Methylation-specific PCR Method Research Articles

Hypermethylation at 45S rDNA promoter in cancers.

The ribosomal genes (rDNA genes) encode 47S rRNA which accounts for up to 80% of all cellular RNA. At any given time, no more than 50% of rDNA genes are actively transcribed, and the other half is silent by forming heterochromatin structures through DNA methylation. In cancer cells, upregulation of ribosome biogenesis has been recognized as a hallmark feature, thus, the reduced methylation of rDNA promoter has been thought to support conformational changes of chromatin accessibility and the subsequent increase in rDNA transcription. However, an increase in the heterochromatin state through rDNA hypermethylation can be a protective mechanism teetering on the brink of a threshold where cancer cells rarely successfully proliferate. Hence, clarifying hypo- or hypermethylation of rDNA will unravel its additional cellular functions, including organization of genome architecture and regulation of gene expression, in response to growth signaling, cellular stressors, and carcinogenesis. Using the bisulfite-based quantitative real-time methylation-specific PCR (qMSP) method after ensuring unbiased amplification and complete bisulfite conversion of the minuscule DNA amount of 1 ng, we established that the rDNA promoter was significantly hypermethylated in 107 breast, 65 lung, and 135 colon tumour tissue samples (46.81%, 51.02% and 96.60%, respectively) as compared with their corresponding adjacent normal samples (26.84%, 38.26% and 77.52%, respectively; p < 0.0001). An excessive DNA input of 1 μg resulted in double-stranded rDNA remaining unconverted even after bisulfite conversion, hence the dramatic drop in the single-stranded DNA that strictly required for bisulfite conversion, and leading to an underestimation of rDNA promoter methylation, in other words, a faulty hypomethylation status of the rDNA promoter. Our results are in line with the hypothesis that an increase in rDNA methylation is a natural pathway protecting rDNA repeats that are extremely sensitive to DNA damage in cancer cells.

Discovery and validation of colorectal cancer tissue-specific methylation markers: a dual-center retrospective cohort study

Background and purposeEarly detection, diagnosis, and treatment of colorectal cancer and its precancerous lesions can significantly improve patients’ survival rates. The purpose of this research is to identify methylation markers specific to colorectal cancer tissues and validate their diagnostic capability in colorectal cancer and precancerous changes by measuring the level of DNA methylation in stool samples.MethodWe analyzed samples from six cancer tissues and adjacent normal tissues and fecal samples from 758 participants, including 62 patients with interfering diseases. Bioinformatics databases were used to screen for candidate biomarkers for CRC, and quantitative methylation-specific PCR methods were applied for identification. The methylation levels of the candidate biomarkers in fecal and tissue samples were measured. Logistic regression and random forest models were built and validated using fecal sample data from one of the centers, and the independent or combined diagnostic value of the candidate biomarkers in fecal samples for CRC and precancerous lesions was analyzed. Finally, the diagnostic capability and stability of the model were validated at another medical center.ResultsThis study identified two colorectal cancer CpG sites with tissue specificity. These two biomarkers have certain diagnostic power when used individually, but their diagnostic value for colorectal cancer and colorectal adenoma is more significant when they are used in combination.ConclusionThe results indicate that a DNA methylation biomarker combined diagnostic model based on two CpG sites, cg13096260 and cg12587766, has the potential for screening and diagnosing precancerous lesions and colorectal cancer. Additionally, compared to traditional diagnostic models, machine learning algorithms perform better but may yield more false-positive results, necessitating further investigation.

PDX1 Methylation, NGN3 and Pax6 Expression Levels in Pregnant Women with Gestational Diabetes Mellitus and their association with neonatal blood sugars and birth weight.

