Association between DNA methylation levels of thioredoxin-interacting protein (TXNIP) and changes in glycemic traits: a longitudinal population-based study.

Thioredoxin-interacting protein (TXNIP) plays an important role in glucose metabolism, and its expression is regulated by DNA methylation (DNAm). Although the association between TXNIP DNAm and type 2 diabetes mellitus has been demonstrated in studies with a cross-sectional design, prospective studies are needed. We therefore examined the association between TXNIP DNAm levels and longitudinal changes in glycemic traits by conducting a longitudinal study involving 169 subjects who underwent two health checkups in 2015 and 2019. We used a pyrosequencing assay to determine TXNIP DNAm levels in leukocytes (cg19693031). Logistic regression analyses were performed to assess the associations between dichotomized TXNIP DNAm levels and marked increases in glycemic traits. At four years, the TXNIP DNA hypomethylation group had a higher percentage of changes in fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) compared to those in the hypermethylation group. The adjusted odds ratios for FPG and HbA1c levels were significantly higher in the TXNIP DNA hypomethylation group than in the hypermethylation group. We found that TXNIP DNA hypomethylation at baseline was associated with a marked increase in glycemic traits. Leukocyte TXNIP DNAm status could potentially be used as an early biomarker for impaired glucose homeostasis.

Understanding the role of DNA methylation in colorectal cancer: Mechanisms, detection, and clinical significance.

Colorectal cancer (CRC) is one of the deadliest malignancies worldwide, ranking third in incidence and second in mortality. Remarkably, early stage localized CRC has a 5-year survival rate of over 90%; in stark contrast, the corresponding 5-year survival rate for metastatic CRC (mCRC) is only 14%. Compounding this problem is the staggering lack of effective therapeutic strategies. Beyond genetic mutations, which have been identified as critical instigators of CRC initiation and progression, the importance of epigenetic modifications, particularly DNA methylation (DNAm), cannot be underestimated, given that DNAm can be used for diagnosis, treatment monitoring and prognostic evaluation. This review addresses the intricate mechanisms governing aberrant DNAm in CRC and its profound impact on critical oncogenic pathways. In addition, a comprehensive review of the various techniques used to detect DNAm alterations in CRC is provided, along with an exploration of the clinical utility of cancer-specific DNAm alterations.

Bacterial Outer Membrane Vesicles and Immune Modulation of the Host.

This article reviews the role of outer membrane vesicles (OMVs) in mediating the interaction between Gram-negative bacteria and their human hosts. OMVs are produced by a diverse range of Gram-negative bacteria during infection and play a critical role in facilitating host-pathogen interactions without requiring direct cell-to-cell contact. This article describes the mechanisms by which OMVs are formed and subsequently interact with host cells, leading to the transport of microbial protein virulence factors and short interfering RNAs (sRNA) to their host targets, exerting their immunomodulatory effects by targeting specific host signaling pathways. Specifically, this review highlights mechanisms by which OMVs facilitate chronic infection through epigenetic modification of the host immune response. Finally, this review identifies critical knowledge gaps in the field and offers potential avenues for future OMV research, specifically regarding rigor and reproducibility in OMV isolation and characterization methods.

Methylation quantitative trait loci are largely consistent across disease states in Crohn's disease.

Recently, we identified 1,189 CpG sites whose DNA methylation level in blood associated with Crohn's disease. Here, we examined associations between DNA methylation and genetic variants to identify methylation quantitative trait loci across disease states in (1) 402 blood samples from 164 newly diagnosed pediatric Crohn's disease patients taken at 2 time points (diagnosis and follow-up), and 74 non-inflammatory bowel disease controls, (2) 780 blood samples from a non-Crohn's disease adult population, and (3) 40 ileal biopsies (17 Crohn's disease cases and 23 non-inflammatory bowel disease controls) from group (1). Genome-wide DNAm profiling and genotyping were performed using the Illumina MethylationEPIC and Illumina Multi-Ethnic arrays. SNP-CpG associations were identified via linear models adjusted for age, sex, disease status, disease subtype, estimated cell proportions, and genotype-based principal components. In total, we observed 535,448 SNP-CpG associations between 287,881 SNPs and 12,843 CpG sites (P < 8.21 × 10-14). Associations were highly consistent across different ages, races, disease states, and tissue types, suggesting that the majority of these methylation quantitative trait loci participate in common gene regulation. However, genes near CpGs associated with inflammatory bowel disease SNPs were enriched for 18 KEGG pathways relevant to inflammatory bowel disease-linked immune function and inflammatory responses. We observed suggestive evidence for a small number of tissue-specific associations and disease-specific associations in ileum, though larger studies will be needed to confirm these results. Our study concludes that the vast majority of blood-derived methylation quantitative trait loci are common across individuals, though a subset may be involved in processes related to Crohn's disease. Independent cohort studies will be required to validate these findings.

