dna methylation

The important roles of aberrant DNA methylation on semen abnormality have been demonstrated. Alternatively, 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a vital enzyme to regulate the sperm DNA methylation patterns. This study aimed to investigate the methylation of MTHFR at differentially methylated regions (DMR) and the correlation between sperm DNA methylation patterns with semen quality parameters for assessing reproductive health and fertility status. In this case-control study, semen samples were collected from 30 infertile (asthenosperm ia and oligoasthenoteratospermia), and 15 healthy men. Following the modification of DNAs by sodium bisulfite treatment, the methylation status of the MTHFR gene at MDRs was evaluated by the quantitative methylation specific PCR (qMSP) method. Men with oligoasthenoteratospermia showed a statistically significant difference in mean MTHFR-DMR methylation levels compared to controls (P=0.047) and asthenospermia (P=0.034). Moreover, significant trends of decreasing values were observed in all parameters of the ejaculate (sperm concentration, their overall motility or vital ity, and morphology) in men with asthenospermia and oligoasthenoteratospermia. These findings suggest a potential association between increased MTHFR-DMR methylation and reduced semen quality such as spermatozoa count (P=0.002), spermatozoa concentration (P=0.003), progressive (P=0.019), and normal morphology (P=0.003). We found that abnormal DNA methylation of MTHFR at DMR region was correlated with decreased sperm parameters and therefore male infertility. Further research is needed to explore the mechanisms affecting of MTHFR methylation on male infertility.

It is well established that tissues exposed to sulfur mustard (SM) generate high levels of reactive oxygen species. This leads to oxidative stress and, ultimately, damage to DNA molecules over the course of time. Additionally, SM, through its alkylating effects, is capable of directly damaging DNA on its own. In cells, these damages trigger a variety of DNA repair pathways, including the base excision repair (BER) pathway. Even so, in the long run, it remains unclear how the BER repair pathways will react.

The purpose of this study was to assess the promoter DNA methylation and the mRNA expression of 8-oxoguanine glycosylase (OGG1), one of the key components of the BER pathway, in patient PBMCs that were exposed to SM 27 years ago using methylation-sensitive high resolution melting and qPCR. The study was conducted on three groups of participants exposed to SM with mild (n = 20), moderate (n = 24), and severe (n = 20) lung complications.

Our results showed significant OGG1 mRNA overexpression was observed in moderate groups compared to mild groups (P = 0.036). DNA methylation was also altered in mild-moderate and moderate-severe groups (P < 0.0001 and 0.023, respectively). Although aging was significantly associated with OGG1 mRNA expression, promoter DNA methylation of OGG1 was not associated with its mRNA expression.

This study revealed differences in OGG1 mRNA expression and DNA methylation among the severity groups of long-term pulmonary complications associated with SM exposure. However, there was no correlation between OGG1 DNA methylation and mRNA expression. Therefore, it appears that other mechanisms may be contributing to the dysregulation of OGG1 mRNA expression.

Growing evidence supports that changes in the expression of the suppressor of cytokine signaling 3 (SOCS3) protein contribute to the pathogenesis of types of inflammatory bowel diseases (IBDs), including ulcerative colitis (UC). Despite the importance of the currently known genetic risk map, an increasing number of observations reveal that epigenetic modifications, including DNA methylation, are considered as or even more important for IBD pathogenesis than genetic predisposition. We investigated the hypothesis that alterations in DNA methylation status at the promoter region within the SOCS3 gene in colorectal tissue specimens may be involved in the susceptibility to UC.

We studied extracted DNA from colorectal biopsies of 15 ulcerative colitis cases and 15 age—and sex-matched healthy controls and performed genotype analyses of the promoter methylation status of the SOCS3 gene, using the real-time quantitative multiplex methylation-specific PCR (QM-MSP) assay to show evidence of differential methylation between cases of ulcerative colitis and healthy controls.

Based on methylation assay data profiling, we found that the mean CpG island methylation levels at the SOCS3 gene promoter region in colorectal mucosa of patients with UC was significantly higher than mucosa from healthy controls (ulcerative colitis vs. healthy controls; 0.00007±0.0018 vs. 0.07±0.142, p<0.000).