To investigate the correlation of maternal PDX1 methylation, NGN3 and Pax6 expression levels with neonatal blood sugars and birth weight in pregnant women with GDM and non GDM. This was a prospective cohort study. Total 80 pregnant women who were examined and delivered in the Department of Obstetrics of Affiliated Hospital of Hebei University from January 2019 to June 2022 were recruited and divided into two groups according to the results of oral glucose tolerance test (OGTT): the control group and the observation group, with 40 cases in each group. PDXl methylation rate was measured by the methylation-specific PCR method, whereas gene expression levels of PDX1, NGN3 and Pax6 were measured by RT-PCR meanwhile, neonatal blood glucose and hemoglobin A1c (HbA1c) levels were also measured. In comparison with the control group, the observation group had higher levels of FBG, 2-hour postprandial blood glucose (2hPBG) and HbA1c (P<0.05). Neonatal birth weight and insulin levels in the observation group were significantly higher than those in the control group, while Apgar scores and blood glucose were lower than those in the control group(P<0.05). Moreover, the observation group had significantly lower gene expression levels of PDX1, NGN3 and Pax6, and a higher PDX1 methylation rate than the control group (P<0.05). Correlation analysis revealed a negative correlation between neonatal blood glucose levels and PDX1, NGN3 and Pax6 levels in the observation group, with statistical significance (P<0.05). Changes in maternal PDX1 methylation, NGN3 and Pax6 expression levels may lead to abnormal glucose metabolism in neonates, which has a close bearing on neonatal hypoglycemia and blood glucose levels caused by GDM.

Effects of 5-Fluorouracil on the Expression of Epigenetic Enzymes and Promoter Methylation of Selected Genes in Monolayer and Spheroid Cultures of Colorectal Cancer Cells

Background: Emerging evidence suggests that epigenetic mechanisms contribute to 5-fluorouracil (5-FU) resistance in colorectal cancer (CRC). However, there is limited research on the direct impact of 5-FU on epigenetic alterations in CRC. This study aimed to investigate how 5-FU treatment affects the expression of enzymes involved in epigenome regulation and promoter DNA methylation in human CRC cells. Methods: The viability of CRC cell lines (SW48, HCT116, LS180, and HT29) was evaluated after 48 hours of 5-FU treatment using MTT assay in both monolayer and hanging drop spheroid cultures. The cells were treated with an IC20 concentration of 5-FU and then the relative expressions of histone deacetylases (HDAC) and DNA methyltransferas1 (DNMT1) in 5-FU-treated and untreated cells were measured by quantitative RT-PCR (qRT-PCR). The status of promoter methylation of selected genes was analyzed using the methylation-specific PCR (MSP) method. Results: The 3D cultures of cells were more resistant to 5-FU than their 2D counterparts. The effect of 5-FU on HDAC1 expression was greater in 3D cultures compared to 2D cultures. 5-FU downregulated SIRT1 and DNMT1 in 2D culture of HCT116 and SW48 and upregulated them in 3D cultures of HT29 and LS180 cells. In both monolayer and spheroid cultures, 5-FU downregulated HDAC2 in HCT116, LS180, and HT29 and HDAC4 in HCT116, LS180, and SW48 cells. 5-FU primarily changed promoter methylation in monolayer cultures. Conclusion: The epigenetic response to 5-FU is cell line-specific and depends on the culture method. 5-FU modulates epigenome in CRC cells by regulating DNMT1 and HDAC expressions. 3D cultures were found to be considerably more resistant to 5-FU-induced cytotoxicity and promoter DNA methylation changes than 2D cultures. 5-FU downregulated HDAC and DNMT1, particularly in the drug-sensitive cells, and increased the levels of DNMT1 in the drug-resistant cells.

Evaluating Gene Expression and Methylation Profiles of TCF4, MBP, and EGR1 in Peripheral Blood of Drug-Free Patients with Schizophrenia: Correlations with Psychopathology, Intelligence, and Cognitive Impairment.

Discovery and validation of new, reliable diagnostic and predictive biomarkers for schizophrenia (SCZ) are an ongoing effort. Here, we assessed the mRNA expression and DNA methylation of the TCF4, MBP, and EGR1 genes in the blood of patients with SCZ and evaluated their relationships to psychopathology and cognitive impairments. Quantitative real-time PCR and quantitative methylation-specific PCR methods were used to assess the expression level and promoter DNA methylation status of these genes in 70 drug-free SCZ patients and 72 healthy controls. The correlation of molecular changes with psychopathology and cognitive performance of participants was evaluated. We observed downregulation of TCF4 and upregulation of MBP mRNA levels in SCZ cases, relative to controls in our study. DNA methylation status at the promoter region of TCF4 demonstrated an altered pattern in SCZ as well. Additionally, TCF4 mRNA levels were inversely correlated with PANSS and Stroop total errors and positively correlated with WAIS total score and working memory, consistent with previous studies by our group. In contrast, MBP mRNA level was significantly positively correlated with PANSS and Stroop total errors and inversely correlated with WAIS total score and working memory. These epigenetic and expression signatures can help to assemble a peripheral biomarker-based diagnostic panel for SCZ.

