DNA methylation, one of the most studied epigenetic mechanisms, when present in the promoter region of genes, causes inhibition of gene expression, and conversely, hypomethylation of these regions enables gene expression. DNA methylation is susceptible to nutritional and environmental influences, and undesirable alterations in methylation patterns manifested in changes in the expression of relevant genes can lead to pathological consequences. In the present work, we studied the methylation status of the bovine GSTP1 gene under the influence of pesticide Mospilan 20SP alone and in combination with pesticide Orius 25EW in in vitro proliferating bovine lymphocytes. We employed methylation-specific PCR, and when studying the effect of pesticide combinations, we also used its real-time version followed by a melting procedure. Our results showed that Mospilan 20SP alone at 5, 25, 50, and 100 µg.ml-1 and 5, 10, 25, and 50 µg.ml-1 for the last 4 and 24 hours of culture with in vitro proliferating bovine lymphocytes, respectively, did not induce methylation of the bovine GSTP1 gene. The same results were revealed when studying the effect of the combination of the pesticides added to the lymphocyte cultures for the last 24 hours of cultivation in the following amounts: 1.25, 2.5, 5, 10, and 25 µg.ml-1 of Mospilan 20SP and 1.5, 3, 6, 15, and 30 µg.ml-1 of Orius 25EW. We have also revealed that the less laborious real-time MSP followed by a melting procedure may replace MSP for studying the methylation status of the GSTP1 gene.

Background One of the most prevalent aberrant epigenetic modifications found in hepatocellular carcinoma (HCC) is abnormal DNA methylation. Our study aimed to evaluate serum Ras association domain family 1A (RASSF1A) gene promoter methylation in patients with chronic viral hepatitis C (HCV)-associated liver cirrhosis with and without HCC as a potential new marker for the early detection of HCC. Methodology The 60 participants who participated in the trial were divided into the following three groups: 20 patients with newly diagnosed primary HCC on top of HCV-related liver cirrhosis, 20 patients with HCV-related liver cirrhosis, and 20 age- and sex-matched healthy individuals as a control group. All participants underwent methylation-specific polymerase chain reaction testing to detect the blood level of the RASSF1A gene\'s methylated promoter. Results Methylated RASSF1A was found in 30% of patients with liver cirrhosis caused by HCV and in 65% of patients with HCC, but not in any of the controls. It was discovered that the serum methylation RASSF1A had an accuracy of 82.50% and an area under the curve (AUC) of 0.825 for separating HCC patients from healthy controls. With an AUC of 0.675 and an accuracy of 67.50%, it was able to differentiate patients with HCC from those with HCV-related liver cirrhosis. Additionally, there was no statistically significant association between RASSF1A methylation status and HCC mass size (p = 0.449). Conclusions Serum RASSF1A promoter methylation status detection could be useful for detecting HCC early, especially in high-risk individuals such as those with HCV.

The hypermethylation status of the promoter region of the breast cancer 1 (BRCA1), a well-known tumor suppressor gene, has been extensively investigated in the last two decades as a potential biomarker for breast cancer. In this retrospective study, we investigated the prevalence of BRCA1 promoter methylation in 84 human breast tissues, and we correlated this epigenetic silencing with the clinical and histopathological parameters of breast cancer. We used methylation-specific PCR (MSP) to analyze BRCA1 promoter hypermethylation in 48 malignant breast tumors (MBTs), 15 normal adjacent tissues (NATs), and 21 benign breast lesions (BBLs). The results showed that BRCA1 promoter hypermethylation was higher in MBTs (20/48; 41.67%) and NATs (7/15; 46.67%) compared to BBLs (4/21; 19.05%). The high percentage of BRCA1 hypermethylation in the histologically normal adjacent tissues to the tumors (NATs) suggests the involvement of this epigenetic silencing as a potential biomarker of the early genomic instability in NATs surrounding the tumors. The detection of BRCA1 promoter hypermethylation in BBLs reinforces this suggestion, knowing that a non-negligible rate of benign breast lesions was reported to evolve into cancer. Moreover, our results indicated that the BRCA1 promoter hypermethylated group of MBTs exhibited higher rates of aggressive features, as indicated by the SBR III grade (14/19; 73.68%), elevated Ki67 levels (13/16; 81.25%), and Her2 receptor overexpression (5/20; 25%). Finally, we observed a concordance (60%) in BRCA1 promoter hypermethylation status between malignant breast tumors and their paired histologically normal adjacent tissues. This study highlights the role of BRCA1 promoter hypermethylation as a potential useful biomarker of aggressiveness in MBTs and as an early marker of genomic instability in both histological NATs and BBLs.