Our data provide an important insight into the influence of epigenetic aberrances in the SOCS3 gene such that the inactivation of the SOCS3 gene by promoter hypermethylation might be a risk factor for inflamed mucosa of UC. It might also fundamentally contribute to the initiation of the inflammatory process and development of UC.

Multiple sclerosis (MS), an autoimmune chronic inflammatory, demyelinating disease, has affected over 2.5 million people in the world, who are mostly in young adulthood ages. As the burden of this disease would highly influence the socioeconomic status of the societies, as well as the patient’s quality of life, any progress in better understanding the pathophysiology of this disease would be valuable. MS is caused by a series of cell-mediated immune mechanisms involving CD4+ T-cell reactivation against CNS. Also, as the involvement of both innate and acquired immunities, different risk factors have been proposed for MS. Environmental factors such as smoking, Epstein-Barr virus infection, sun exposure and vitamin D, body mass index, gut microbiota, and melatonin disturbance may affect gene expression patterns through epigenetic changes, and therefore, play roles in disease occurrence. These epigenetic changes could be categorized as alterations in DNA methylation, histone modifications and non-coding RNAs. Moreover, the reversibility of these epigenetic changes could be potentially considered as therapeutic targets. Therefore, several experimental and preclinical studies have investigated medications for reversing the pathologic epigenetic changes in MS. Accordingly, the current review was conducted to gather the current findings on the role of epigenetics in the pathophysiology and also treatment of MS.

Aberrant methylation and expression of various noncoding RNAs, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), confer a great potential as tumor markers. This study aimed to investigate miR-30b DNA methylation and metastasis associated lung adenocarcinoma transcript 1 (MALAT-1) expression patterns as potential diagnostic biomarkers for non-small cell lung cancer (NSCLC). In this cross-sectional study, miR-30b DNA methylation and MALAT-1 expression patterns were first explored using microarray data retrieved from the NSCLC dataset in the Cancer Genome Atlas (TCGA)-LUNG. Then, the obtained results were further validated in internal samples. Subsequently, genomic DNA was extracted and modified by sodium bisulfite to determine DNA methylation using q-MSP. Total RNA was extracted and transcribed to cDNA to measure transcription level by quantitative real-time polymerase chain reaction. GraphPad 6 Prism v.8 was used to perform the statistical analyses. Comparisons between groups in internal samples were conducted by paired student's t-test, while Mann-Whitney U test was used to analyze TCGA-LUNG data (P < 0.05). Our results indicated miR-30b hypermethylation, miR-30b downregulation and lncRNA MALAT-1 overexpression in NSCLC tumor samples compared with marginal normal samples. These changes were significantly associated with the stage of malignancy like lymph node metastasis. Also, using receiver operating characteristic curve analysis, MALAT-1 expression, and miR-30b methylation and expression patterns were found as possible diagnostic biomarkers for NSCLC (area under the curve was 0.70, 0.67, and 0.74, respectively). We found involvement of miR-30b hypermethylation and downregulation as well as lncRNA MALAT-1 overexpression with tumor outcomes of NSCLC patients.

Lipoprotein lipase (LPL) is a critical enzyme in lipid metabolism that hydrolyzes triglyceride-rich lipoproteins. While its role in mature adipose tissue is well understood, the epigenetic regulation of LPL during mesenchymal stem cell (MSC) differentiation into adipocytes remains poorly characterized. This study aimed to investigate the temporal expression pattern of LPL and its relationship with DNA methylation during adipogenic differentiation of human bone marrow-derived MSCs.

Human bone marrow MSCs were isolated and characterized using flow cytometry for specific surface markers (CD34, CD31, CD90, and CD105). Cells were differentiated into adipocytes over 14 days and osteoblasts over 21 days using specific differentiation media. Differentiation was confirmed through Oil Red O and Alizarin Red staining respectively. LPL gene expression was analyzed using both qualitative RT-PCR and quantitative real-time PCR at day 0 (undifferentiated) and day 14 (differentiated) timepoints. DNA methylation patterns were assessed using methylation-specific PCR (MSP) following bisulfite conversion, with collagen gene serving as an internal control.

