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1
Cottrell, S. E. "A real-time PCR assay for DNA-methylation using methylation-specific blockers." Nucleic Acids Research 32, no. 1 (January 2, 2004): 10e—10. http://dx.doi.org/10.1093/nar/gnh008.
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Pu, Robert T., Zong-Mei Sheng, Claire W. Michael, Michael G. Rhode, Douglas P. Clark, and Timothy J. O'Leary. "Methylation profiling of mesothelioma using real-time methylation-specific PCR: A pilot study." Diagnostic Cytopathology 35, no. 8 (2007): 498–502. http://dx.doi.org/10.1002/dc.20692.
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Bonanno, Cinzia, Erlet Shehi, Daniel Adlerstein, and G. Mike Makrigiorgos. "MS-FLAG, a Novel Real-Time Signal Generation Method for Methylation-Specific PCR." Clinical Chemistry 53, no. 12 (December 1, 2007): 2119–27. http://dx.doi.org/10.1373/clinchem.2007.094011.
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Abstract Background: Aberrant promoter methylation is a major mechanism for silencing tumor suppressor genes in cancer. Detection of hypermethylation is used as a molecular marker for early cancer diagnosis, as a prognostic index, or to define therapeutic targets for reversion of aberrant methylation. We report on a novel signal generation technology for real-time PCR to detect gene promoter methylation. Methods: FLAG (fluorescent amplicon generation) is a homogeneous signal generation technology based on the exceptionally thermostable endonuclease PspGI. FLAG provides real-time signal generation during PCR by PspGI-mediated cleavage of quenched fluorophores at the 5′ end of double-stranded PCR products. Methylation-specific PCR (MSP) applied on bisulfite-treated DNA was adapted to a real-time format (methylation-specific FLAG; MS-FLAG) for quantifying methylation in the promoter of CDKN2A (p16), GATA5, and RASSF1. We validated MS-FLAG on plasmids and genomic DNA with known methylation status and applied it to detection of methylation in a limited number of clinical samples. We also conducted bisulfite sequencing on these samples. Results: Real-time PCR results obtained via MS-FLAG agreed with results obtained via conventional, gel-based MSP. The new technology showed high specificity, sensitivity (2–3 plasmid copies), and selectivity (0.01% of methylated DNA) on control samples. It enabled correct prediction of the methylation status of all 3 gene promoters in 21 lung adenocarcinoma samples, as confirmed by bisulfite sequencing. We also developed a multiplex MS-FLAG assay for GATA5 and RASSF1 promoters. Conclusion: MS-FLAG provides a new, quantitative, high-throughput method for detecting gene promoter methylation and is a convenient alternative to agarose gel-based MSP for screening methylation. In addition to methylation, FLAG-based real-time signal generation may have broad applications in DNA diagnostics.
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Bruce, Sara, Katariina Hannula-Jouppi, Cecilia M. Lindgren, Marita Lipsanen-Nyman, and Juha Kere. "Restriction Site–Specific Methylation Studies of Imprinted Genes with Quantitative Real-Time PCR." Clinical Chemistry 54, no. 3 (March 1, 2008): 491–99. http://dx.doi.org/10.1373/clinchem.2007.098491.
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Abstract Background: Epigenetic studies, such as the measurement of DNA methylation, are important in the investigation of syndromes influenced by imprinted genes. Quick and accurate quantification of methylation at such genes can be of appreciable diagnostic aid. Methods: We first digested genomic DNA with methylation-sensitive restriction enzymes and used DNA without digestion as a control and nonmethylated λ DNA as an internal control for digestion efficiency. We then performed quantitative real-time PCR analyses with 6 unique PCR assays to investigate 4 imprinting control regions on chromosomes 7 and 11 in individuals with uniparental disomy of chromosome 7 (UPD7) and in control individuals. Results: Our validation of the method demonstrated both quantitative recovery and low methodologic imprecision. The imprinted loci on chromosome 7 behaved as expected in maternal UPD7 (100% methylation) and paternal UPD7 (<10% methylation). In controls, the mean (SD) for percent methylation at 2 previously well-studied restriction sites were 46% (6%) for both H19 and KCNQ1OT1, a result consistent with the previously observed methylation rate of approximately 50%. The methylation percentages of all investigated imprinted loci were normally distributed, implying that the mean and SD can be used as a reference for screening methylation loss or gain. Conclusion: The investigated loci are of particular importance for investigating the congenital Silver–Russell and Beckwith–Wiedemann syndromes; however, the method can also be applied to other imprinted regions. This method is easy to set up, has no PCR bias, requires small amounts of DNA, and can easily be applied to large patient populations for screening the loss or gain of methylation.
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Yoshioka, Masaki, Tomoo Matsutani, Ayaka Hara, Seiichiro Hirono, Takaki Hiwasa, Masaki Takiguchi, and Yasuo Iwadate. "Real-time methylation-specific PCR for the evaluation of methylation status of MGMT gene in glioblastoma." Oncotarget 9, no. 45 (June 12, 2018): 27728–35. http://dx.doi.org/10.18632/oncotarget.25543.
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Mehta, Akanksha, Anna Mielnik, DariusA Paduch, and PeterN Schlegel. "Novel methylation specific real-time PCR test for the diagnosis of Klinefelter syndrome." Asian Journal of Andrology 16, no. 5 (2014): 684. http://dx.doi.org/10.4103/1008-682x.125914.
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Vlassenbroeck, Ilse, Stéphane Califice, Annie-Claire Diserens, Eugenia Migliavacca, Josef Straub, Ivano Di Stefano, Fabrice Moreau, et al. "Validation of Real-Time Methylation-Specific PCR to Determine O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation in Glioma." Journal of Molecular Diagnostics 10, no. 4 (July 2008): 332–37. http://dx.doi.org/10.2353/jmoldx.2008.070169.
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Kondo, Masahiro, Hirofumi Aboshi, Masaaki Yoshikawa, Ayano Ogata, Ryosuke Murayama, Masami Takei, and Shin Aizawa. "A newly developed age estimation method based on CpG methylation of teeth-derived DNA using real-time methylation-specific PCR." Journal of Oral Science 63, no. 1 (2021): 54–58. http://dx.doi.org/10.2334/josnusd.20-0138.
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Kristensen, L. S., T. Mikeska, M. Krypuy, and A. Dobrovic. "Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection." Nucleic Acids Research 36, no. 7 (February 16, 2008): e42-e42. http://dx.doi.org/10.1093/nar/gkn113.
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Kristensen, Lasse Sommer, and Lise Lotte Hansen. "PCR-Based Methods for Detecting Single-Locus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment." Clinical Chemistry 55, no. 8 (August 1, 2009): 1471–83. http://dx.doi.org/10.1373/clinchem.2008.121962.
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Abstract Background: DNA methylation is a highly characterized epigenetic modification of the human genome that is implicated in cancer. The altered DNA methylation patterns found in cancer cells include not only global hypomethylation but also discrete hypermethylation of specific genes. In particular, numerous tumor suppressor genes undergo epigenetic silencing because of hypermethylated promoter regions. Some of these genes are considered promising DNA methylation biomarkers for early cancer diagnostics, and some have been shown to be valuable for predicting prognosis or the response to therapy. Content: PCR-based methods that use sodium bisulfite–treated DNA as a template are generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at single loci. A number of new methods, such as methylation-specific fluorescent amplicon generation (MS-FLAG), methylation-sensitive high-resolution melting (MS-HRM), and sensitive melting analysis after real-time methylation-specific PCR (SMART-MSP), now complement the traditional PCR-based methods and promise to be valuable diagnostic tools. In particular, the HRM technique shows great potential as a diagnostic tool because of its closed-tube format and cost-effectiveness. Summary: Numerous traditional and new PCR-based methods have been developed for detecting DNA methylation at single loci. All have characteristic advantages and disadvantages, particularly with regard to use in clinical settings.
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Nakamura, Kazunorii, Horomichi Sawaki, Keishi Yamashita, Masahiko Watanabe, and Hisashi Narimatsu. "Identification of epigenetic silencing of GCNT2 expression by comprehensive real-time PCR screening in colorectal cancer." Journal of Clinical Oncology 32, no. 3_suppl (January 20, 2014): 506. http://dx.doi.org/10.1200/jco.2014.32.3_suppl.506.