DNA methylation of KIFC1 gene in determination of histological diagnosis, prognosis and metastasis of lung cancer

BackgroundOne of the main features of cancer, especially lung cancer (LC), is abnormal cell division. Abnormal expression of kinesin family member C1 (KIFC1/HSET), which is involved in mitotic cell division and ensures equatorial alignment of chromosomes during division, is observed in both premalignant and malignant lesions. There are no studies in the literature addressing the role of KIFC1 in the diagnosis and follow-up of LC. In this study, we investigated the epigenetic role of KIFC1 in the diagnosis, stage, and prognosis of various histological subtypes diagnosed with LC. Material and MethodsThe expression and methylation status of the KIFC1 gene were examined after DNA/RNA isolation in tumor, conjugate normal tissue, and blood samples from 39 patients diagnosed with LC and in blood samples from 39 healthy controls. Changes in KIFC1 gene expression were examined by the Quantitative Real Time-PCR (qRT-PCR) method after cDNA synthesis following RNA isolation. The Methylation-Specific PCR (MSP) method was used to determine the methylation status of the KIFC1 gene. In this study, the expression/methylation profiles of the KIFC1 gene and the clinical and pathological characteristics of the patients were analyzed by statistical methods. ResultHypomethylation was detected in 95.8% of the 62.1% of patients’ tissues with increased KIFC1 gene expression. The expression level of the KIFC1 gene was found to be increased 3.2-fold in the tumor tissues of the patients compared with the conjugated normal tissues and 2.4-fold in the serum of the patients compared with the healthy serum. Statistical comparison of patients' clinical parameters and methylation and expression results revealed statistical significance between KIFC1 expression and metastasis, tumor stage and tumor grade. ConclusionIn conclusion, the increase in the expression level of the KIFC1 gene is higher in patients diagnosed with LC than in the healthy population, and therefore, the increase in the expression level of the KIFC1 gene due to hypomethylation can be used as a screening biomarker in LC. It can also be considered that the methylation profile of the KIFC1 gene may be a potential biomarker for determining the subtype of squamous cell carcinoma in LC. The results of the study need to be analyzed and continued with a larger number of patients.

Novel DNA methylation biomarkers in stool and blood for early detection of colorectal cancer and precancerous lesions

BackgroundEarly detection and prevention of precancerous lesions can significantly reduce the morbidity and mortality of colorectal cancer (CRC). Here, we developed new candidate CpG site biomarkers for CRC and evaluated the diagnostic value of their expression in blood and stool samples of CRC and precancerous lesions.MethodsWe analyzed 76 pairs of CRC and adjacent normal tissue samples, 348 stool samples, and 136 blood samples. Candidate biomarkers for CRC were screened using a bioinformatics database and identified using a quantitative methylation-specific PCR method. The methylation levels of the candidate biomarkers were validated using blood and stool samples. The divided stool samples were used to construct and validate a combined diagnostic model and to analyze the independent or combined diagnostic value of candidate biomarkers in stool samples of CRC and precancerous lesions.ResultsTwo candidate CpG site biomarkers for CRC, cg13096260 and cg12993163, were identified. Although both biomarkers demonstrated diagnostic performance to a certain extent when using blood samples, they showed better diagnostic value for different stages of CRC and AA with stool samples.Conclusionscg13096260 and cg12993163 detection in stool samples could be a promising approach for screening and early diagnosis of CRC and precancerous lesions.

