Identification of DNA methylation characteristics associated with metastasis and prognosis in colorectal cancer.

Colorectal cancer (CRC) is prone to metastasis and recurrence after surgery, which is one of the main causes for its poor treatment and prognosis. Therefore, it is essential to identify biomarkers associated with metastasis and recurrence in CRC. DNA methylation has a regulatory role in cancer metastasis, tumor immune microenvironment (TME), and prognosis and may be one of the most valuable biomarkers for predicting CRC metastasis and prognosis. We constructed a diagnostic model and nomogram that can effectively predict CRC metastasis based on the differential methylation CpG sites (DMCs) between metastatic and non-metastatic CRC patients. Then, we identified 17 DMCs associated with progression free survival (PFS) of CRC and constructed a prognostic model. The prognosis model based on 17 DMCs can predict the PFS of CRC with medium to high accuracy. The results of immunohistochemical analysis indicated that the protein expression levels of the genes involved in prognostic DMCs were different between normal and colorectal cancer tissues. According to the results of immune-related analysis, we found that the low-risk patients had better immunotherapy response. In addition, high risk scores were negatively correlated with high tumor mutation burden (TMB) levels, and patients with low TMB levels in the high-risk group had the worst PFS. Our work shows the clinical value of DNA methylation in predicting CRC metastasis and PFS, as well as their correlation with TME, immunotherapy, and TMB, which helps understand the changes of DNA methylation in CRC metastasis and improving the treatment and prognosis of CRC.

Novel genome-wide DNA methylation profiling reveals distinct epigenetic landscape, prognostic model and cellular composition of early-stage lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) has been a leading cause of cancer-related mortality worldwide. Early intervention can significantly improve prognosis. DNA methylation could occur in the early stage of tumor. Comprehensive understanding the epigenetic landscape of early-stage LUAD is crucial in understanding tumorigenesis. METHODS: Enzymatic methyl sequencing (EM-seq) was performed on 23 tumors and paired normal tissue to reveal distinct epigenetic landscape, for compared with The Cancer Genome Atlas (TCGA) 450K methylation microarray data. Then, an integrative analysis was performed combined with TCGA LUAD RNA-seq data to identify significant differential methylated and expressed genes. Subsequently, the prognostic risk model was constructed and cellular composition was analyzed. RESULTS: Methylome analysis of EM-seq comparing tumor and normal tissues identified 25 million cytosine-phosphate-guanine (CpG) sites and 30,187 differentially methylated regions (DMR) with a greater number of untraditional types. EM-seq identified a significantly higher number of CpG sites and DMRs compared to the 450K microarray. By integrating the differentially methylated genes (DMGs) with LUAD-related differentially expressed genes (DEGs) from the TCGA database, we constructed prognostic model based on six differentially methylated-expressed genes (MEGs) and verified our prognostic model in GSE13213 and GSE42127 dataset. Finally, cell deconvolution based on the in-house EM-seq methylation profile was used to estimate cellular composition of early-stage LUAD. CONCLUSIONS: This study firstly delves into novel pattern of epigenomic DNA methylation and provides a multidimensional analysis of the role of DNA methylation revealed by EM-seq in early-stage LUAD, providing distinctive insights into its potential epigenetic mechanisms.

Epigenetics of the non-coding RNA nc886 across blood, adipose tissue and skeletal muscle in offspring exposed to diabetes in pregnancy.

