Journal articles on the topic 'Late stage methylation'
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Feng, Kaibo, Raundi E. Quevedo, Jeffrey T. Kohrt, Martins S. Oderinde, Usa Reilly, and M. Christina White. "Late-stage oxidative C(sp3)–H methylation." Nature 580, no. 7805 (March 16, 2020): 621–27. http://dx.doi.org/10.1038/s41586-020-2137-8.
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Deshmukh, R. S., O. Svarcova, J. Li, H. Callesen, G. Vajta, and P. Maddox-Hyttel. "85 DEOXYRIBONUCLEIC ACID METHYLATION IN PORCINE PARTHENOGENETIC PREIMPLANTATION EMBRYOS." Reproduction, Fertility and Development 21, no. 1 (2009): 143. http://dx.doi.org/10.1071/rdv21n1ab85.
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DNA methylation is one of the most important epigenetic mechanisms involved in gene silencing. Waves of DNA de- and re-methylation occur during mammalian preimplantation development. Whether the same happens in porcine parthenogenetic embryos has never been determined, and we set out to investigate this question. Porcine oocytes were aspirated from antral follicles, matured in vitro for 42 h, parthenogenetically activated and fixed in 4% paraformaldehyde at the 1-, 2-, 4-, and 8-cell stage as well as at the early and late blastocyst stage. The degree of DNA methylation was assessed by immunocytochemical staining (Anti-5MetC mouse monoclonal; Abcam, Copenhagen, Denmark) and DNA was counterstained with Hoechst 33258. Porcine fetal fibroblasts were used as standard. The fluorescent signals were detected using an epifluorescence microscope (Leica) and a Leica camera set at fixed exposure times. Signals were quantified using NIH ImageJ sofware. Total means of intensities (methylation and DNA) were calculated and exponential curves were obtained using Microsoft Excel-based statistics. DNA methylation and DNA content were highly correlated in porcine fetal fibroblasts demonstrating the effect of an immediate maintenance methylation taking place during the DNA S-phase of the cell cycle. A similar correlation was observed in the parthenogenetic embryos at all the developmental stages. The level of DNA methylation increased slightly from the early to the late 1-cell stage, and a pronounced increase in DNA methylation level was noted at the 2-cell stage. At the late 1-cell stage, the DNA methylation level of the two pronuclei was similar probably due to the maternal origin of both pronuclei. At the 4-cell stage, DNA methylation had decreased again but was higher compared with other developmental stages, except the 2-cell stage, and at the 8-cell stage, the level of DNA methylation reached a minimum. Subsequently, the level of DNA methylation increased slightly at the blastocyst stages. In conclusion, DNA methylation and DNA content were correlated in porcine fetal fibroblasts and parthenogenetic embryos. Furthermore, the levels of DNA methylation in parthenogenetic embryos exhibited an increase to the 2-cell stage followed by a decrease to the 8-cell stage and a final increase to the blastocyst stage. The initial increase in methylation to the 2-cell stage is different from what has been reported previously for in vivo derived porcine embryos. We are thankful to H. Holm and J. Nielsen for excellent technical assistance. This project was supported by Marie Currie Action project MRTN-CT-2006-35468 (CLONET).
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Yang, Xiaosong, Rui Wu, Weiguang Shan, Liqing Yu, Bingzhong Xue, and Hang Shi. "DNA Methylation Biphasically Regulates 3T3-L1 Preadipocyte Differentiation." Molecular Endocrinology 30, no. 6 (June 1, 2016): 677–87. http://dx.doi.org/10.1210/me.2015-1135.
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Abstract Better understanding the mechanisms underlying adipogenesis may provide novel therapeutic targets in the treatment of obesity. Most studies investigating the mechanisms underlying adipogenesis focus on highly regulated transcriptional pathways; little is known about the epigenetic mechanisms in this process. Here, we determined the role of DNA methylation in regulating 3T3-L1 adipogenesis in early and late stage of differentiation. We found that inhibiting DNA methylation pharmacologically by 5-aza-2′-deoxycytidine (5-aza-dC) at early stage of 3T3-L1 differentiation markedly suppressed adipogenesis. This inhibition of adipogenesis by 5-aza-dC was associated with up-regulation of Wnt10a, an antiadipogenic factor, and down-regulation of Wnt10a promoter methylation. In contrast, inhibiting DNA methylation by 5-aza-dC at late stage of differentiation enhanced the lipogenic program. The differential effects of 5-aza-dC on adipogenesis were confirmed by gain or loss of function of DNA methyltransferase 1 using genetic approaches. We further explored the molecular mechanism underlying the enhanced lipogenesis by inhibition of DNA methylation at late stage of differentiation. The Srebp1c promoter is enriched with CpG sites. Chromatin immunoprecipitation assays showed that DNA methyltransferase 1 bound to the methylation region at the Srebp1c promoter. Pyrosequencing analysis revealed that the DNA methylation at the key cis-elements of the Srebp1c promoter was down-regulated in adipogenesis. Further, luciferase reporter assays showed that the Srebp1c promoter activity was dramatically up-regulated by the unmethylated promoter compared with the fully methylated promoter. Thus DNA methylation appears to exert a biphasic regulatory role in adipogenesis, promoting differentiation at early stage while inhibiting lipogenesis at late stage of 3T3-L1 preadipocyte differentiation.
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Li, Y., H. D. Morgan, L. Ganeshan, and C. O'Neill. "124. DNA METHYLATION IN THE 2-CELL MOUSE EMBRYO IS THE RESULT OF DNMT ACTIVITY DURING DEVELOPMENT FROM THE ZYGOTE TO THE 2-CELL STAGE." Reproduction, Fertility and Development 21, no. 9 (2009): 43. http://dx.doi.org/10.1071/srb09abs124.
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In an accompanying abstract we show for the first time that global demethylation of both paternally- and maternally-derived genomes occurs prior to syngamy. It is commonly considered that new methylation of the genome does not commence until late in the preimplantation stage. Yet embryos during cleavage stage are known to show DNA methylation. This creates a paradox, if global demethylation occurs by the time of syngamy yet remethylation does not occur until the blastocysts stage, how can cleavage stage embryos possess methylated DNA. We examined this paradox. We examined DNA methylation in 2-cell embryos by confocal microscopy of anti-methylcytosine immunofluorescence and propidium iodide co-staining of whole mounts. We confirmed that DNA in late zygotes was substantially demethylated in both the male and female pronuclei. By the 2-cell stage, embryos collected direct from the oviduct showed high levels of cytosine methylation. We assessed whether this accumulation of cytosine methylation during the early 2-cell stage was a consequence of DNA methyltransferase (DNMT) activity. This was achieved by treating late stage zygotes with the DNMT inhibitor RG108 (5 μM) for the period of development spanning pronuclear stage 5 to early 2-cell stage. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (P<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. Our results show for the first time that de novo methylation of the genome occurs as early as the 2-cell stage of development and that this is mediated by a RG108-sensitive DNMT activity. The results substantially change our understanding of epigenetic reprogramming in the early embryo.
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Friis, Stig D., Magnus J. Johansson, and Lutz Ackermann. "Cobalt-catalysed C–H methylation for late-stage drug diversification." Nature Chemistry 12, no. 6 (May 29, 2020): 511–19. http://dx.doi.org/10.1038/s41557-020-0475-7.
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Ekwattanakit, Supachai, Suchada Riolueang, and Vip Viprakasit. "Epigenetic Analysis of Beta-Globin Gene Cluster during Hematopoiesis." Blood 112, no. 11 (November 16, 2008): 1863. http://dx.doi.org/10.1182/blood.v112.11.1863.1863.
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Abstract Hemoglobin (Hb) switching is described as temporal, tissue- and stage-specific patterns of globin gene expression; from embryonic to fetal and adult Hb in parallel to developmental stages of erythropoiesis. DNA methylation, one of the epigenetic mechanisms, was associated with inactivated chromatin domain and repressive transcription. To study the role of the DNA methylation on the beta (β)-globin genes, we analyzed CpG dinucleotides in 87 kb regions around β-globin gene cluster, including 5’upstream locus control regions (LCR; DNAse I Hypersensitive site (HS) 1–5), 3’HS1, the promoter regions of the G-and A-gamma (Gγ and Aγ), and β-globin genes, in several representative cells. These cells were primary adult erythroid cells culture (three different stages: early, intermediate, and late), fetal cord blood DNA, and neutrophil cell line (non-erythroid). Using bisulphite modification, followed by nested PCR and in vitro translation, the cleavage products were analysed by MALDI-TOF Mass Spectrometry to quantify the DNA methylation level. The results were consistent with bisulphite sequencing. We found that the promoters of Gγ and Aγ-globin genes were significantly hypomethylated in fetal cells (44% and 47% global methylation), when γ-globin genes were fully expressed, while they were heavily methylated in non-erythroid (86% and 95%). There was also a decreasing trend of the DNA methylation level at Gγ and Aγ-globin genes during adult erythroid differentiation from 80% and 82%, in early stage, to 67% and 66% in late stage (p=0.12 and 0.04). At β-globin promoter, the global methylation level changed from 90% in non-erythroid to 81%, 42%, and 26% in fetal, early and late adult erythroid cells, respectively. Moreover, we found the significant changes at 5’HS4, 3, and 1 as all erythroid cells were hypomethylated compare to non-erythroid. While at the insulators, 5’HS5 and 3’HS1, all tested CpG dinucleotides were heavily methylated in all cells. This is the first report that demonstrates the differences in DNA methylation at β-globin LCR between erythroid and non-erythroid cells. These epigenetic marks were associated with globin genes expression and might be useful to predict clinical severity in patients with β-thalassemia intermedia.
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Buckingham, Lela, Gregory Pelkey, Mary J. Fidler, and Philip D. Bonomi. "DNA repair gene promoter methylation in non-small cell lung cancer (NSCLC) patients treated with DNA damaging agents." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e22130-e22130. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e22130.
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e22130 Background: Approximately 75% of NSCLC patients are advanced at time of diagnosis. Treatments include regimens of DNA-damaging and alkylating agents. Previous in vitro studies have shown that loss of BRCA1 and BRCA2 function through promoter methylation increases sensitivity to sapacitabine, suggesting that tumor cells may be protected by repair systems including BRCA-RAD50 and MGMT pathways induced by DNA damage. The purpose of this study is to investigate the effect of DNA repair gene promoter methylation on outcome of NSCLC patients treated with DNA damaging agents. DNA repair gene promoter hypermethylation may sensitize tumor cells to DNA damaging agents. Methods: The patient group of 240 patients included 135 early stage and 105 late stage (III/IV) cases. Chemotherapy regimens of late stage patients included carboplatin, cisplatin and gemcitabine, as well as other non-DNA damaging agents. Mean overall survival (OS) was 43.7 months. DNA was extracted from micro-dissected primary tumor, bisulfite converted and assessed by pyrosequencing to quantify methylation of cytosine nucleotides along the MGMT, BRCA1 and BRCA2 promoters. Results: Overall average methylation values were 8.4%, 7.3% and 8.3% for MGMT, BRCA1, BRCA2 respectively (compared to 3.2%, 3.6% and 7.3%, respectively, in nonmalignant lung tissue). Percent promoter methylation levels were not significantly correlated with age, gender, smoking status nor histology, with the exception of higher MGMT promoter methylation in smokers. Hypermethylation (greater than 10%) of BRCA1 and BRCA2 promoter was not significantly associated with survival in late stage patients (6.6 mos vs 6.8 mos; p=0.533) in this patient group, however, superior survival was observed with BRCA2 promoter hypermethylation when early stage patients were included (29.3 mos vs median not reached; p=0.045). Promoter hypermethylation of MGMTwas significantly associated with lower five-year survival rate in early stage patients (p=0.022). Conclusions: These data suggest epigenetic control of DNA repair gene expression can affect response to therapy. Further studies on specific treatment regimens will be required to definitively assess these effects.