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506 Background: Glycoprotein expression profile has been proved to be dramatically altered in human cancers, however specific glycogenes which are aberrant in expression in cancer cells has not been fully identified. Recent accumulated evidence supported notion that the reduced expression of tumor suppressor genes is explained by DNA promoter methylation in human cancer. Methods: We used Comprehensive Real time PCR system (CRPS) for glycogenes (189 genes) to identify genes aberrantly expressed in colorectal cancer tissues (CRC) as compared to the corresponding normal mucosa tissues. GCNT2 was of particular interest among the identified genes in CRC. Results: (1) GCNT2 harbors 3 isoforms which have different promoter regions. (2) All of the 3 isoforms of GCNT2 genes were remarkably decreased in CRC as compared to the corresponding normal mucosa, and each isoform expression was strongly associated with other 2 isoforms in primary cancer tissues by TaqMan real time PCR (R = 0.99-995, p < 0.0001). (3) Among the 5 CRC cell lines (DLD1, HCT116, CACO2, LOVO), those which were silenced in expression were reactivated by demethylating agents such as 5-aza-2’ deoxycytidine and trichostatin A. (4) Promoter region of the variant 2 of GCNT2 was consistent with its silenced expression in CRC cell lines by cloned sequence, so we examined DNA methylation status of the promoter of the GCNT2 variant 2 in 50 primary cancer tissues and the corresponding normal tissues. Quantitative MSP revealed that almost half of normal tissues have methylation as high as tumor tissues, while, in the primary CRC with less methylation in the corresponding normal tissues, DNA methylation was higher in primary CRC tissues than in the corresponding normal tissues. Finally, GCNT2 variant 2 stable transfection induced expression of other 2 isoform variants. Conclusions: We identified novel methylation gene GCNT2 among the glycoenes. Glycoenes that were altered in genomic or epigenetic manner have been few, so GCNT2 may play a critical role in cancer progression through glycan change.
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Zhao, Ning-Hui, Yu Qian, Chen-Si Wu, Jing-Wen Wang, Yu Fang, Xiao-Peng Fan, Shuai Gao, Yu-Chen Fan, and Kai Wang. "Diagnostic value of NKG2D promoter methylation in hepatitis B virus-associated hepatocellular carcinoma." Biomarkers in Medicine 13, no. 13 (September 2019): 1093–105. http://dx.doi.org/10.2217/bmm-2019-0102.
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Aim: Natural killer cell receptor group 2D (NKG2D) plays an important role in the immune regulation of tumors. We speculate that DNA methylation are involved in the regulation of NKG2D gene. Methods: We investigated the methylation status of the NKG2D promoter in peripheral blood mononuclear cells of hepatocellular carcinoma (HCC) patients, chronic hepatitis B patients and healthy controls by methylation-specific PCR and the mRNA expression level was examined by real-time quantitative PCR. Results: The methylation frequency of NKG2D promoter in HCC patients was higher than that of chronic hepatitis B patients and healthy controls. NKG2D promoter methylation has a good predictive value for HCC diagnosis. Conclusion: NKG2D promoter methylation can be used as a noninvasive marker for detecting HCC.
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Hoshino, Koyu, Hui Yang, Blanca Sanchez-Gonzalez, and Guillermo Garcia-Manero. "Detection of Aberrant DNA Methylation in Acute Lymphocytic Leukemia (ALL) Using a Real-Time Polymerase Chain Reaction (PCR) Assay." Blood 104, no. 11 (November 16, 2004): 998. http://dx.doi.org/10.1182/blood.v104.11.998.998.
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Abstract Aberrant DNA methylation of promoter-associated CpG islands is a frequent event in ALL. DNA methylation of specific subsets of genes is associated with poor prognosis and is stable in a majority of patients (pts) at the time of relapse (Clinical Cancer Res2002;8:1897), and therefore tracking these epigenetic marks during remission may predict for relapse risk. Most commonly used methods to detect DNA methylation exploit the availability of sodium bisulfite that transforms unmethylated C into A/T leaving methylated C as such. This has allowed the development of several techniques that use conventional PCR or sequencing to detect methylated alleles. Although there are significant differences in the sensitivity and specificity of these assays, with bisulfite sequencing considered the gold standard, most of them are labor intensive and require several days to be performed. To circumvent some of these problems, we have developed a real-time bisulfite PCR assay to detect methylation of p57KIP2, p73, and p15 in samples obtained from bone marrows in pts with ALL at remission. Methylation of these genes has been shown to be common in ALL and to predict for poor prognosis at initial presentation (Blood2003;101:4131). This method allows for the simultaneous analysis of multiple samples in less than 24 hours, is quantitative, and requires less than 0.2μg of DNA for each target gene. To amplify bisulfite treated DNA, we designed primer sets and probes for all three genes in regions known to be inversely correlated with gene expression. To quantify methylation density, we used the interferon γ (INFG) gene as an internal control because it has very rare CpG sites, is a single copy gene and has no homology with other known genes. Methylation density is defined as: Methylation (%) = 2CT of target gene / 2 CT of EFM x 100. CT is the number of cycles threshold, and EFM the estimated 100% fully methylation (EFM) of the target gene in control experiments. Using DNA artificially methylated with SssI in dilution experiments with unmethylated DNA, p57, p15 and p73 methylation density could be detected at dilutions of 0.2%, 1.2% and 0.1%, respectively. We then studied the methylation status of 30 cell lines (22 hematopoietic and 8 no-hematopoietic). Methylation of p57, p15 and p73 gene was detected in 17(57%), 19(63%) and 16(53%) cell lines respectively. Methylation of 3 genes, 2 genes and 1 gene was observed in 9(30%), 8(27%) and 7(23%) cell lines respectively. DNA methylation of p15 and p73 was not detected in the marrow from 6 healthy volunteers but p15 was detected (0.1% methylation) in 1 out of 6 of these specimens. Subsequently, we studied 50 pts with ALL at the time of remission. We found the frequencies of p57, p15 and p73 gene to be 2(4%), 26(52%) and 10(20%), respectively. There was a trend towards a better overall survival for pts with methylation of 0 or 1 gene (209 weeks) compared with those with methylation of 2 or more genes (71 weeks), p=0.1. In conclusion, the real-time bisulfite PCR described here allows for the rapid detection of aberrant DNA methylation in samples obtained at the time of remission in pts with ALL and may have a role in the development of techniques to assess minimal residual disease in this group of pts.
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Kawakami, Y., K. Miyamoto, K. Takehara, M. Kumagai, O. Samura, H. Nakamura, T. Mizunoe, H. Yoshida, K. Taniyama, and F. Saji. "Down-regulation of MDR1 by epigenetic alteration in human epithelial ovarian cancer cells." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): e16556-e16556. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.e16556.
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e16556 Background: Ovarian cancer is one of the most lethal malignancies in women. Despite recent studies of many oncogenes and tumor-suppressor genes concerning about its progression, the details of ovarian cancer biology still remains to be unclear. P-glycoprotein (P-gp) encoded by the MDR1 gene is a membrane protein that can export many kinds of antineoplastic agents from cells, and overexpression of P-gp has been reported to be implicated in treatment failure in cancer. DNA methylation, a major epigenetic process, can affect every step in carcinogenesis as well as genetic alterations. The contribution of such epigenetic alteration to the expression of MDR1 remains largely unexplored in human ovarian cancer. Methods: In this study, we evaluated the DNA methylation status of the MDR1 gene by methylation-specific PCR. Then, the expression of MDR-1 mRNA and protein in primary epithelial ovarian cancer specimens were evaluated by real-time RT-PCR and immunohistochemistry using anti-mouse P-gp F4 monoclonal antibody, respectively. The correlation between these results and clinicopathological features was examined. Results: MDR1 was hypermethylated in 12 of 12 (100%) ovarian cancer cell lines, and 5 of 13 (38%) primary ovarian cancers by methylation-specific PCR analysis. MDR1 mRNA expression was subsequently found to be lost in ovarian cancer cell lines with methylation by both real-time RT-PCR and immunohistochemistry. Thus, MDR1expression was associated with the DNA methylation status of the MDR1 gene. Conclusions: In conclusion, MDR1 was frequently hypermethylated in human ovarian cancers. Our results suggest that epigenetic regulation might play a role in the expression of MDR1 and clinical treatment outcomes in human ovarian cancer. No significant financial relationships to disclose.
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Parrella, Paola, Antonella la Torre, Massimiliano Copetti, Vanna M. Valori, Raffaela Barbano, Angelo Notarangelo, Michele Bisceglia, et al. "High Specificity of Quantitative Methylation-Specific PCR Analysis forMGMTPromoter Hypermethylation Detection in Gliomas." Journal of Biomedicine and Biotechnology 2009 (2009): 1–8. http://dx.doi.org/10.1155/2009/531692.
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Normal brain tissue from 28 individuals and 50 glioma samples were analyzed by real-time Quantitative Methylation-Specific PCR (QMSP). Data from this analysis were compared with results obtained on the same samples by MSP. QMSP analysis demonstrated a statistically significant difference in both methylation level (P=.000009Mann Whitney Test) and frequencies (P=.0000007, Z-test) in tumour samples as compared with normal brain tissues. Although QMSP and MSP showed similar sensitivity, the specificity of QMSP analysis was significantly higher (93%; CI95%: 84%–100%) as compared with MSP (64%; 95%CI: 46%–82%). Our results suggest that QMSP analysis may represent a powerful tool to identify glioma patients that will benefit from alkylating agents chemotherapy.
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Deligezer, Ugur, Ebru E. Akisik, Nilgün Erten, and Nejat Dalay. "Sequence-Specific Histone Methylation Is Detectable on Circulating Nucleosomes in Plasma." Clinical Chemistry 54, no. 7 (July 1, 2008): 1125–31. http://dx.doi.org/10.1373/clinchem.2007.101766.