BACKGROUND: Diabetes in pregnancy is associated with increased risk of long-term metabolic disease in the offspring, potentially mediated by in utero epigenetic variation. Previously, we identified multiple differentially methylated single CpG sites in offspring of women with gestational diabetes mellitus (GDM), but whether stretches of differentially methylated regions (DMRs) can also be identified in adolescent GDM offspring is unknown. Here, we investigate which DNA regions in adolescent offspring are differentially methylated in blood by exposure to diabetes in pregnancy. The secondary aim was to characterize the RNA expression of the identified DMR, which contained the nc886 non-coding RNA. METHODS: To identify DMRs, we employed the bump hunter method in samples from young (9-16 yr, n = 92) offspring of women with GDM (O-GDM) and control offspring (n = 94). Validation by pyrosequencing was performed in an adult offspring cohort (age 28-33 years) consisting of O-GDM (n = 82), offspring exposed to maternal type 1 diabetes (O-T1D, n = 67) and control offspring (O-BP, n = 57). RNA-expression was measured using RT-qPCR in subcutaneous adipose tissue and skeletal muscle. RESULTS: One significant DMR represented by 10 CpGs with a bimodal methylation pattern was identified, located in the nc886/VTRNA2-1 non-coding RNA gene. Low methylation status across all CpGs of the nc886 in the young offspring was associated with maternal GDM. While low methylation degree in adult offspring in blood, adipose tissue, and skeletal muscle was not associated with maternal GDM, adipose tissue nc886 expression was increased in O-GDM compared to O-BP, but not in O-T1D. In addition, adipose tissue nc886 expression levels were positively associated with maternal pre-pregnancy BMI (p = 0.006), but not with the offspring's own adiposity. CONCLUSIONS: Our results highlight that nc886 is a metastable epiallele, whose methylation in young offspring is negatively correlated with maternal obesity and GDM status. The physiological effect of nc886 may be more important in adipose tissue than in skeletal muscle. Further research should aim to investigate how nc886 regulation in adipose tissue by exposure to GDM may contribute to development of metabolic disease.

Hyper-physiologic mechanical cues, as an osteoarthritis disease-relevant environmental perturbation, cause a critical shift in set points of methylation at transcriptionally active CpG sites in neo-cartilage organoids.

BACKGROUND: Osteoarthritis (OA) is a complex, age-related multifactorial degenerative disease of diarthrodial joints marked by impaired mobility, joint stiffness, pain, and a significant decrease in quality of life. Among other risk factors, such as genetics and age, hyper-physiological mechanical cues are known to play a critical role in the onset and progression of the disease (Guilak in Best Pract Res Clin Rheumatol 25:815-823, 2011). It has been shown that post-mitotic cells, such as articular chondrocytes, heavily rely on methylation at CpG sites to adapt to environmental cues and maintain phenotypic plasticity. However, these long-lasting adaptations may eventually have a negative impact on cellular performance. We hypothesize that hyper-physiologic mechanical loading leads to the accumulation of altered epigenetic markers in articular chondrocytes, resulting in a loss of the tightly regulated balance of gene expression that leads to a dysregulated state characteristic of the OA disease state. RESULTS: We showed that hyper-physiological loading evokes consistent changes in CpGs associated with expression changes (ML-tCpGs) in ITGA5, CAV1, and CD44, among other genes, which together act in pathways such as anatomical structure morphogenesis (GO:0009653) and response to wound healing (GO:0042060). Moreover, by comparing the ML-tCpGs and their associated pathways to tCpGs in OA pathophysiology (OA-tCpGs), we observed a modest but particular interconnected overlap with notable genes such as CD44 and ITGA5. These genes could indeed represent lasting detrimental changes to the phenotypic state of chondrocytes due to mechanical perturbations that occurred earlier in life. The latter is further suggested by the association between methylation levels of ML-tCpGs mapped to CD44 and OA severity. CONCLUSION: Our findings confirm that hyper-physiological mechanical cues evoke changes to the methylome-wide landscape of chondrocytes, concomitant with detrimental changes in positional gene expression levels (ML-tCpGs). Since CAV1, ITGA5, and CD44 are subject to such changes and are central and overlapping with OA-tCpGs of primary chondrocytes, we propose that accumulation of hyper-physiological mechanical cues can evoke long-lasting, detrimental changes in set points of gene expression that influence the phenotypic healthy state of chondrocytes. Future studies are necessary to confirm this hypothesis.