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Pimson, Charinya, Tipaya Ekalaksananan, Chamsai Pientong, Supannee Promthet, Nuntiput Putthanachote, Krittika Suwanrungruang, and Surapon Wiangnon. "Aberrant methylation ofPCDH10andRASSF1Agenes in blood samples for non-invasive diagnosis and prognostic assessment of gastric cancer." PeerJ 4 (June 9, 2016): e2112. http://dx.doi.org/10.7717/peerj.2112.
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Background.Assessment of DNA methylation of specific genes is one approach to the diagnosis of cancer worldwide. Early stage detection is necessary to reduce the mortality rate of cancers, including those occurring in the stomach. For this purpose, tumor cells in circulating blood offer promising candidates for non-invasive diagnosis. Transcriptional inactivation of tumor suppressor genes, likePCDH10andRASSF1A, by methylation is associated with progression of gastric cancer, and such methylation can therefore be utilized as a biomarker.Methods.The present research was conducted to evaluate DNA methylation in these two genes using blood samples of gastric cancer cases. Clinicopathological data were also analyzed and cumulative survival rates generated for comparison.Results.High frequencies ofPCDH10andRASSF1Amethylations in the gastric cancer group were noted (94.1% and 83.2%, respectively, as compared to 2.97% and 5.45% in 202 matched controls). Most patients (53.4%) were in severe stage of the disease, with a median survival time of 8.4 months after diagnosis. Likewise, the patients with metastases, orRASSF1AandPCDH10methylations, had median survival times of 7.3, 7.8, and 8.4 months, respectively. A Kaplan–Meier analysis showed that cumulative survival was significantly lower in those cases positive for methylation ofRASSF1Athan in their negative counterparts. Similarly, whereas almost 100% of patients positive forPCDH10methylation had died after five years, none of the negative cases died over this period. Notably, the methylations ofRASSF1AandPCDH10were found to be higher in the late-stage patients and were also significantly correlated with metastasis and histology.Conclusions.PCDH10andRASSF1Amethylations in blood samples can serve as potential non-invasive diagnostic indicators in blood for gastric cancer. In addition toRASSF1Amethylation, tumor stage proved to be a major prognostic factor in terms of survival rates.
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Weis, Erik, Martin A. Hayes, Magnus J. Johansson, and Belén Martín-Matute. "Iridium-catalyzed C−H methylation and d3-methylation of benzoic acids with application to late-stage functionalizations." iScience 24, no. 5 (May 2021): 102467. http://dx.doi.org/10.1016/j.isci.2021.102467.
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Kempster, S., W. A. Phillips, S. Baindur-Hudson, R. J. S. Thomas, C. Dow, and S. P. Rockman. "Methylation of exon 2 of p16 is associated with late stage oesophageal cancer." Cancer Letters 150, no. 1 (March 2000): 57–62. http://dx.doi.org/10.1016/s0304-3835(99)00372-9.
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Gao, Y., V. Hall, and P. Hyttel. "127 REGULATION OF H3K27me3 AND H3K4me3 DURING INITIAL PORCINE EMBRYONIC DEVELOPMENT." Reproduction, Fertility and Development 22, no. 1 (2010): 222. http://dx.doi.org/10.1071/rdv22n1ab127.
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During mammalian development, gene expression is partly regulated by posttranslational modifications of histones. In particular, H3K27me3 and H3K4me3 are involved in transcriptional repression and activation, respectively. In this study, we examined the global levels of H3K27me3 and H3K4me3, as well as the expression levels of their specific methylases and demethylases during porcine pre-implantation embryonic development. Global histone methylation was analyzed by immunocytochemical quantification within in vivo porcine embryos at 1-cell (Day 1), 4-cell (Day 3), morula (Day 5), and late blastocyst (containing the epiblast; Day 9). The numbers of embryos analyzed for H3K27me3 at the 1-cell, 4-cell, morula, and late blastocyst stage were 7, 8, 6, and 5, respectively, and for H3K4me3 at these four stages were 7, 6, 6, and 5, respectively. At the same developmental stages, mRNA expression of methylases (EZH2, EED, and SUZ12, three core components of PRC2) and demethylases (JMJD3 and UTX) of H3K27me3 was performed on pooled embryos (n = 10), as well as expression of methylases (MLL1 and ASH1L) and demethylase (RBP2) of H3K4me3, by comparative RT-PCR. Expression was compared with pooled embryos from the limb bud stage (Day 21). GAPDH was used as the reference gene, and expression was normalized to Day 21 embryos. Our results show that the levels of global histone methylation of H3K27me3 and H3K4me3 decrease gradually from 1-cell to morula, but both were increased in late blastocysts. The levels of H3K27me3 methylase (EZH2, EED, and SUZ12) transcripts increased from 1-cell to late blastocyst stage. Low expression of the H3K27me3 demethylase JMJD3 was found at 1-cell stage and high expression at the 4-cell stage from when it decreased gradually to the late blastocyst. UTX expression was low but peaked at the 4-cell stage. Expression of H3K4me3 methylase MLL1, was low, whereas ASH1L expression was high at the 4-cell stage. RBP2, a demethylase of H3K4me3, was highly expressed at the late blastocyst stage. In conclusion, at the major genome activation (the 4-cell stage), H3K27me3 and H3K4me3 have decreased to moderate levels, which apparently balance each other with respect to gene repression and activation allowing for genome activation. At the 4-cell stage the activation of H3K4me3 is favored as a consequence of low levels of H3K27 methylases and high levels of H3K27 demethylases combined with high levels of H3K4 methylases and low levels of H3K4 demethylases. Interestingly, at the late blastocyst stage of development, high expression of H3K27me3 methylases and the H3K4me3 demethylase, RBP2, are observed, indicating repression of gene expression, which is counterintuitive to accelerating development. We speculate other factors, such as microRNA or other kinds of epigenetic mechanisms, might play a critical role at this developmental stage. Thus, further research is required to explain these phenomena occurred during early porcine development.
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Fulka, Helena, Milan Mrazek, Olga Tepla, and Josef Fulka. "DNA methylation pattern in human zygotes and developing embryos." Reproduction 128, no. 6 (December 2004): 703–8. http://dx.doi.org/10.1530/rep.1.00217.
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We report on observations of the global methylation/demethylation pattern of both pronuclei in human zygotes and in early embryos up to the blastocyst stage. Our results demonstrate that in about half of the zygotes examined the paternal chromatin was less methylated than the maternal chromatin. In the other half, both pronuclei exhibited the same intensity of labeling. The nuclei in developing embryos were intensively labeled for up to the four-cell stage; thereafter, a decline of labeling intensity was detected. Remethylation in some nuclei starts in late morulae. Surprisingly, and unlike the mouse, at the blastocyst stage the inner cell mass showed a weaker intensity of labeling than the trophectodermal cells.
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Gong, Pei-Xue, Fangning Xu, Lu Cheng, Xu Gong, Jie Zhang, Wei-Jin Gu, and Wei Han. "Iron-catalyzed domino decarboxylation-oxidation of α,β-unsaturated carboxylic acids enabled aldehyde C–H methylation." Chemical Communications 57, no. 48 (2021): 5905–8. http://dx.doi.org/10.1039/d1cc01536b.
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The iron-catalyzed domino decarboxylation-oxidation of α,β-unsaturated carboxylic acids enabled aldehyde C–H methylation for preparing diversified methyl ketones is developed and can be applied to the late-stage modification of complex small molecules.
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Lepikhov, K., F. Yang, C. Wrenzycki, V. Zakhartchenko, H. Niemann, E. Wolf, and J. Walter. "131 DYNAMICS OF HISTONE H3 METHYLATION AT POSITIONS K4 AND K9 IN MOUSE, RABBIT, AND BOVINE PRE-IMPLANTATION EMBRYOS." Reproduction, Fertility and Development 18, no. 2 (2006): 174. http://dx.doi.org/10.1071/rdv18n2ab131.
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In mammals, upon the penetration of sperm into the oocyte, the paternal genome undergoes dramatic epigenetic changes. Protamin packaging of DNA is replaced by histones that acquire specific modifications. In mouse zygotes, paternal DNA gets rapidly demethylated by an active mechanism. In bovine zygotes the methylation from paternal DNA is erased only partially, and in rabbit zygotes it persists at the initial level. To understand whether these reprogramming differences are also reflected in histone modifications, we examined the dynamic changes of histone H3 methylation at positions K4 and K9 in mouse, bovine, and rabbit zygotes and in preimplantation embryos using an immunofluorescence staining procedure (Lepikhov and Walter 2004 BMC Dev. Biol. 4, 12). In zygotes, maternal chromatin contains both types of histone H3 methylation. After fertilization protamines in sperm are very quickly replaced by histones. After the formation of nucleosomes, histone H3 acquires methylation at position K4 in a stepwise manner: first as mono-methylated form and later as tri-methylated. In the late zygote, both paternal and maternal pronuclei show equal levels of histone H3 methylation at position K4. Regardless of the differences in DNA reprogramming in these 3 species, H3/K9 di-methylation is not detected on paternal genomes and is only associated with maternal genomes. During the subsequent cleavage stages, H3/K9 di-methylation decreases gradually and becomes hardly detectable in 4-cell bovine and rabbit embryos. In mouse embryos, it is detectable through all the stages. Bovine embryos reacquire this type of modification at the 8-16 cell stage, and it remains at the very low levels in rabbit, embryos until the blastocyst stage. In conclusion, mouse, rabbit and bovine zygotes show similar patterns of H3/K4triMe and H3/K9diMe distribution despite the difference in paternal DNA demethylation. The dynamics of H3/K9diMe distribution patterns in cleavage stage embryos from all embryos do not correlate with embryonic genomic activation events.
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Zhou, Dan, Weiwei Tang, Wenyi Wang, Xiaoyan Pan, Han-Xiang An, and Yun Zhang. "Association between aberrant APC promoter methylation and breast cancer pathogenesis: a meta-analysis of 35 observational studies." PeerJ 4 (July 14, 2016): e2203. http://dx.doi.org/10.7717/peerj.2203.
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Background.Adenomatous polyposis coli (APC) is widely known as an antagonist of the Wnt signaling pathway via the inactivation ofβ-catenin. An increasing number of studies have reported that APC methylation contributes to the predisposition to breast cancer (BC). However, recent studies have yielded conflicting results.Methods.Herein, we systematically carried out a meta-analysis to assess the correlation between APC methylation and BC risk. Based on searches of the Cochrane Library, PubMed, Web of Science and Embase databases, the odds ratio (OR) with 95% confidence interval (CI) values were pooled and summarized.Results.A total of 31 articles involving 35 observational studies with 2,483 cases and 1,218 controls met the inclusion criteria. The results demonstrated that the frequency of APC methylation was significantly higher in BC cases than controls under a random effect model (OR= 8.92, 95% CI [5.12–15.52]). Subgroup analysis further confirmed the reliable results, regardless of the sample types detected, methylation detection methods applied and different regions included. Interestingly, our results also showed that the frequency of APC methylation was significantly lower in early-stage BC patients than late-stage ones (OR= 0.62, 95% CI [0.42–0.93]).Conclusion.APC methylation might play an indispensable role in the pathogenesis of BC and could be regarded as a potential biomarker for the diagnosis of BC.