Flow cytometry confirmed MSC identity through positive expression of CD166, CD13, CD105, and CD44. Successful adipogenic differentiation was demonstrated by Oil Red O-positive lipid droplet accumulation, while osteogenic differentiation was confirmed by Alizarin Red S staining of calcium deposits. LPL gene expression was absent in undifferentiated MSCs but showed significant expression in differentiated adipocytes at day 14, coinciding with morphological changes and lipid accumulation.

This study demonstrates that LPL expression is epigenetically regulated during MSC differentiation into adipocytes, with significant changes in both gene expression and DNA methylation patterns. The temporal correlation between LPL expression, methylation status, and adipogenic differentiation suggests that LPL serves as a key molecular switch in this process.

Colorectal cancer (CRC) is a common malignancy with high mortality. Despite advancements in understanding its molecular causes and improved drug therapies, patient survival rates remain low. The main reasons for the high mortality rate are cancer metastasis and the emergence of drug-resistant cancer cell populations. While genetic changes are recognized as the main driver of CRC occurrence and progression, recent studies suggest that epigenetic regulation is a crucial marker in cancer, influencing the interplay between genetics and the environment. Research has shown the significant regulatory roles of non-coding RNAs (ncRNAs) in CRC development. This review explores epigenetically regulated ncRNAs and their functions, aiming to understand key regulatory mechanisms that impact CRC development. Additionally, it discusses the potential use of these ncRNAs in CRC diagnosis, prognosis, and targeted treatments.

The Pediatric Buccal Epigenetic (PedBE) and Neonatal Epigenetic Estimator of Age (NEOage) clocks provide a novel method for assessing the biological age of young individuals, enhancing our comprehension of their health and development. By analyzing DNA methylation patterns, these clocks identify risk factors for various health conditions and guide personalized interventions to promote optimal growth in children and infants. With ongoing research and validation, PedBE and NEOage could revolutionize pediatric and neonatal healthcare by facilitating early detection of age-related changes and targeted interventions to improve long-term outcomes. In pediatric oncology, PedBE is particularly promising for evaluating biological age in children with cancer, as it accurately estimates DNA methylation age in buccal cells, revealing the effects of cancer and its treatments on biological aging. Additionally, PedBE can detect DNA methylation changes associated with environmental exposures and childhood adversities, making it a valuable tool for studying the impact of cancer on the epigenetic age of pediatric patients. The NEOage clock, designed to predict gestational age in newborns, complements the PedBE clock, offering a comprehensive assessment of biological age from infancy to adolescence, which is vital for understanding pediatric oncology’s influence on aging. This paper examines the complexities of both clocks, highlighting their potential for accurately determining the age of children and infants through DNA methylation analysis.

Lung cancer is the most lethal malignancy in the world due to its poor prognosis. DNA methylation change has been identified as a valuable target for cancer, diagnosis, and prognosis. Ferritin heavy chain 1 (FTH-1) and SHOX homeobox 2 (SHOX2) DNA methylation were investigated in non-small cell lung cancer (NSCLC) as novel epigenetic biomarkers. In this case-control study, we initially evaluated the diagnostic value of FTH-1 and SHOX2 DNA methylation, and the Cancer Genome Atlas (TCGA) data on the methylation profile of NSCLC was analyzed. Whole DNA was extracted and bisulfite modification was performed. Then, the methylation status of FTH-1 and SHOX2 was evaluated using quantitative methylation specific polymerase chain reaction (PCR) (qMSP). We used GraphPad Prism version 6.00 program for statistical analysis. Mann-Whitney U test (TCGA-LUNG), paired t-test (internal samples) and receiver operating characteristic (ROC) curve analysis were used to evaluate the statistical differences of DNA methylation between NSCLC tissues samples and adjacent normal specimens (P < 0.05, mean ± SD). TCGA and q-MSP results showed significant FTH-1 hypomethylation and SHOX2 hypermethylation in NSCLC tissues in comparison with margin specimens. Also, FTH-1 and SHOX2 methylation levels were significantly associated with the clinical stage of malignancy. Furthermore, The ROC curve analysis revealed that the area under the curve values for FTH-1 and SHOX2 were determined to be 0.751 and 0.8676, respectively. This indicates the importance of FTH-1 and SHOX2 as diagnostic biomarkers for NSCLC. This study indicates that FTH-1 and SHOX2 methylation could be promising targets for liquid biopsy application of lung cancer.

