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Saadatmand, Forough, Muneer Abbas, Victor Apprey, Krishma Tailor, and Bernard Kwabi-Addo. "Sex differences in saliva-based DNA methylation changes and environmental stressor in young African American adults." PLOS ONE 17, no. 9 (2022): e0273717. http://dx.doi.org/10.1371/journal.pone.0273717.
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Background Low socioeconomic status neighborhood exposure to stress and violence may be sources of negative stimuli that poses significant health risks for children, adolescents and throughout the life course of an individual. The study aims to investigate if aberrant epigenetic DNA methylation changes may be a potential mechanism for regulating neighborhood exposures and health outcomes. Methods Exposure to environmental stressors identified in 98 young African American (AA) adults aged 18–25 years old from the Washington D.C., area were used in the study. We correlated the association between stress markers; cortisol, CRP, IgG, IGA, IgM, and self-reported exposure to violence and stress, with quantitative DNA methylation changes in a panel of gene-specific loci using saliva DNA. Results In all participants studied, the exposure to violence was significant and negatively correlated with DNA methylation of MST1R loci (p = 0.032; r = -0.971) and nominally significant with NR3C1 loci (p = 0.053; r = -0.948). In addition, we observed significant and negative correlation of DNA methylation changes of LINE1 (p = 0.044; r = -0.248); NR3C1 (p = 0.017; r = -0.186); MSTR1 (p = 0.022; r = -0.192); and DRD2 (p = 0.056; r = -0.184; albeit nominal significant correlation) with IgA expression. On the other hand, we observed a significant and position correlation of DNA methylation changes in DRD2 (p = 0.037; r = 0.184) with IgG expression. When participants were stratified by sex, we observed in AA young male adults, significant DNA methylation changes of MST1R (p< 0.05) and association with exposure to violence and IgG level. We also observed significant DNA methylation levels of DRD2 (p< 0.05) and association with IgA, IgG, and cortisol level. Furthermore, we observed significant DNA methylation changes of NR3C1 (p< 0.05) with stress, IgA, and IgG in the male participants only. On the other hand, we only observed significant and a positive association of IgG with DNA methylation levels of ESR1 (p = 0.041) in the young AA female participants. Conclusion Our preliminary observation of significant DNA methylation changes in neuronal and immune genes in saliva samples supports our recently published genome-wide DNA methylations changes in blood samples from young AA male adults indicating that saliva offers a non-invasive means for DNA methylation prediction of exposure to environmental stressors in a gender-specific manner.
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Abula, Abudureyimu, Xiaona Li, Xing Quan, et al. "Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation." Nucleic Acids Research 49, no. 8 (2021): 4738–49. http://dx.doi.org/10.1093/nar/gkab202.
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Abstract RNA 2′-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3′-5′ exoribonuclease and is particularly sensitive to RNA 2′-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2′-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR’s ribonuclease activity and significant sensitivity to RNA 2′-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2′-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2′-O-methylations research in biological and clinical samples.
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Walker, Elsbeth L. "Paramutation of the r1 Locus of Maize Is Associated With Increased Cytosine Methylation." Genetics 148, no. 4 (1998): 1973–81. http://dx.doi.org/10.1093/genetics/148.4.1973.
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Abstract In paramutation two alleles of a gene interact so that one of the alleles is epigenetically silenced. The silenced state is then genetically transmissible for many generations. The large (220 kbp) multigenic complex R-r is paramutable: its level of expression is changed during paramutation. R-r was found to exhibit increases in its level of cytosine methylation (C-methylation) following paramutation. These C-methylation changes are localized to the 5′ portions of the two genes in the complex that are most sensitive to paramutation. These methylation changes flank a small region called σ that is thought to have been derived from a transposon named doppia. A mutant derivative of R-r that has a deletion of the σ region fails to become methylated under conditions in which R-r is heavily methylated. This suggests that the presence of σ sequences at the locus is required for the methylation changes that are observed following paramutation.
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Vertino, Paula M., and Paul A. Wade. "R Loops: Lassoing DNA Methylation at CpGi." Molecular Cell 45, no. 6 (2012): 708–9. http://dx.doi.org/10.1016/j.molcel.2012.03.014.
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Li, Xiao-Hong, Mei-Yin Lu, Jia-Li Niu, Dong-Yan Zhu, and Bin Liu. "cfDNA Methylation Profiles and T-Cell Differentiation in Women with Endometrial Polyps." Cells 11, no. 24 (2022): 3989. http://dx.doi.org/10.3390/cells11243989.