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Winter, Jean M., Lorraine Sheehan-Hennessy, Susanne Kartin Pedersen, Graeme P. Young, and Erin L. Symonds. "Detection of circulating DNA methylated BCAT1 and IKZF1 in pancreatic adenocarcinoma." Journal of Clinical Oncology 40, no. 4_suppl (February 1, 2022): 597. http://dx.doi.org/10.1200/jco.2022.40.4_suppl.597.
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597 Background: Pancreatic adenocarcinoma patients have poor survival outcomes and there are no non-invasive tests to aid diagnosis or therapy. Detection of circulating tumour DNA (ctDNA) methylated for BCAT1 and/or IKZF1 is over 60% sensitive for colorectal adenocarcinoma. Both pancreatic and colorectal adenocarcinomas are of endodermal origin, therefore, these methylated biomarkers might also have utility in detecting pancreatic cancer. The aim of this study was to investigate whether individuals with pancreatic adenocarcinoma have detectable methylated BCAT1 and IKZF1 DNA in circulation. Methods: In this pilot study, pre-treatment blood and clinicopathological findings were collected from 21 patients diagnosed with pancreatic adenocarcinoma. DNA isolated from plasma was bisulfite-converted and assayed for methylated BCAT1, IKZF1 and a non-methylated region in ACTB (for yield estimates). Samples with methylation in either gene was deemed positive. Chi -squared test was used to compare positivity between Stage I/II and Stage III/IV cases. The sum of percent (%) methylation ([average BCAT1]+[average IKZF1])/average ACTB) ± standard error was compared between stages using Kruskal-Wallis rank test. Results: 10/21 (47.6%) patients were positive for methylated BCAT1 and/or IKZF1. There was a trend of increasing positivity with advancing stage (Stage I/II 2/8 (25.0%) vs Stage III/IV 8/13 (61.5%, p=0.104)), and for higher % methylation with more advanced disease (Stage II 0.01%±0.004 vs Stage IV 5.0%±3.7, p=0.06). No other comorbidities or demographics were associated with positivity. Conclusions: Assay for methylated BCAT1 and IKZF1 ctDNA detects approximately two-thirds of late-stage pancreatic adenocarcinoma. Future studies are warranted to assess the clinical utility of these biomarkers for detection and monitoring of pancreatic cancer. Clinical trial information: 12616001138471.
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Gore, Athurva J., Justin Dang, Alan Fung, Bin Xie, and Rui Liu. "Abstract 1421: Non-invasive early detection of pancreatic ductal adenocarcinoma using methylation signatures in circulating tumor DNA." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1421. http://dx.doi.org/10.1158/1538-7445.am2022-1421.
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Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a low overall survival rate primarily due to late onset of symptoms and rapid progression. Early symptoms, which include abdominal and back pain, diarrhea, weight loss, and jaundice are non-specific for PDAC and may be associated with other gastrointestinal diseases. PDAC lacks reliable and accurate tests for early detection. We aimed to discover biomarkers carrying a PDAC-specific DNA methylation signature in blood cell-free DNA, and to develop and validate a non-invasive test for PDAC detection. Methods: Reduced representative bisulfite sequencing (RRBS) was previously performed on tissue and plasma samples from PDAC patients and healthy individuals to identify PDAC-specific methylation markers. These markers were combined with previously reported cancer methylation markers for a second round of marker screening. The resulting markers were utilized to develop a targeted methylation sequencing assay for plasma DNA, designated as PDACatch. The PDACatch assay was validated on an independent sample set to demonstrate its efficacy for pancreatic cancer methylation signature detection. Results: The PDACatch assay was performed on 91 PDAC plasma and 84 healthy plasma samples obtained from commercial biobanks. In this independent verification sample set, the PandaX assay predicted PDAC from healthy with AUC of 0.91; accuracy remained high for classification of both stage I and early-stage (I/IIa) samples. Conclusion: The PDACatch assay, a ctDNA methylation based blood test, achieved a high accuracy for detecting PDAC at early stages, indicating its potential in screening effectiveness. Citation Format: Athurva J. Gore, Justin Dang, Alan Fung, Bin Xie, Rui Liu. Non-invasive early detection of pancreatic ductal adenocarcinoma using methylation signatures in circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1421.
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Zheng, Louise D., Leah E. Linarelli, Joseph Brooke, Cayleen Smith, Sarah S. Wall, Mark H. Greenawald, Richard W. Seidel, Paul A. Estabrooks, Fabio A. Almeida, and Zhiyong Cheng. "Mitochondrial Epigenetic Changes Link to Increased Diabetes Risk and Early-Stage Prediabetes Indicator." Oxidative Medicine and Cellular Longevity 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/5290638.
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Type 2 diabetes (T2D) is characterized by mitochondrial derangement and oxidative stress. With no known cure for T2D, it is critical to identify mitochondrial biomarkers for early diagnosis of prediabetes and disease prevention. Here we examined 87 participants on the diagnosis power of fasting glucose (FG) and hemoglobin A1c levels and investigated their interactions with mitochondrial DNA methylation. FG and A1c led to discordant diagnostic results irrespective of increased body mass index (BMI), underscoring the need of new biomarkers for prediabetes diagnosis. Mitochondrial DNA methylation levels were not correlated with late-stage (impaired FG or A1c) but significantly with early-stage (impaired insulin sensitivity) events. Quartiles of BMI suggested that mitochondrial DNA methylation increased drastically from Q1 (20 < BMI < 24.9, lean) to Q2 (30 < BMI < 34.9, obese), but marginally from Q2 to Q3 (35 < BMI < 39.9, severely obese) and from Q3 to Q4 (BMI > 40, morbidly obese). A significant change was also observed from Q1 to Q2 in HOMA insulin sensitivity but not in A1c or FG. Thus, mitochondrial epigenetic changes link to increased diabetes risk and the indicator of early-stage prediabetes. Further larger-scale studies to examine the potential of mitochondrial epigenetic marker in prediabetes diagnosis will be of critical importance for T2D prevention.
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Zhao, Grace Q., Yun Bao, Heng Wang, Jianmin Wang, and Shengrong Lin. "The development and validation of a novel targeted methylation sequencing technology detecting 0.003 percent tumor signal in early-stage cancer plasma." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 4092. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.4092.
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4092 Background: Methylation analysis in cell-free DNA holds great potential for early cancer detection. In the plasma of early stage cancer patient, the tumor content is estimated to be less than 0.1%, therefore demands a highly sensitive assay. Targeted Methylation Sequencing (TMS) is the most promising approach; however, the current sensitivity and specificity are compromised by low efficiency and low recovery of target enrichment, and further hampered by background noise associated with large panels. The ideal solution would be an in-depth analysis using a focused small cancer-specific methylation biomarker panel, but is not supported by existing technologies. Methods: Here we present a new technology designed for TMS analysis in cfDNA: Point-n-Seq, featuring an enrichment of target molecules directly from cfDNA before bisulfite conversion and amplification. Particularly, this technology enables small focused panel that interrogates the methylation status of 1 to ~1000 markers. We designed a CRC panel covering 100 methylation markers in 3 steps: identify ~1000 CRC-specific markers from public databases; eliminate makers with high background signal in baseline cfDNA of healthy population; finalize the list with the most differentiating markers between patient and healthy cfDNA. Results: The capture of Point-n-Seq CRC panel is highly efficient resulting in high uniformity (94% > 0.5X) and on-target rate ( > 80%). For 20 ng cfDNA input, more than 1000 deduped informative reads were obtained for each marker on average, despite the high GC content ( > 80%). The output of informative reads was linear to the cfDNA input ranging from 1 ng to 40 ng. In titration studies, 0.6 pg (0.2X genome equivalent) methylated DNA in 20 ng cfDNA (0.003%) was reliably detected over cfDNA background. Using plasma samples from patients with CRC - early stage (I, n = 7; II, n = 7), late stage (III, n = 11; IV, n = 3), and control individuals (n = 105), the average fractions of methylated signal are 0.0034%, 0.013%, 0.09%, 0.17%, 0.29% for control, stage I, II, III, IV accordingly. With a simple cut-off using methylation fraction, Point-n Seq CRC panel achieved a sensitivity of 86% for stage I, 100% for stage (II-IV) at a specificity of 91%, with AUC = 0.96. Conclusions: Point-n-Seq TMS is the first hybridization based NGS technology enables the small focused methylation panel (e.g. 100 markers) sequencing using cfDNA, and it will greatly facilitate the development of practical and cost-effective methylation assays for clinical use.
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Canal-Noguer, Pol, Alejandro Requena Bermejo, Špela Zavodnik, Vivian Erklavec Zajec, Francesco Mattia Mancuso, Marina Manrique López, Primož Knap, et al. "Use of methylation and fragmentation signals in the detection of early-stage colorectal cancer." Journal of Clinical Oncology 41, no. 4_suppl (February 1, 2023): 201. http://dx.doi.org/10.1200/jco.2023.41.4_suppl.201.
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201 Background: Colorectal cancer (CRC) remains a leading cause of cancer related mortality worldwide. We utilized cell-free DNA (cfDNA) methylation and fragmentation characteristics of selected cancer-related biomarker regions and applied tumor-derived signal deduction and a machine learning algorithm to refine a blood test for the early detection of CRC. Methods: This was a prospective, international (Spain, Ukraine, Germany and USA [part of NCT04792684 study] population), observational cohort study. Plasma samples were collected either prior to a scheduled screening colonoscopy or prior to colonic surgery for primary CRC. 95 cfDNA samples from 48 early stage (I-II), 47 late-stage (III-IV) CRC patients (mean age 65 [48-83], female 45%, distal cancers 51%) and 204 age, gender and country of origin matched colonoscopy-checked controls were analyzed. 79 of the control patients had a negative colonoscopy finding (cNEG), 96 had benign findings of diverticulosis, hemorrhoids and/or hyperplastic polyps (BEN), 29 had non-advanced adenomas (NAA). Samples were analyzed utilizing previously described hybrid-capture based sequencing methodology. Panel of targeted biomarkers was previously identified through tissue- and plasma-based discovery and further narrowed down through cancer-related biological pathways analysis workflow. Individual cfDNA fragments belonging to each biomarker region were scored for cancer-specific signal. Finally, calculated scores were used in prediction model building and testing for establishing panel accuracy for CRC detection. Results: Prediction model utilizing a panel of methylation and fragmentation scores originating from cfDNA biomarkers belonging to relevant cancer development and progression related pathways, such as axonal guidance, ephrin receptor signaling, epithelial-mesenchymal transition and FGF signaling, correctly classified 92% (87/95) of CRC patients. Sensitivity per cancer stage ranged from 91% (21/23) for stage I, 92% (23/25) for stage II, 91% (30/33) for stage III and 93% (13/14) for stage IV. Fragmentation signals contributed most to early-stage cancers (I-II), while methylation signals were more significant for late stage (III-IV) detection. Specificity of the model was 94% (199/204), with 97% (28/29) NAA, 91% (87/96) BEN and 96% (76/79) cNEG patients correctly identified. Lesion location, gender, age and country of origin were not significantly correlated to prediction outcome. Conclusions: Use of methylation and fragmentation characteristics of cancer-related cfDNA regions, combined with a machine-learning algorithm is highly accurate for early-stage (I-II) CRCs (92% sensitivity at 94% specificity). The study is being further expanded on larger cohort for validation of a highly accurate and minimally invasive blood-based CRC screening test.