Aggressive periodontitis is an inflammation of the periodontal tissue that usually affects adolescents and young adults aged <30 years, caused by attachment loss and fast bone degradation. The correlation between the epigenetic status and the initiation and progression of numerous acquired diseases was documented. Consequently, targeting epigenetic factors within periodontal tissues stands as an appealing prospect for both the diagnosis and treatment of periodontitis. In addition to the role of pathogenic bacteria and their products,  alterations in gene expression due to extrinsic and intrinsic factors can cause disturbances in the host’s immune response. Epigenetic changes, whether DNA methylation or microRNA (miRNA) dysregulation, can cause changes in gene expression in aggressive periodontitis and lead to more severe and rapid loss of the periodontal tissues. This study aimed to elucidate the relationships between oral hygiene, pathogenic bacteria, and genetics in periodontitis development to promote targeted prevention and treatment for enhanced oral health in individuals at risk of aggressive periodontitis. The method employed in this study entailed a comprehensive review and analysis of scholarly literature on the relationship between epigenetic mechanisms and the development of aggressive periodontitis. In conclusion, epigenetic regulation plays an important role in the pathogenesis of periodontitis through DNA methylation mechanisms that begin with Toll-like receptors (TLRs), cytokine signaling pathways, promoter genes, and progress to pro-inflammatory cells. When periodontal tissue inflammation occurs, miRNA inhibits protein translation from messenger ribonucleic acid (mRNA), which contributes to its aggressiveness.

Changes in the expression of nucleotide-binding oligomerization domain containing 2 (NOD2) play an important role in the pathogenesis of a variety of autoimmune diseases including inflammatory bowel diseases (IBDs). Epigenetic modifications, including DNA methylation, are considered an important mechanism in the suppression of gene activity. In this study, we investigated the relationship between DNA methylation patterns of the promoter region of the NOD2 gene and the pathogenesis of Crohn’s disease (CD).

Colonic mucosa samples were obtained from 15 Iranian patients with IBD and 15 age- and sex-matched healthy controls with no history of autoimmune disease. After the bisulfite conversion of genomic DNA, the DNA methylation status of three CpG sites in the promoter region of the NOD2 gene was determined by the real-time quantitative multiplex methylation-specific PCR (QM-MSP) assay.

Using this approach, we identified that IBD patients showed a decreased level of methylation of the NOD2 promoter in the colonic mucosa than did the healthy controls. (Unmethylated DNA in Crohn's disease vs. healthy controls; 0.128±0.093 vs. 0.025±0.016, P<0.000).

According to our findings, promoter hypomethylation of the NOD2 gene in the colonic mucosa might contribute to the development and severity of CD. Furthermore, aberrant DNA methylation levels are expected to serve as a clinically useful risk marker.

A systematic review was conducted to summarize the methylated circulating tumor DNA (ctDNA) markers reported over the last decade for early detection of colorectal cancer (CRC) and to identify the main technical challenges that are impeding their clinical implementation.

CRC is a major cause of cancer deaths worldwide, but early detection is key for successful treatment. Non-invasive methods such as methylated ctDNA testing show promise for improving detection and monitoring of CRC.

A comprehensive search was performed using Web of Science, PubMed, and Scopus up to December 30, 2023, limited to articles published in the last 10 years (after 2012), while including advanced adenoma/stage 0 or stage I/II samples in biomarker validation.