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DNA methylation is a part of the regulatory mechanisms of gene expression, including chromatin remodeling and the activity of microRNAs, which are involved in the regulation of T-cell differentiation and function. However, the role of cfDNA methylation in T-cell differentiation is entirely unknown. In patients with endometrial polyps (EPs), we have found an imbalance of T-cell differentiation and an aberrant cfDNA methylation profile, respectively. In this study, we investigated the relationship between cfDNA methylation profiles and T-cell differentiation in 14 people with EPs and 27 healthy controls. We found that several differentially methylated genes (DMGs) were associated with T-cell differentiation in people with EPs (ITGA2-Naïve CD4, r = −0.560, p = 0.037; CST9-EMRA CD4, r = −0.626, p = 0.017; and ZIM2-CM CD8, r = 0.576, p = 0.031), but not in healthy controls (all p > 0.05). When we combined the patients’ characteristics, we found a significant association between ITGA2 methylation and polyp diameter (r = 0.562, p = 0.036), but this effect was lost when adjusting the level of Naïve CD4 T-cells (r = 0.038, p = 0.903). Moreover, the circulating sex hormone levels were associated with T-cell differentiation (estradiol-Naïve CD4, r = −0.589, p = 0.027), and the cfDNA methylation profile (testosterone-ZIM2, r = −0.656, p = 0.011). In conclusion, this study has established a link between cfDNA methylation profiles and T-cell differentiation among people with EPs, which may contribute to the etiology of EPs. Further functional studies are warranted.
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Su, Shian, Quentin Gouil, Marnie E. Blewitt, Dianne Cook, Peter F. Hickey, and Matthew E. Ritchie. "NanoMethViz: An R/Bioconductor package for visualizing long-read methylation data." PLOS Computational Biology 17, no. 10 (2021): e1009524. http://dx.doi.org/10.1371/journal.pcbi.1009524.
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A key benefit of long-read nanopore sequencing technology is the ability to detect modified DNA bases, such as 5-methylcytosine. The lack of R/Bioconductor tools for the effective visualization of nanopore methylation profiles between samples from different experimental groups led us to develop the NanoMethViz R package. Our software can handle methylation output generated from a range of different methylation callers and manages large datasets using a compressed data format. To fully explore the methylation patterns in a dataset, NanoMethViz allows plotting of data at various resolutions. At the sample-level, we use dimensionality reduction to look at the relationships between methylation profiles in an unsupervised way. We visualize methylation profiles of classes of features such as genes or CpG islands by scaling them to relative positions and aggregating their profiles. At the finest resolution, we visualize methylation patterns across individual reads along the genome using the spaghetti plot and heatmaps, allowing users to explore particular genes or genomic regions of interest. In summary, our software makes the handling of methylation signal more convenient, expands upon the visualization options for nanopore data and works seamlessly with existing methylation analysis tools available in the Bioconductor project. Our software is available at https://bioconductor.org/packages/NanoMethViz.
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Jiang, Xinyin, Chauntelle Jack-Roberts, Kaydine Edwards, Ella Gilboa, Ikhtiyor Djuraev, and Mudar Dalloul. "Association of Methylation-Related Nutrient Intake and Status with Offspring DNA Methylation in Pregnant Women with and Without Gestational Diabetes Mellitus." Current Developments in Nutrition 4, Supplement_2 (2020): 1016. http://dx.doi.org/10.1093/cdn/nzaa054_088.
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Abstract Objectives Gestational diabetes mellitus (GDM) is associated with alterations in DNA methylation in the placenta and offspring tissues. Nutrients participating in the methionine cycle (e.g., choline, betaine, folate, vitamin B12, methionine) influence the supply of methyl groups. The objective of this research was to determine whether maternal intake and status of these nutrients during pregnancy may interact with the GDM status to shape the offspring epigenome. Methods We conducted 3-day dietary recalls and collected blood samples from pregnant women with and without GDM (n = 22/group) to quantify methylation-related nutrient intakes and status. At delivery, we collected cord blood samples and measured global DNA methylation. Results GDM was associated with a 25% increase (P = 0.041) in global DNA methylation in the cord blood. Maternal choline intake (r = −0.602, P = 0.006) as well as cord blood methionine (r = −0.553, P = 0.014) and betaine (r = −0.566, P = 0.011) levels were negatively correlated with cord blood DNA methylation only in non-GDM women, while intakes and maternal blood levels of other methylation-related nutrients were not related to cord blood DNA methylation. Conclusions GDM and methyl nutrient intake/status interact to modify offspring DNA methylation in humans. Funding Sources Egg Nutrition Center.
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Jintaridth, Pornrutsami, and Apiwat Mutirangura. "Distinctive patterns of age-dependent hypomethylation in interspersed repetitive sequences." Physiological Genomics 41, no. 2 (2010): 194–200. http://dx.doi.org/10.1152/physiolgenomics.00146.2009.