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Wang, Wei, Wanqiu Li, Xueliang Ge, Kaige Yan, Chandra Sekhar Mandava, Suparna Sanyal, and Ning Gao. "Loss of a single methylation in 23S rRNA delays 50S assembly at multiple late stages and impairs translation initiation and elongation." Proceedings of the National Academy of Sciences 117, no. 27 (June 22, 2020): 15609–19. http://dx.doi.org/10.1073/pnas.1914323117.
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Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2′-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from anEscherichia coliΔrrmJstrain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from ΔrrmJstrain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the ΔrrmJ50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the ΔrrmJ70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.
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Zhang, Y., N. Fukui, M. Yahata, Y. Katsuragawa, T. Tashiro, S. Ikegawa, and M. T. Michael Lee. "Genome-wide DNA methylation profile implicates potential cartilage regeneration at the late stage of knee osteoarthritis." Osteoarthritis and Cartilage 24, no. 5 (May 2016): 835–43. http://dx.doi.org/10.1016/j.joca.2015.12.013.
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Chen, L., Y. Wang, and Z. Yu. "Identification of epigenetic aberrant promoter methylation of RASSF1A in serum DNA and its clinicopathological significance in lung cancer." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 21014. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.21014.
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21014 Objective: To detect the hypermethylation status of RASSF1A promoter in serum DNA of lung cancer patients and to analyze its correlation with clinicopathological features. Methods: Serum DNA was extracted from peripheral blood from 80 primary lung cancer patients, 35 benign pulmonary disease patients and 15 healthy donors. The methylation status of RASSF1A promoter was determined using methylation-specific PCR technique, and the correlation between methylation profiles and clinicopathological parameters was statistically analyzed. Results: Aberrant methylation of RASSF1A was detected in 27 of the 80 (33.8%) patients with lung cancer but no benign pulmonary disease patients or healthy donors (p<0.001). RASSF1A was preferentially observed in small cell lung cancer (p=0.042), while no statistical difference was found among methylation frequencies of different subtypes of non-small cell lung cancer. The methylation status was also found to be associated with relative poor differentiation (p=0.009) and late stage (p=0.013), but not with gender, age or treatment. Conclusion: RASSF1A promoter is frequently hypermethylated in serum DNA of primary lung cancer patients, and RASSF1A is a promising novel biomarker for lung cancer diagnosis. No significant financial relationships to disclose.
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Schütt, Sabine, Andrea R. Florl, Wei Shi, Myriam Hemberger, Annie Orth, Sabine Otto, Wolfgang A. Schulz, and Reinald H. Fundele. "DNA Methylation in Placentas of Interspecies Mouse Hybrids." Genetics 165, no. 1 (September 1, 2003): 223–28. http://dx.doi.org/10.1093/genetics/165.1.223.
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Abstract Interspecific hybridization in the genus Mus results in several hybrid dysgenesis effects, such as male sterility and X-linked placental dysplasia (IHPD). The genetic or molecular basis for the placental phenotypes is at present not clear. However, an extremely complex genetic system that has been hypothesized to be caused by major epigenetic changes on the X chromosome has been shown to be active. We have investigated DNA methylation of several single genes, Atrx, Esx1, Mecp2, Pem, Psx1, Vbp1, Pou3f4, and Cdx2, and, in addition, of LINE-1 and IAP repeat sequences, in placentas and tissues of fetal day 18 mouse interspecific hybrids. Our results show some tendency toward hypomethylation in the late gestation mouse placenta. However, no differential methylation was observed in hyper- and hypoplastic hybrid placentas when compared with normal-sized littermate placentas or intraspecific Mus musculus placentas of the same developmental stage. Thus, our results strongly suggest that generalized changes in methylation patterns do not occur in trophoblast cells of such hybrids.
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Ham, Juyeon, Seungyeon Lee, Hyunkyung Lee, Dawoon Jeong, Sungbin Park, and Sun Jung Kim. "Genome-Wide Methylation Analysis Identifies NOX4 and KDM5A as Key Regulators in Inhibiting Breast Cancer Cell Proliferation by Ginsenoside Rg3." American Journal of Chinese Medicine 46, no. 06 (January 2018): 1333–55. http://dx.doi.org/10.1142/s0192415x18500702.
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Ginsenoside Rg3 is a key metabolite of ginseng and is known to inhibit cancer cell growth. However, the epigenetics of CpG methylation and its regulatory mechanism have yet to be determined. Genome-wide methylation analysis of MCF-7 breast cancer cells treated with Rg3 was performed to identify epigenetically regulated genes and pathways. The effect of Rg3 on apoptosis and cell proliferation was examined by a colony formation assay and a dye-based cell proliferation assay. The association between methylation and gene expression was monitored by RT-PCR and Western blot analysis. Genome-wide methylation analysis identified the “cell morphology”-related pathway as the top network. Rg3 induced late stage apoptosis but inhibited cell proliferation up to 60%. Hypermethylated TRMT1L, PSMC6 and NOX4 were downregulated by Rg3, while hypomethylated ST3GAL4, RNLS and KDM5A were upregulated. In accordance, downregulation of NOX4 by siRNA abrogated the cell growth effect of Rg3, while the effect was opposite for KDM5A. Notably, breast cancer patients with a higher expression of NOX4 and KDM5A showed poor and good prognosis of survival, respectively. In conclusion, Rg3 deregulated tumor-related genes through alteration of the epigenetic methylation level leading to growth inhibition of cancer cells.
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Novaes, Luiz F. T., Justin S. K. Ho, Kaining Mao, Kaida Liu, Mayank Tanwar, Matthew Neurock, Elisia Villemure, Jack A. Terrett, and Song Lin. "Exploring Electrochemical C(sp3)–H Oxidation for the Late-Stage Methylation of Complex Molecules." Journal of the American Chemical Society 144, no. 3 (January 11, 2022): 1187–97. http://dx.doi.org/10.1021/jacs.1c09412.
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Baranova, Ivana, Helena Kovarikova, Jan Laco, Iva Sedlakova, Filip Vrbacky, Dalibor Kovarik, Petr Hejna, Vladimir Palicka, and Marcela Chmelarova. "Identification of a four-gene methylation biomarker panel in high-grade serous ovarian carcinoma." Clinical Chemistry and Laboratory Medicine (CCLM) 58, no. 8 (July 28, 2020): 1332–40. http://dx.doi.org/10.1515/cclm-2019-1319.
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AbstractBackgroundThe lack of effective biomarkers for the screening and early detection of ovarian cancer (OC) is one of the most pressing problems in oncogynecology. Because epigenetic alterations occur early in the cancer development, they provide great potential to serve as such biomarkers. In our study, we investigated a potential of a four-gene methylation panel (including CDH13, HNF1B, PCDH17 and GATA4 genes) for the early detection of high-grade serous ovarian carcinoma (HGSOC).MethodsFor methylation detection we used methylation sensitive high-resolution melting analysis and real-time methylation specific analysis. We also investigated the relation between gene hypermethylation and gene relative expression using the 2−ΔΔCt method.ResultsThe sensitivity of the examined panel reached 88.5%. We were able to detect methylation in 85.7% (12/14) of early stage tumors and in 89.4% (42/47) of late stage tumors. The total efficiency of the panel was 94.4% and negative predictive value reached 90.0%. The specificity and positive predictive value achieved 100% rates. Our results showed lower gene expression in the tumor samples in comparison to control samples. The more pronounced downregulation was measured in the group of samples with detected methylation.ConclusionsIn our study we designed the four-gene panel for HGSOC detection in ovarian tissue with 100% specificity and sensitivity of 88.5%. The next challenge is translation of the findings to the less invasive source for biomarker examination, such as plasma. Our results indicate that combination of examined genes deserve consideration for further testing in clinical molecular diagnosis of HGSOC.
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Yang, J. F., S. H. Yang, Y. Y. Niu, Q. Zhou, and W. Z. Ji. "80 EFFECT OF DNA METHYLATION ON SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT IN RHESUS MONKEY." Reproduction, Fertility and Development 18, no. 2 (2006): 148. http://dx.doi.org/10.1071/rdv18n2ab80.
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Up to now, no primate animals have been successfully cloned with somatic cell nuclear transfer (SCNT) and little is known about molecular events occurring in SCNT embryos. DNA methylation reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Epigenetic characteristics of donor cell nuclei and their epigenetic reprogramming in oocyte cytoplasm have been supposed as major factors influencing the development of SCNT embryos. In Experiment 1, on donor cells used in a previous SCNT at our laboratory, global DNA methylation and histone 3 lysine 9 acetylation (H3K9ac) of three cell lines (S11, S1-04, and S1-03) derived from ear skin were examined after serum starvation by immunofluorescence with monoclonal antibody to 5-methyl cytosine (Oncogene, Science, Inc., Cambridge, MA, USA) and anti-acetyl-Histone H3 (Lys 9) (Upstate Jingmei Biotech, Ltd., Shenzhen, China). In the results, two cells lines, S11 and S1-04, supporting higher blastocyst development (about 20%) than that (7.8%) of S1-03, showed a higher level of H3K9ac than the S1-03 cell line. Global DNA methylation levels in the three cell lines were decreased after serum starvation, but no obvious correlation between the level and SCNT embryo developmental potential was found among the three cell lines. In Experiment 2, on SCNT and IVF embryos, global DNA methylation reprogramming during pre-implantation development was investigated with immunofluorescence and laser scanning microscopy techniques. In IVF embryos, active demethylation of paternal genome occurred soon after fertilization; subsequently, passive demethylation resulted in remarkably reduced global methylation level at the 8-cell stage and the morula stage. Thereafter, genomewide remethylation started at the late morula stage and an asymmetric methylation pattern was formed in blastocysts, with higher methylated trophectoderm than inner cell mass (ICM). Compared with IVF embryos, most SCNT 2-cell embryos and ICM in blastocysts showed higher methylation levels, and the asymmetric methylation pattern was not as evident as that in IVF blastocysts. Some SCNT 8-cell embryos showed higher methylation, but others were slightly stained, even lower than IVF embryos. In conclusion, the higher global H3K9 acetylation level of donor cells may benefit chromatin remolding and development of SCNT embryos. Abnormal methylation reprogramming in most SCNT embryos, especially in ICM of blastocysts, may be one main obstacle for primate cloning, although relatively high blastocyst development rate was obtained. DNA methylation reprogramming in rhesus monkey pre-implantation embryos, on the whole, was as conservative as that reported in other mammals.
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Kinross, James M., Pol Canal-Noguer, Marko Chersicola, Primož Knap, Marko Bitenc, Alexandre Perera-Lluna, Michael H. A. Roehrl, and Kristi Kruusmaa. "Accurate early-stage colorectal cancer detection through analysis of cell-free circulating tumor DNA (ctDNA) methylation patterns." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 3606. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.3606.