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AbstractBackground: Alterations in DNA methylation and histone modifications have been implicated in carcinogenesis. Although tumor-specific alterations in DNA methylation can be detected in the serum and plasma of cancer patients, no data are available on the presence of histone modifications in circulating blood. We investigated whether histone methylation, as a model of histone modifications, is detectable in plasma. Because methylation at histone 3 lysine 9 (H3K9) has been demonstrated to be enriched at sites of repetitive ALU elements, we addressed the specificity of histone-methylation detection and hypothesized that if monomethylated H3K9 (H3K9me1) is detectable in plasma, the concentrations in mononucleosomes and oligonucleosomes would be different. We also analyzed a single-copy gene, CDKN2A.Methods: We enrolled 21 multiple myeloma patients in the study. We used ELISA and real-time PCR analysis to evaluate nucleosomes and cell-free DNA, respectively, as evidence of the presence of histones and associated DNA in circulating blood. H3K9me1 was analyzed by chromatin immunoprecipitation.Results: ELISA and real-time PCR assays indicated the presence of free nucleosomes and DNA in plasma, and the results were quantitatively correlated (P < 0.001). The detection of histone methylation on free nucleosomes was sequence dependent. Fragments representing mono- and oligonucleosomes differed with respect to H3K9me1 concentrations (P = 0.004), in accordance with our hypothesis. In addition, the detection rate and concentrations of H3K9me1 were significantly higher on the fragment covering both mononucleosomes and oligonucleosomes than on the CDKN2A promoter (P < 0.001).Conclusions: If validated in further studies, our findings may be a basis for investigations of cancer-specific alterations in histone modifications in the circulation.
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Utriainen, Pauliina, Jianqi Liu, Tiina Kuulasmaa, and Raimo Voutilainen. "Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R." Journal of Endocrinology 188, no. 2 (February 2006): 305–10. http://dx.doi.org/10.1677/joe.1.06392.
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Activin affects adrenocortical steroidogenesis and increases apoptosis, while follistatin (FS) acts as an activin antagonist by binding to activin, preventing attachment to its receptors. The regulation of FS expression in the adrenal cortex is poorly understood. Adrenocortical tumors often display aberrant methylation. In the present study, we investigated the effect of DNA methylation on FS mRNA expression and peptide secretion in adrenocortical cells. We treated human NCI-H295R adrenocortical cells with the methylation inhibitor 5-Aza-2′deoxycytidine (Azad; 0.1–100 μM for 1, 4 or 7 days) and measured FS mRNA expression by Northern blot and quantitative real time RT-PCR analyses as well as FS secretion by specific ELISA. Methylation-specific PCR showed decreased methylation in the FS promoter region after Azad treatment. A significant (P < 0.05) time- and dose-dependent increase in FS mRNA expression (up to 4.6-fold) and peptide secretion (up to 17.1-fold) was detected after Azad treatment. We conclude that FS gene expression and peptide secretion in NCI-H295R adrenocortical cells are regulated by DNA methylation. Thus, variable methylation in different adrenocortical tumors may influence activin bioactivity and its consequences in steroidogenesis and cell proliferation/apoptosis.
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La Torre, Annamaria, Lucia Anna Muscarella, Paola Parrella, Teresa Balsamo, Michele Bisceglia, Vanna Maria Valori, Antonella La Torre, et al. "Aberrant Genes Promoter Methylation in Neural Crest-Derived Tumors." International Journal of Biological Markers 27, no. 4 (October 2012): 389–94. http://dx.doi.org/10.5301/jbm.2012.9766.
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Disturbances in the epigenetic landscape by aberrant methylation of CpG islands can lead to inactivation of cancer-related genes in solid tumors. We analyzed the promoter methylation status of 6 genes previously reported as cancer-specific methylated (MCAM, SSBP2, NISCH, B4GALT1, KIF1A and RASSF1A) in 38 neural crest-derived tumors by quantitative methylation-specific real-time PCR (QMSP). The results demonstrated that the determination of the methylation status of RASSF1A is able to distinguish between normal and tumor samples in cutaneous melanomas, lung carcinoids and small bowel carcinoids. MCAM methylation levels were significantly higher in lung carcinoids tumors (p=0.001), suggesting that this alteration may represent a molecular biomarker in this tumor type.
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Longo Machado de Almeida, Marina, Paulo Henrique Pires de Aguiar, Katharyna De Gois, Flavia De Sousa Gehrke, and Fernando Fonseca. "The importance of MGMT Promoter Methylation Status for Glioblastomas Prognosis: meta-analysis." JBNC - JORNAL BRASILEIRO DE NEUROCIRURGIA 29, no. 4 (October 23, 2019): 595–611. http://dx.doi.org/10.22290/jbnc.v29i4.1795.
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The authors discussed a meta-analysis on MGMT and glioblastomas and the positivity of methylation expression in patients with these tumors. The review included articles published between 2000 and 2018. As keywords we used: glioblastoma, MGMT or O-6- methylguanine DNA methyltransferase and methylation for the search. A meta-analysis was performed. The survey included only papers written in English, resulting in 34 articles in the review. In the articles, 16,426 glioblastoma samples were analyzed by means of pyrosequencing, methylation-specific polymerase chain reaction, immunohistochemistry or quantitative real time polymerase chain reaction (PCR) to determine the MGMT promoter methylation status. The authors found 2,414 tumor samples having the methylated promoter and 14,012 unmethylated. This article is very impressive, proving the real importance of MGMT methylation and its low positivity rate.
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Ruszova, E., M. Chmelarova, M. Senkerikova, and S. Stefackova. "Duplex Methylation-Specific Semi-Quantitative Real-Time PCR for Cost-Effective & Time-Efficient Diagnostic Screening of Chromosome 15 and 14 Imprinted Regions." Acta Medica Martiniana 15, no. 3 (December 1, 2015): 5–12. http://dx.doi.org/10.1515/acm-2015-0012.
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AbstractPurpose: Our goal was to develop two-tier strategy based onin house-designed methylation specific-duplex polymerase chain reactions (MS-PCRs) that could serve as a relatively simple, cost effective, time efficient approach for molecular screening of imprinted regions on chromosomes 15 and 14.Patients and methods: Patients were referred to examination during infancy due to hypotonia and motor development delay. Duplex MS-PCRs were performed that enabled detection of methylated/unmethylated DNA inNDNandMEG3CpG islands via plurality of detection channels on PCR instrument Rotor Gene 6000.Results and discussion: Both, copy number variations as well as methylation changes, were revealed by ourin house-designed methodology by focusingNDNgene. No imprinting aberrations were yet discovered inMEG3gene. Clinical features of the patients were compared. In agree with literature no typical facial features were observed in PWS patient with imprinting defect and AS UPD patient seems to have a relatively better development and language ability in comparison to deletional form of the disease.Conclusion: In conclusion we were able to establish new, throughput and robust diagnostic approach to PWS/AS.
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Lofton-Day, Catherine, Fabian Model, Theo DeVos, Reimo Tetzner, Juergen Distler, Matthias Schuster, Xiaoling Song, et al. "DNA Methylation Biomarkers for Blood-Based Colorectal Cancer Screening." Clinical Chemistry 54, no. 2 (February 1, 2008): 414–23. http://dx.doi.org/10.1373/clinchem.2007.095992.
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Abstract Background: Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma. Methods: We first used restriction enzyme–based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range. Results: Restriction enzyme–based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%–73%] of plasma samples from CRC patients and not detected in 69% (62%–76%) of the controls. The corresponding results for NGFR were 51% (42%–60%) and 84% (77%–89%); for SEPT9, the values were 69% (60%–77%) and 86% (80%–91%). Conclusions: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.
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Rahimzadeh, Sevda, Reza Rahbarghazi, Somayeh Aslani, Hadi Rajabi, Zeinab Latifi, Majid Farshdousti Hagh, Alireza Nourazarian, Hojjatollah Nozad Charoudeh, Mohammad Nouri, and Alireza Abhari. "Promoter methylation and expression pattern of DLX3, ATF4, and FRA1 genes during osteoblastic differentiation of adipose-derived mesenchymal stem cells." BioImpacts 10, no. 4 (November 25, 2019): 243–50. http://dx.doi.org/10.34172/bi.2020.31.
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Introduction: Nowadays, mesenchymal stem cells are touted as suitable cell supply for the restoration of injured bone tissue. The existence of osteogenic differentiation makes these cells capable of replenishing damaged cells in the least possible time. It has been shown that epigenetic modifications, especially DNA methylation, contribute to the regulation of various transcription factors during phenotype acquisition. Hence, we concentrated on the correlation between the promoter methylation and the expression of genes DLX3, ATF4, and FRA1 during osteoblastic differentiation of adipose-derived mesenchymal stem cells in vitro after 21 days. Methods: Adipose-derived mesenchymal stem cells were cultured in osteogenesis differentiation medium supplemented with 0.1 µM dexamethasone, 10 mM β-glycerol phosphate, and 50 µM ascorbate-2-phosphate for 21 days. RNA and DNA extraction was done on days 0, 7, 14, and 21. Promoter methylation and expression levels of genes DLX3, ATF4, and FRA1 were analyzed by methylation-specific quantitative PCR and real-time PCR assays, respectively. Results: We found an upward expression trend with the increasing time for genes DLX3, ATF4, and FRA1 in stem cells committed to osteoblast-like lineage compared to the control group (P<0.05). On the contrary, methylation-specific quantitative PCR displayed decreased methylation rates of DLX3 and ATF4 genes, but not FRA1, over time compared to the non-treated control cells (P<0.05). Bright-field images exhibited red-colored calcified deposits around Alizarin Red S-stained cells after 21 days compared to the control group. Statistical analysis showed a strong correlation between the transcription of genes DLX3 and ATF4 and methylation rate (P<0.05). Conclusion: In particular, osteoblastic differentiation of adipose-derived mesenchymal stem cells enhances DLX3 and ATF4 transcriptions by reducing methylation rate for 21 days.