Methylation of melatonin receptors in patients with unipolar and bipolar depression

Disturbances of melatonin secretion alter the circadian rhythm and sleep-wake cycle, which is observed among patients with depression. Melatonin acts via melatonin receptors MT1 and MT2, which are present in many tissues, including peripheral blood mononuclear cells (PBMC). We assume that disturbances of the melatonin pathway in the brain may be reflected by molecular changes in peripheral organs. The study objective was to evaluate the methylation profile of CpG island in the promoter region of melatonin receptor genes MTNR1A and MTNR1B in PBMC of patients with depression and compare it with healthy volunteers. The study group comprised 85 patients with unipolar (UP) and bipolar disorders (BP) and 83 controls. The methylation pattern of CpG island in the promoter region was analyzed using the quantitative methylation-specific real-time PCR (qMSP-PCR) method. We found that the methylation profile of the patients with depression varied in comparison to the control group. The methylation level of MTNR1A was significantly lower among depressed patients compared to controls. Additionally, melatonin concentration was negatively correlated with MTNR1B methylation level among the UP patients. The study may suggest that the methylation profile of melatonin receptors in PBMC may be used as a complementary molecular marker in depression diagnosis.

Influence of Disease Severity and Gender on HLA-C Methylation in COVID-19 Patients.

In the pathophysiology of COVID-19, immunomodulatory factors play a vital role. Viruses have epigenetic effects on various genes, particularly methylation. Explaining the changes in immunological factor methylation levels during viral infections requires substantial consideration. HLA-C is a crucial determinant of immune function and NK cell activity and is primarily implicated in viral infections. This research focused on studying HLA-C methylation in COVID-19 patients with different severity. Peripheral blood samples were collected from 470 patients (235 men and 235 women) with RT-qPCR-confirmed COVID-19 test and classified into moderate, severe, and critical groups based on WHO criteria. Also, one hundred (50 men and 50 women) healthy subjects were selected as the control group. Peripheral blood mononuclear cells were used for DNA extraction, and the methylation-specific PCR (MSP) method and gel electrophoresis were used to determine the methylation status of the HLA-C. Significant statistical differences in HLA-C methylation were observed among cases and controls and various stages of the disease. HLA-C methylation in men and women has decreased in all stages (p < 0.05). In comparison with control, HLA-C methylation in both genders were as follows: moderate (women: 41.0%, men: 52.33%), severe (women: 43.42%, men: 64.86%), critical (women: 42.33%, men: 60.07%), and total patients (women: 45.52%, men: 56.97%). Furthermore, the methylation levels in men were higher than in women in all groups (p < 0.05). Significantly, among all groups, the severe group of men participants showed the highest methylation percentage (p < 0.05). No significant differences were detected for different disease severity in the women group (p > 0.1). This study found that HLA-C methylation was significantly lower in COVID-19 patients with different disease severity. There were also significant differences in HLA-C methylation between men and women patients with different severity. Therefore, during managing viral infections, particularly COVID-19, it is critical to consider patient gender and disease severity.

Quantitative detection of methylated SOCS-1 in schizophrenia and bipolar disorder considering SOCS-1 -1478 CA/del polymorphism and clinical parameters

IntroductionSuppressor of cytokine signaling (SOCS) proteins are the critical negative regulators of immune responses that exert their effects by inhibiting the Jak/Stat signaling pathway.ObjectivesTo investigate quantitative detection of methylated SOCS-1 in schizophrenia and bipolar disorder considering SOCS-1 -1478CA/del polymorphism and clinical parameters.Methods 114 patients with SCZ, 86 patients with BD, and 80 healthy volunteers were included in the case-control study. The patients were consecutively admitted to the outpatient clinic for three months and were evaluated with some scales for clinical parameters. To measure the methylation level of the SOCS-1 gene, bisulfite-converted DNA samples were analyzed using the real-time quantitative methylation-specific PCR method. SOCS-1 -1478CA/del gene polymorphism was evaluated by using the PCR-RFLP.ResultsThe SOCS-1 promoter methylation levels of SCZ (p = .001) and BD (p = .024) were found to be significantly different from the control group. SOCS-1 methylation was significantly different between SCZ groups due to the age of onset (p = .009). Again, SOCS-1 methylation was significantly different between BD groups due to YMRS scale scores (p = .027). While the SOCS-1 genotype distributions of SCZ patients were not found to be statistically different from the control group, a significant difference in genotype distribution between BD patients and healthy controls was found (p = .013).ConclusionsThe methylated SOCS-1 quantity in DNA samples of both SCZ and BD patients was significantly lower than in control samples. Whereas the SOCS-1 -1478CA/del polymorphism was not related to SCZ, it may be associated with the BD.DisclosureNo significant relationships.

