modification. Previous work has characterized the binding affinity and sequence selectivity of MBD-containing proteins toward palindromes of 5-methylcytosine (5mC) containing 5mCpG dinucleotides, often referred to as single symmetrically methylated CpG sites. However, little is known about how MBD binding is influenced by the prototypical local clustering of methylated CpG sites and the presence of DNA structural motifs encountered, e.g., during DNA replication and transcription. Here, we use Single-Molecule Kinetics through Equilibrium Poisson Sampling (SiMKEPS) to measure precise binding and dissociation rate constants of the MBD of human protein MBD1 to DNAs with varying patterns of multiple methylated CpG sites and diverse structural motifs. MBD binding is promoted by two major properties of its DNA substrates: 1) tandem (consecutive) symmetrically methylated CpG sites in double-stranded DNA and secondary structures in single-stranded DNA; and 2) DNA forks. Based on our findings, we propose a mechanistic model for how MBD proteins contribute to epigenetic boundary maintenance between transcriptionally silenced and active genome regions.

Prenatal cannabis exposure is associated with alterations in offspring DNA methylation at genes involved in neurodevelopment, across the life course.

Prenatal cannabis exposure (PCE) is of increasing concern globally, due to the potential impact on offspring neurodevelopment, and its association with childhood and adolescent brain development and cognitive function. However, there is currently a lack of research addressing the molecular impact of PCE, that may help to clarify the association between PCE and neurodevelopment. To address this knowledge gap, here we present epigenome-wide association study data across multiple time points, examining the effect of PCE and co-exposure with tobacco using two longitudinal studies, the Avon Longitudinal Study of Parents and Children (ALSPAC) and the Christchurch Health and Development Study (CHDS) at birth (0 y), 7 y and 15-17 y (ALSPAC), and ~27 y (CHDS). Our findings reveal genome-wide significant DNA methylation differences in offspring at 0 y, 7 y, 15-17 y, and 27 y associated with PCE alone, and co-exposure with tobacco. Importantly, we identified significantly differentially methylated CpG sites within the genes LZTS2, NPSR1, NT5E, CRIP2, DOCK8, COQ5, and LRP5 that are shared between different time points throughout development in offspring. Notably, functional pathway analysis showed enrichment for differential DNA methylation in neurodevelopment, neurotransmission, and neuronal structure pathways, and this was consistent across all timepoints in both cohorts. Given the increasing volume of epidemiological evidence that suggests a link between PCE and adverse neurodevelopmental outcomes in exposed offspring, this work highlights the need for further investigation into PCE, particularly in larger cohorts.

DNAm scores for serum GDF15 and NT-proBNP levels associate with a range of traits affecting the body and brain

BackgroundPlasma growth differentiation factor 15 (GDF15) and N‐terminal proB‐type natriuretic peptide (NT‐proBNP) are cardiovascular biomarkers that associate with a range of diseases. Epigenetic scores (EpiScores) for GDF15 and NT-proBNP may provide new routes for risk stratification.ResultsIn the Generation Scotland cohort (N ≥ 16,963), GDF15 levels were associated with incident dementia, ischaemic stroke and type 2 diabetes, whereas NT-proBNP levels were associated with incident ischaemic heart disease, ischaemic stroke and type 2 diabetes (all PFDR < 0.05). Bayesian epigenome-wide association studies (EWAS) identified 12 and 4 DNA methylation (DNAm) CpG sites associated (Posterior Inclusion Probability [PIP] > 95%) with levels of GDF15 and NT-proBNP, respectively. EpiScores for GDF15 and NT-proBNP were trained in a subset of the population. The GDF15 EpiScore replicated protein associations with incident dementia, type 2 diabetes and ischaemic stroke in the Generation Scotland test set (hazard ratios (HR) range 1.36–1.41, PFDR < 0.05). The EpiScore for NT-proBNP replicated the protein association with type 2 diabetes, but failed to replicate an association with ischaemic stroke. EpiScores explained comparable variance in protein levels across both the Generation Scotland test set and the external LBC1936 test cohort (R2 range of 5.7–12.2%). In LBC1936, both EpiScores were associated with indicators of poorer brain health. Neither EpiScore was associated with incident dementia in the LBC1936 population.ConclusionsEpiScores for serum levels of GDF15 and Nt-proBNP associate with body and brain health traits. These EpiScores are provided as potential tools for disease risk stratification.