The association between neighborhood deprivation and DNA methylation in an autopsy cohort.

Previous research has found that living in a disadvantaged neighborhood is associated with poor health outcomes. Living in disadvantaged neighborhoods may alter inflammation and immune response in the body, which could be reflected in epigenetic mechanisms such as DNA methylation (DNAm). We used robust linear regression models to conduct an epigenome-wide association study examining the association between neighborhood deprivation (Area Deprivation Index; ADI), and DNAm in brain tissue from 159 donors enrolled in the Emory Goizueta Alzheimer's Disease Research Center (Georgia, USA). We found one CpG site (cg26514961, gene PLXNC1) significantly associated with ADI after controlling for covariates and multiple testing (p-value=5.0e-8). Effect modification by APOE ε4 was statistically significant for the top ten CpG sites from the EWAS of ADI, indicating that the observed associations between ADI and DNAm were mainly driven by donors who carried at least one APOE ε4 allele. Four of the top ten CpG sites showed a significant concordance between brain tissue and tissues that are easily accessible in living individuals (blood, buccal cells, saliva), including DNAm in cg26514961 (PLXNC1). Our study identified one CpG site (cg26514961, PLXNC1 gene) that was significantly associated with neighborhood deprivation in brain tissue. PLXNC1 is related to immune response, which may be one biological pathway how neighborhood conditions affect health. The concordance between brain and other tissues for our top CpG sites could make them potential candidates for biomarkers in living individuals.

Evaluating the link between DIO3-FA27 promoter methylation, biochemical indices, and heart failure progression.

BACKGROUND: Heart failure (HF) is a disease that poses a serious threat to individual health, and DNA methylation is an important mechanism in epigenetics, and its role in the occurrence and development of the disease has attracted more and more attention. The aim of this study was to evaluate the link between iodothyronine deiodinase 3 promoter region fragment FA27 (DIO3-FA27) methylation levels, biochemical indices, and HF. RESULTS: The methylation levels of DIO3-FA27_CpG_11.12 and DIO3-FA27_CpG_23.24 significantly differed in HF patients with different degrees. Multivariate logistic regression analysis indicated that the relative HF risk in the third and fourth quartiles of activated partial thromboplastin time and fibrin degradation products. The results of the restricted cubic spline model showed that the methylation levels of DIO3-FA 27_CpG_11.12 and DIO3-FA 27_CpG_23.24 were associated with coagulation indicators, liver function, renal function, and blood routine. CONCLUSIONS: Based on the differential analysis of CpG methylation levels based on DIO3-FA27, it was found that biochemical indicators combined with DIO3-FA27 promoter DNA methylation levels could increase the risk of worsening the severity classification of HF patients, which provided a solid foundation and new insights for the study of epigenetic regulation mechanisms in patients with HF.

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

BACKGROUND: While 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. RESULTS: We 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. CONCLUSIONS: This 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.

Characterization of the genetic determinants of context-specific DNA methylation in primary monocytes.

To better understand inter-individual variation in sensitivity of DNA methylation (DNAm) to immune activity, we characterized effects of inflammatory stimuli on primary monocyte DNAm (n = 190). We find that monocyte DNAm is site-dependently sensitive to lipopolysaccharide (LPS), with LPS-induced demethylation occurring following hydroxymethylation. We identify 7,359 high-confidence immune-modulated CpGs (imCpGs) that differ in genomic localization and transcription factor usage according to whether they represent a gain or loss in DNAm. Demethylated imCpGs are profoundly enriched for enhancers and colocalize to genes enriched for disease associations, especially cancer. DNAm is age associated, and we find that 24-h LPS exposure triggers approximately 6 months of gain in epigenetic age, directly linking epigenetic aging with innate immune activity. By integrating LPS-induced changes in DNAm with genetic variation, we identify 234 imCpGs under local genetic control. Exploring shared causal loci between LPS-induced DNAm responses and human disease traits highlights examples of disease-associated loci that modulate imCpG formation.