Quantitative detection of methylated SOCS-1 in schizophrenia and bipolar disorder considering SOCS-1 -1478CA/del polymorphism and clinical parameters.

We aimed to investigate the quantitative detection of methylated suppressor of cytokine signaling-1 (SOCS-1) in schizophrenia (SCZ) and bipolar disorder (BD), considering SOCS-1 -1478CA/del polymorphism and clinical parameters. Our research is a case-control study in which 114 patients with SCZ, 86 patients with BD, and 80 volunteers as a healthy group participated. Bisulfite-converted DNA samples were analyzed using the real-time quantitative methylation-specific PCR (qMS-PCR) method to measure the methylation level of the SOCS-1 gene. In addition, SOCS-1 -1478CA/del gene polymorphism was analyzed with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). When the SOCS-1 promoter methylation levels of SCZ and BD patients were compared with the control group, the methylation levels of SCZ and BD were significantly lower than the control group. An earlier age of illness onset was significantly related to the SOCS-1 promoter hypermethylation in DNA samples of SCZ patients. Again, SOCS-1 promoter hypermethylation was significantly associated with the higher Young Mania Rating Scale (YMRS) score in BD patients. While the SOCS-1 CA/CA genotype frequency was significantly higher in the control group than in the BD group, the del/del genotype was significantly related to a higher frequency of rapid cycling and a lower frequency of family history in the BD patient group. In summary, the methylated SOCS-1 quantity in DNA samples of SCZ and BD patients were significantly lower than in control samples. Whereas the SOCS-1 -1478CA/del polymorphism was not related to SCZ, it may be associated with the BD.

Expression and methylation status of BTG2, PPP1CA, and PEG3 genes in colon adenocarcinoma cell lines: promising treatment targets.

This study investigated the association between methylation status and expression levels of BTG2, PPP1CA, and PEG3 genes in colon cancer. Aberrant DNA methylation is one of the most important epigenetic modifications in the development of cancer. Evidence indicates that hypermethylation of various tumor suppressor genes could be a potential mechanism of colon tumorigenesis. The expression levels of BTG2, PPP1CA, and PEG3 genes were evaluated in HT-29/219, HCT116, SW48, SW742, SW480, and LS180 cell lines using quantitative Real-Time PCR. The methylation status of BTG2 and PPP1CA was determined by methylation-specific PCR (MSP) method, and the methylation pattern of PEG3 was evaluated by bisulfite sequencing PCR (BSP). To investigate the effect of methylation on the expression of these genes, all colon cancer cell lines were treated by 5-Azacitidine (5-Aza) and/or Trichostatin A (TSA). The expression levels of BTG2, PPP1CA, and PEG3 were highly heterogeneous and quantitatively correlated to their promoter methylation status in the studied colon cancer cell lines. Treatment by 5-Aza and/or TSA increased the expression of the above-named genes in colon cancer cell lines. Overall, it seems that BTG2, PPP1CA, and PEG3 act as tumor suppressor genes in colon cancer, and methylation is a potential mechanism for their loss of expression. Therefore, these genes may be considered as suitable targets for demethylation approaches and, eventually, colon cancer treatment. Combined treatment by 5-Aza and TSA may be a promising therapeutic strategy for colon cancer treatment. Further studies may contribute to confirm these results.

Long non-coding RNA LINC00607 silencing exerts antioncogenic effects on thyroid cancer through the CASP9 Promoter methylation.