The association between cumulative exposure to PM2.5 and DNA methylation measured using methyl-capture sequencing among COPD patients

BackgroundParticulate matter with a diameter of < 2.5 μm (PM2.5) influences gene regulation via DNA methylation; however, its precise mechanism of action remains unclear. Thus, this study aimed to examine the connection between personal PM2.5 exposure and DNA methylation in CpG islands as well as explore the associated gene pathways.MethodsA total of 95 male patients with chronic obstructive pulmonary disease (COPD) were enrolled in this study. PM2.5 concentrations were measured for 12 months, with individual exposure recorded for 24 h every 3 months. Mean indoor and estimated individual PM2.5 exposure levels were calculated for short-term (7 days), mid-term (35 days), and long-term (90 days). DNA methylation analysis was performed on the blood samples, which, after PCR amplification and hybridization, were finally sequenced using an Illumina NovaSeq 6000 system. Correlation between PM2.5 exposure and CpG methylation sites was confirmed via a mixed-effects model. Functional enrichment analysis was performed on unique CpG methylation sites associated with PM2.5 exposure to identify the relevant biological functions or pathways.ResultsThe number of CpG sites showing differential methylation was 36, 381, and 182 for the short-, mid-, and long-term indoor models, respectively, and 3, 98, and 28 for the short-, mid-, and long-term estimated exposure models, respectively. The representative genes were TMTC2 (p = 1.63 × 10-3, R2 = 0.656), GLRX3 (p = 1.46 × 10-3, R2 = 0.623), DCAF15 (p = 2.43 × 10-4, R2 = 0.623), CNOT6L (p = 1.46 × 10-4, R2 = 0.609), BSN (p = 2.21 × 10-5, R2 = 0.606), and SENP6 (p = 1.59 × 10-4, R2 = 0.604). Functional enrichment analysis demonstrated that the related genes were mostly associated with pathways related to synaptic transmission in neurodegenerative diseases and cancer.ConclusionA significant association was observed between PM2.5 exposure and DNA methylation upon short-term exposure, and the extent of DNA methylation was the highest upon mid-term exposure. Additionally, various pathways related to neurodegenerative diseases and cancer were associated with patients with COPD.ClinicalTrials.gov identifierNCT04878367.

Causal association between epilepsy and its DNA methylation profile and atrial fibrillation

BackgroundThe "epileptic heart" concept is emerging, but the causal relationship between epilepsy and atrial fibrillation (AF) remains unclarified. ObjectiveThis study explores the genetic correlations and bidirectional causality between various epilepsy phenotypes and AF. MethodsGenome-wide association study (GWAS) statistics for 10 epilepsy subtypes (29,944 cases, 52,538 controls) and AF (60,620 cases, 970,216 controls) were sourced from the International League Against Epilepsy (ILAE) and HGRI-EBI Catalog-GWAS, respectively. Linkage disequilibrium score regression (LDSC) and genome-wide Mendelian Randomization (MR) evaluated genetic correlations and bidirectional causal relationships. Epilepsy-related DNA methylation data (N= ∼800) from EWAS catalog were analyzed to identify causal CpG sites influencing AF risk through epigenetic MR. ResultsLDSC revealed significant genetic correlations between four epilepsy subtypes and AF (rg from 0.116 to 0.241). Forward MR suggested a significant causal effect of focal epilepsy with hippocampal sclerosis (FE with HS) on AF risk (IVW and MR-PRESSO: OR = 1.046, P ≤ 0.004), with results robust against heterogeneity, horizontal pleiotropy, and outliers. Epigenetic MR indicated that lower methylation at cg06222062 (OR = 0.994, P = 3.16E-04) mapped to PLA2G5 and cg08461451 mapped to SPPL2B gene (OR = 0.954, P = 1.19E-03), and higher cg10541930 in the C10orf143 promoter (OR = 1.043, P = 4.18E-22) increases AF risk. Sensitivity analyses affirmed no pleiotropic bias. ConclusionFE with HS significantly increases AF risk, highlighting the natural neural-cardiac connection and the need for cardiac monitoring in epilepsy patients. Specific methylated CpG sites may serve as biomarkers and preventive targets for AF susceptibility.