After identifying 694 articles, removing duplicates and screening titles, abstracts, and full texts, a total of 62 articles were found to meet the inclusion criteria. Among the single biomarkers, MYO1-G, SEPT9, SDC2, and JAM3 revealed the highest sensitivity for polyps and stage I/II CRC. For multi-biomarkers with suitable sensitivity, combinations of SFRP1, SFRP2, SDC2, PRIMA1, or ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9, VIM or ZFHX4, ZNF334, ELOVL2, UNC5C, LOC146880, SFMBT2, GFRA1 were identified for polyps and stage I/II CRC.

Enhancing sensitivity and specificity of molecular screening methods is crucial for improving CRC detection. Identifying a select few valuable biomarkers is key to reducing costs, despite challenges posed by low ctDNA levels in plasma, particularly in early-stage cancers.

Children born with low birth weight (LBW) often face an increased risk of developing cardiovascular disease (CVD) earlier in life, but the reasons behind this connection aren't fully clear. This study seeks to explore how being born with LBW might lead to heart problems down the road, with a focus on the subtle changes in our genes and the role of tiny molecules called microRNAs (miRNAs). We believe that certain genes involved in heart and blood vessel health might be altered by difficult conditions in the womb, making LBW individuals more vulnerable to CVD. Interestingly, we also propose that miRNAs might step in to protect these individuals during childhood, though this protective effect seems to fade as they get older, leading to early signs of heart disease. By identifying these key miRNAs and understanding their role, we hope to discover new ways to intervene early, possibly during adolescence, to prevent or delay heart issues. This research could lead to more personalized approaches to healthcare, helping those born with LBW live healthier lives by addressing their unique risks sooner.

Aberrant DNA methylation is a key epigenetic alteration observed in multiple cancers. Acute myeloid leukemia (AML), a prominent form of hematopoietic cancer, is characterized by abnormal proliferation and differentiation of myeloid progenitor cells. This study focuses on examining the methylation status of the CpG islands in the DNMT1 and CDX2 promoter regions and exploring their correlation with prognostic hematological laboratory parameters across three phases of AML: newly diagnosed, undergoing treatment, and in remission.

This follow-up case-control study recruited 11 new cases of confirmed AML admitted to Shariati Hospital in Tehran. All patients received AML treatment according to FDA protocol. The samples (peripheral blood) were collected before medication (new case phase), during medication (under treatment phase), and in the remission phase. Then, genomic DNA was extracted and treated with the bisulfite treatment method. Then, methylation-specific PCR (MSP) was conducted to amplify treated DNAs using two methylated and unmethylated primers related to their promoters' DNMT1 and CDX2 CpG- islands. All statistical analysis was performed using SPSS v.25.

The results of the methylation pattern of DNMT1 gene promoter CpG islands in the present study show that the hemimethylated pattern of the DNMT1 gene promoter is predominant in control (100%), new case phase (90.9%), under treatment phase (72.7%), and remission phase (100%). In the case of the CDX2 gene, the unmethylated pattern is predominant in control (57.14%), new case phase (72.7%), under-treatment phase (90.9%), and remission phase (81.8%). These differences were not statistically significant. No methylated pattern was observed in the control group, and different phases of AML were used for DNMT1 and CDX2. Also, the methylation status of DNMT1 and CDX2 were not correlated with prognostic hematological laboratory parameters.

The methylation patterns of CDX2 and DNMT1 are not different in healthy individuals and AML patients, as well as in different phases of AML. Also, the methylation patterns of CDX2 and DNMT1 cannot help determine the prognosis of AML patients through changes in hematological laboratory parameters.