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3606 Background: Colorectal cancer (CRC) screening programs suffer from poor uptake and biomarkers have limited diagnostic accuracy. The measurement of the methylation status of tumor-derived cell-free DNA in plasma may address these challenges. We used a targeted methylation panel, tumor-derived signal deduction and machine learning algorithm to refine a blood test for the detection of early-stage CRC. Methods: This was a prospective, international multicenter observational cohort study. Plasma samples were collected either prior to a scheduled colonoscopy as part of standard colorectal cancer screening or prior to colonic surgery for primary CRC. Differentially methylated regions (DMRs) were initially selected by analyzing CRC and control tissue samples with whole genome bisulfite sequencing. A targeted sequencing assay was designed to capture these DMRs in plasma ctDNA. Individual sequencing reads were evaluated for cancer-specific methylation signal and scores calculated for each DMR in a sample. A panel of methylation scores originating from 203 DMRs was used in a prediction model building and validated in a test cohort of patients. Results: Calculated scores were used to train a machine learning model on 68 ctDNA samples from 18 early stage (I-II) and 16 late-stage (III-IV) CRC patients and 34 age, BMI, gender and country of origin matched neoplasia-free controls (median age 63 [50-74], mean BMI 27 [19.5-37], female 50%, Spanish and Ukrainian population, distal cancers 50%). This model was then applied to an independent set of subjects from Spanish, Ukraine and Germany, including 36 stage I-IV cancer patients (median age 61.5 [55-82], BMI 28 [16-39], female 47%, 42% of the tumors were distal) and 159 age and sex matched controls. 87 of the control patients had a negative colonoscopy finding (cNEG), 19 had hyperplastic polyps (HP), 37 had small non-advanced adenomas (NAA) and 16 were diagnosed with other benign gastrointestinal diseases (GID). The model correctly classified 92% (33/36) of CRC patients. Sensitivity per cancer stage ranged from 83% (5/6) for stage I, 92% (11/12) for stage II, 92% (12/13) for stage III to 100% (5/5) for stage IV. Specificity of the model was 97% (154/159), with 100% (37/37) NAA, 94% (15/16) GID, 95% (18/19) HP and 97% cNEG patients correctly identified. Lesion location, gender, BMI, age and country of origin were not significantly correlated to prediction outcome. Conclusions: Methylation sequencing data analyzed using read-wise scoring approach combined with a machine-learning algorithm is highly diagnostic for early-stage (I-II) CRCs (89% sensitivity at 97% specificity). This method could serve as the basis for a highly accurate and minimally invasive blood-based CRC screening test with significant implications for the clinical utility of ctDNA in early-stage cancer detection.
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Pápai, Imre, Petri M. Pihko, Juha H. Siitonen, and Dániel Csókás. "Total Synthesis of Stemoamide, 9a-epi-Stemoamide, and 9a,10-epi-Stemoamide: Divergent Stereochemistry of the Final Methylation Steps." Synlett 31, no. 16 (July 27, 2020): 1581–86. http://dx.doi.org/10.1055/s-0040-1707201.
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Total syntheses of stemoamide, 9a-epi-stemoamide, and 9a,10-epi-stemoamide by a convergent A + B ring-forming strategy is reported. The synthesis required a diastereoselective late-stage methylation of the ABC stemoamide core that successfully enabled access to three of the four possible diastereomeric structures. For the natural stemoamide series, the diastereoselectivity can be rationalized both by kinetic and thermodynamic arguments, whereas for the natural 9a-epi-stemoamide series, the kinetic selectivity is explained by the prepyramidalization of the relevant enolate.
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Riojas, Marco A., Mingzhou Guo, Sabine C. Glöckner, Emi Ota Machida, Stephen B. Baylin, and Nita Ahuja. "Methylation-induced silencing of ASC/TMS1, a pro-apoptotic gene, is a late-stage event in colorectal cancer." Cancer Biology & Therapy 6, no. 11 (November 2007): 1710–16. http://dx.doi.org/10.4161/cbt.6.11.4829.
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Kulis, Marta, Simon Heath, Giancarlo Castellano, Renée Beekman, Angelika Merkel, Emanuele Raineri, Anna Esteve, et al. "Characterization of the DNA Methylome during Human B-Cell Differentiation." Blood 124, no. 21 (December 6, 2014): 4346. http://dx.doi.org/10.1182/blood.v124.21.4346.4346.
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Abstract Introduction: Modulation of the DNA methylation landscape during cell differentiation is a well-established phenomenon. The B-cell lineage represents a paradigmatic cellular model to study the dynamic epigenome during cell development and specification because major B-cell maturation stages are well defined and display differential phenotypic and gene expression features. Furthermore, different B-cell subpopulations show different proliferation abilities, microenvironmental influences and life spans, providing a window of opportunity to study the epigenome in the context of multiple processes. Methods: We performed whole-genome bisulfite sequencing (WGBS), high-density methylation microarrays and gene expression profiling of ten purified human B-cell subpopulations spanning the entire differentiation program, ranging from uncommitted progenitors to terminally-differentiated plasma cells. Results: The results of both WGBS and methylation microarrays indicate that B-cell ontogenesis involves an extensive and gradual reconfiguration of the DNA methylome. We uncovered that non-CpG methylation at CpApC trinucleotides is present in progenitor cells and disappears upon B-cell commitment independently of CpG demethylation. CpG methylation, in contrast, changed extensively during the entire B-cell maturation program, with one quarter of all measured CpGs showing dynamic methylation. B-cell enhancers suffered more extensive methylation changes than promoter regions, especially in the early differentiation steps up to the germinal center B-cell (gcBC) stage, and their demethylation seemed to be mediated by binding of lineage-specific transcription factors. Enhancers with dynamic methylation were related to genes involved in a large B-cell network that showed high gene expression variability throughout differentiation. In highly proliferative gcBCs, we observed a shift of dynamic methylation from regulatory towards non-functional elements; gcBCs start to undergo global demethylation of late-replicating heterochromatic regions and methylation of polycomb-repressed regions. This signature becomes particularly extensive in long-lived memory B cells and plasma cells, indicating that these changes start in highly proliferative cells and then accumulate in non-proliferative cells with extended lifespan. Conclusion: Our epigenomic analysis of the B-cell differentiation program extends our knowledge on how the DNA methylome is modulated during cell specification and maturation and offers a resource for researchers in the field, both at global and single gene levels. Disclosures No relevant conflicts of interest to declare.
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Ashapkin, Vasily V., Lyudmila I. Kutueva, Nadezhda I. Aleksandrushkina, and Boris F. Vanyushin. "Epigenetic Regulation of Plant Gametophyte Development." International Journal of Molecular Sciences 20, no. 12 (June 22, 2019): 3051. http://dx.doi.org/10.3390/ijms20123051.
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Unlike in animals, the reproductive lineage cells in plants differentiate from within somatic tissues late in development to produce a specific haploid generation of the life cycle—male and female gametophytes. In flowering plants, the male gametophyte develops within the anthers and the female gametophyte—within the ovule. Both gametophytes consist of only a few cells. There are two major stages of gametophyte development—meiotic and post-meiotic. In the first stage, sporocyte mother cells differentiate within the anther (pollen mother cell) and the ovule (megaspore mother cell). These sporocyte mother cells undergo two meiotic divisions to produce four haploid daughter cells—male spores (microspores) and female spores (megaspores). In the second stage, the haploid spore cells undergo few asymmetric haploid mitotic divisions to produce the 3-cell male or 7-cell female gametophyte. Both stages of gametophyte development involve extensive epigenetic reprogramming, including siRNA dependent changes in DNA methylation and chromatin restructuring. This intricate mosaic of epigenetic changes determines, to a great extent, embryo and endosperm development in the future sporophyte generation.
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Liu, Xin, Gengtai Ye, Chunhui Cui, Hui Li, Ting Yang, Xuetao Lei, Guofan Zhang, et al. "A noninvasive gastric cancer Her2 test using surrogate methylation markers." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e16084-e16084. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e16084.
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e16084 Background: There are 10-20% of gastric cancer (GC) with overexpressed Her2. Her2 status remains an essential biomarker for guiding the trastuzumab (Herceptin) therapy, a monoclonal antibody approved for the first-line treatment of late-stage Her2-positive GC. Although IHC, together with FISH, is comprehensively applied to verify Her2 status on tissue samples, an accurate blood test is highly desirable due to the inaccessibility of tissue samples, especially in very late stage GC patients as well as tumor heterogeneity of tissue biopsy. Detecting copy number aberration of Her2 gene in cell-free DNA (cfDNA) gains a lot of interest for its non-invasive approach. However, the limited signal-to-noise ratio poses a great challenge for the accuracy and robustness of the tests (either targeted sequencing or ddPCR). Here, we report a non-invasive test for Her2 status verification based on novel surrogate DNA methylation markers. Methods: Genome-wide DNA methylation sequencing was performed in 30 Her2-negative (IHC 0/1+) and 44 Her2-positive (IHC 3+) tissue samples to identify Her2-overexpression-specific methylation markers. Then we analyzed the performance of these candidate markers using methylation-specific quantitative PCR (qMSP) in plasma samples collected from 102 GC patients before surgical treatment. A Her2-status diagnostic model was built and further validated in a multi-center, prospective cohort (n = 150). The concordance of Her2 status between GC plasma and matching tissue samples (IHC/FISH) was determined. Results: We first discovered 102 statistically significant methylation markers of Her2 status in tissue. Out of these candidate markers, a 3-marker diagnostic model was built and validated on plasma samples, which could discriminate Her2-positive from Her2-negative GC patients with high sensitivity (86.7%) and specificity (96.8%). The overall plasma-tissue concordance of this liquid biopsy test was 95.3%. Furthermore, the Her2-status test was able to classify Her2 2+ status (IHC) into either Her2-negative or Her2-positive status, which was confirmed by conventional FISH test. Conclusions: Overall, the cfDNA-based test is a novel, accurate and noninvasive approach for determining Her2 status in GC patients. The high concordance with IHC/FISH results of this blood test holds great promise as an auxiliary method for guiding Her2-targeted therapy in GC patients. A clinical trial is undergoing to validate this test in the phase-2 clinical trial of a Her2-targeted drug (for GC) in China.
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Mattern, F., J. Heinzmann, D. Herrmann, A. Lucas-Hahn, T. Haaf, and H. Niemann. "Gene-specific profiling of DNA methylation and mRNA expression in bovine oocytes derived from follicles of different size categories." Reproduction, Fertility and Development 29, no. 10 (2017): 2040. http://dx.doi.org/10.1071/rd16327.