Multi-omics analysis reveals epigenetically regulated processes and patient classification in lung adenocarcinoma.

Aberrant DNA methylation is a hallmark of many cancer types. Despite our knowledge of epigenetic and transcriptomic alterations in lung adenocarcinoma (LUAD), we lack robust multi-modal molecular classifications for patient stratification. This is partly because the impact of epigenetic alterations on lung cancer development and progression is still not fully understood. To that end, we identified disease-associated processes under epigenetic regulation in LUAD. We performed a genome-wide expression-methylation Quantitative Trait Loci (emQTL) analysis by integrating DNA methylation and gene expression data from 453 patients in the TCGA cohort. Using a community detection algorithm, we identified distinct communities of CpG-gene associations with diverse biological processes. Interestingly, we identified a community linked to hormone response and lipid metabolism; the identified CpGs in this community were enriched in enhancer regions and binding regions of transcription factors such as FOXA1/2, GRHL2, HNF1B, AR, and ESR1. Furthermore, the CpGs were connected to their associated genes through chromatin interaction loops. These findings suggest that the expression of genes involved in hormone response and lipid metabolism in LUAD is epigenetically regulated through DNA methylation and enhancer-promoter interactions. By applying consensus clustering on the integrated expression-methylation pattern of the emQTL-genes and CpGs linked to hormone response and lipid metabolism, we further identified subclasses of patients with distinct prognoses. This novel patient stratification was validated in an independent patient cohort of 135 patients and showed increased prognostic significance compared to previously defined molecular subtypes.

CpG Islands, Gene Expression and Pseudogenization: A Case for a Potential Trilogy.

BACKGROUND: The promoters of mammalian genes contain clusters of CG dinucleotides known as CpG islands. Most mammalian housekeeping genes predominantly contain CpG islands (CGIs), facilitating gene transcription. Numerous studies have explored the physiological implications of the relationship between CGIs and gene expression. However, the evolutionary implications of this relationship remain largely unexplored. Pseudogenes, in contrast, are genomic remnants that have lost their function over evolutionary time. METHODS: In our current research, we employed comparative genomic techniques to demonstrate a correlation between the absence of gene expression due to a lack of CGIs in the gene promoters and pseudogenization. RESULTS: We showed that there is a significant enrichment of tissue-specific genes in the functional orthologs of pseudogenes. We also found a significant correlation between the lack of CGIs and enriched tissue specificity in these functional orthologs of pseudogenes. CONCLUSIONS: We inferred that perhaps tissue-specific genes are more prone to the process of pseudogenization. In this way, because of their impact on gene expression, CGIs may affect the fate of a gene. To our knowledge, this is the first study to propose a connection between CGIs, gene expression, and the pseudogenization process and discuss the evolutionary implications of this potential trilogy.

Investigating the potential of single-cell DNA methylation data to detect allele-specific methylation and imprinting.

Allele-specific methylation (ASM) is an epigenetic modification whereby one parental allele becomes methylated and the other unmethylated at a specific locus. ASM is most often driven by the presence of nearby heterozygous variants that influence methylation, but also occurs somatically in the context of genomic imprinting. In this study, we investigate ASM using publicly available single-cell reduced representation bisulfite sequencing (scRRBS) data on 608 B cells sampled from six healthy B cell samples and 1,230 cells from 11 chronic lymphocytic leukemia (CLL) samples. We developed a likelihood-based criterion to test whether a CpG exhibited ASM, based on the distributions of methylated and unmethylated reads both within and across cells. Applying our likelihood ratio test, 65,998 CpG sites exhibited ASM in healthy B cell samples according to a Bonferroni criterion (p < 8.4 × 10-9), and 32,862 CpG sites exhibited ASM in CLL samples (p < 8.5 × 10-9). We also called ASM at the sample level. To evaluate the accuracy of our method, we called heterozygous variants from the scRRBS data, which enabled variant-based calls of ASM within each cell. Comparing sample-level ASM calls to the variant-based measures of ASM, we observed a positive predictive value of 76%-100% across samples. We observed high concordance of ASM across samples and an overrepresentation of ASM in previously reported imprinted genes and genes with imprinting binding motifs. Our study demonstrates that single-cell bisulfite sequencing is a potentially powerful tool to investigate ASM, especially as studies expand to increase the number of samples and cells sequenced.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