Thyroid cancer (TC) was the most frequent thyroid malignant tumour, accounting for about 1% of all malignant tumours. Some long non‐coding RNAs (lncRNAs) have been reported to exert essential tumour promotion effects, while caspase‐9 (CASP9) gene could play a promotive role in the cell apoptosis in TC. However, whether they have a specific effect on TC remains unclear. Hence, this study aims to explore the relationship between LINC00607 and CASP9, and its effect in TC. LINC00607 expression in the TC tissues and cell lines was determined. Then, we explored the combination effect between a LINC00607 and a methylation inhibitor 5‐Aza‐dc in doxorubicin‐resistant ARO cells using colony formation assay, flow cytometry, WST‐1 and EdU assay, as well as in vivo tumour growth assay. Besides, the dual‐luciferase reporter gene assay, RIP, ChIP, methylation‐specific PCR and BSP method were employed to detect the relationship between LINC00607 and CASP9 and its methylation. LINC00607 expression was up‐regulated in the doxorubicin‐resistant TC cell lines and tissues and negatively correlated to the poor prognosis of TC patients. Knockdown of LINC00607 suppressed doxorubicin resistance, proliferation and colony formation, and promoted cell apoptosis of TC cells in vitro, as well as suppressed tumour growth in vivo, whereas LINC00607 overexpression was observed to exercise the opposite effects. Notably, it was also revealed that LINC00607 down‐regulated the CASP9 expression by promoting CASP9 promoter methylation. In conclusion, LINC00607 could inhibit the apoptosis and augment the doxorubicin resistance of TC cells by decreasing CASP9 expression, which might provide a novel therapeutic target for TC treatment.

MGMT methylation alterations in brain cancer following organochlorine pesticides exposure

Background: Alterations in the methylation levels of tumor suppressor genes are considered as one of the essential aspects of malignancies. The present study explored the association of O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation, as a tumor suppressor, with some organochlorine pesticides (OCPs) in primary brain tumor (PBT) patients. Methods: The present study was conducted on a total of 73 PBT patients. The patients’ serum was analyzed using gas chromatography for seven OCP derivatives. The methylation-specific PCR (MSP) method was also used to determine the methylation status of the MGMT promoter. Results: The current findings demonstrated that the methylation of MGMT promoter occurred in 22 out of 34 glioma cases (64%), but in only one out of 35 meningioma cases. No MGMT promoter methylation was observed in other PBT, hemangioma, and anaplastic medulloblastoma stages. Besides, there were significant associations between MGMT methylation and γ-HCH (odds ratio [OR]: 1.50; 95% CI: 1.03- 2.40, P=0.04), 4,4DDE (OR: 1.44; 95% CI: 1.01- 2.05, P=0.02), 2,4 DDT (OR: 1.23; CI: 1.04- 1.45, P=0.03), and 4,4DDT (OR: 1.46; CI: 1.23- 2.15, P=0.02) in glioma patients. Conclusion: The results of the study suggested that the hypermethylation of the MGMT promoter in glioma patients is associated with increased OCPs in their serum, especially γ- HCH, 4,4DDE, 2,4DDT, and 4,4DDT. Moreover, it may lead to the hypermethylation of the MGMT promoter gene. Hence, it can be concluded that exposure to OCPs may potentially induce glioma.

Underexpression of hsa-miR-449 family and their promoter hypermethylation in infertile men: A case-control study.

BackgroundPost-transcriptional microRNAs (miRNAs) have a impotrant pattern in the spermatogenesis process.Objective Study of the expression and methylation of hsa-miR-449 family in sperm samples of infertile men.Materials and Methods In this case-control study, we recruited 74 infertile men (with asthenozoospermia, teratozoospermia, asthenoteratozoospermia, and oligoasthenoteratozoospermia) and 30 control samles. Methylation-specific PCR (MSP) method was used for methylation evaluation of hsa-miR-449 a, b, c promoter. By Real time PCR (qRT-PCR) method,we showed downregulation of hsa-miR-449 a, b, c in the sperm samples of infertile men and compared it to their fertile counterparts.ResultsThere was significant underexperssion, in hsa-miR-449-b in oligoasthenoteratospermic samples (p = 0.0001, F = 2.9). About the methylation pattern, infertile men showed high frequency of methylation in the promoter of hsa-miR-449 a, b, c in comparison to controls (60.8% vs 23.3%), the highest amount of methylation was observed in oligoasthenoteratospermia samples (81.2%).Conclusion In this study, low expression and high methylation of hsa-miR-449-b were observed in infertile men in compared to control samples, which can be one of the causes of defective spermatogenesis.