UNASSIGNED: Autoimmune hemolytic anemia (AIHA) is caused by auto-antibodies, secreted by overactivated B cells, directed against self-red blood cells, resulting in hemolysis. It found that aberrant DNA methylation in B cells can induce the production of autoantibodies. Therefore, we attempted to explore if similar aberrant DNA methylation occur in AIHA patients.
UNASSIGNED: A 49-year-old female wAIHA patient and a 47-year-old female healthy control (HC) were enrolled. Peripheral blood (PB) B cells DNA was extracted. After constructing genomic libraries, bisulfite genomic sequencing (BSP) and DNA methylation profiles were analyzed. BSP was verified using PB B cells from 10 patients with hemolysis, 10 patients with hemolytic remission, and 10 healthy controls (HCs) by Methylation-specific PCR.
UNASSIGNED: Total DNA methylation of whole-genome C bases (4.8%) and CG type bases (76.8%) in wAIHA patient were lower than those in the HC (5.3 and 82.5%, respectively) (p = 0.022 and p < 0.001). DNA methylation of C bases and CG type bases in whole-genome regulatory elements, such as coding sequence, up2Kb and down2Kb in the patient were also lower than those in the HC (p = 0.041, p = 0.038, and p = 0.029). 30,180 DNA-methylated regions (DMRs) on all 23 chromosomes were identified. DMR-related genes were mainly involved in the Rap1, phospholipase D, HIF-1, calcium, vascular endothelial growth factor (VEGF) and Ras signaling pathways.
UNASSIGNED: The DNA methylation spectrum of B cells in AIHA patients is different from that of HC, and the proportion of hypo-methylation regions is higher than that of HC. DMR-related genes are mainly related to some signaling pathways.

Glioblastoma (GBM) remains a treatment-resistant malignant brain tumor in large part because of its genetic heterogeneity and epigenetic plasticity. In this study, we investigated the epigenetic heterogeneity of GBM by evaluating the methylation status of the O6 -methylguanine methyltransferase (MGMT) promoter in individual clones of a single cell derived from GBM cell lines. The U251 and U373 GBM cell lines, from the Brain Tumour Research Centre of the Montreal Neurological Institute, were used for the experiments. To evaluate the methylation status of the MGMT promoter, pyrosequencing and methylation-specific PCR (MSP) were used. Moreover, mRNA and protein expression levels of MGMT in the individual GBM clones were evaluated. The HeLa cell line, which hyper-expresses MGMT, was used as control. A total of 12 U251 and 12 U373 clones were isolated. The methylation status of 83 of 97 CpG sites in the MGMT promoter were evaluated by pyrosequencing, and 11 methylated CpG sites and 13 unmethylated CpG sites were evaluated by MSP. The methylation status by pyrosequencing was relatively high at CpG sites 3-8, 20-35, and 7-83, in both the U251 and U373 clones. Neither MGMT mRNA nor protein was detected in any clone. These findings demonstrate tumor heterogeneity among individual clones derived from a single GBM cell. MGMT expression may be regulated, not only by methylation of the MGMT promoter but by other factors as well. Further studies are needed to clarify the mechanisms underlying the epigenetic heterogeneity and plasticity of GBM.

DNA methylation of promoter CpG islands silences their downstream genes, and enhancer methylation can be associated with decreased or increased gene expression. DNA methylation alterations in normal and diseased cells provide rich information, such as tissue origin, disease risk, patient response, and prognosis. DNA methylation status is detected by bisulfite conversion, which converts unmethylated cytosines into uracils but methylated cytosines very inefficiently. A genome-wide DNA methylation analysis is conducted by a BeadChip microarray or next-generation sequencing (NGS) of bisulfite-treated DNA. A region-specific DNA methylation analysis can be conducted by various methods, such as methylation-specific PCR (MSP), quantitative MSP, and bisulfite sequencing. This chapter provides protocols for bisulfite-mediated conversion, a BeadChip array-based method (Infinium), quantitative MSP, and bisulfite sequencing.

Objective. Nowadays, type 2 diabetes mellitus (T2D) is the most common chronic endocrine disorder affecting an estimated 5-10% of adults worldwide, and this disease also rapidly increased among the population in the Kurdistan region. This research aims to identify DNA methylation change in the TCF7L2 gene as a possible predictive T2D biomarker. Methods. One hundred and thirteen participants were divided into three groups: diabetic (47), prediabetic (36), and control (30). The study was carried out in patients who visited the private clinical sector between August and December 2021 in Koya city (Iraq Kurdistan region) to determine DNA methylation status using a methylation-specific PCR (MSP) with paired primers for each methylated and non-methylated region. In addition, the X2 Kruskal-Wallis statistical and Wilcoxon signed-rank tests were used, p<0.05 was considered significant. Results. The results showed hypermethylation of DNA in the promoter region in diabetic and prediabetic groups compared to the healthy controls. Different factors affected the DNA methylation level, including body max index, alcohol consumption, family history, and physical activity with the positive Coronavirus. Conclusion. The results obtained indicate that DNA methylation changes in the TCF7L2 promoter region may be used as a potential predictive biomarker of the T2D diagnosis. However, the findings obtained in this study should be supported by additional data.