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Interspersed repetitive sequences (IRSs) are a major contributor to genome size and may contribute to cellular functions. IRSs are subdivided according to size and functionally related structures into short interspersed elements, long interspersed elements (LINEs), DNA transposons, and LTR-retrotransposons. Many IRSs may produce RNA and regulate genes by a variety of mechanisms. The majority of DNA methylation occurs in IRSs and is believed to suppress IRS activities. Global hypomethylation, or the loss of genome-wide methylation, is a common epigenetic event not only in senescent cells but also in cancer cells. Loss of LINE-1 methylation has been characterized in many cancers. Here, we evaluated the methylation levels of peripheral blood mononuclear cells of LINE-1, Alu, and human endogenous retrovirus K (HERV-K) in 177 samples obtained from volunteers between 20 and 88 yr of age. Age was negatively associated with methylation levels of Alu (r = −0.452, P < 10−3) and HERV-K (r = −0.326, P < 10−3) but not LINE-1 (r = 0.145, P = 0.055). Loss of methylation of Alu occurred during ages 34–68 yr, and loss of methylation of HERV-K occurred during ages 40–63 yr and again during ages 64–83 yr. Interestingly, methylation of Alu and LINE-1 are directly associated, particularly at ages 49 yr and older (r = 0.49, P < 10−3). Therefore, only some types of IRSs lose methylation at certain ages. Moreover, Alu and HERV-K become hypomethylated differently. Finally, there may be several mechanisms of global methylation. However, not all of these mechanisms are age-dependent. This finding may lead to a better understanding of not only the biological causes and consequences of genome-wide hypomethylation but also the role of IRSs in the aging process.
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He, Y., R. Zhang, J. Chen, J. Tan, M. Wang, and X. Wu. "The ability of arsenic metabolism affected the expression of lncRNA PANDAR, DNA damage, or DNA methylation in peripheral blood lymphocytes of laborers." Human & Experimental Toxicology 39, no. 5 (2019): 605–13. http://dx.doi.org/10.1177/0960327119897101.
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Arsenic has been associated with significant effects on human health. Exposure to inorganic arsenic has been associated with the changes in gene expression. Promoter of CDKN1A antisense DNA damage activated RNA (PANDAR) expression is induced by p53 protein and DNA damage response. Here, we investigated whether the ability of arsenic metabolism in individuals affected the expression of PANDAR, DNA damage, and DNA methylation. Levels of gene expression and DNA damage were examined by the quantitative polymerase chain reaction and DNA methylation was measured by the methylation-sensitive high-resolution melting curve. In our study, we demonstrated that arsenic exposure increased PANDAR expression and DNA damage among arsenic smelting plant laborers. The PANDAR expression and DNA damage were positively linked to monomethylarsonic acid % ( R = 0.25, p < 0.05 and R = 0.32, p < 0.01) and negatively linked to dimethylarsinic acid % ( R = −0.21, p < 0.05 and R = −0.31, p < 0.01). Subjects with low primary methylation index had increased levels of DNA damage (51.62 ± 2.96 vs. 60.93 ± 3.10, p < 0.05) and methylation (17.14 (15.88–18.51) vs. 15.83 (14.82–18.00), p < 0.05). Subjects with low secondary methylation index had increased levels of PANDAR expression (4.88 ± 0.29 vs. 4.07 ± 0.23, p < 0.01) and DNA damage (17.38 (15.88–19.29) vs. 15.83 (14.82–17.26), p < 0.01). DNA methylation of PANDAR gene was linked to the regulation of its expression in peripheral blood lymphocytes among laborers ( Y = −2.08 × X + 5.64, p < 0.05). These findings suggested arsenic metabolism ability and exposure affected the expression of PANDAR, DNA damage, and DNA methylation.
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He, Shiwei, Yuan Wu, Shuidi Yan, et al. "Methylation of CYP1A1 and VKORC1 promoter associated with stable dosage of warfarin in Chinese patients." PeerJ 9 (June 22, 2021): e11549. http://dx.doi.org/10.7717/peerj.11549.
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Objective To investigate the association between DNA methylation and the stable warfarin dose through genome-wide DNA methylation analysis and pyrosequencing assay. Method This study included 161 patients and genome-wide DNA methylation analysis was used to screen potential warfarin dose-associated CpGs through Illumina Infinium HumanMethylation 450 K BeadChip; then, the pyrosequencing assay was used to further validate the association between the stable warfarin dose and alterations in the methylation of the screened CpGs. GenomeStudio Software and R were used to analyze the differentially methylated CpGs. Results The methylation levels of CpGs surrounding the xenobiotic response element (XRE) within the CYP1A1 promoter, differed significantly between the different dose groups (P < 0.05), and these CpGs presented a positive correlation (r> 0, P < 0.05) with an increase in the stable dose of warfarin. At the VKORC1 promoter, two CpGs methylation levels were significantly different between the differential dose groups (P < 0.05), and one CpG (Chr16: 31106793) presented a significant negative correlation (r < 0, P < 0.05) among different dose (low, medium, and high) groups. Conclusion This is a novel report of the methylation levels of six CpGs surrounding the XRE within the CYP1A1 promoter and one differential CpG at the VKORC1 promoter associated with stable warfarin dosage; these methylation levels might be applied as molecular signatures for warfarin.