Numerous suspect genes associated with type 1 diabetes mellitus (T1DM) have been identified, suggesting a need to focus on the disease's causal genes and mechanisms. This necessitates an update to raise public awareness. This review articulates genes with mutations that predispose individuals to T1DM. We conducted a comprehensive search of academic databases, including Web of Science, Scopus, PubMed, and Google Scholar, for relevant materials. Available information indicates that at least 70 genes are suspected in the pathogenesis of T1DM. However, the most frequently implicated genes include human leukocyte antigen (HLA), insulin (INS), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and protein tyrosine phosphatase non-receptor type 22 (PTPN22). Mutations or variants in these genes may lead to insulin insufficiency and, consequently, T1DM by tricking immune cells, such as T-cells and B-cells, into attacking self-antigens and triggering the autoimmunity of beta cells. Furthermore, this pathophysiology can be mediated through aberrant epigenetic modifications, including DNA methylation, histone post-translational modifications, and non-coding RNAs, in the mentioned genes. Some of these pathophysiologies are gene-specific and may have an epigenetic origin that is reversible. In the event of an epigenetic origin, a treatment for T1DM that addresses the causal genes or reverses epigenetic changes and their mechanisms could yield improved outcomes. Medical professionals are encouraged to design therapeutic regimens that specifically target the mentioned genes and address the identified epigenetic alterations in individuals expressing such etiologies.

Ankylosing spondylitis (AS) is a chronic autoimmune disorder characterized by the fusion of vertebral joints and axial arthritis. The programmed death-1 (PD-1) inhibitory receptor has a pivotal role in controlling T cell function and may have a significant impact on the pathogenesis of autoimmune diseases such as AS pathogenesis.

To investigate PD-1 gene expression and its epigenetic regulation by detecting methylated CpG islands in the regulatory sites of the gene. This will provide insight into the mechanisms involved in the disease.

30 AS patients and 30 healthy individuals were examined to detect the 16 CpG islands in intron 1 using bisulfite conversion and methylation-specific PCR technique. In addition, RNA samples were isolated from fresh peripheral blood mononuclear cells (PBMCs), and after complementary DNA (cDNA) synthesis, the expression level of the PD-1 gene was evaluated using Real-Time PCR.

The CpG islands located in the intronic zone of the PD-1 gene were hyper-methylated in both the patients with AS and the healthy controls. The gene expression of PD-1 was significantly downregulated in AS patients compared with the controls (p=0.017). A negative correlation between the Bath Ankylosing Spondylitis Disease Activity Index and PD-1 gene expression was also revealed.

The low level of PD-1 gene expression is implicated in the pathogenesis of AS. However, in both groups, the methylation level of the intron 1 CpG islands of the PD-1 gene suggests that other regulatory mechanisms are more relevant to PD-1 gene expression than methylation in the intron.

 Epigenetics is crucial in differentiating mesenchymal stem cells (MSCs) into adipocytes. Specifically, DNA methylation, an epigenetic modification, regulates the expression of genes involved in this process. The peroxisome proliferator-activated receptor gamma (PPARγ) gene is a critical player in adipocyte differentiation, with epigenetic changes affecting its expression.

We isolated mesenchymal stem cells (MSCs) from the human bone marrow. The isolated MSCs were expanded and cultured in a differentiation medium for two weeks. DNA extraction was performed on undifferentiated and differentiated adipocytes after the culturing process. The methylation status of the promoter region of the PPARγ gene was assessed using methylation-specific primers (M for methylated and U for unmethylated) in a methylation-specific PCR (MSP) assay. This analysis involved the treatment of DNA samples with sodium bisulfite to convert unmethylated cytosine to uracil, thereby enabling the differentiation between methylated and unmethylated regions of the gene.

 The successful differentiation of MSCs into adipocytes was confirmed by the accumulation of lipid droplets within the differentiated cells, as visualized by the oil Red O dye staining. This observation provides strong evidence of the commitment of MSCs towards the adipogenic lineage and their ability to undergo adipocyte differentiation. Surprisingly, the MSP analysis revealed no significant changes in the methylation pattern of this gene following differentiation. The PPARγ gene promoter region exhibited an unmethylated status in both undifferentiated and differentiated states.

Our study revealed that additional genetic or epigenetic mechanisms control the expression of PPARγ during the adipogenic differentiation of mesenchymal stem cells. These findings highlight the regulatory role of PPARγ in the differentiation pathway from mesenchymal stem cells to adipocytes.