Epigenetic Modification in Methylene Tetrahydrofolate Reductase (MTHFR) Gene of Women with Pre-eclampsia

Genetic and epigenetic factors play significant roles in the aetio-pathogenesis of pre-eclampsia (PE). The effects may vary across racial and geographical boundaries. The role of epigenetic modification in pre-eclampsia was studied among African populations in Lagos, Nigeria.This study aimed to determine the pattern of Methylene tetrahydrofolate reductase gene (MTHFR) CpG island methylation in pre-eclampsia, and evaluate associated covariates.This study was an observational, cross-sectional, study conducted at the Lagos University Teaching Hospital and the Lagos State Island Maternity Hospital. A total of 400 pregnant women consisting of 200 pregnant women diagnosed with pre-eclampsia (study group) and 200 pregnant normotensive and apparently healthy women (control group) were recruited for the study. Demographic and clinical histories were obtained through questionnaires. The DNA Methylation status of the CpG Island in promoter region of the MTHFR gene was assessed using bisulphite conversion and methylation specific PCR method. The biochemical parameters measured in the study were: red cell folate, vitamin B12, plasma homocysteine (Hcy) and methylene tetrahydrofolate reductase enzyme level.Homozygous MTHFR CpG island hypomethylation pattern was significantly associated with pre-eclampsia (χ2 = 22.96; p = 0.000), Mean values of plasma homocysteine in PE women with homozygous hypomethylation (26.1 ± 9.1 umol/L) were significantly higher than (20.1 ± 4.2 umol/L) observed in PE subjects with homozygous hypermethylation (p = 0.008). Homozygous CpG island hypomethylated pattern of the MTHFR promoter region, was associated with the lowest median MTHFR enzyme level (72.8 ± 39.8 pmol/L) compared with heterozygous methylated pattern (91.3 ± 60.9 pmol/L; p = 0.047) and homozygous methylated pattern (82.3 ± 31.0 pmol/L; 0.047). Red cell folate and Vitamin B12 levels were not significantly associated with CpG island methylation status.Epigenetic modification plays significant role in the pathogenesis of pre-eclampsia.

Epigenetic silencing of the DAPK1 gene in Egyptian patients with chronic myeloid Leukemia

BackgroundEpigenetics play a crucial role in the carcinogenesis of hematological malignancies. Alterations of the methylation status in the promoter region of tumor suppressor genes, such as DAPK1, cause loss of gene function and subsequent progression of Chronic Myelogenous Leukemia (CML) to more advanced stages. This study aimed to analyze the methylation status of the DAPK1 gene promoter in different phases of CML patients. Material and methodsThe study included 200 CML patients positive for BCR/ABL and 50 as control. Clinicopathological examination was done for all patients. DAPK1 gene methylation was determined by the Methylation Specific-PCR method. ResultsDAPK1 promoter methylation was evident in 37.0% of CML patients and it was more frequent in affected females than males (p = 0.014). Platelet count and bone marrow blast cells were significantly different in the methylated group compared to the unmethylated group (p = 0.001 and p = 0.034 respectively). Out of the methylated group, fifteen patients had a hemimethylated pattern. Unmethylation was more frequent in patients in the chronic phase (66.5%; p = 0.001). In contrast, methylation was found in 75.0% of patients in the accelerated phase (p = 0.040) with an odds ratio (OR) of 5.470 (95% CI: 1.074–27.849). Similarly, methylation was found in 85.7% of patients in the blast phase (p = 0.027) where the OR increased to 11.029 (95% CI: 1.300–93.513). By logistic regression analysis, there was significant association between methylation pattern and CML disease clinical outcome. ConclusionCML progression is closely associated with methylation changes in the DAPK1 promoter region. Identification of the underlying epigenetic mechanisms in CML is valuable in predicting disease prognosis and offering suitable management for each patient.