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Schafer, Eric S., Rumen Kostadinov, Peter Murakami, et al. "Lineage, Fusion Partner and Age Differences in the Methylome of MLL-r Leukemias." Blood 120, no. 21 (2012): 3506. http://dx.doi.org/10.1182/blood.v120.21.3506.3506.
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Abstract Abstract 3506 Background: MLL gene rearrangements (MLL-r) are seen in all ages in both acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). The most common fusion partners are AF4, AF9 and ENL/ELL. Murine models suggest that the expression of MLL fusion proteins is necessary but not sufficient for leukemogenesis, but true cooperating lesions have been difficult to identify. Our lab and others have previously shown that MLL-r infant ALL can be defined by a unique signature characterized by genome-wide hypermethylated CpG islands leading to tumor suppressor gene silencing. In this study we aimed to discover the driving mechanism of MLL-r infant ALL epigenetic signature and whether that signature is intrinsic to the MLL-r or possibly one of the elusive cooperating leukemogenic events. Methods: We measured DNA methylation levels at roughly 225,000 CpG sites covering over 19,000 genes using the HELP (HpaII-tiny fragment Enrichment by Ligation-mediated PCR) assay to evaluate 102 leukemia samples and 12 normal controls. Gene expression using Illumina HT-12 arrays were concurrently performed for future paired expression-methylation analyses. Leukemia samples were primary diagnostic patient samples representing ages infancy to adulthood and included 60 ALL samples (36 MLL-r and 24 MLL-wt) and 42 AML samples (29 MLL-r and 13 MLL-wt). We used unsupervised hierarchical clustering and principal component analysis (PCA) to evaluate whether the methylation patterns in the 102 samples discriminate between lineage (ALL vs. AML), MLL fusion status (MLL-r vs. MLL-wt), fusion partners, and age (infant, pediatric, adult). Results: Hierarchical clustering, using the top 50% of genes that show highest variance in methylation level across all 102 samples, discriminated leukemia samples into distinct lineage specific groups (ALL vs. AML). Within the ALL cluster, there were distinct sub-clusters separating T- and B- cell disease. In addition, In B- cell disease only, MLL-r and MLL-wt formed distinct sub-clusters while no MLL-r vs MLL-wt subclustering was noticed in AML. PCA recapitulated descriptive findings. Regression analysis of the first principal component of all 102 samples showed significant linear separation of AML vs. ALL (p<0.001), but not by age or MLL-r status. However, analysis of the second principal component (diminishing the effect of lineage) showed that there was a secondary MLL-r specific signature among all samples (p<0.001). Regression analysis of the first principal component in the ALL-only samples showed significant linear separation of MLL-r vs. MLL-wt (p<0.001) and showed distinct separation by fusion partner group defined as AF4, AF9 and all other MLL-r (p<0.01). In addition, PCA showed significant distinction between infant ALL and childhood ALL (p<0.01) and trended towards significance when compared to adult disease (p=0.07). These patterns were not duplicated in AML where PCA showed no significant linear separation of MLL-r vs. MLL-wt (p=0.42) or separation by age(p>0.4). While there was no intrinsic MLL-r specific signature in AML, there was a weaker but significant clustering of MLL-r samples into two distinct groups; one consisting of AF9 samples and the other containing all other MLL-r fusion partners (p<0.03). Finally, we explored linage differences controlling for fusion partner, using false discovery rate analysis. Enumerating regions with q-values of <0.05, 29,736 probes showed differential methylation patterns when comparing infant MLL-AF9 ALL and infant MLL-AF9 AML while there were 0 probes were isolated when comparing infant MLL-AF9 AML to adult MLL-AF9 AML. Conclusions: Methylation patterns in MLL-r ALL and AML are distinct. The primary determinant of methylation signatures in MLL-r leukemia is lineage, not MLL status, even when controlling for fusion partners, suggesting that the MLL-r does not drive methylation patterns observed. However, within lineages MLL status is an important contributor to methylation patterns, but this influence is different in ALL and AML. In B-ALL, MLL-r defines a unique methylation signature, subgrouped by fusion partner and additionally, but less so, by age. In AML, MLL fusions AF9 and other MLL-r cluster together in restricted analyses but there is no intrinsic MLL-r signature. However, while lineage is the principle driver of methylation, MLL status does define a secondary significant signature irrespective of lineage. Disclosures: No relevant conflicts of interest to declare.
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Zhang, Lei, Rui Ma, Qingxiang Yu, and Lihua Sun. "Cyclin-dependent kinase 6 body methylation and association with prognosis of patients with acute myeloid leukemia." Journal of Clinical Oncology 40, no. 16_suppl (2022): e19000-e19000. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e19000.