Relationships between serotonin 1A receptor DNA methylation, self-reported history of childhood abuse and gray matter volume in major depression

BackgroundEarly life adversity is a risk factor for psychopathology and is associated with epigenetic alterations in the 5-HT1A receptor gene promoter. The 5-HT1A receptor mediates neurotrophic effects, which could affect brain structure and function. We examined relationships between self-reported early childhood abuse, 5-HT1A receptor promoter DNA methylation, and gray matter volume (GMV) in Major Depressive Disorder (MDD). MethodsPeripheral DNA methylation of 5-HT1A receptor promoter CpG sites −681 and −1007 was assayed in 50 individuals with MDD, including 18 with a history of childhood abuse. T1-weighted structural magnetic resonance imaging (MRI) was performed. Voxel-based morphometry (VBM) was quantified in amygdala, hippocampus, insula, occipital lobe, orbitofrontal cortex, temporal lobe, parietal lobe, and at the voxel level. ResultsNo relationship was observed between DNA methylation and history of childhood abuse. We observed regional heterogeneity comparing −681 CpG site methylation and GMV (p = 0.014), with a positive relationship to GMV in orbitofrontal cortex (p = 0.035). Childhood abuse history was associated with higher GMV considering all ROIs simultaneously (p < 0.01). In whole-brain analyses, childhood abuse history was positively correlated with GMV in multiple clusters, including insula and orbitofrontal cortex (pFWE = 0.005), and negatively in intracalcarine cortex (pFWE = 0.001). LimitationsSmall sample size, childhood trauma assessment instrument used, and assay of peripheral, rather than CNS, methylation. ConclusionsThese cross-sectional findings support hypotheses of 5-HT1A receptor-related neurotrophic effects, and of increased regional GMV as a potential regulatory mechanism in the setting of childhood abuse. Orbitofrontal cortex was uniquely associated with both childhood abuse history and 5-HT1A receptor methylation.

DNA Methylation of PXDN Is Associated with Early-Life Adversity in Adult Mental Disorders.

Early-life adversity (ELA) is characterized by exposure to traumatic events during early periods of life, particularly involving emotional, sexual and/or physical adversities during childhood. Mental disorders are strongly influenced by environmental and lifestyle-related risk factors including ELA. However, the molecular link between ELA and the risk of an adult mental disorder is still not fully understood. Evidence is emerging that long-lasting changes in the epigenetic processes regulating gene expression, such as DNA methylation, play an important role in the biological mechanisms linking ELA and mental disorders. Based on a recent study, we analyzed the DNA methylation of a specific CpG site within the gene PXDN-cg10888111-in blood in the context of ELA across a set of psychiatric disorders, namely Borderline Personality Disorder (BPD), Major Depressive Disorder (MDD) and Social Anxiety Disorder (SAD), and its potential contribution to their pathogenesis. We found significant hypermethylation in mentally ill patients with high levels of ELA compared to patients with low levels of ELA, whereas cg10888111 methylation in healthy control individuals was not affected by ELA. Further investigations revealed that this effect was driven by the MDD cohort. Providing a direct comparison of cg10888111 DNA methylation in blood in the context of ELA across three mental disorders, our results indicate the role of PXDN regulation in the response to ELA in the pathogenesis of mental disorders, especially MDD. Further studies will be needed to validate these results and decipher the corresponding biological network that is involved in the transmission of ELA to an adult mental disorder in general.

Epigenome-wide differential methylation and differential variability as predictors of high-grade cervical intraepithelial neoplasia (cin2+).

CpG site methylation patterns have potential to improve differentiation of high-grade screening-detected cervical abnormalities. We assessed CpG differential methylation (DM) and differential variability (DV) in high-grade (CIN2+) vs. low-grade (≤CIN1) lesions. In ≤CIN1 (n=117) and CIN2+ (n=31) samples, cervical sample DNA underwent testing with Illumina HumanMethylation arrays. We assessed DM and DV of CpG methylation M values among nine cervical cancer-associated genes. We fit CpG-specific linear models and estimated empirical Bayes standard errors and false discovery rates (FDR). An exploratory epigenome-wide association study (EWAS) aimed to detect novel DM and DV CpGs (FDR<0.05) and Gene Ontology (GO) term enrichment. Compared to ≤CIN1, CIN2+ exhibited greater methylation at CCNA1 Cluster 1 (M value difference 0.24; 95% CI 0.04, 0.43) and RARB Cluster 2 (0.16; 95% CI 0.05, 0.28), and lower methylation at CDH1 Cluster 1 (-0.15; 95% CI -0.26, -0.04). CIN2+ exhibited lower variability at CDH1 Cluster 2 (variation difference -0.24; 95% CI -0.41, -0.05) and FHIT Cluster 1 (-0.30; 95% CI -0.50, -0.09). EWAS detected 3,534 DM and 270 DV CpGs. Forty-four GO terms were enriched with DM CpGs related to transcriptional, structural, developmental, and neuronal processes. Methylation patterns may help triage screening-detected cervical abnormalities and inform US screening algorithms.