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Kim, Sollip, Hye Won Lee, Woochang Lee, Sail Chun, and Won-Ki Min. "Validation of New Allele-Specific Real-Time PCR System for Thiopurine Methyltransferase Genotyping in Korean Population." BioMed Research International 2013 (2013): 1–4. http://dx.doi.org/10.1155/2013/305704.
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Introduction. Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered.TPMTgenotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population.Materials and Methods. Three majorTPMTsingle-nucleotide polymorphisms (TPMT*2, *3B, and *3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing.Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency ofTPMT*3Cwas 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. NoTPMT*2, *3A, or *3Balleles were observed in this Korean population.Conclusion. This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice.
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Zou, Hongzhi, Hatim Allawi, Xiaoming Cao, Mike Domanico, Jonathan Harrington, William R. Taylor, Tracy Yab, David A. Ahlquist, and Graham Lidgard. "Quantification of Methylated Markers with a Multiplex Methylation-Specific Technology." Clinical Chemistry 58, no. 2 (February 1, 2012): 375–83. http://dx.doi.org/10.1373/clinchem.2011.171264.
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Abstract BACKGROUND Aberrantly methylated genes represent important markers for cancer diagnosis. We describe a multiplex detection approach to efficiently quantify these markers for clinical applications such as colorectal cancer screening. METHODS Quantitative allele-specific real-time target and signal amplification (QuARTS) combines a polymerase-based target amplification with an invasive cleavage-based signal amplification. The fluorescence signal is detected in a fashion similar to real-time PCR. We measured the dynamic range and analytical sensitivity of multiplex QuARTS reactions with titrated plasmid DNA. We used the QuARTS technology to quantify methylated BMP3, NDRG4, VIM, and TFPI2 genes on 91 DNA samples extracted from colorectal tissues, including 37 cancers, 25 adenomas, and 29 healthy epithelia. The assays were designed in triplex format that incorporated ACTB as a reference gene. Percent methylation was calculated by dividing methylated strands over ACTB strands and multiplying by 100. RESULTS The QuARTS method linearly detected methylated or unmethylated VIM gene down to 10 copies. No cross-reactivity was observed when methylated assays were used to amplify 105 copies of unmethylated gene and vice versa. The multiplex assay detected methylated genes spiked in unmethylated genes at a 0.01% ratio and vice versa. At a diagnostic specificity cutoff of 95%, methylated BMP3, NDRG4, VIM, and TFPI2 detected 84%, 92%, 86%, and 92% of colorectal cancers and 68%, 76%, 76%, and 88% of adenomas, respectively. CONCLUSIONS The QuARTS technology provides a promising approach for quantifying methylated markers. The markers assayed highly discriminated colorectal neoplasia from healthy epithelia.
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Shen, Zhisen, Chongchang Zhou, Jinyun Li, Dong Ye, Hongxia Deng, Bin Cao, Wenjuan Hao, Lexi Lin, and Yuna Zhang. "SHISA3Promoter Methylation Is a Potential Diagnostic and Prognostic Biomarker for Laryngeal Squamous Cell Carcinoma." BioMed Research International 2017 (2017): 1–8. http://dx.doi.org/10.1155/2017/9058749.
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The purpose of this study was to evaluate the contribution ofSHISA3promoter methylation to laryngeal squamous cell carcinoma (LSCC).SHISA3promoter methylation status and expression were determined using methylation-specific polymerase chain reaction (MSP) and quantitative real-time PCR (qRT-PCR) in 93 paired LSCC and adjacent normal tissues, respectively. Furthermore, the regulatory function of theSHISA3promoter fragment was analyzed using a luciferase reporter assay. The results reveal that there is a significant increase inSHISA3methylation in LSCC tissues compared with corresponding nontumor tissuesP=4.58E-12. The qRT-PCR results show a significant association betweenSHISA3methylation and expression in LSCCP=1.67E-03. In addition, the area under the receiver operating characteristic curve was 0.91. Consequently, a log-rank test and multivariate Cox analysis suggest thatSHISA3promoter hypermethylation is a predictor of poor overall survival for LSCC (log-rankP= 0.024; HR = 2.71; 95% CI = 1.024–7.177;P= 0.047). The results indicate thatSHISA3promoter hypermethylation might increase the risk of LSCC through regulation of gene expression and is a potential diagnostic and prognostic biomarker for LSCC.
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FAWZI, Mohammed, Ahmed TAIFI, and Zahraa Kamil Kadhim LAWI. "SOMATOSTATIN (SST) PROMOTER HYPERMETHYLATION IN ASSOCIATION WITH COLORECTAL CANCER." Periódico Tchê Química 17, no. 36 (December 20, 2020): 1075–82. http://dx.doi.org/10.52571/ptq.v17.n36.2020.1090_periodico36_pgs_1075_1082.pdf.
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Colorectal cancer (CRC) is one of the most common diagnosis malignancies with different risk factors, including environmental and genetic. Several genes, called tumor suppressor genes, play an essential role in inhibiting these risk factors by preventing tumor development. One of these genes is somatostatin (SST). Somatostatin is an antiproliferative peptide with pro-apoptotic effects that enhance cell death to prevent tumor growth. This study aimed to investigate the association relationship between DNA methylation in SST promotor and colorectal cancer progression. After DNA bisulfite conversion, SST promoter methylation was examined using quantitative methylation‐specific PCR (qMSP) in 71 cases (19 metastasis CRC, 28 early-stage CRC, and 24 healthy controls). Quantitative methylation‐specific PCR (qMSP) is a real-time PCR method used to determine the unmethylated and methylated cytosine residues using a specific set of primers. The percentage of hypermethylation in SST promoter was 17%, 60%, and 79% for healthy controls, early-stage, and metastasis CRC groups. The results showed a significant association between DNA hypermethylation of SST promoter and CRC progression. P-values were 0.0364 for the early-stage group and 0.0138 for the metastasis group. The results also supported that the DNA hypermethylation block the expression of SST, which in turn induce carcinogenesis. The detection of SST promoter hypermethylation at early stage of cancer could be used as a biomarker for screening and prognosis of CRC.
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Hou, Yaping, Huayun Chen, Qiong He, Wei Jiang, Tao Luo, Jinhai Duan, Nan Mu, Yunshao He, and Huaqiao Wang. "Changes in methylation patterns of multiple genes from peripheral blood leucocytes of Alzheimer's disease patients." Acta Neuropsychiatrica 25, no. 2 (February 21, 2013): 66–76. http://dx.doi.org/10.1111/j.1601-5215.2012.00662.x.
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BackgroundEfforts aiming at identifying biomarkers and corresponding methods for early diagnosis of Alzheimer's disease (AD) might be the most appropriate strategy to initiate promising new treatments and/or prevention of ADObjectiveThe aim of our study is to assess the association of DNA methylation pattern of various leucocyte genes with AD pathogenesis in order to find potential biomarkers and corresponding methods for molecular diagnosis of AD.MethodsDNA methylation level of various genes in AD patients and normal population were compared by bisulphite sequencing PCR and methylation-specific PCR (MSP). Furthermore, real-time PCR was used to explore the effects of DNA methylation on the expression of target genes.ResultsResults showed significant hypermethylation of mammalian orthologue of Sir2 (SIRT1) gene in AD patients compared with normal population. Meanwhile, changes in methylation level of SIRT1 gene between different severities of AD were also found. Specific primers were designed from the SIRT1 CpG islands to differentiate AD and control group by MSP method. Besides, significant demethylation of β-amyloid precursor protein (APP) gene was observed in AD patients, whereas no difference was observed in other AD-related genes. Moreover, significant decrease in expression of SIRT1 gene and increase in expression of APP gene were also found in AD patients. In addition, the expression level of SIRT1/APP genes was associated with the severity, but not with the age or gender, of AD patients.Conclusion:SIRT1 and APP might be the interesting candidate biomarkers and valuable for clinical diagnosis or treatment of AD.
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Yang, Min, Ping Chen, Hong Peng, Hongliang Zhang, Yan Chen, Shan Cai, Qianjin Lu, and Chaxiang Guan. "Cigarette smoke extract induces aberrant cytochrome-c oxidase subunit II methylation and apoptosis in human umbilical vascular endothelial cells." American Journal of Physiology-Cell Physiology 308, no. 5 (March 1, 2015): C378—C384. http://dx.doi.org/10.1152/ajpcell.00197.2014.