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e19000 Background: Cyclin-dependent kinase 6 (CDK6) plays critical roles in leukemogenesis and leukemia stem cells activation, and its inhibition has emerged as a novel therapeutic strategy in acute myeloid leukemia (AML) management. However, association between CDK6 methylation and prognosis in patients with AML remains unclear. Methods: Methylation profile, somatic alterations, and CDK6 gene expression data of 200 clinically annotated patients with de novo AML were retrieved from the TCGA Pan-Cancer Atlas project deposited in the UCSC Xena database. Specifically, methylation levels of all 64 CpGs across CDK6 were extracted, and then methylation abundances of promoter and gene body regions were calculated as average levels of CpGs located in the respective regions. Patients were categorized into two subgroups (hypo- and hyper-methylated) as per the median methylation level for either region or single CpG site. Log-rank tests were conducted to compare overall survival (OS) of patients stratified by methylation status. A Cox proportional hazards model was fitted to control confounding factors, including age, white blood cell count, cytogenetic risk classification (favorable, intermediate, and poor risk), and TP53 mutations, as reported previously. Associations of methylation status with cytogenetic risk and somatic alterations were examined using the Wilcoxon and Kruskal-Wallis rank sum tests, respectively. Spearman rank correlation coefficient ( R) was used to evaluate correlations between methylation and expression. Results: Of the 200 patients, 109 (54.5%) were men, and median (interquartile range [IQR]) age was 57.5 (44.75-67) years. Median (IQR) methylations of promoter and body were 5.2% (4.8%-5.7%) and 55.6% (53.7%-57.9%), respectively. Body methylation was observed to correlate with gene expression ( R, -0.22; P = .005), but not promoter methylation. In Kaplan-Meier analysis, low methylation of body rather than promoter predicted prolonged OS (median, 27.4 vs 9.2 months; hazard ratio [HR], 0.43; 95% CI, 0.29-0.63; P < .001). Notably, the OS improvement persisted with multivariable Cox model adopted (HR, 0.64; 95% CI, 0.42-0.98; P = .038). In addition, the body hypomethylation was strongly associated with early age, favorable cytogenetic risk, mutations in TP53, NPM1, and IDH1/ 2, as well as PML- RARA and MYH11- CBFB fusions (all P < .05). Further analysis identified that cg08300570, cg09653641, cg10481072, and cg13590558 in the body were all prognostic for OS (all P < .05). Intriguingly, all these CpGs resides in the enhancer. Conclusions: This is the first study to unravel clinical significance of CDK6 body methylation in AML, and it may serve as an independent prognostic marker. Given that azacitidine and decitabine are widely used, our findings may have implications for combination therapy of CDK4/6 inhibitors with hypomethylating agents in AML. Prospective studies are warranted.
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Fujiyoshi, Sunao, Shohei Honda, Eiso Hiyama, and Akinobu Taketomi. "Investigation of aberrant DNA methylation in association with cisplatin-resistant hepatoblastoma." Journal of Clinical Oncology 37, no. 4_suppl (2019): 258. http://dx.doi.org/10.1200/jco.2019.37.4_suppl.258.
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258 Background: Cisplatin (CDDP) is a key-drug of mainstream treatment for hepatoblastoma (HB), but 22.3% of HB patients have resistance to CDDP, and their prognoses are poor. We investigated methylation status of HB patients and identified CDDP resistant candidate genes in HB. Methods: First, we performed a genome-wide methylation array analysis of 11 resected HB tumors. These cases included six cases of RECIST (Response Evaluation Criteria In Solid Tumors) CR or PR (S group) and five cases of SD or PD (R group). We analyzed methylation status of these patients, comparing with clinical course, and selected significant differentially methylated genes. Next, We made CDDP-resistant HepG2 cells by repeated CDDP treatments for a year, then compared expression levels between the CDDP-resistant cells and original cells, using cDNA microarray analysis. We selected CDDP resistant candidate genes from these data. Results: In methylation array analysis data, unsupervised hierarchical clustering analysis showed that methylation profiles were separable into 2 groups in accord with clinical S group and R group. In principal component analysis, the S group also showed a different methylation pattern to the R group. We compared survival rates using Kaplan-Meier method, and results showed that the R group had poorer prognosis significantly. The R group showed 336 hypermethylated genes and 106 hypomethylated genes around the transcriptional start site compared with S group significantly. And in cDNA microarray analysis, 1271 genes were downregulated and 1571 genes were upregulated significantly in CDDP-resistant cells. From among these genes, we selected 15 suppressor and 8 promoter candidate genes that could be related to CDDP-resistance. Conclusions: We identified methylation status associated with CDDP resistance and poor prognosis, and 23 candidates genes in HB. Further refining might lead us to find out the pathogenesis of chemoresistance or new target of therapies and biomarkers in HB.