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Epigenetic changes, such as DNA methylation, play an essential role in the acquisition of full developmental competence by mammalian oocytes during the late follicular growth phase. Here we used the bovine model to investigate the DNA methylation profiles of seven candidate genes (imprinted: bH19, bSNRPN; non-imprinted: bZAR1, bDNMT3A, bOCT4, bDNMT3 Lo and bDNMT3 Ls) and the mRNA expression of nine candidate genes (imprinted: bSNRPN, bPEG3, bIGF2R; non-imprinted: bPRDX1, bDNMT1B, bDNMT3A, bZAR1, bHSF1 and bNLRP9) in oocytes from antral follicles of three different size classes (≤2 mm, 3–5 mm, ≥6 mm) to unravel the epigenetic contribution to this process. We observed an increased number of aberrantly methylated alleles in bH19, bSNRPN and bDNMT3 Lo of oocytes from small antral follicles (≤2 mm), correlating with lower developmental competence. Furthermore, we detected an increased frequency of CpG sites with an unclear methylation status for DNMT3 Ls, specifically in oocytes from follicles ≥6 mm, predominantly at three CpG positions (CpG2, CpG7 and CpG8), of which CpG7 is a potential regulatory site. No major differences in mRNA expression were observed, indicating that the transcriptional machinery may not yet be active during the follicular growth phase. Our results support the notion that a follicle diameter of ~2 mm is a critical stage for establishing DNA methylation profiles and indicate a link between DNA methylation and the acquisition of oocyte developmental competence.
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Zhao, Grace Q., Yun Bao, Heng Wang, Wanping Hu, John Coller, and Shengrong Lin. "A dual target sequencing solution to assess genomic and epigenomic alterations in cell-free DNA with no sample splitting." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 3043. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.3043.
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3043 Background: Assessing the genomic and epigenomic changes on plasma cell-free DNA (cfDNA) using next-generation sequencing (NGS) has become increasingly important for cancer detection and treatment selection guidance. However, two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS), let alone one that analyzes both mutation and methylation information in one assay. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for conducting multi-omic analysis. Thus, an assay that is capable of both genomic and epigenomic analysis would be advantageous for clinical research and future diagnostic assays. Methods: Here, we report the performance of Point-n-SeqTM dual analysis, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both genomic and epigenomic analysis without sample splitting. With custom panels of tens to thousands of markers designed with > 99% first-pass success rate, we conducted both performance validation and multi-center, multi-operator, reproducibility studies. Using spike-in titration of cancer cell-line gDNA with known mutation and methylation profiles, Point-n-Seq assay achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions. Benchmarked with conventional targeted sequencing and methylation sequencing, Point-n-Seq solution also demonstrated improved performance, speed and shortened hands-on time. Results: In a pilot clinical study, a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with > 350 hotspot mutations in 22 genes were used in the dual assay. Using 1ml of plasma from late-stage CRC patients, cancer-specific methylation signals were detected in all samples tested, and oncogenic mutations. In an early-stage cohort (33 stage I/II CRC patient ), comparison of the analysis between tumor-informed, personalized-mutation panels (̃100 private SNVs) for each patient and the tumor-independent CRC methylation panels were conducted. The initial results showed that tumor-independent TMS assay achieved a comparable detection compared to the personalized tumor-informed approach. Moreover, cfDNA size information (fragmentome) is also integrated into the analysis of the same Point-n-Seq workflow to improve the assay sensitivity. Conclusions: Point-n-Seq dual analysis is poised to advance both research and clinical applications of early cancer detection, minimal residual disease (MRD), and monitoring.
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Zhang, Wei, Jinke Sui, Xianrui Wu, Fuao Cao, Guanyu Yu, Chenyang Wang, Zhihong Zhang, et al. "DNA methylation profiling from circulating tumor DNA for early-detection of colorectal cancer." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e15076-e15076. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e15076.
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e15076 Background: Colorectal cancer (CRC) develops as a result of neoplastic progression, which often takes decades, providing a window for early detection. Unfortunately, there has been little success in developing blood-based screening method due to the low amount of ctDNA present in the circulation, especially in patients with early stage disease. The role of aberrant DNA methylation, occurring very early in tumorigenesis, has been well elucidated. In this prospective study, we evaluated the potentiality of DNA methylation status obtained from ctDNA as an early detection method. Methods: Panel Design: Methylation data of tumor samples (12 types, n = 4,772), adjacent normal (8 types, n = 411), and normal white blood cells (n = 656) from TCGA and GSE were compared. Differentially methylated sites were extracted using modified wald-test with an adjusted p-value < 0.05 and fold-change > 2. Our panel covers 80,672 CpG sites, spanning 1.05Mb of human genome. We performed targeted bisulfite sequencing on plasma samples of 67 (stage I: 13, II:29, III: 23, IV: 2) Chinese CRC patients and 144 healthy individuals to construct a model for deriving markers that are differentially methylated and their associated weight. The model was validated in 2 independent cohorts. Results: We constructed a model using a support vector machine (SVM)-based machine learning classifier based on top 4,000 differentially methylated regions (DMRs) selected by random forest between tumor and normal plasma samples. Subsequently, 5-fold cross-validation with 100-time repeats were performed to gain a robust estimation of model performance, achieving a sensitivity of 91%, specificity of 98% and area under curve (AUC) of 98.6%. The model was subsequently validated in 2 independent cohorts: one consisted of 57 stage I-III CRC patients and 74 healthy individuals and another one with 47 stage IV patients and the same 74 healthy individuals. The model yielded a sensitivity of 83% and 95% for the early and late stage cohorts, respectively. A specificity of 95% was obtained for both cohorts. Conclusions: Our findings demonstrated the potential of profiling DNA methylation, which can effectively distinguish cancerous from healthy, for the purpose of screening. This method has potential to serve as a supplementary or alternative approach in early detection.
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Chen, Sai, Shile Zhang, Tingting Jiang, Jennifer Yen, Yupeng He, Ariel Jaimovich, Yvonne Kim, et al. "Abstract 3763: Detection of tumor-associated gene inactivation in clinical blood draws via cell-free DNA methylation profiling." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3763. http://dx.doi.org/10.1158/1538-7445.am2022-3763.
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Abstract Introduction: The ability to detect genome-wide epigenetic changes, such as DNA methylation, has expanded translational applications in oncology settings. Because these changes occur early in carcinogenesis, they can be used for early cancer detection when genomic technologies fall short due to lower sensitivity, and in the early and late-stage cancer setting for minimal residual disease detection, disease monitoring and therapy selection. In this analysis, we demonstrated our detection of differential methylations that classify cancer from healthy normals, as well as the quantification of promoter methylation, using a highly sensitive targeted assay that simultaneously captures both genomic alterations and methylation signatures in cell-free DNA (cfDNA). Methods: Methylation signals were profiled with a broad genomic panel (15.2 Mb) targeting regions that are unmethylated in plasma cfDNA from cancer-free donors. The panel covers the promoter regions of 925 out of 1,217 known tumor suppressor genes (TSGs) (e.g. TP53, APC, RB1, PTEN), homologous recombination and repair (HRR) genes (e.g. ATM, BRCA1/2, CDK12, RAD51C/D). We applied our genomic and epigenomic assay on cfDNA from 1,968 colorectal cancer (CRC) patients, 480 patients with other 6 common cancers, and 2,037 cancer-free donors. To test the sensitivity of our epigenomic assay, we generated an in-silico dataset by computationally mixing reads from the cancer patients with those from cancer-free donors at a low tumor fraction (TF) of 0.1%. Results: Among 62 clinically relevant TSG and HRR genes, 56 (90%) were differentially methylated (Wilcoxon p<0.05) between the 2,448 cancer vs. 2,037 cancer-free donor samples. In the in-silico dataset, 51 of the 56 differentially methylated genes remained statistically significant (Wilcoxon p<0.05) at 0.1% TF. In the 1,968 CRC patients, we observed a significant association (Fisher’s p<1e-05) between MLH1 promoter methylation and microsatellite instability (MSI-H): 70% of MSI-H samples has MLH1 promoter methylation above a predefined “high methylation” threshold, while only 7% of microsatellite-stable samples has MLH1 promoter methylation above this threshold. Our result is consistent with previous studies that 54-100% of CRC patients with MSI-H tumors harbor MLH1 promoter methylation. Conclusion: We demonstrate that our assay can accurately detect cancer-driven DNA methylation across the genome in clinical plasma samples. Detection of differential methylation in cancerous versus non-cancerous tissues could allow for early cancer detection, leading to better survival and prediction of recurrence prior to imaging. The highly sensitive detection of promoter methylation shown with TSG, HRR, and MLH1 genes may provide orthogonal information to oncologists in therapeutic selection with high confidence. Citation Format: Sai Chen, Shile Zhang, Tingting Jiang, Jennifer Yen, Yupeng He, Ariel Jaimovich, Yvonne Kim, Dustin Ma, Giao Tran, Daniel P. Gaile, Rebecca J. Nagy, Elena Helman, Han-Yu Chuang. Detection of tumor-associated gene inactivation in clinical blood draws via cell-free DNA methylation profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3763.
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Linger, G. D., C. L. Bormann, M. D. Peoples, M. C. Golding, and C. R. Long. "189 TRANSCRIPTIONAL PROFILING OF HISTONE-MODIFYING GENES DURING BOVINE PRE-IMPLANTATION EMBRYO DEVELOPMENT IN VITRO." Reproduction, Fertility and Development 21, no. 1 (2009): 193. http://dx.doi.org/10.1071/rdv21n1ab189.
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The proper removal of gametic epigenetic marks and coordinated re-establishment of the epigenome is critical to mammalian embryonic development. This global reprogramming of the embryonic genome includes fluctuations in both DNA methylation and histone modifications that are necessary to control chromatin structure and thus gene expression. In the bovine model, epigenetic changes occur from fertilization through blastocyst stages; in particular, and concurrent with the maternal-embryonic transition, de novo DNA methylation begins at the 8-cell stage. In order to understand which factors might be playing key roles in this epigenetic process, we used quantitative real-time PCR to characterize the temporal expression profiles of several genes involved in DNA and/or histone methylation: G9a, SetB1, Suv39h1, Suv420h1, SmyD3, Suz12, and LSH. Bovine ova and embryos were produced via in vitro maturation, fertilization, and culture from multiple pools of ova. Groups of 12–25 bovine ova or embryos, pooled at the 2-, 4 to 7-, mid 8-, late 8-, 12 to 16-cell, morula, and blastocyst stages, were washed twice through 1X PBS and stored in RNA lysis buffer at –80°C until further use. RNA was isolated from each sample using the RNeasy® Mini kit (Qiagen, Valencia, CA, USA), optimized for isolating RNA from single embryos, and treated to remove any contaminating genomic DNA. cDNA was generated with iScript™ reverse transcriptase (Bio-Rad Laboratories, Hercules, CA, USA) and diluted 1:10 with RNase/DNase-free water for further use in real-time PCR. Relative gene expression from each RNA sample was calculated in triplicate using the SYBR Green comparative Ct method (Applied Biosystems, Foster City, CA, USA) adjusted for individual PCR efficiencies (Bustin 2003) and normalized to the geometric mean Ct of 3 endogenous controls (GAPDH, YWHAZ, and SDHA) in order to account for differences in both cell number and amount of total mRNA present in each sample (Goossens et al. 2005). G9a and SetB1, both lysine-specific methyltransferases, were expressed at their highest levels in the metaphase II (MII) oocyte and 2-cell stage, before expression decreased gradually to basal levels by the morula and blastocyst stages. Suv39h1, Suv420h1, and SmyD3, also lysine-specific methyltransferases, all shared a similar pattern of expression: transcript levels were fairly high in the MII oocyte, increased at the 2-cell stage, then gradually dropped off around the 8–16-cell stage to basal levels by the morula stage. Interestingly, Suz12 and LSH both showed low expression from the MII oocyte until the 4 to 7-cell stage, increased dramatically at the 8-cell stage, then decreased again by the morula stage. Suz12 is a member of several Polycomb group complexes (PRCs); LSH associates with PRC-mediated gene silencing as well as DNMT3a and 3b. These data suggest that Suz12 and LSH may be implicated in bovine embryonic genome activation, while the latter genes are active during earlier cleavage events. Ongoing studies will evaluate the role of each of these epigenetic modifiers in bovine pre-implantation embryos by selective silencing via RNA interference.