Cardiovascular health is associated with the epigenetic clock in the Berlin Aging Study II (BASE-II).

The epigenetic clock parameter DNAm age acceleration is a promising biomarker of aging. We have recently described an epigenetic clock based on only seven cytosine-phosphate-guanine sites, which is highly associated with chronological age. The aim of this study was to examine this epigenetic clock with respect to its relationship with cardiovascular health (CVH) in older adults. We used data from the Berlin Aging Study II (BASE-II; 1,671 participants; 68.8 ± 3.7 years old). CVH was operationalized using two different CVH scores, the Framingham Risk Score (FRS), and the Life's simple 7 (LS7). To adjust for potential confounding, e.g. by sex, we performed regression analyses. The LS7 score was higher, i.e. more favorable, in woman than in men (8.8 ± 2 vs. 8.2 ± 2, p < 0.001). DNAm age acceleration was associated with the FRS (ß = 0.122, p = 0.028) and with the LS7 (ß = -0.804, p = 0.032). In more detail, physical activity (ß = -0.461, p = 0.05), HDL-cholesterol (ß = 0.343, p = 0.03) and total cholesterol (ß = -0.364, p = 0.002) were associated with epigenetic age acceleration. We present evidence suggesting that better CVH is associated with decelerated biological aging measured by the epigenetic clock.

A Blood-Bone-Tooth Model for Age Prediction in Forensic Contexts.

The development of age prediction models (APMs) focusing on DNA methylation (DNAm) levels has revolutionized the forensic age estimation field. Meanwhile, the predictive ability of multi-tissue models with similar high accuracy needs to be explored. This study aimed to build multi-tissue APMs combining blood, bones and tooth samples, herein named blood-bone-tooth-APM (BBT-APM), using two different methodologies. A total of 185 and 168 bisulfite-converted DNA samples previously addressed by Sanger sequencing and SNaPshot methodologies, respectively, were considered for this study. The relationship between DNAm and age was assessed using simple and multiple linear regression models. Through the Sanger sequencing methodology, we built a BBT-APM with seven CpGs in genes ELOVL2, EDARADD, PDE4C, FHL2 and C1orf132, allowing us to obtain a Mean Absolute Deviation (MAD) between chronological and predicted ages of 6.06 years, explaining 87.8% of the variation in age. Using the SNaPshot assay, we developed a BBT-APM with three CpGs at ELOVL2, KLF14 and C1orf132 genes with a MAD of 6.49 years, explaining 84.7% of the variation in age. Our results showed the usefulness of DNAm age in forensic contexts and brought new insights into the development of multi-tissue APMs applied to blood, bone and teeth.

Predominant DNMT and TET mediate effects of allergen on the human bronchial epithelium in a controlled air pollution exposure study.

BACKGROUND: Epidemiological data show that traffic-related air pollution contributes to the increasing prevalence and severity of asthma. DNA methylation (DNAm) changes may elucidate adverse health effects of environmental exposures. OBJECTIVES: We sought to assess the effects of allergen and diesel exhaust (DE) exposures on global DNAm and its regulation enzymes in human airway epithelium. METHODS: A total of 11 participants, including 7 with and 4 without airway hyperresponsiveness, were recruited for a randomized, double-blind crossover study. Each participant had 3 exposures: filtered air + saline, filtered air + allergen, and DE + allergen. Forty-eight hours postexposure, endobronchial biopsies and bronchoalveolar lavages were collected. Levels of DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) enzymes, 5-methylcytosine, and 5-hydroxymethylcytosine were determined by immunohistochemistry. Cytokines and chemokines in bronchoalveolar lavages were measured by electrochemiluminescence multiplex assays. RESULTS: Predominant DNMT (the most abundant among DNMT1, DNMT3A, and DNMT3B) and predominant TET (the most abundant among TET1, TET2, and TET3) were participant-dependent. 5-Methylcytosine and its regulation enzymes differed between participants with and without airway hyperresponsiveness at baseline (filtered air + saline) and in response to allergen challenge (regardless of DE exposure). Predominant DNMT and predominant TET correlated with lung function. Allergen challenge effect on IL-8 in bronchoalveolar lavages was modified by TET2 baseline levels in the epithelium. CONCLUSIONS: Response to allergen challenge is associated with key DNAm regulation enzymes. This relationship is generally unaltered by DE coexposure but is rather dependent on airway hyperresponsiveness status. These enzymes therefore warranted further inquiry regarding their potential in diagnosis, prognosis, and treatment of asthma.