DNA Methylation Effects on Van der Woude Syndrome Phenotypic Variability.

Van der Woude Syndrome (VWS) presents with combinations of lip pits (LP) and cleft lip and/or cleft palate (CL/P, CPO). VWS phenotypic heterogeneity even amongst relatives, suggests that epigenetic factors may act as modifiers. IRF6, causal for 70% of VWS cases, and TP63 interact in a regulatory loop coordinating epithelial proliferation and differentiation in palatogenesis. We hypothesize that differential DNA methylation within IRF6 and TP63 regulatory regions underlie VWS phenotypic discordance. DNA methylation of CpG sites in IRF6 and TP63 promoters and in an IRF6 enhancer element was compared amongst blood or saliva DNA samples of 78 unrelated cases. Analyses were done separately for blood and saliva, within each sex and in combination, and to address cleft type (CL/P ± LP vs. CPO ± LP) and phenotypic severity (any cleft + LP vs. any cleft only). For cleft type, blood samples showed higher IRF6 and TP63 promoter methylation on males with CPO ± LP compared to CL/P ± LP and on individuals with CPO ± LP compared to those with CL/P ± LP, respectively. Saliva samples showed higher IRF6 enhancer methylation on individuals with CPO ± LP compared to CL/P ± LP and contrary to above, lower TP63 promoter methylation on CPO ± LP compared to CL/P ± LP. For phenotypic severity, blood samples showed no differences; however, saliva samples showed higher IRF6 promoter methylation in individuals with any cleft + LP compared to those without lip pits. We observed differential methylation in IRF6 and TP63 regulatory regions associated with cleft type and phenotypic severity, indicating that epigenetic changes in IRF6 and TP63 can contribute to phenotypic heterogeneity in VWS.

Blood Leukocyte DNA Methylation Markers of Periodontal Disease and Risk of Lung Cancer in a Case-Control Study Nested in the CLUE II Cohort.

Periodontal disease and DNA methylation markers have separately been associated with lung cancer risk. Examining methylation levels at genomic regions previously linked to periodontal disease may provide insights on the link between periodontal disease and lung cancer. In a nested case-control study drawn from the CLUE II cohort, we measured DNA methylation levels in 208 lung cancer cases and 208 controls. We examined the association between 37 DNA-methylated 5'-C-phosphate-G-3' (CpG) sites at three genomic regions, homeobox 4 (HOXA4), zinc finger protein (ZFP57), and a long noncoding RNA gene located in Chr10 (ENSG00000231601), and lung cancer risk. Statistically significant associations with lung cancer risk were observed for all 14 CpG sites from HOXA4 (OR ranging 1.41-1.62 for 1 SD increase in the DNA methylation level, especially within 15 years) and 1 CpG site on gene ENSG00000231601 (OR = 1.34 for 1 SD increase in the DNA methylation level). Although CpG sites on gene ZFP57 were not associated with lung cancer risk overall, statistically significant inverse associations were noted for six CpG sites when restricting follow-up to 15 years (OR = 0.73-0.77 for 1 SD increase in the DNA methylation level). Key methylation levels associated with periodontal disease are also associated with lung cancer risk. For both HOXA4 and ZFP57, the associations were stronger within 15 years of follow-up, which suggest that, if causal, the impact of methylation is acting late in the natural history of lung cancer. Identifying biological pathways that link periodontal disease and lung cancer could provide new opportunities for lung cancer detection and prevention.

Detection of differentially methylated CpGs between tumour and adjacent benign cells in diagnostic prostate cancer samples

Differentially methylated CpG sites (dmCpGs) that distinguish prostate tumour from adjacent benign tissue could aid in the diagnosis and prognosis of prostate cancer. Previously, the identification of such dmCpGs has only been undertaken in radical prostatectomy (RP) samples and not primary diagnostic tumour samples (needle biopsy or transurethral resection of the prostate). We interrogated an Australian dataset comprising 125 tumour and 43 adjacent histologically benign diagnostic tissue samples, including 41 paired samples, using the Infinium Human Methylation450 BeadChip. Regression analyses of paired tumour and adjacent benign samples identified 2,386 significant dmCpGs (Bonferroni p < 0.01; delta-β ≥ 40%), with LASSO regression selecting 16 dmCpGs that distinguished tumour samples in the full Australian diagnostic dataset (AUC = 0.99). Results were validated in independent North American (npaired = 19; AUC = 0.87) and The Cancer Genome Atlas (TCGA; npaired = 50; AUC = 0.94) RP datasets. Two of the 16 dmCpGs were in genes that were significantly down-regulated in Australian tumour samples (Bonferroni p < 0.01; GSTM2 and PRKCB). Ten additional dmCpGs distinguished low (n = 34) and high Gleason (n = 88) score tumours in the diagnostic Australian dataset (AUC = 0.95), but these performed poorly when applied to the RP datasets (North American: AUC = 0.66; TCGA: AUC = 0.62). The DNA methylation marks identified here could augment and improve current diagnostic tests and/or form the basis of future prognostic tests.