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Shao, Zonghong, Yue Ren, and Rong Fu. "Preliminary Study on the Abnormal DNA Methylation in T Cells from the Patients with Immune Related Pancytopenia." Blood 124, no. 21 (2014): 5156. http://dx.doi.org/10.1182/blood.v124.21.5156.5156.
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Abstract Objective To explore the global DNA methylation and the expression of regulatory genes for methylation in CD4 + T cells of the patients with immune related pancytopenia (IRP) and explore the role of methylation in pathogenesis of IRP. Methods Thirty IRP patients (untreated, n=15; remission, n=15) and 15 healthy donors as controls were enrolled from December 2012 to December 2013. CD4+ T cells were sorted by immunomagnetic separation. The global DNA methylation was tested with enzyme-linked immunosorbent assay (ELISA). The mRNA levels of DNA methylation-related regulating genes, DNA methyltransferases (DNMTs) and methylated CpG binding proteins (MBDs), were measured by real-time quantitative polymerase chain reaction (RT-PCR). Results The level of global DNA methylation in peripheral blood CD4+ T cells of untreated IRP patients (3.525%±2.046%)and remission patients (4.790%±1.471%) were significantly lower than that of normal controls (10.101%±3.449%) respectively (both P<0.05). DNMT3b mRNA level of untreated IRP patients (1.332±0.785) was significantly lower than that of normal controls (2.077±1.059) in CD4+T cells (P<0.05). The mRNA expression of MBD2 was significantly higher in CD4+ T cells from untreated and remission IRP patients (2.999±1.601, 2.055±1.576) than that in controls (0.581±0.247) (both P<0.05). The MBD4 mRNA level was significantly higher in CD4+ T cells from untreated IRP patients (2.736±2.719) compared to that in normal controls (1.167±1.006) (p<0.05). DNMT3b mRNA expression and CD4+ T cell DNA methylation to be positive correlated within IRP patients (r=0.569, p<0.01). The MBD2 mRNA expression negatively correlated with CD4+ T cell DNA methylation and the ratio of Th1/Th2 (r=-0.763, p<0.001; r = -0.652, p<0.05). The global methylation of CD4+ T cells negatively related to the ratio of CD5+ B cells (r= -0.439, p<0.05). Conclusions The globe DNA hypomethylation and abnormal expression of DNA methylation-related enzymes in peripheral blood CD4+ T cells may be related with the pathogenesis of IRP. Disclosures No relevant conflicts of interest to declare.
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Maugeri, Andrea, Martina Barchitta, Matteo Fallico, Niccolò Castellino, Michele Reibaldi, and Antonella Agodi. "Characterization of SIRT1/DNMTs Functions and LINE-1 Methylation in Patients with Age-Related Macular Degeneration." Journal of Clinical Medicine 8, no. 2 (2019): 159. http://dx.doi.org/10.3390/jcm8020159.
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Previous studies proposed the application of DNA methylation signatures as clinical biomarkers of age-related macular degeneration (AMD). However, the characterization of Long Interspersed Nuclear Element-1 (LINE-1) methylation levels—a surrogate marker of global DNA methylation—in AMD patients has not been investigated so far. In the present study, we first characterized DNA methyltransferases (DNMTs) and Sirtuin 1 (SIRT1) functions in blood samples of 40 AMD patients and 10 age- and sex-matched controls. Then, we evaluated whether changes in DNMTs functions were associated with different LINE-1 methylation levels in leukocyte DNA. We demonstrated that total DNMTs activity was 48% higher in AMD patients than in controls (p = 0.005). AMD patients also exhibited up-regulation of DNMT1 and DNMT3B expression (FC = 2.6; p = 0.003 and FC = 2.4; p = 0.018, respectively). In line with increased DNMTs functions, the LINE-1 methylation level was higher in AMD patients than in controls (mean = 69.10%; SE = 0.68 vs. mean = 65.73%; SE = 0.59; p = 0.020). All p-values were adjusted by Bonferroni correction. In AMD patients, LINE-1 methylation level was positively associated with total DNMTs activity (r = 0.694; p < 0.001), DNMT1 (r = 0.579; p < 0.001), and DNMT3B (r = 0.521; p = 0.001) expression. Our results encourage further large-size prospective research to understand the relationship between LINE-1 methylation and AMD aetiology, and its usefulness in the clinical setting.
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Schafer, Eric, Rafael Irizarry, Sandeep Negi, et al. "Promoter hypermethylation in MLL-r infant acute lymphoblastic leukemia: biology and therapeutic targeting." Blood 115, no. 23 (2010): 4798–809. http://dx.doi.org/10.1182/blood-2009-09-243634.