DNA Methylation at Birth Predicts Intellectual Functioning and Autism Features in Children with Fragile X Syndrome.

Fragile X syndrome (FXS) is a leading single-gene cause of intellectual disability (ID) with autism features. This study analysed diagnostic and prognostic utility of the Fragile X-Related Epigenetic Element 2 DNA methylation (FREE2m) assessed by Methylation Specific-Quantitative Melt Analysis and the EpiTYPER system, in retrospectively retrieved newborn blood spots (NBS) and newly created dried blood spots (DBS) from 65 children with FXS (~2-17 years). A further 168 NBS from infants from the general population were used to establish control reference ranges, in both sexes. FREE2m analysis showed sensitivity and specificity approaching 100%. In FXS males, NBS FREE2m strongly correlated with intellectual functioning and autism features, however associations were not as strong for FXS females. Fragile X mental retardation 1 gene (FMR1) mRNA levels in blood were correlated with FREE2m in both NBS and DBS, for both sexes. In females, DNAm was significantly increased at birth with a decrease in childhood. The findings support the use of FREE2m analysis in newborns for screening, diagnostic and prognostic testing in FXS.

Identification of DNA methylation patterns and biomarkers for clear-cell renal cell carcinoma by multi-omics data analysis.

BACKGROUND: Tumorigenesis is highly heterogeneous, and using clinicopathological signatures only is not enough to effectively distinguish clear cell renal cell carcinoma (ccRCC) and improve risk stratification of patients. DNA methylation (DNAm) with the stability and reversibility often occurs in the early stage of tumorigenesis. Disorders of transcription and metabolism are also an important molecular mechanisms of tumorigenesis. Therefore, it is necessary to identify effective biomarkers involved in tumorigenesis through multi-omics analysis, and these biomarkers also provide new potential therapeutic targets. METHOD: The discovery stage involved 160 pairs of ccRCC and matched normal tissues for investigation of DNAm and biomarkers as well as 318 cases of ccRCC including clinical signatures. Correlation analysis of epigenetic, transcriptomic and metabolomic data revealed the connection and discordance among multi-omics and the deregulated functional modules. Diagnostic or prognostic biomarkers were obtained by the correlation analysis, the Least Absolute Shrinkage and Selection Operator (LASSO) and the LASSO-Cox methods. Two classifiers were established based on random forest (RF) and LASSO-Cox algorithms in training datasets. Seven independent datasets were used to evaluate robustness and universality. The molecular biological function of biomarkers were investigated using DAVID and GeneMANIA. RESULTS: Based on multi-omics analysis, the epigenetic measurements uniquely identified DNAm dysregulation of cellular mechanisms resulting in transcriptomic alterations, including cell proliferation, immune response and inflammation. Combination of the gene co-expression network and metabolic network identified 134 CpG sites (CpGs) as potential biomarkers. Based on the LASSO and RF algorithms, five CpGs were obtained to build a diagnostic classifierwith better classification performance (AUC > 99%). A eight-CpG-based prognostic classifier was obtained to improve risk stratification (hazard ratio (HR) > 4; log-rank test, p-value < 0.01). Based on independent datasets and seven additional cancers, the diagnostic and prognostic classifiers also had better robustness and stability. The molecular biological function of genes with abnormal methylation were significantly associated with glycolysis/gluconeogenesis and signal transduction. CONCLUSION: The present study provides a comprehensive analysis of ccRCC using multi-omics data. These findings indicated that multi-omics analysis could identify some novel epigenetic factors, which were the most important causes of advanced cancer and poor clinical prognosis. Diagnostic and prognostic biomarkers were identified, which provided a promising avenue to develop effective therapies for ccRCC.

Exploring the Relationship of Relative Telomere Length and the Epigenetic Clock in the LipidCardio Cohort.

Telomere length has been accepted widely as a biomarker of aging. Recently, a novel candidate biomarker has been suggested to predict an individual's chronological age with high accuracy: The epigenetic clock is based on the weighted DNA methylation (DNAm) fraction of a number of cytosine-phosphate-guanine sites (CpGs) selected by penalized regression analysis. Here, an established methylation-sensitive single nucleotide primer extension method was adapted, to estimate the epigenetic age of the 1005 participants of the LipidCardio Study, a patient cohort characterised by high prevalence of cardiovascular disease, based on a seven CpGs epigenetic clock. Furthermore, we measured relative leukocyte telomere length (rLTL) to assess the relationship between the established and the promising new measure of biological age. Both rLTL (0.79 ± 0.14) and DNAm age (69.67 ± 7.27 years) were available for 773 subjects (31.6% female; mean chronological age= 69.68 ± 11.01 years; mean DNAm age acceleration = -0.01 ± 7.83 years). While we detected a significant correlation between chronological age and DNAm age (n = 779, R = 0.69), we found neither evidence of an association between rLTL and the DNAm age (ß = 3.00, p = 0.18) nor rLTL and the DNAm age acceleration (ß = 2.76, p = 0.22) in the studied cohort, suggesting that DNAm age and rLTL measure different aspects of biological age.