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Cigarette smoke-induced apoptosis of vascular endothelial cells contributes to the pathogenesis of chronic obstructive pulmonary disease. However, the mechanisms responsible for endothelial apoptosis remain poorly understood. We conducted an in vitro study to investigate whether DNA methylation is involved in smoking-induced endothelial apoptosis. Human umbilical vascular endothelial cells (HUVECs) were exposed to cigarette smoke extract (CSE) at a range of concentrations (0–10%). HUVECs were also incubated with a demethylating reagent, 5-aza-2′-deoxycytidinem (AZA), with and without CSE. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and flow cytometry using annexin V-FITC/propidium iodide staining. We found that CSE treatment significantly increased HUVEC apoptosis in a dose- and time-dependent manner. Quantitative real-time RT-PCR and immunoblot revealed that CSE treatment decreased cytochrome- c oxidase subunit II (COX II) mRNA and protein levels and decreased COX activity. Methylation-specific PCR and direct bisulfite sequencing revealed positive COX II gene methylation. AZA administration partly increased mRNA and protein expressions of COX II, and COX activity decreased by CSE and attenuated the toxic effects of CSE. Our results showed that CSE induced aberrant COX II methylation and apoptosis in HUVECs.
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Bujnowska, Magda, Jiacheng Zhang, Qing Dai, Emily M. Heideman, and Jingyi Fei. "Deoxyribozyme-based method for absolute quantification of N6-methyladenosine fractions at specific sites of RNA." Journal of Biological Chemistry 295, no. 20 (April 8, 2020): 6992–7000. http://dx.doi.org/10.1074/jbc.ra120.013359.
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N6-Methyladenosine (m6A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA. Although candidate sites for the m6A modification are identified at the transcriptomic level, methods for site-specific quantification of absolute m6A modification levels are still limited. Herein, we present a facile method implementing a deoxyribozyme, VMC10, which preferentially cleaves the unmodified RNA. We leveraged reverse transcription and real-time quantitative PCR along with key control experiments to quantify the methylation fraction of specific m6A sites. We validated the accuracy of this method with synthetic RNA in which methylation fractions ranged from 0 to 100% and applied our method to several endogenous sites that were previously identified in sequencing-based studies. This method provides a time- and cost-effective approach for absolute quantification of the m6A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications.
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Wojtczyk-Miaskowska, Anita, Malgorzata Presler, Jerzy Michajlowski, Marcin Matuszewski, Beata Schlichtholz, and Medical University of Gdansk. "Gene Expression, DNA Methylation and Prognostic Significance of DNA Repair Genes in Human Bladder Cancer." Cellular Physiology and Biochemistry 42, no. 6 (2017): 2404–17. http://dx.doi.org/10.1159/000480182.
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Background/Aims: This study investigated the gene expression and DNA methylation of selected DNA repair genes (MBD4, TDG, MLH1, MLH3) and DNMT1 in human bladder cancer in the context of pathophysiological and prognostic significance. Methods: To determine the relationship between the gene expression pattern, global methylation and promoter methylation status, we performed real-time PCR to quantify the mRNA of selected genes in 50 samples of bladder cancer and adjacent non-cancerous tissue. The methylation status was analyzed by methylation-specific polymerase chain reaction (MSP) or digestion of genomic DNA with a methylation-sensitive restriction enzyme and PCR with gene-specific primers (MSRE-PCR). The global DNA methylation level was measured using the antibody-based 5-mC detection method. Results: The relative levels of mRNA for MBD4, MLH3, and MLH1 were decreased in 28% (14/50), 34% (17/50) and 36% (18/50) of tumor samples, respectively. The MBD4 mRNA expression was decreased in 46% of non-muscle invasive tumors (Ta/T1) compared with 11% found in muscle invasive tumors (T2-T4) (P<0.003). Analysis of mRNA expression for TDG did not show any significant differences between Ta/T1 and T2-T4 tumors. The frequency of increased DNMT1 mRNA expression was higher in T2-T4 (52%) comparing to Ta/T1 (16%). The overall methylation rates in tumor tissue were 18% for MBD4, 25% for MLH1 and there was no evidence of MLH3 promoter methylation. High grade tumors had significantly lower levels of global DNA methylation (P=0.04). There was a significant association between shorter survival and increased expression of DNMT1 mRNA (P=0.002), decreased expression of MLH1 mRNA (P=0.032) and the presence of MLH1 promoter methylation (P=0.006). Conclusion: This study highlights the importance of DNA repair pathways and provides the first evidence of the role of MBD4 and MLH3 in bladder cancer. In addition, our findings suggest that DNMT1 mRNA and MLH1 mRNA expression, as well as the status of MLH1 promoter methylation, are attractive prognostic markers in this pathology.
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Röhrs, Sonja, Julia Romani, Margarete Zaborski, Andreas Rosenwald, Hans G. Drexler, and Hilmar Quentmeier. "Hypermethylation of CD44 Is Characteristic of Specific Lymphoma Subtypes." Blood 114, no. 22 (November 20, 2009): 3469. http://dx.doi.org/10.1182/blood.v114.22.3469.3469.
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Abstract Abstract 3469 Poster Board III-357 Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a common hallmark of cancer. It is generally agreed, that CpG island hypermethylation profiles are specific for different tumor types [Costello et al., 2000, Nat Genet 25:132-138]. Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification), methylation of 24 different TSG was analyzed in 40 lymphoma cell lines representing Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). On average 8 ± 2.8 TSG out of the 24 analyzed were methylated per lymphoma cell line, whereas 0/24 TSG were methylated in healthy donor tonsils. Methylation frequencies decreased from HL, ALCL, BL, FL, DLBCL to MCL cell lines. The TSG methylation status of the most relevant genes was verified by methylation-specific PCR. Moreover, TSG hypermethylation correlated with transcriptional silencing as assessed by quantitative real time PCR. While our studies on the methylation status of TSG in lymphoma cell lines support previous methylation analyses performed on primary lymphoma patient material for many of the genes analyzed here, MS-MLPA screening also revealed a new interesting candidate: CD44. Hypermethylation of CD44 was characteristic of ALCL, BL, FL and DLBCL cell lines and allowed their discrimination from MCL and HL cell lines. In CD44 hypermethylated cell lines expression of CD44 was re-inducible at mRNA and protein levels by treatment with the demethylating agent 5-Aza-2'-deoxycytidine, confirming its epigenetic regulation. Furthermore, CD44 ligation with an anti-CD44 antibody induced apoptosis in CD44+ (CD44 unmethylated) lymphoma cell lines whereas CD44 hypermethylated cell lines showed no response. Thus, CD44 might be an interesting new epigenetic marker and a potential molecular target for the diagnosis and treatment of specific lymphoma subtypes. Disclosures No relevant conflicts of interest to declare.
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Bergallo, Massimiliano, Ilaria Galliano, Paola Montanari, Cristina Calvi, Valentina Daprà, Diana Carli, Silvia Russo, Alessandro Mussa, and Giovanni Battista Ferrero. "Comparison of Quantitative Analysis of Methylated Alleles Real-Time PCR and Methylation-Specific MLPA for Molecular Diagnosis of Beckwith-Wiedemann Syndrome." Pathobiology 86, no. 4 (2019): 217–24. http://dx.doi.org/10.1159/000500627.
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Kordi-Tamandani, Dor Mohammad, Abdolkarim Moazeni-Roodi, Mohammad Ayoub Rigi Ladez, Mohammad Hashemi, Elnaz Birjandian, and Adam Torkamanzehi. "Analysis of Methylation Patterns and Expression Profiles of P14Arf Gene in Patients with Oral Squamous Cell Carcinoma." International Journal of Biological Markers 25, no. 2 (April 2010): 99–103. http://dx.doi.org/10.1177/172460081002500207.
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Aims To analyze the promoter methylation profile and mRNA expression of the p14ARF gene in oral squamous cell carcinoma (OSCC). Methods Promoter methylation of the p14ARF gene was investigated by methylation-specific polymerase chain reaction in paraffin-embedded tissues from 76 patients with OSCC and 57 oral tissues used as healthy controls. Expression of p14ARF mRNA was also determined using real-time quantitative reverse-transcription PCR. The methylation status and mRNA level profile of the gene and their relationship with clinical data were analyzed. Results Methylation of the p14ARF gene in OSCC was significantly increased compared to normal control tissues (χ2 = 16.73, p<0.0001). The relative expression of p14ARF mRNA in OSCC was not significantly different from that in healthy control samples. Conclusion Promoter methylation of p14ARF may be an important mechanism in OSCC, and its determination may be considered an important tool in the early diagnosis and treatment of OSCC.
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Pan, Zhangyuan, Xiangyu Wang, Ran Di, Qiuyue Liu, Wenping Hu, Xiaohan Cao, Xiaofei Guo, et al. "A 5-Methylcytosine Site of Growth Differentiation Factor 9 (GDF9) Gene Affects Its Tissue-Specific Expression in Sheep." Animals 8, no. 11 (November 7, 2018): 200. http://dx.doi.org/10.3390/ani8110200.
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Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (−1453 to −1854) of GDF9 promoter in ovary and heart was determined. In this region (−1987 to −1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.
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Aftab, Ayma, Samia Afzal, Muhammad Idrees, and Ahmad Ali Shahid. "p53 and rb promoter methylation in hepatitis C virus-related chronic hepatitis and hepatocellular carcinoma." Future Virology 16, no. 1 (January 2021): 15–25. http://dx.doi.org/10.2217/fvl-2020-0154.