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Abstract Cooperating leukemogenic events in MLL-rearranged (MLL-r) infant acute lymphoblastic leukemia (ALL) are largely unknown. We explored the role of promoter CpG island hypermethylation in the biology and therapeutic targeting of MLL-r infant ALL. The HELP (HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction [PCR]) assay was used to examine genome-wide methylation of a cohort of MLL-r infant leukemia samples (n = 5), other common childhood ALLs (n = 5), and normals (n = 5). Unsupervised analysis showed tight clustering of samples into their known biologic groups, indicating large differences in methylation patterns. Global hypermethylation was seen in the MLL-r cohort compared with both the normals and the others, with ratios of significantly (P < .001) hypermethylated to hypomethylated CpGs of 1.7 and 2.9, respectively. A subset of 7 differentially hypermethylated genes was assayed by quantitative reverse-transcription (qRT)–PCR, confirming relative silencing in 5 of 7. In cell line treatment assays with the DNA methyltransferase inhibitor (DNMTi) decitabine, MLL-r (but not MLL wild-type cell lines) showed dose- and time-dependent cytotoxicity and re-expression of 4 of the 5 silenced genes. Methylation-specific PCR (MSP) confirmed promoter hypermethylation at baseline, and a relative decrease in methylation after treatment. DNMTi may represent a novel molecularly targeted therapy for MLL-r infant ALL.
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Houde, A. A., J. St-Pierre, M. F. Hivert, et al. "Placental lipoprotein lipase DNA methylation levels are associated with gestational diabetes mellitus and maternal and cord blood lipid profiles." Journal of Developmental Origins of Health and Disease 5, no. 2 (2014): 132–41. http://dx.doi.org/10.1017/s2040174414000038.
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Placental lipoprotein lipase (LPL) is crucial for placental lipid transfer. ImpairedLPLgene expression and activity were reported in pregnancies complicated by gestational diabetes mellitus (GDM) and intra-uterine growth restriction. We hypothesized that placentalLPLDNA methylation is altered by maternal metabolic status and could contribute to fetal programming. The objective of this study was thus to assess whether placentalLPLDNA methylation is associated with GDM and both maternal and newborn lipid profiles. Placenta biopsies were sampled at delivery from 126 women including 27 women with GDM diagnosed following a post 75 g-oral glucose tolerance test (OGTT) between weeks 24 and 28 of gestation. PlacentalLPLDNA methylation and expression levels were determined using bisulfite pyrosequencing and quantitative real-time PCR, respectively. DNA methylation levels withinLPLproximal promoter region (CpG1) and intron 1 CpG island (CpGs 2 and 3) were lower in placenta of women with GDM. DNA methylation levels atLPL-CpG1 and CpG3 were also negatively correlated with maternal glucose (2-h post OGTT;r=–0.22;P=0.02) and HDL-cholesterol levels (third trimester of pregnancy;r=–0.20;p=0.03), respectively. Moreover, we report correlation betweenLPL-CpG2 DNA methylation and cord blood lipid profile. DNA methylation levels within intron 1 CpG island explained up to 26% (r⩽–0.51;P<0.001) of placentalLPLmRNA expression variance. Overall, we showed that maternal metabolic profile is associated with placentalLPLDNA methylation dysregulation. Our results suggest that site-specificLPLepipolymorphisms in the placenta are possibly functional and could potentially be involved in determining the future metabolic health of the newborn.
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Barve, Vega, Shah, et al. "Perturbation of Methionine/S-adenosylmethionine Metabolism as a Novel Vulnerability in MLL Rearranged Leukemia." Cells 8, no. 11 (2019): 1322. http://dx.doi.org/10.3390/cells8111322.
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Leukemias bearing mixed lineage leukemia (MLL) rearrangement (MLL-R) resulting in expression of oncogenic MLL fusion proteins (MLL-FPs) represent an especially aggressive disease subtype with the worst overall prognoses and chemotherapeutic response. MLL-R leukemias are uniquely dependent on the epigenetic function of the H3K79 methyltransferase DOT1L, which is misdirected by MLL-FPs activating gene expression, driving transformation and leukemogenesis. Given the functional necessity of these leukemias to maintain adequate methylation potential allowing aberrant activating histone methylation to proceed, driving leukemic gene expression, we investigated perturbation of methionine (Met)/S-adenosylmethionine (SAM) metabolism as a novel therapeutic paradigm for MLL-R leukemia. Disruption of Met/SAM metabolism, by either methionine deprivation or pharmacologic inhibition of downstream metabolism, reduced overall cellular methylation potential, reduced relative cell numbers, and induced apoptosis selectively in established MLL-AF4 cell lines or MLL-AF6-expressing patient blasts but not in BCR-ABL-driven K562 cells. Global histone methylation dynamics were altered, with a profound loss of requisite H3K79 methylation, indicating inhibition of DOT1L function. Relative occupancy of the repressive H3K27me3 modification was increased at the DOT1L promoter in MLL-R cells, and DOT1L mRNA and protein expression was reduced. Finally, pharmacologic inhibition of Met/SAM metabolism significantly prolonged survival in an advanced, clinically relevant patient–derived MLL-R leukemia xenograft model, in combination with cytotoxic induction chemotherapy. Our findings provide support for further investigation into the development of highly specific allosteric inhibitors of enzymatic mediators of Met/SAM metabolism or dietary manipulation of methionine levels. Such inhibitors may lead to enhanced treatment outcomes for MLL-R leukemia, along with cytotoxic chemotherapy or DOT1L inhibitors.