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Gao, Qiang, Yuzi Zhang, Jiayue Xu, Guoqiang Wang, Jing Zhao, Xiaofang Wen, Bingsi Li, et al. "Clinical validation of a multicancer detection blood test by circulating cell-free DNA (cfDNA) methylation sequencing: The THUNDER study." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): 10544. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.10544.
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10544 Background: The development of ultra-sensitive genomic and epigenomic assays enables the early detection of multiple cancers in parallel. However, large-scale prospective clinical validation data are rare. Here, we report clinical validation data from the THUNDER (The Unintrusive Detection of EaRly-stage cancers, NCT04820868) study, which evaluates the performance of ELSA-seq among 6 cancer types in lung, colorectum, liver, esophagus, pancreas and ovary, which account for 50% of cancer morbidity and 62% of cancer mortality. Methods: This prospective case-control study consists of four stages: marker discovery, model training, validation, and independent validation. A customized panel covering 161,984 CpG sites was established using public data and in-house data. Cancer patients were pre-specified into the training and validation sets, and healthy controls (HC) were age-matched. Two multi-cancer detection blood test (MCDBT-1 and MCDBT-2) models with different cut-offs were established from the retrospectively collected training set and tested in the validation set. An independent validation set was enrolled prospectively, matched by age, and tested with the locked MCDBT-1/2 models. An interception model was then applied based on the model performance and China cancer incidence data to infer potential positive predictive value (PPV) and clinical utility in real-world practice. Results: In total, the training set consisted of 399 cases and 626 HC; the validation set consisted of 301 cases and 123 HC; and the independent validation set consisted of 505 cases and 505 HC. In the training set, the specificities were 98.9% (95% confidence interval [CI], 97.7%‒99.5%) and 99.7% (98.8%‒100.0%) for MCDBT-1/2 models, respectively. In the independent validation set ( n = 856, the rest to be sequenced and reported), MCDBT-1 and MCDBT-2 yielded sensitivity of 76.2% (72.0%‒80.0%) and 70.2% (65.8%‒74.4%) in 6 cancers with specificity of 96.3% (93.9%‒97.9%) and 99.3% (97.8%‒99.8%), respectively. Stage I‒III sensitivity was 68.5% (63.1%‒73.5%) and 60.8% (55.3%‒66.2%) for MCDBT-1/2 models, respectively. The prediction accuracy of top predicted origin was 79.1% (74.5%‒83.2%) and 83.0% (78.4%‒87.0%) for MCDBT-1/2, respectively. The interception model projected an estimated PPV of 3.1% and 12.4% for MCDBT-1/2 models, respectively. MCDBT-1 could reduce 5-year cancer mortality by 20.3%‒24.6% and reduce late-stage incidence by 51.7%‒61.7%, and MCDBT-2 could reduce 5-year cancer mortality by 15.9%‒19.9% and reduce late-stage incidence by 40.9%‒49.2%. Conclusions: cfDNA methylation-based MCDBT-1/2 models can effectively identify multiple cancers simultaneously at early stages with promising sensitivity, specificity, and accuracy of predicted origin. Their performances are to be further validated in a prospective interventional study (NCT05227534).
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Gao, Qiang, Yuzi Zhang, Jiayue Xu, Guoqiang Wang, Jing Zhao, Xiaofang Wen, Bingsi Li, et al. "Clinical validation of a multicancer detection blood test by circulating cell-free DNA (cfDNA) methylation sequencing: The THUNDER study." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): 10544. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.10544.
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10544 Background: The development of ultra-sensitive genomic and epigenomic assays enables the early detection of multiple cancers in parallel. However, large-scale prospective clinical validation data are rare. Here, we report clinical validation data from the THUNDER (The Unintrusive Detection of EaRly-stage cancers, NCT04820868) study, which evaluates the performance of ELSA-seq among 6 cancer types in lung, colorectum, liver, esophagus, pancreas and ovary, which account for 50% of cancer morbidity and 62% of cancer mortality. Methods: This prospective case-control study consists of four stages: marker discovery, model training, validation, and independent validation. A customized panel covering 161,984 CpG sites was established using public data and in-house data. Cancer patients were pre-specified into the training and validation sets, and healthy controls (HC) were age-matched. Two multi-cancer detection blood test (MCDBT-1 and MCDBT-2) models with different cut-offs were established from the retrospectively collected training set and tested in the validation set. An independent validation set was enrolled prospectively, matched by age, and tested with the locked MCDBT-1/2 models. An interception model was then applied based on the model performance and China cancer incidence data to infer potential positive predictive value (PPV) and clinical utility in real-world practice. Results: In total, the training set consisted of 399 cases and 626 HC; the validation set consisted of 301 cases and 123 HC; and the independent validation set consisted of 505 cases and 505 HC. In the training set, the specificities were 98.9% (95% confidence interval [CI], 97.7%‒99.5%) and 99.7% (98.8%‒100.0%) for MCDBT-1/2 models, respectively. In the independent validation set ( n = 856, the rest to be sequenced and reported), MCDBT-1 and MCDBT-2 yielded sensitivity of 76.2% (72.0%‒80.0%) and 70.2% (65.8%‒74.4%) in 6 cancers with specificity of 96.3% (93.9%‒97.9%) and 99.3% (97.8%‒99.8%), respectively. Stage I‒III sensitivity was 68.5% (63.1%‒73.5%) and 60.8% (55.3%‒66.2%) for MCDBT-1/2 models, respectively. The prediction accuracy of top predicted origin was 79.1% (74.5%‒83.2%) and 83.0% (78.4%‒87.0%) for MCDBT-1/2, respectively. The interception model projected an estimated PPV of 3.1% and 12.4% for MCDBT-1/2 models, respectively. MCDBT-1 could reduce 5-year cancer mortality by 20.3%‒24.6% and reduce late-stage incidence by 51.7%‒61.7%, and MCDBT-2 could reduce 5-year cancer mortality by 15.9%‒19.9% and reduce late-stage incidence by 40.9%‒49.2%. Conclusions: cfDNA methylation-based MCDBT-1/2 models can effectively identify multiple cancers simultaneously at early stages with promising sensitivity, specificity, and accuracy of predicted origin. Their performances are to be further validated in a prospective interventional study (NCT05227534).
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Zhao, A. Z., A. M. Colin, J. Bell, M. Baker, B. R. Char, and R. Maxson. "Activation of a late H2B histone gene in blastula-stage sea urchin embryos by an unusual enhancer element located 3' of the gene." Molecular and Cellular Biology 10, no. 12 (December 1990): 6730–41. http://dx.doi.org/10.1128/mcb.10.12.6730-6741.1990.
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In the sea urchin embryo, late histone genes are transcribed at low levels during cleavage and blastula formation and at substantially higher levels in later stages of embryogenesis. To investigate the molecular basis of the stage-specific expression of a late H2B histone gene, we injected mutant genes lacking portions of 5'- and 3'-flanking regions into Lytechinus pictus embryos and monitored their expression by RNase protection. A 200-bp region located 489 bp downstream of the mRNA 3' terminus was necessary for the increase in transcription of the late H2B gene at the mid-blastula stage of development. DNase I and methylation interference footprint analyses located only one factor-binding site in this region, and gel mobility shift experiments showed that the DNA-binding activity of this factor (designated H2B abp 1) paralleled the transcriptional activity of the L1 H2B gene. Additional mutagenesis and microinjection experiments located the activator element to a 32-bp DNA segment that includes the H2B abp 1-binding site. These experiments also showed that the 32-bp fragment functions independently of position and orientation and therefore has the hallmarks of an enhancer. That this fragment contains most or all of the L1 H2B gene transcription-stimulatory activity makes it unusual among enhancerlike elements, which generally consist of several clustered factor-binding sites that act additively or cooperatively to affect transcription. The nucleotide sequence of the L1 H2B enhancer element suggests that the trans-acting factor that interacts with it is a member of the antennapedia or engrailed class of homeodomain proteins.
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Zhao, A. Z., A. M. Colin, J. Bell, M. Baker, B. R. Char, and R. Maxson. "Activation of a late H2B histone gene in blastula-stage sea urchin embryos by an unusual enhancer element located 3' of the gene." Molecular and Cellular Biology 10, no. 12 (December 1990): 6730–41. http://dx.doi.org/10.1128/mcb.10.12.6730.
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In the sea urchin embryo, late histone genes are transcribed at low levels during cleavage and blastula formation and at substantially higher levels in later stages of embryogenesis. To investigate the molecular basis of the stage-specific expression of a late H2B histone gene, we injected mutant genes lacking portions of 5'- and 3'-flanking regions into Lytechinus pictus embryos and monitored their expression by RNase protection. A 200-bp region located 489 bp downstream of the mRNA 3' terminus was necessary for the increase in transcription of the late H2B gene at the mid-blastula stage of development. DNase I and methylation interference footprint analyses located only one factor-binding site in this region, and gel mobility shift experiments showed that the DNA-binding activity of this factor (designated H2B abp 1) paralleled the transcriptional activity of the L1 H2B gene. Additional mutagenesis and microinjection experiments located the activator element to a 32-bp DNA segment that includes the H2B abp 1-binding site. These experiments also showed that the 32-bp fragment functions independently of position and orientation and therefore has the hallmarks of an enhancer. That this fragment contains most or all of the L1 H2B gene transcription-stimulatory activity makes it unusual among enhancerlike elements, which generally consist of several clustered factor-binding sites that act additively or cooperatively to affect transcription. The nucleotide sequence of the L1 H2B enhancer element suggests that the trans-acting factor that interacts with it is a member of the antennapedia or engrailed class of homeodomain proteins.
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Rein, Brandon J. D., Sajal Gupta, Rima Dada, Joelle Safi, Chad Michener, and Ashok Agarwal. "Potential Markers for Detection and Monitoring of Ovarian Cancer." Journal of Oncology 2011 (2011): 1–17. http://dx.doi.org/10.1155/2011/475983.
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This paper reviews current screening techniques as well as novel biomarkers and their potential role in early detection of ovarian cancer. Ovarian cancer is one of the most common reproductive cancers and has the highest mortality rate amongst gynecologic cancers. Because most ovarian cancer diagnoses occur in the late stages of the disease, five-year survival rates fall below 20%. To improve survival rates and to lower mortality rates for ovarian cancer, improved detection at early stages of the disease is needed. Current screening approaches include tumor markers, ultrasound, or a combination. Efforts are underway to discover new biomarkers of ovarian cancer in order to surmount the obstacles in early-stage diagnosis. Among serum protein markers, HE4 and mesothelin can augment CA125 detection providing higher sensitivity and specificity due to the presence of these proteins in early-stage ovarian cancer. Detection testing that includes methylation of the MCJ gene and increased expression of vascular endothelial growth factor is correlated to poor prognosis and may predict patient survival outcome. Detection testing of biomarkers with long-term stability and combination panels of markers, will likely lead to effective screening strategies with high specificity and sensitivity for early detection of ovarian cancer.