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Aim: To identify methylation in p53 and rb during hepatitis C virus (HCV) infection in individuals in Pakistan. Materials & methods: Methylation-specific PCR was used on liver biopsies from hepatocellular carcinoma and chronic hepatitis C patients and on blood samples from healthy individuals. Real-time PCR was used to assess changes in the expression of p53 and rb in Huh-7 cells transfected with HCV-3a. Results: The p53 and rb promoters were methylated in hepatocellular carcinoma patients. The presence of HCV-3a- Core (p = 0.03), HCV-3a- NS-3 (p = 0.01) and HCV-3a- NS-5a (p = 0.02) downregulated p53 expression. Exposure to HCV-3a- Core (p = 0.04) downregulated rb expression. Conclusion: It can be hypothesized that HCV-induced epigenetic modifications may lead to the development of hepatic cancer that in turn inactivates p53 and rb.
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Hagiwara, Kazumi, Hirokazu Nagai, Toshiya Yokozawa, CHiaki Kato, Motohiro Hamaguchi, Tomomitsu Hotta, and Keizo Horibe. "Detection of p57KIP2 Gene Methylation Is Useful Marker for Minimal Residual Disease in Diffuse Large B Cell Lymphoma." Blood 110, no. 11 (November 16, 2007): 4415. http://dx.doi.org/10.1182/blood.v110.11.4415.4415.
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Abstract Background: The t(14;18)(q32;q21) translocation, which causes a BCL2/IgH gene rearrangement, is a characteristic chromosomal abnormality found mainly in B cell lymphomas, and its detection and quantification is useful for monitoring of minimal residual disease (MRD) in follicular lymphoma (FL). However, in diffuse large B cell lymphoma (DLBCL), the frequency of this rearrangement is relatively low. We revealed that the promoter region of p57KIP2 gene is frequently methylated in B cell lymphomas. Aberrant DNA methylation in the promoter regions of genes is known as the major mechanism for inactivation of tumor suppressor genes, so the detection of the methylation is expected to be used as a promising tumor specific marker. Therefore, we investigated the possibility of p57KIP2 gene promoter methylation as a biomarker for MRD detection in B cell lymphoma, especially in DLBCL. Methods: We analyzed lymphoid cell line DNA, tumor DNA from 64 patients with DLBCL, and 4 patients with FL. The bisulfite-modified DNA was used as a template for conventional methylation-specific PCR (MSP) and real-time quantitative MSP (Q-MSP). Results: In clinical samples (tumor DNA), p57KIP2 gene methylation was detected by Q-MSP in 73% (47/64) DLBCL, and 50% (2/4) FL. Using cell line DNA, which was fully methylated in promoter region of p57KIP2 gene, we determined the detection limit of Q-MSP assay. The methylated DNA could be detected in the presence of a 1000-fold excess of unmethylated DNA by Q-MSP. We could validate the level of methylated DNA in each sample by Q-MSP evaluating the p57KIP2/b-actin ratio. The sensitivity to detect MRD in bone marrow by this method was found to be equivalent to real time quantitative PCR for BCL2/IgH major breakpoint region. Conclusion: The methylation of p57KIP2 gene is detected at high frequency in DLBCL. This biomarker is thought to be conventional and widely applicable to the detection of MRD in DLBCL.
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Watanabe, Atsushi, Takeshi Inukai, Minori Tamai, Tamao Shinohara, Shinpei Somazu, Hiroko Oshiro, Koshi Akahane, et al. "Involvement of Allele-Specific Methylation of Asparagine Synthetase Gene in Asparaginase Sensitivity of BCP-ALL." Blood 128, no. 22 (December 2, 2016): 3966. http://dx.doi.org/10.1182/blood.v128.22.3966.3966.
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Abstract Asparaginase is one of the most important components for the treatment of ALL. ALL cells are supposed to be unable to synthesize adequate amounts of Asparagine (Asn), and, therefore, depend on extracellular source of Asn to survive. Asparaginase therapy induces the depletion of serum Asn by catalyzing the deamination of Asn and leads to cell death of ALL cells. Asparagine synthetase (ASNS) is an enzyme that produces Asn from Aspartic acid. Thus, silencing of the ASNS gene in ALL cells could be crucial for complete starving ALL cells of the Asn. Considering that the ASNS gene has a CpG island in its promotor, aberrant methylation of CpG island could be one of epigenetic mechanisms for silencing of ASNS gene in ALL cells. Previous qualitative analysis of ALL samples using methylation-specific restriction enzyme revealed frequent methylation of CpG island in the ASNS gene. However, associations of methylation status of ASNS gene with its expression level and sensitivity to asparaginase in ALL cells remain unknown. Moreover, little is known about mechanisms for leukemia-specific ASNS gene silencing by methylation. To shed light on these issues, we analyzed a large panel of BCP-ALL cell lines. We quantified ASNS gene expression level by real time RT-PCR in 79 BCP-ALL cell lines cultured in the presence or the absence of L-asparaginase (L-asp), and determined IC50 values of L-asp using alamar blue assay. In the majority of cell lines, although degree of the induction was highly variable, ASNS gene expression level was upregulated in the presence of L-asp. IC50 value of L-asp showed significant correlation with ASNS gene expression level cultured in the presence of L-asp (r=0.222, p=0.049) rather than that in the absence of L-asp (r=0.193, p=0.089). We next analyzed methylation status of the ASNS gene in 79 BCP-ALL cell lines by bisulfite PCR sequencing using a next-generation sequencer (NGS). Strong correlation was confirmed between mean % methylation by NGS and Sanger sequencing in representative cell lines. Of importance, mean % methylation in 79 BCP-ALL cell lines showed significant negative correlation with ASNS gene expression level cultured in the presence of L-asp (r=-0.482, p=6.73x10-6) and, subsequently, IC50 value of L-asp (r=-0.39, p=3.86x10-4). Unexpectedly, % methylation of 79 cell lines distributed in three clusters; 15 cell lines (19%) were highly methylated (>66%, median; 89%), 26 cell lines (32.9%) were moderately methylated (33-66%, median; 40%), and 38 cell lines (48.1%) were weakly methylated (<33%, median; 3.7%). In the majority of moderately methylated cell lines, histograms of % methylation in each read of NGS showed two peaks of high and low methylation, suggesting an allele-specific methylation. In the middle of CpG island, tandem repeat polymorphism of 14bp nucleotides is located adjacent to methylation-specific restriction enzyme site of Aor13HI. Of note, in 7 out of 8 moderately methylated cell lines with heterozygous tandem repeat genotype, only single PCR product was detectable when PCR was performed after Aor13HI treatment, whereas two PCR products derived from two- and three-repeat alleles was detectable when PCR was performed without treatment, indicating an allele-specific methylation. We next analyzed a possible one-allele-loss of the ASNS gene in highly methylated (>66%; 8 cell lines) and weakly methylated (<20%; 12 cell lines) cell lines. We directly sequenced genotype in a portion of introns 2 and 4 and exon 5 based on the imputated SNP genotypes, and confirmed heterozygous genotype in every cell lines at least in one of eight SNPs analyzed, demonstrating that loss-of-heterozygosity is not the mechanism for high or low methylation of the ASNS gene. Similar pattern of methylation was observed in 52 BCP-ALL samples. Taken together, these observations indicate that stepwise allele-specific methylation of ASNSgene is critically involved in the sensitivity to L-asp of BCP-ALL. Disclosures No relevant conflicts of interest to declare.
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Sauer, Julia, Hyeran Jang, Ella M. Zimmerly, Kyong-chol Kim, Zhenhua Liu, Aurelie Chanson, Donald E. Smith, Joel B. Mason, Simonetta Friso, and Sang-Woon Choi. "Ageing, chronic alcohol consumption and folate are determinants of genomic DNA methylation, p16 promoter methylation and the expression of p16 in the mouse colon." British Journal of Nutrition 104, no. 1 (March 8, 2010): 24–30. http://dx.doi.org/10.1017/s0007114510000322.
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Older age, dietary folate and chronic alcohol consumption are important risk factors for the development of colon cancer. The present study examined the effects of ageing, folate and alcohol on genomic and p16-specific DNA methylation, and p16 expression in the murine colon. Old (aged 18 months; n 70) and young (aged 4 months; n 70) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18 % of energy), a Lieber-DeCarli diet with alcohol (18 %) and reduced folate (0·25 mg folate/l) or an isoenergetic control diet (0·5 mg folate/l) for 5 or 10 weeks. Genomic DNA methylation, p16 promoter methylation and p16 gene expression were analysed by liquid chromatography–MS, methylation-specific PCR and real-time RT-PCR, respectively. Genomic DNA methylation was lower in the colon of old mice compared with young mice (P < 0·02) at 10 weeks. Alcohol consumption did not alter genomic DNA methylation in the old mouse colon, whereas it tended to decrease genomic DNA methylation in young mice (P = 0·08). p16 Promoter methylation and expression were higher in the old mouse colon compared with the corresponding young groups. There was a positive correlation between p16 promoter methylation and p16 expression in the old mouse colon (P < 0·02). In young mice the combination of alcohol and reduced dietary folate led to significantly decreased p16 expression compared with the control group (P < 0·02). In conclusion, ageing and chronic alcohol consumption alter genomic DNA methylation, p16 promoter methylation and p16 gene expression in the mouse colon, and dietary folate availability can further modify the relationship with alcohol in the young mouse.