The function of ABC transporters in the body is manifold; such as maintenance of homeostasis, effect on multi-drug resistance and their role in tumor initiation & progression. Evidence pointing towards the direct or indirect role of ABC transporter genes in particular; ABCB1 and ABCG2 in cancer genesis is increasing. However, their role in gallbladder cancer is unexplored. Therefore, we investigated the methylation status and expression pattern of ABCB1 and ABCG2in gallbladder carcinogenesis. The methylation and expression study of ABCB1/MDR1 and ABCG2/BCRP was performed in tumour and normal fresh tissue samples collected from 61 histopathologically diagnosed gallbladder cancer patients. The methylation status was analysed by Methylation-Specific PCR and expression was determined by Real-Time PCR and Immunohistochemistry. Hypomethylation of ABCB1 and ABCG2 was found in 44 (72.13%) and 48 (78.6%) cases, respectively. ABCB1 hypomethylation pattern showed association with female patients (p = 0.040) and GradeII tumors (p = 0.036) while, ABCG2 hypomethylation was more frequent in early tumors (T1-T2). The mRNA expression ofABCB1 and ABCG2 was up-regulated in 33 (54.10%) and 41 (67.21%) patients with fold change of 4.7 and 5.5, respectively. The mRNA expression of both genes showed association with Grade II tumours and the increased fold change of ABCG2 was higher in (T1-T2) depth of invasion (p = 0.02) and Stage I-II disease (p = 0.08). The protein expression on IHC was strongly positive for ABCB1/MDR1and ABCG2/BCRP in 32 (52.46%) and 45 (73.77%) patients, respectively. The protein expression in ABCG2 showed association with patients age > 50 years (p = 0.04) and GradeII differentiation (p = 0.07). Interestingly, the hypomethylation of both the genes showed significant correlation with increased expression. ABCB1/MDR1 and ABCG2/BCRP hypomethylation and overexpression could have a potential role in gallbladder cancer tumorigenesis especially in early stages. The epigenetic change might be a plausible factor for altered gene expression of ABCB1 and ABCG2 in gallbladder cancer.

BACKGROUND: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, caused by CGG-repeats expansion (> 200 repeats). Premutation alleles (PM) (55-200 CGG repeats) are associated with tremor ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), and autistic problems.
OBJECTIVE: To screen the frequency of premutation carriers using molecular diagnostic assays, in a cohort of Egyptian males with suspected clinical features of (FXS) checking for the presence of premutation alleles.
METHODS: The current study comprised 192 Egyptian male children, 92 participants presented with intellectual disability, delayed language development, autistic-like features, behavioral difficulties, anxiety, seizures, and depression compared to 100 healthy males. All cases were subjected to clinical and neuroimaging assessments, when indicated as well as molecular analysis using methylation-specific PCR (MS-PCR) and quantitative real-time PCR (qRT-PCR).
RESULTS: Thirty-four premutation carriers out of 92 Egyptian males (37%) of CGG repeats (55 to 200) were illustrated with elevated FMR1 mRNA expression level (p-value < 0.001). Additionally, 2 intermediate (IM) cases (0.03%) (45-55 CGG repeats) showed poor increase in expression level (p-value = 0.02838) plus 6 full mutation (FM) patients (0.07%) with (> 200 CGG repeats) (p-value < 0.001) resulted in FMR1 gene silence.
CONCLUSIONS: Molecular diagnostic assay including (MS-PCR) and (qRT-PCR) proved to be a sensitive and rapid screening tool for the detection of premutation cases. Furthermore, the presence of positive correlation between FMR1 mRNA expression levels with CGG repeats in premutation cases could serve as a potential diagnostic marker. Application of these diagnostic tools on larger number clinically suspected cases is recommended.

RAD21 plays multiple roles in numerous cancers. In breast cancer (BC), a high level of RAD21 correlates with poor disease outcomes and resistance to chemotherapy. However, data regarding RAD21 promoter methylation in BC tissue and its correlation with clinical outcomes in patients with BC remain limited. Here, we investigated the clinicopathological features associated with the methylation status of RAD21 in BC to figure out its possible role in pathogenesis and the formation of breast carcinogenesis. The methylation status of the RAD21 gene was significantly associated with better clinical outcomes in patients with BC.