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Chen, Qing, Kingsley Okechukwu Nwozor, Maarten van den Berge, Dirk-Jan Slebos, Alen Faiz, Marnix R. Jonker, H. Marike Boezen, Irene H. Heijink, and Maaike de Vries. "From Differential DNA Methylation in COPD to Mitochondria: Regulation of AHRR Expression Affects Airway Epithelial Response to Cigarette Smoke." Cells 11, no. 21 (October 29, 2022): 3423. http://dx.doi.org/10.3390/cells11213423.
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Cigarette smoking causes hypomethylation of the gene Aryl Hydrocarbon Receptor Repressor (AHRR), which regulates detoxification and oxidative stress-responses. We investigated whether AHRR DNA methylation is related to chronic obstructive pulmonary disease (COPD) and studied its function in airway epithelial cells (AECs). The association with COPD was assessed in blood from never and current smokers with/without COPD, and in AECs from ex-smoking non-COPD controls and GOLD stage II-IV COPD patients cultured with/without cigarette smoke extract (CSE). The effect of CRISPR/Cas9-induced AHRR knockout on proliferation, CSE-induced mitochondrial membrane potential and apoptosis/necrosis in human bronchial epithelial 16HBE cells was studied. In blood, DNA methylation of AHRR at cg05575921 and cg21161138 was lower in smoking COPD subjects than smoking controls. In vitro, AHRR DNA methylation at these CpG-sites was lower in COPD-derived than control-derived AECs only upon CSE exposure. Upon AHRR knockout, we found a lower proliferation rate at baseline, stronger CSE-induced decrease in mitochondrial membrane potential, and higher CSE-induced late apoptosis/necroptosis. Together, our results show lower DNA methylation of AHRR upon smoking in COPD patients compared to non-COPD controls. Our data suggest that higher airway epithelial AHRR expression may lead to impaired cigarette smoke-induced mitochondrial dysfunction and apoptosis/necroptosis, potentially promoting unprogrammed/immunogenic cell death.
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Horrell, Scott A., and Douglas L. Chalker. "LIA4Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila." Eukaryotic Cell 13, no. 10 (August 1, 2014): 1300–1311. http://dx.doi.org/10.1128/ec.00125-14.
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ABSTRACTExtensive DNA elimination occurs as part of macronuclear differentiation duringTetrahymenasexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway.LIA4knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, withoutLIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus duringTetrahymenamacronuclear differentiation.
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Lunardelli, Paula Alvares, Luciana Simões Rafagnin Marinho, Camila Oliveira Rosa, Amauri Alcindo Alfieri, and Marcelo Marcondes Seneda. "Effect of follicular diameter, time of first cleavage and H3K4 methylation on embryo production rates of Bos indicus cattle." Semina: Ciências Agrárias 37, no. 5 (October 26, 2016): 3189. http://dx.doi.org/10.5433/1679-0359.2016v37n5p3189.
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This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699) from small follicles (? 2 mm) and 639 oocytes from large follicles (4-8 mm) were punched from 1,982 Bos indicus’ slaughterhouse ovaries. After maturation and in vitro fertilization (IVF), the cultured embryos were separated into early (? 28 h post-IVF), intermediate (> 28 h and ? 34 h post-IVF) and late (> 34 h and ? 54 h post-IVF) cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3%) was higher than that for small follicles (22.9%, P < 0.05). In addition, blastocyst rates for early and intermediate cleavage groups (45.3% and 33.8%, respectively) were higher than that for late cleavage group (13.5%, P < 0.05). The blastocysts from all groups displayed H3K4me staining by immunofluorescence, particularly intense in what seemed to be trophectoderm cells and weak or absent in cells seemingly from the inner cell mass. For the first time for indicus embryos, data from this study demonstrate that higher blastocyst embryo rates are obtained from embryos that cleave within 34 h after fertilization and from those produced from follicles of 4-8 mm in diameter, indicating a greater ability of these embryos to develop to the stage of embryonic preimplantation. This is the first article demonstrating the occurrence of H3K4me in cattle embryos; its presence in all the evaluated blastocysts suggests that this histone modification plays a key role in maintaining embryo viability at preimplantation stage.
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Howlett, S. K., and W. Reik. "Methylation levels of maternal and paternal genomes during preimplantation development." Development 113, no. 1 (September 1, 1991): 119–27. http://dx.doi.org/10.1242/dev.113.1.119.
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The methylation status of three highly repeated sequences was studied in sperm, eggs and preimplantation embryos with different combinations of parental chromosomes. High levels of methylation of the IAP and MUP sequence families were found in sperm and in eggs, whereas the L1 repeat was found to be highly methylated in sperm but only about 42% methylated in eggs. To assess how the two parental genomes behaved during preimplantation development, normal, fertilised embryos were compared with parthenogenetic embryos where the chromosomes are exclusively of maternal origin. It was observed that the high levels of methylation at the IAP and MUP sequences were retained through early development, with the first signs of demethylation at the IAP sequences apparent on both parental chromosomes in the blastocyst. Methylation at the sperm-derived L1 sequences dropped to about the same level as that of the egg-derived sequences by the late 2-cell stage, both then remain at this intermediate level until around the time of cavitation when levels fell to about 10% in the blastocyst. High levels of DNA methylase were detected in germinal vesicle and metaphase II oocytes; these high levels were maintained in fertilised and parthenogenetic embryos through into the morula and then declined to be undetectable in the blastocyst. Our comparison of maternal and paternal genomes suggests that methylation levels at repeat sequences are remarkably similar at the time of fertilisation or, as in the case of the L1 sequences, they become so during the first few cell cycles. Hence, there do not appear to be global methylation differences between the genomes that are retained through preimplantation development.(ABSTRACT TRUNCATED AT 250 WORDS)
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Hasselbalch, Hans Carl, Helene Myrtue Nielsen, Fazila Asmar, Christen Lykkegaard Andersen, Lasse Sommer Kristensen, Kruse A. Torben, Mads Thomassen, et al. "DNA Methylation Profiling of Sorted Cells from Myelofibrosis Patients reveals Aberrant Epigenetic Regulation of Immune Pathways and identifies Early MPN Driver Genes." Blood 124, no. 21 (December 6, 2014): 4576. http://dx.doi.org/10.1182/blood.v124.21.4576.4576.
Full textAbstract:
Abstract Introduction: Primary myelofibrosis (PMF) belongs to the heterogeneous group of chronic myeloproliferative neoplasms (MPN) together with essential thrombocytosis (ET) and polycythemia vera (PV). It has been suggested that these neoplasms represent a biological continuum from early cancer stage (ET,PV) to advanced MF. Multiple studies report frequent mutations in epigenetic regulators. However, the association to epigenetic changes and the role of epigenetic aberrations in different cell populations is still unknown. We therefore performed DNA methylation profiling of sorted cells from MF patients to unravel pathways contributing to disease phenotype and gain insight into MF pathogenesis. As an aberrant DNA methylation pattern may be an early event in tumorigenesis and may be crucial for progression of the malignant clone towards the more aggressive forms of MPN, we further aimed to identify methylated candidate driver genes. Material and methods: Peripheral blood samples from 16 MF patients were together with BM (bone marrow) and peripheral blood from 3 healthy age matched controls sorted in CD34+ cells, granulocytes and mononuclear cells, and analysed for differential methylated regions using Illumina Infinium HumanMethylation 450K BeadChip. Candidate genes were validated by pyrosequencing in a second cohort of 30 MF patients. To identify potential driver genes the DNA methylation status of candidate genes were likewise analyzed in a larger cohort consisting of 60 ET and PV patients. Results: The number of differential methylated CpG sites between MF cells and the healthy counterparts differed extensively among the three cell populations analyzed. In MF CD34+ cells 1628 CpG sites were differential methylated compared to normal CD34+ cells, and 519 and 213 differential methylated CpG sites were observed in MF granulocytes and MF mononuclear cells, respectively (Δβ was set to 0.2 with an adjusted p-value < 0.05, T-test). Differentially methylated genes were mainly involved in cancer and embryogenic pathways in both the MF CD34+ and mononuclear cells, while mononuclear cells also showed aberrant methylation of genes involved in the inflammatory disease pathways. MF granulocytes showed significant aberrations in pathways involving immunological diseases, cell death and survival. Candidate genes have been identified and validation is ongoing. Interestingly, a gradual increase of the DNA methylation level of TRIM59 was observed from the healthy controls (31%) over ET (53%) to PV (64%) and MF (65%). ET patients could be distinguished from both healthy controls (P= 0.0004, Mann-Whitney test) and from the more progressed stages PV and MF (P=0.0132, Mann-Whitney test) based on the TRIM59 DNA methylation level. TRIM59 promoter methylation could, however, not discriminate between PV and MF (P=0.4721, Mann-Whitney test). Conclusion: Genome-wide DNA methylation profiling of sorted MF blood cells provided an exclusive insight into which pathways that are contributing to MF disease phenotype at a cell specific level. The MF CD34+ cells had the highest number of differential methylated CpG sites (n=1628) when comparing to granulocytes (n=519) and mononuclear cells (n=213) and should be cells of choice when exploring new treatment strategies. Interestingly, the mononuclear compartment show aberrant methylation of inflammatory genes supporting a role of aberrant immune regulation in the pathogenesis of MPN. Earlier studies have failed to identify aberrant methylation in early ET and PV, however, the preliminary data on the methylation of individual genes (TRIM59 promoter methylation) shows that it might be possible to identify early driver genes, and that it may be possible to select a panel of genes that can discriminate early MPN from the late MF stage. Disclosures No relevant conflicts of interest to declare.
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E. A. R., Engku Nur Syafirah, Ahmad Adebayo Irekeola, and Chan Yean Yean. "Diagnostic and Prognostic Indications of Nasopharyngeal Carcinoma." Diagnostics 10, no. 9 (August 19, 2020): 611. http://dx.doi.org/10.3390/diagnostics10090611.
Full textAbstract:
Nasopharyngeal carcinoma (NPC) is a disease that is highly associated with the latent infection of Epstein–Barr virus. The absence of obvious clinical signs at the early stage of the disease has made early diagnosis practically impossible, thereby promoting the establishment and progression of the disease. To enhance the stride for a reliable and less invasive tool for the diagnosis and prognosis of NPC, we synopsize biomarkers belonging to the two most implicated biological domains (oncogenes and tumor suppressors) in NPC disease. Since no single biomarker is sufficient for diagnosis and prognosis, coupled with the fact that the known established methods such as methylation-specific polymerase chain reaction (PCR), multiplex methylation-specific PCR, microarray assays, etc., can only accommodate a few biomarkers, we propose a 10-biomarker panel (KIT, LMP1, PIKC3A, miR-141, and miR-18a/b (oncogenic) and p16, RASSF1A, DAP-kinase, miR-9, and miR-26a (tumor suppressors)) based on their diagnostic and prognostic values. This marker set could be explored in a multilevel or single unified assay for the diagnosis and prognosis of NPC. If carefully harnessed and standardized, it is hoped that the proposed marker set would help transform the diagnostic and prognostic realm of NPC, and ultimately, help prevent the life-threatening late-stage NPC disease.