Genetic and epigenetic changes in clonal descendants of irradiated human fibroblasts.

To study delayed genetic and epigenetic radiation effects, which may trigger radiation-induced carcinogenesis, we have established single-cell clones from irradiated and non-irradiated primary human fibroblasts. Stable clones were endowed with the same karyotype in all analyzed metaphases after 20 population doublings (PDs), whereas unstable clones displayed mosaics of normal and abnormal karyotypes. To account for variation in radiation sensitivity, all experiments were performed with two different fibroblast strains. After a single X-ray dose of 2 Gy more than half of the irradiated clones exhibited radiation-induced genome instability (RIGI). Irradiated clones displayed an increased rate of loss of chromosome Y (LOY) and copy number variations (CNVs), compared to controls. CNV breakpoints clustered in specific chromosome regions, in particular 3p14.2 and 7q11.21, coinciding with common fragile sites. CNVs affecting the FHIT gene in FRA3B were observed in independent unstable clones and may drive RIGI. Bisulfite pyrosequencing of control clones and the respective primary culture revealed global hypomethylation of ALU, LINE-1, and alpha-satellite repeats as well as rDNA hypermethylation during in vitro ageing. Irradiated clones showed further reduced ALU and alpha-satellite methylation and increased rDNA methylation, compared to controls. Methylation arrays identified several hundred differentially methylated genes and several enriched pathways associated with in vitro ageing. Methylation changes in 259 genes and the MAP kinase signaling pathway were associated with delayed radiation effects (after 20 PDs). Collectively, our results suggest that both genetic (LOY and CNVs) and epigenetic changes occur in the progeny of exposed cells that were not damaged directly by irradiation, likely contributing to radiation-induced carcinogenesis. We did not observe epigenetic differences between stable and unstable irradiated clones. The fact that the DNA methylation (DNAm) age of clones derived from the same primary culture varied greatly suggests that DNAm age of a single cell (represented by a clone) can be quite different from the DNAm age of a tissue. We propose that DNAm age reflects the emergent property of a large number of individual cells whose respective DNAm ages can be highly variable.

Profiling placental and fetal DNA methylation in human neural tube defects.

BACKGROUND: The incidence of neural tube defects (NTDs) declined by about 40 % in Canada with the introduction of a national folic acid (FA) fortification program. Despite the fact that few Canadians currently exhibit folate deficiency, NTDs are still the second most common congenital abnormality. FA fortification may have aided in reducing the incidence of NTDs by overcoming abnormal one carbon metabolism cycling, the process which provides one carbon units for methylation of DNA. We considered that NTDs persisting in a folate-replete population may also occur in the context of FA-independent compromised one carbon metabolism, and that this might manifest as abnormal DNA methylation (DNAm). Second trimester human placental chorionic villi, kidney, spinal cord, brain, and muscle were collected from 19 control, 22 spina bifida, and 15 anencephalic fetuses in British Columbia, Canada. DNA was extracted, assessed for methylenetetrahydrofolate reductase (MTHFR) genotype and for genome-wide DNAm using repetitive elements, in addition to the Illumina Infinium HumanMethylation450 (450k) array. RESULTS: No difference in repetitive element DNAm was noted between NTD status groups. Using a false discovery rate <0.05 and average group difference in DNAm ≥0.05, differentially methylated array sites were identified only in (1) the comparison of anencephaly to controls in chorionic villi (n = 4 sites) and (2) the comparison of spina bifida to controls in kidney (n = 3342 sites). CONCLUSIONS: We suggest that the distinctive DNAm of spina bifida kidneys may be consequent to the neural tube defect or reflective of a common etiology for abnormal neural tube and renal development. Though there were some small shifts in DNAm in the other tested tissues, our data do not support the long-standing hypothesis of generalized altered genome-wide DNAm in NTDs. This finding may be related to the fact that most Canadians are not folate deficient, but it importantly opens the field to the investigation of other epigenetic and non-epigenetic mechanisms in the etiology of NTDs.