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Buchynska, L. G., N. P. Iurchenko, N. P. Verko, K. A. Nekrasov, and V. I. Kashuba. "FOXP3 gene promoter methylation in endometrial cancer cells." Experimental Oncology 37, no. 4 (December 22, 2015): 246–49. http://dx.doi.org/10.31768/2312-8852.2015.37(4):246-249.
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Aim - to determine the methylation level of promoter region of the FOXP3 gene promoter depending on the heterogeneity of intracellular localization of its protein product in endometrial cancer (EC) cells and assess its relation to the clinical and morphological features of tumor. Samples of surgical material of 40 EC patients who have not received any specific treatment before the surgery, were studied. Real time methylation-specific PCR (MSP) as well as morphological and immunohis-tochemical methods were used in the study. Methylation of promoter region of the FOXP3 gene was determined in all EC cases, but variability of the methylation level in EC cells from 45,0 % to 85,0 % was observed. With tumor progression and in tumors with deep (<$Esymbol У~1 "/" 2>) invasion in myometrium, an increase of the methylation level of the FOXP3 and of cell number with cytoplasmic FOXP3 localization was observed. In EC patients the correlation between of methylation level of the FOXP3 gene and the number of FOXP3<^>+ tumor cells with cytoplasmic expression (r = 0,41) was determined. Conclusion: The methylation level of FOXP3 gene promoter region and intracellular localization of its protein product are associated with tumor differentiation grade and the depth of myometrial invasion.
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Shirkavand, Atefeh, Zahra N. Boroujeni, and Seyed A. Aleyasin. "Solanum nigrum Anticancer Effect Through Epigenetic Modulations in Breast Cancer Cell Lines." Current Cancer Therapy Reviews 16, no. 2 (June 9, 2020): 121–26. http://dx.doi.org/10.2174/1573394715666190101114541.
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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<0.05). Conclusion: Our findings reveal for the first time the impact of SNE on the methylation of breast cancer cells.
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Attia, Hanaa RM, Mona Hamed Ibrahim, Shereen H. Abd El-Aziz, Naglaa M. Hassan, Randa A. Osman, Heba A. Hagag, Marianne E. Yassa, Amany H. Abdelrahman, Iman I. Salama, and Mohamed Emam Sobeih. "ITGA4 gene methylation status in chronic lymphocytic leukemia." Future Science OA 6, no. 7 (August 1, 2020): FSO583. http://dx.doi.org/10.2144/fsoa-2020-0034.
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Background: We aimed to investigate ITGA4 gene expression pattern and to explore its methylation heterogeneity in chronic lymphocytic leukemia (CLL). Patients & methods: Eighty one CLL patients and 75 healthy subjects were enrolled and prognostic evaluation of patients was assessed. ITGA4 q-realtime PCR was performed using Applied Biosystems, TaqMan gene expression assay. ITGA4 gene-specific CpG methylation was investigated in real time using pyrosequencing technology. Results: ITGA4 was differentially expressed in CLL patients. The CpG sites-1, 2 and 3 showed significantly higher mean levels than healthy controls (p = <0.001, 0.007 and 0.009). Significant association between CpG site-1 and CLL has been detected using age-adjusted logistic regression (p < 0.001). Conclusion: Hypermethylation at ITGA4 gene CpG sites (1,2,3) is a characteristic feature in CLL.
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Hopfer, Olaf J., Martina Komor, Ina S. Koehler, Matthias Schulze, Claudia Freitag, Dieter Hoelzer, Eckhard Thiel, and Wolf-Karsten Hofmann. "Expression of DNMT Isoforms Is Differentially Associated with Aberrant Promotor Methylation in MDS Hematopoietic Progenitor Cells during Lineage Specific Differentiation." Blood 108, no. 11 (November 16, 2006): 2628. http://dx.doi.org/10.1182/blood.v108.11.2628.2628.
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Abstract Recent findings suggest that in myelodysplastic syndrome (MDS) several key regulatory genes are affected by aberrant promotor methylation. To explore the molecular basis of this impairment we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Promotor methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite treated genomic DNA for each lineage at each time point. In addition, expression of DNMT1 (maintenance DNA methyltransferase), DNMT3a and DNMT3b (both de novo DNA methyltransferase) was analyzed by real time RT-PCR and correlated with gene promotor methylation at any time point. DNMT1 expression was increased during erythropoiesis in both, normal controls and MDS patients. On the other hand, expression of de novo DNMTs was elevated during thrombopoiesis at all time points. During erythropoiesis hypermethylation of p73, hMLH1 and RARb was associated with elevated DNMT1, hypermethylation of p15, p16, p73 and survivin was positively associated with increasing DNMT3 expression. Interestingly, DNMT1 was only elevated in low risk MDS, but not further increased in high risk MDS patients. Surprisingly, MDS specific survivin promotor methylation was inverse correlated with DNTM1 and DNMT3a expression. However, a negative correlation of DNMT3a with survivin expression was found in low risk MDS but not in high risk MDS. In summary our data indicate that all mammalian DNMT isoforms may be involved in the aberrant DNA-methylation phenotype in MDS. Elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in low risk MDS. DNMT3a and 3b were elevated during megakaryopoiesis and their expression was inversely correlated with MDS disease risk (IPSS). We conclude that the knowledge about distinct expression patterns of DNMT isoforms in hematopoiesis may be of help for further strategies to implicate DNMT-inhibitors in the treatment of patients with MDS.
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Luo, Hao-Lun, Hui-Ying Liu, Yin-Lun Chang, Ming-Tse Sung, Po-Yen Chen, Yu-Li Su, Chun-Chieh Huang, and Jei-Ming Peng. "Hypomethylated RRBP1 Potentiates Tumor Malignancy and Chemoresistance in Upper Tract Urothelial Carcinoma." International Journal of Molecular Sciences 22, no. 16 (August 16, 2021): 8761. http://dx.doi.org/10.3390/ijms22168761.
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Ribosome-binding protein 1 (RRBP1) is a potential oncogene in several cancer types. However, the correlation between RRBP1 expression and the prognosis of patients with upper tract urothelial carcinoma (UTUC) remains unclear. In this study, we identified that RRBP1 is associated with carcinogenesis and metastasis in UTUC using a methylation profiling microarray. High correlations between RRBP1 and cancer stages, nodal metastasis status, molecular subtypes, and prognosis in bladder urothelial cancer (BLCA) were found. Aberrant DNA methylation in the gene body region of RRBP1 was determined in UTUC tissues by methylation-specific PCR. RRBP1 expression was significantly increased in UTUC tissues and cell lines, as determined by real-time PCR and immunohistochemistry. RRBP1 depletion significantly reduced BFTC909 cell growth induced by specific shRNA. On the other hand, molecular subtype analysis showed that the expression of RRBP1 was associated with genes related to cell proliferation, epithelial–mesenchymal transition, and basal markers. A patient-derived organoid model was established to analyze patients’ responses to different drugs. The expression of RRBP1 was related to chemoresistance. Taken together, these results provide the first evidence that RRBP1 gene body hypomethylation predicts RRBP1 high expression in UTUC. The data highlight the importance of RRBP1 in UTUC malignancy and chemotherapeutic tolerance.
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Pérez Pereda, Sara, María Toriello Suárez, Vicente González Quintanilla, and Agustín Oterino. "Methylation analysis of NPTX2 and SH2D5 genes in chronic migraine: A case–control study." Cephalalgia Reports 3 (January 1, 2020): 251581632092359. http://dx.doi.org/10.1177/2515816320923592.
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Background: Methylation of two CpG sites related to neuronal pentraxin II protein (NPTX2) and SH2 domain containing 5 protein (SH2D5), corresponding to two neuroplasticity genes, has been associated to headache chronification. We aimed to investigate the epigenetic modification of these two genes in chronic migraine (CM). Methods: We conducted a case–control study in which the DNA of 305 age- and sex-matched subjects classified according to the International Classification of Headache Disorders version beta (ICHD-III β) in CM (109), episodic migraine (EM; n = 98), and healthy controls (HC; 98) was analyzed. Real-time quantitative methylation-specific PCR was performed using specific methylation primers for two representative CpG sites within these genes. Results: We found no significant differences in methylation level between CM, EM, and HC in the first exon of the NPTX2 gene nor in the 5′ upstream region of the SH2D5 gene. Methylation level in the first exon of the NPTX2 showed a low correlation with age ( r = 0.266; p < 0.005). Conclusion: We did not find methylation level differences in analyzed regions related to NPTX2 and SH2D5 in our CM sample. Despite the potential relevance of neuroplasticity genes in headache chronification, we conclude that CM is a more heterogeneous clinical diagnosis than desired and that an epigenetic marker remains elusive.
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Najafi, Maryam, Dor Mohammad Kordi-Tamandani, and Mohammad Arish. "Evaluation ofLATS1andLATS2Promoter Methylation with the Risk of Pterygium Formation." Journal of Ophthalmology 2016 (2016): 1–5. http://dx.doi.org/10.1155/2016/5431021.