Solanum nigrum Anticancer Effect Through Epigenetic Modulations in Breast Cancer Cell Lines

Background: DNA methylation plays an important role in the regulation of gene expression in mammalian cells and often occurs at CpG islands in the genome. It is more reversible than genetic variations and has therefore attracted much attention for the treatment of many diseases, especially cancer. In the present study, we investigated the effect of Solanum nigrum Extract (SNE) on the methylation status of the VIM and CXCR4 genes in breast cancer cell lines. Methods: The Trypan blue assay was used to study the effect of SNE at various concentrations of 0, 0.1, 1.5, 2.5, 3.5 and 5 mg/ml for 48 h on the survival of three human breast cancer cell lines MCF7, MDA-MB-468, MDA-MB-231. Methylation status of VIM and CXCR4 genes in breast cancer cell lines was assessed by Methylation-Specific PCR (MSP) method. Also, methylation changes of VIM and CXCR4 genes in breast cancer cell lines after treatment with 0.1 mg/ml of SNE for 6 days were analyzed by MSP method. To confirm the effect of SNE on methylation of VIM and CXCR4 genes, Real-Time PCR was performed. Results: The Trypan blue assay results indicated that treatment with SNE reduced cell viability in a dose-dependent manner in breast cancer cells. Our results showed that treatment of breast cancer cells with 0.1 mg/ml of SNE hypermethylated the VIM, CXCR4 genes and significantly reduced the expression levels of their mRNA (P&lt;0.05). Conclusion: Our findings reveal for the first time the impact of SNE on the methylation of breast cancer cells.

DNA Methylation-Based Testing in Liquid Biopsies as Detection and Prognostic Biomarkers for the Four Major Cancer Types.

Lung, breast, colorectal, and prostate cancers are the most incident worldwide. Optimal population-based cancer screening methods remain an unmet need, since cancer detection at early stages increases the prospects of successful and curative treatment, leading to a lower incidence of recurrences. Moreover, the current parameters for cancer patients’ stratification have been associated with divergent outcomes. Therefore, new biomarkers that could aid in cancer detection and prognosis, preferably detected by minimally invasive methods are of major importance. Aberrant DNA methylation is an early event in cancer development and may be detected in circulating cell-free DNA (ccfDNA), constituting a valuable cancer biomarker. Furthermore, DNA methylation is a stable alteration that can be easily and rapidly quantified by methylation-specific PCR methods. Thus, the main goal of this review is to provide an overview of the most important studies that report methylation biomarkers for the detection and prognosis of the four major cancers after a critical analysis of the available literature. DNA methylation-based biomarkers show promise for cancer detection and management, with some studies describing a “PanCancer” detection approach for the simultaneous detection of several cancer types. Nonetheless, DNA methylation biomarkers still lack large-scale validation, precluding implementation in clinical practice.

Methylation profiling in promoter sequences of ATM and CDKN2A (p14ARF/p16INK4a ) genes in blood and cfDNA from women with impalpable breast lesions.

The objective of the present study was to evaluate the epigenetic changes occurring in early stages of breast cancer. The present study investigated the methylation profile of the ATM, p14ARF and p16INK4a promoters in total blood and plasma cell-free DNA (cfDNA) from women with impalpable breast lesions compared with in total blood of a control cohort of women without breast lesions. The samples were evaluated using the methylation-specific PCR method. The Fisher's exact test was used to evaluate statistical significance between the methylation and clinical variables. A total of 111 women were evaluated, including 56 women with impalpable breast cancer (39/56 also had paired plasma cfDNA) and 55 women in the control cohort (55 blood DNA). For blood DNA from women with malignant impalpable breast lesions, p16INK4a exhibited the greatest percentage of methylation (48%), followed by ATM (37.5%) and p14ARF (27%) promoters, regardless of age variation. For plasma cfDNA, the methylation rates for ATM, p14ARF and p16INK4a were 26, 26 and 10%, respectively. The methylation rates for the blood DNA of controls were the lowest for ATM (9%), p14ARF (7%) and p16INK4a (7%). The women with impalpable breast lesions (benign and malignant lesions) exhibited the highest methylation rate, regardless of age, compared with the paired plasma cfDNA and controls. This epigenetic change was statistically significant for the promoters of ATM (P=0.009) and p16INK4a (P=0.001) (impalpable breast lesions vs. control). The present study demonstrated that epigenetic changes occurring in the ATM and CDKN2A genes detectable in liquid biopsy were associated with the development of impalpable breast lesions.