CmirC update 2024: a multi-omics database for clustered miRNAs

Clustered miRNAs consist of two or more miRNAs transcribed together and may coordinately regulate gene expression. Differential expression of clustered miRNAs is found to be controlled by crosstalk of genetic or epigenetic mechanisms. It has been demonstrated that clustered miRNA expression patterns greatly impact cancer cell progression. With the CmirC initiative, we initially developed a comprehensive database to identify copy number variation (CNV) driven clustered miRNAs in cancer. Now, we extended the analysis and identified three miRNAs, mir-96, mir-183, and mir-21, were found to be significantly upregulated in 17 cancer types. Further, CmirC is now upgraded to determine the impact of changes in the DNA methylation status at clustered miRNAs by utilizing The Cancer Genomic Atlas (TCGA) cancer datasets. We examined specific methylation datasets from 9,639 samples, pinpointing 215,435 methylation sites and 27,949 CpG islands with miRNA cluster information. The integrated analysis identified 34 clusters exhibiting differentially methylated CpG sites across 14 cancer types. Furthermore, we determined that CpG islands in the promoter region of 20 miRNA clusters could play a regulatory role. Along with ensuring a straightforward and convenient user experience, CmirC has been updated with improved data browsing and analysis functionalities, as well as enabled hyperlinks to literature and miR-cancer databases. The enhanced version of CmirC is anticipated to play an important role in providing information on the regulation of clustered miRNA expression, and their targeted oncogenes and tumor suppressors. The newly updated version of CmirC is available at https://slsdb.manipal.edu/cmirclust/.

The correlation between CpG methylation and gene expression is driven by sequence variants

Gene promoter and enhancer sequences are bound by transcription factors and are depleted of methylated CpG sites (cytosines preceding guanines in DNA). The absence of methylated CpGs in these sequences typically correlates with increased gene expression, indicating a regulatory role for methylation. We used nanopore sequencing to determine haplotype-specific methylation rates of 15.3 million CpG units in 7,179 whole-blood genomes. We identified 189,178 methylation depleted sequences where three or more proximal CpGs were unmethylated on at least one haplotype. A total of 77,789 methylation depleted sequences (~41%) associated with 80,503 cis-acting sequence variants, which we termed allele-specific methylation quantitative trait loci (ASM-QTLs). RNA sequencing of 896 samples from the same blood draws used to perform nanopore sequencing showed that the ASM-QTL, that is, DNA sequence variability, drives most of the correlation found between gene expression and CpG methylation. ASM-QTLs were enriched 40.2-fold (95% confidence interval 32.2, 49.9) among sequence variants associating with hematological traits, demonstrating that ASM-QTLs are important functional units in the noncoding genome.

Molecular subtypes explain lupus epigenomic heterogeneity unveiling new regulatory genetic risk variants

The heterogeneity of systemic lupus erythematosus (SLE) can be explained by epigenetic alterations that disrupt transcriptional programs mediating environmental and genetic risk. This study evaluated the epigenetic contribution to SLE heterogeneity considering molecular and serological subtypes, genetics and transcriptional status, followed by drug target discovery. We performed a stratified epigenome-wide association studies of whole blood DNA methylation from 213 SLE patients and 221 controls. Methylation quantitative trait loci analyses, cytokine and transcription factor activity - epigenetic associations and methylation-expression correlations were conducted. New drug targets were searched for based on differentially methylated genes. In a stratified approach, a total of 974 differential methylation CpG sites with dependency on molecular subtypes and autoantibody profiles were found. Mediation analyses suggested that SLE-associated SNPs in the HLA region exert their risk through DNA methylation changes. Novel genetic variants regulating DNAm in disease or in specific molecular contexts were identified. The epigenetic landscapes showed strong association with transcription factor activity and cytokine levels, conditioned by the molecular context. Epigenetic signals were enriched in known and novel drug targets for SLE. This study reveals possible genetic drivers and consequences of epigenetic variability on SLE heterogeneity and disentangles the DNAm mediation role on SLE genetic risk and novel disease-specific meQTLs. Finally, novel targets for drug development were discovered.