AIMS/HYPOTHESIS: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion. METHODS: Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging. RESULTS: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation. CONCLUSIONS/INTERPRETATION: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. DATA AVAILABILITY: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.

Epigenetics as a Key Factor in Prostate Cancer.

Nowadays, prostate cancer is one of the most common forms of malignant neoplasms in men all over the world. Against the background of increasing incidence, there is a high mortality rate from prostate cancer, which is associated with an inadequate treatment strategy. Such a high prevalence of prostate cancer requires the development of methods that can ensure early detection of the disease, improve the effectiveness of treatment, and predict the therapeutic effect. Under these circumstances, it becomes crucial to focus on the development of effective diagnostic and therapeutic approaches. Due to the development of molecular genetic methods, a large number of studies have been accumulated on the role of epigenetic regulation of gene activity in cancer development, since it is epigenetic changes that can be detected at the earliest stages of cancer development. The presence of epigenetic aberrations in tumor tissue and correlations with drug resistance suggest new therapeutic approaches. Detection of epigenetic alterations such as CpG island methylation, histone modification, and microRNAs as biomarkers will improve the diagnosis of the disease, and the use of these strategies as targets for therapy will allow for greater personalization of prostate cancer treatment.

Epigenome-wide association study of total nicotine equivalents in multiethnic current smokers from three prospective cohorts.

The impact of tobacco exposure on health varies by race and ethnicity and is closely tied to internal nicotine dose, a marker of carcinogen uptake. DNA methylation is strongly responsive to smoking status and may mediate health effects, but study of associations with internal dose is limited. We performed a blood leukocyte epigenome-wide association study (EWAS) of urinary total nicotine equivalents (TNEs; a measure of nicotine uptake) and DNA methylation measured using the MethylationEPIC v1.0 BeadChip (EPIC) in six racial and ethnic groups across three cohort studies. In the Multiethnic Cohort Study (discovery, n = 1994), TNEs were associated with differential methylation at 408 CpG sites across >250 genomic regions (p < 9 × 10-8). The top significant sites were annotated to AHRR, F2RL3, RARA, GPR15, PRSS23, and 2q37.1, all of which had decreasing methylation with increasing TNEs. We identified 45 novel CpG sites, of which 42 were unique to the EPIC array and eight annotated to genes not previously linked with smoking-related DNA methylation. The most significant signal in a novel gene was cg03748458 in MIR383;SGCZ. Fifty-one of the 408 discovery sites were validated in the Singapore Chinese Health Study (n = 340) and the Southern Community Cohort Study (n = 394) (Bonferroni corrected p < 1.23 × 10-4). Significant heterogeneity by race and ethnicity was detected for CpG sites in MYO1G and CYTH1. Furthermore, TNEs significantly mediated the association between cigarettes per day and DNA methylation at 15 sites (average 22.5%-44.3% proportion mediated). Our multiethnic study highlights the transethnic and ethnic-specific methylation associations with internal nicotine dose, a strong predictor of smoking-related morbidities.

Tet-mediated DNA methylation dynamics affect chromosome organization.