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Purpose. Pterygium is a serious eye problem in countries with high exposure to UV. However, despite numerous studies, the molecular etiology of pterygium is unclear. Recent studies have indicated thatLATS1andLATS2genes are involved in DDR signaling pathways against continuous UV exposure. Our aim was to evaluate theLATS1andLATS2promoter methylation with the risk of pterygium formation.Methods. We evaluated the promoter methylation status ofLATS1andLATS2using methylation-specific PCR technique. Also, mRNA expression ofLATS1andLATS2was assessed in 14 cases of pterygium and 14 normal specimens by real-time PCR.Results. Promoter methylation ofLATS1andLATS2was detected significantly between pterygium tissues and normal tissues [LATS1; OR = 4.9; 95% CI: 1.54 to 15.48,P=0.003;LATS2; OR = 7.1; 95% CI: 1.53 to 33.19,P=0.004]. The gene expression analysis showed a statistically significant difference between pterygium tissues and healthy controls for bothLATS1andLATS2(P<0.05).Conclusions. The data of this study is the first report regarding the effect of promoter methylation of theLATS1andLATS2in the pterygium. To confirm these data, doing further studies in various genetic populations with large sample sizes using advanced molecular techniques is proposed.
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Zhang, Ying-Ying, Jing-Dong Zhou, Dong-Qin Yang, Pin-Fang He, Dong-Ming Yao, Zhen Qian, Jing Yang, Wen-Rong Xu, Jiang Lin, and Jun Qian. "Intragenic hypomethylation of DNMT3A in patients with myelodysplastic syndrome." Clinical Chemistry and Laboratory Medicine (CCLM) 56, no. 3 (February 23, 2018): 485–91. http://dx.doi.org/10.1515/cclm-2016-0142.
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AbstractBackground:DNMT3Ais a DNA methyltransferase that acts inde novomethylation. Aberrant expression ofDNMT3Ahas been reported in several human diseases, including myelodysplastic syndrome (MDS). However, the pattern ofDNMT3Amethylation remains unknown in MDS.Methods:The present study was aimed to investigate the methylation status ofDNMT3Aintragenic differentially methylated region 2 (DMR2) using real-time quantitative methylation-specific PCR and analyze its clinical significance in MDS.Results:Aberrant hypomethylation ofDNMT3Awas found in 57% (51/90) MDS cases. There were no significant differences in age, sex, white blood cell counts, platelet counts, hemoglobin counts and World Health Organization, International Prognostic Scoring System and karyotype classifications betweenDNMT3Ahypomethylated andDNMT3Ahypermethylated groups. However, the patients withDNMT3Ahypomethylation had shorter overall survival time than those withoutDNMT3Ahypomethylation (11 months vs. 36 months, p=0.033). Multivariate analysis confirmed the independent adverse impact ofDNMT3Ahypomethylation in MDS.Conclusions:Our data suggest thatDNMT3ADMR2 hypomethylation may be a negative prognostic hallmark in MDS.
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Dittharot, Kanthanadon, Sumana Dakeng, Parichat Suebsakwong, Apichart Suksamrarn, Pimpicha Patmasiriwat, and Moltira Promkan. "Cucurbitacin B Induces Hypermethylation of Oncogenes in Breast Cancer Cells." Planta Medica 85, no. 05 (November 21, 2018): 370–78. http://dx.doi.org/10.1055/a-0791-1591.
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AbstractBreast cancer is a complex disease driven by multiple factors including both genetic and epigenetic alterations. Recent studies revealed that abnormal gene expression induced by epigenetic changes including aberrant promoter methylation plays a critical role in human breast carcinogenesis. Cucurbitacin B has antiproliferative activity against various human breast cancer cells, but the molecular mechanism is not completely understood. In this study, we explore the influence of cucurbitacin B from Trichosanthes cucumerina on the methylation status at the promoter of oncogenes c-Myc, cyclin D1, and survivin in breast cancer cell lines. Growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay and colony formation assay. Methylation status of genomic DNA was determined by methylation-specific PCR. Gene and protein expression levels of all genes studied were analyzed by real-time RT-PCR and western blot. The results indicated that cucurbitacin B could inhibit cell growth in breast cancer cells. The oncogene promoters are usually hypomethylated in cancer cells. Upon cucurbitacin B treatment, upregulation of DNMT1 and obvious heavy methylation in the promoters of c-Myc, cyclin D1, and survivin, which consequently downregulated the expression of all these oncogenes, were observed. Hence, cucurbitacin B proved to be a potential cancer therapeutic agent, in part by inducing hypermethylation and silences the oncogenic activation.
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Lai, Zi-Lun, Yung-An Tsou, Shin-Ru Fan, Ming-Hsui Tsai, Hsiao-Ling Chen, Nai-Wen Chang, Ju-Chien Cheng, and Chuan-Mu Chen. "Methylation-Associated Gene Silencing ofRARBin Areca Carcinogens Induced Mouse Oral Squamous Cell Carcinoma." BioMed Research International 2014 (2014): 1–13. http://dx.doi.org/10.1155/2014/378358.
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Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptorβ(RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown thatde novoDNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed thatRARBhypermethylation was involved in the areca-associated oral carcinogenesis.
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Singh, Gurinder Bir, Sanskriti Khanna, Satish K. Raut, Saurabh Sharma, Rajni Sharma, and Madhu Khullar. "DUSP-1 gene expression is not regulated by promoter methylation in diabetes-associated cardiac hypertrophy." Therapeutic Advances in Cardiovascular Disease 11, no. 5-6 (April 16, 2017): 147–54. http://dx.doi.org/10.1177/1753944717704590.
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Background: The exact mechanism causing decreased expression of the dual specific phosphatase-1 ( DUSP-1) gene in diabetes-associated cardiac hypertrophy is not known. DNA promoter methylation is often associated with decreased gene expression in many diseases including cardiovascular diseases. So, we investigated whether epigenetic silencing via promoter methylation is involved in the decreased expression of DUSP-1 in diabetes-associated cardiac hypertrophy. Methods: Real-time polymerase chain reaction (PCR) and Western blotting confirmed the down regulation of the DUSP-1 gene at transcriptional and translational levels. Bisulfite-converted DNA samples from myocardium of rat model of diabetic cardiomyopathy (DCM), high glucose (HG)-treated neonatal rat cardiomyocytes (NRCMs) and cardiac tissues from archived human myocardial DCM autopsies along with their respective controls were analyzed for methylation in the promoter region of the DUSP-1 gene. Results: We observed no methylation in the promoter regions of the DUSP-1 gene in DCM rat hearts, in HG-treated NRCMs (between −355 bp and −174 bp) and in cardiac tissues from archived human myocardial DCM autopsies (between −274 bp and −73 bp). Conclusion: Methylation-mediated silencing of the DUSP-1 promoter does not appear to be associated with reduced expression, indicating the involvement of other factors in specific suppression of DUSP-1 in diabetes-associated cardiac hypertrophy.
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Aldape, K. D., G. Jones, M. Wang, M. Hegi, R. C. Janzer, R. Stupp, M. P. Mehta, and M. R. Gilbert. "MGMT methylation testing in RTOG 0525: A phase III trial of newly diagnosed glioblastoma." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 2051. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.2051.
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2051 Background: MGMT promoter methylation has been described as a prognostic marker in glioblastoma (GBM) and may be associated with chemosensitivity to alkylating agents. This study determined the feasibility of real-time determination of MGMT promoter methylation testing in a large international phase III clinical trial. Methods: Paraffin tumor blocks were obtained from patients registered onto RTOG 0525 then distributed to one of two central pathology labs: MD Anderson (Houston TX, K. Aldape) or CHUV (Lausanne, CH, R. Janzer). After histologic confirmation of GBM, unstained slides (40 microns of tumor tissue) were sent to the testing laboratories. Results were used for randomization into a treatment arm. MGMT methylation was assessed using methylation specific real-time PCR (MSP). The assay determined the number of copies of both methylated MGMT and of beta-actin (ACTB) in the sample. The ACTB copy number was used to assess the quality and quantity of the sample DNA. Results: Results were available for 995 samples. Following MSP samples were categorized into one of five possible results: failed (2, 0.2%), methylated (302, 30.2%), non-methylated (602, 60.2%), indeterminate (62, 6.2%), and invalid (27, 2.7%). Among cases that were evaluable as either methylated or unmethylated (n = 904) the MGMT promoter methylation rate in newly diagnosed GBM was approximately 1/3 (33.4%), a rate that is somewhat lower than prior reports. The average time from receipt of sample at the central pathology laboratory to reporting of results was 9.3 days. The time required decreased over the course of the clinical trial. This was due, in part, to training of the sites to deliver samples just before the start of runs. Conclusions: The results demonstrate the feasibility of performing real-time MGMT methylation testing, a tumor based assay, as a stratification factor in a multinational clinical trial. This study confirms that treatment decisions based on the molecular characteristics of the tumor are feasible, thereby providing opportunities to develop more molecularly-based tumor stratification or selection, a major advance in developing personalized treatment regimens. No significant financial relationships to disclose.