DNA Methylation is a significant epigenetic modification that can modulate chromosome states, but its role in orchestrating chromosome organization has not been well elucidated. Here we systematically assessed the effects of DNA Methylation on chromosome organization with a multi-omics strategy to capture DNA Methylation and high-order chromosome interaction simultaneously on mouse embryonic stem cells with DNA methylation dioxygenase Tet triple knock-out (Tet-TKO). Globally, upon Tet-TKO, we observed weakened compartmentalization, corresponding to decreased methylation differences between CpG island (CGI) rich and poor domains. Tet-TKO could also induce hypermethylation for the CTCF binding peaks in TAD boundaries and chromatin loop anchors. Accordingly, CTCF peak generally weakened upon Tet-TKO, which results in weakened TAD structure and depletion of long-range chromatin loops. Genes that lost enhancer-promoter looping upon Tet-TKO showed DNA hypermethylation in their gene bodies, which may compensate for the disruption of gene expression. We also observed distinct effects of Tet1 and Tet2 on chromatin organization and increased DNA methylation correlation on spatially interacted fragments upon Tet inactivation. Our work showed the broad effects of Tet inactivation and DNA methylation dynamics on chromosome organization.

Investigating FGFR2 gene as a blood-based epigenetic biomarker in gastric cancer.

BACKGROUND: Gastric adenocarcinoma is a prevalent form of cancer that often remains undetected in its early stages due to the lack of specific symptoms. This delayed diagnosis leads to poor clinical outcomes, underscoring the need for an effective and non-invasive method for early detection. Recent advances in cancer epigenetics have led to the identification of biomarkers that have the potential to revolutionize the early detection and monitoring of this disease. One such promising biomarker is the methylation of the FGFR2 promoter. This study aims to measure the methylation levels of a specific CpG site in the FGFR2 promoter gene in DNA extracted from blood leukocytes from patients with intestinal metaplasia, gastric cancer, and healthy control. MATERIAL AND METHODS: The CpG site of the FGFR2 gene promoter was identified in its control region. Methylation alteration of the selected FGFR2 CpG site was determined through the (methylation-sensitive restriction enzyme) MSRE-qPCR. Genomic DNA was extracted from one hundred twenty-five participants. RESULTS: The normal group had mean methylation levels of 93.23 ± 4.929%, while the IM group had a level of 69.85 ± 27.15%. In GC patients, the levels varied, with 25.96 ± 18.98% in the intestinal type and 28.30 ± 16.07% in the diffuse type. The methylation levels in the IM and GC patients were significantly lower than those in the normal control group. However, no significant difference was observed between the methylation status of the intestinal type of GC and the diffuse type. The Receiver operating characteristic (ROC) curve analysis showed that FGFR2 CpG methylation levels in GC patients compared to normal controls had a high sensitivity of 100% and specificity of 100%, with a cut-off of < 74.25%; when GC patients were compared to IM patients, the sensitivity was 85%, and the specificity was 80%, with a cut-off < 44.45%. CONCLUSIONS: The potential of the FGFR2 methylation status as a non-invasive biomarker lies in its ability to be detected in blood leukocytes, which makes it a promising tool for the early detection of intestinal metaplasia and gastric cancer. This could significantly improve the detection and management of these gastric conditions.

Predicting age from blood by droplet digital PCR using a set of three DNA methylation markers.

Evaluation of DNA methylation (DNAm) patterns is a promising tool for age estimation. The duplex droplet digital PCR (ddPCR) method has been recently investigated for DNAm evaluation, revealing to be a potential methodology for DNAm evaluation and molecular age estimation. In this study, we evaluated DNAm levels of CpGs located at the three age-associated genes ELOVL2, FHL2 and PDE4C using ddPCR to develop an age prediction model. Blood-derived DNA samples from 58 healthy individuals (42 women and 16 men; aged 1-93 years old) were submitted to bisulfite conversion followed by ddPCR using dual-labeled probes targeting methylated and unmethylated DNA sequences. Simple linear regression statistics revealed a strong correlation between DNAm levels and chronological age for FHL2 (R = 0.948; P = 1.472 × 10-29) and PDE4C (R = 0.819; P = 3.917 × 10-15), addressing only one CpG for each gene. For the ELOVL2 gene, evaluating five CpG sites in simultaneous, revealed a strong age correlation (R = 0.887; P = 2.099 × 10-20) in a simple linear regression statistics and very strong age correlation (R = 0.926; P = 2.202 × 10-25) when using quadratic regression statistics. The multivariable regression analysis, using methylation information captured on ELOVL2 (squared), FHL2 and PDE4C genes, revealed a very strong age correlation (R = 0.970; P = 5.356 ×10-33), explaining 93.7 % of age variance, displaying a mean absolute deviation (MAD) between chronological and predicted age of 4.657 years (RMSE = 6.044). We postulate that the ddPCR method should be further investigated for DNAm-based age prediction, because it is a relatively simple and an accurate method that can be routinely used in forensic laboratories for testing a few numbers of markers.

Enrichment of Bacteroides fragilis and enterotoxigenic Bacteroides fragilis in CpG island methylator phenotype-high colorectal carcinoma.

OBJECTIVES: Data support that enterotoxigenic Bacteroides fragilis (ETBF) harbouring the Bacteroides fragilis toxin (bft) gene may promote colorectal tumourigenesis through the serrated neoplasia pathway. We hypothesized that ETBF may be enriched in colorectal carcinoma subtypes with high-level CpG island methylator phenotype (CIMP-high), BRAF mutation, and high-level microsatellite instability (MSI-high). METHODS: Quantitative PCR assays were designed to quantify DNA amounts of Bacteroides fragilis, ETBF, and each bft gene isotype (bft-1, bft-2, or bft-3) in colorectal carcinomas in the Health Professionals Follow-up Study and Nurses' Health Study. We used multivariable-adjusted logistic regression models with the inverse probability weighting method. RESULTS: We documented 4476 colorectal cancer cases, including 1232 cases with available bacterial data. High DNA amounts of Bacteroides fragilis and ETBF were positively associated with BRAF mutation (p ≤ 0.0003), CIMP-high (p ≤ 0.0002), and MSI-high (p < 0.0001 and p = 0.01, respectively). Multivariable-adjusted odds ratios (with 95% confidence interval) for high Bacteroides fragilis were 1.40 (1.06-1.85) for CIMP-high and 2.14 (1.65-2.77) for MSI-high, but 1.02 (0.78-1.35) for BRAF mutation. Multivariable-adjusted odds ratios for high ETBF were 2.00 (1.16-3.45) for CIMP-high and 2.86 (1.64-5.00) for BRAF mutation, but 1.09 (0.67-1.76) for MSI-high. Neither Bacteroides fragilis nor ETBF was associated with colorectal cancer-specific or overall survival. DISCUSSION: The tissue abundance of Bacteroides fragilis is associated with CIMP-high and MSI-high, whereas ETBF abundance is associated with CIMP-high and BRAF mutation in colorectal carcinoma. Our findings support the aetiological relevance of Bacteroides fragilis and ETBF in the serrated neoplasia pathway.

Causality-enriched epigenetic age uncouples damage and adaptation.

Machine learning models based on DNA methylation data can predict biological age but often lack causal insights. By harnessing large-scale genetic data through epigenome-wide Mendelian randomization, we identified CpG sites potentially causal for aging-related traits. Neither the existing epigenetic clocks nor age-related differential DNA methylation are enriched in these sites. These CpGs include sites that contribute to aging and protect against it, yet their combined contribution negatively affects age-related traits. We established a new framework to introduce causal information into epigenetic clocks, resulting in DamAge and AdaptAge-clocks that track detrimental and adaptive methylation changes, respectively. DamAge correlates with adverse outcomes, including mortality, while AdaptAge is associated with beneficial adaptations. These causality-enriched clocks exhibit sensitivity to short-term interventions. Our findings provide a detailed landscape of CpG sites with putative causal links to lifespan and healthspan, facilitating the development of aging biomarkers, assessing interventions, and studying reversibility of age-associated changes.