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Chang, Xiaojing, Jinguo Ma, Xiaoying Xue, et al. "DNMT family induces down-regulation of NDRG1 via DNA methylation and clinicopathological significance in gastric cancer." PeerJ 9 (September 16, 2021): e12146. http://dx.doi.org/10.7717/peerj.12146.
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Background Aberrant DNA methylation of tumor suppressor genes is a common event in the development and progression of gastric cancer (GC). Our previous study showed NDRG1, which could suppress cell invasion and migration, was frequently down-regulated by DNA methylation of its promoter in GC. Purpose and Methods To analyze the relationship between the expression and DNA methylation of NDRG1 and DNA methyltransferase (DNMT) family. We performed a comprehensive comparison analysis using 407 patients including sequencing analysis data of GC from TCGA. Results NDRG1 was down-regulated in GC, and was negatively correlative to DNMT1 (r = −0.11, p = 0.03), DNMT3A (r = −0.10, p = 0.01), DNMT3B (r = −0.01, p = 0.88), respectively, whereas the DNA methylation of NDRG1 was positively correlative to DNMT family (DNMT1 r = 0.20, p < 0.01; DNMT3A r = 0.26, p < 0.001; DNMT3B r = 0.03, p = 0.57, respectively). NDRG1 expression was significantly inverse correlated with invasion depth (p = 0.023), but DNMT1 was significantly positive correlated with invasion depth (p = 0.049). DNMT3B was significantly correlated with the degree of tumor cell differentiation (p = 0.030). However, there was no association between the expression of DNMT3A and clinicopathological features. The KM plotter showed that NDRG1 (HR = 0.95, 95% CI [0.8–1.12], p = 0.53) and DNMT1 (HR = 1.04, 95% CI [0.88–1.23], p = 0.67) had no association with prognosis of GC patients, while, DNMT3A (p = 0.0064) and DNMT3B (p = 0.00025) displayed significantly association. But the overall survival of high expression of NDRG1 tended to be prolonged. Conclusion These data suggest that down-regulation of NDRG1expression in GC may be due to its promoter DNA methylation via DNMT family. The demethylating agent maybe a potential target drug for GC patients.
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Mah, Clarence K., Jill P. Mesirov, and Lukas Chavez. "An accessible GenePattern notebook for the copy number variation analysis of Illumina Infinium DNA methylation arrays." F1000Research 7 (December 5, 2018): 1897. http://dx.doi.org/10.12688/f1000research.16338.1.
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Illumina Infinium DNA methylation arrays are a cost-effective technology to measure DNA methylation at CpG sites genome-wide and across cohorts of normal and cancer samples. While copy number alterations are commonly inferred from array-CGH, SNP arrays, or whole-genome DNA sequencing, Illumina Infinium DNA methylation arrays have been shown to detect copy number alterations at comparable sensitivity. Here we present an accessible, interactive GenePattern notebook for the analysis of copy number variation using Illumina Infinium DNA methylation arrays. The notebook provides a graphical user interface to a workflow using the R/Bioconductor packages minfi and conumee. The environment allows analysis to be performed without the installation of the R software environment, the packages and dependencies, and without the need to write or manipulate code.
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Saied, Marwa, Sabah Khaled, Thomas Down, et al. "Genome Wide Study of DNA Methylation In AML." Blood 116, no. 21 (2010): 3618. http://dx.doi.org/10.1182/blood.v116.21.3618.3618.
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Abstract Abstract 3618 DNA methylation is the most stable epigenetic modification and has a major role in cancer initiation and progression. The two main aims for this research were, firstly, to use the genome wide analysis of DNA methylation to better understand the development of acute myeloid leukemia (AML). The second aim was to detect differentially methylated genes/regions between certain subtypes of AML and normal bone marrow (NBM). We used the methylated DNA immunoprecipitation technique followed by high-throughput sequencing by Illumina Genome Analyser II (MeDIP -seq) for 9 AML samples for which ethical approval has been obtained. The selected leukemias included three with the t(8; 21), three with the t(15; 17) translocations and three with normal karyotypes (NK). The control samples were 3 normal bone marrows (NBMs) from healthy donors. The number of reads generated from Illumina ranged between 18– 20 million paired-end reads/lane with a good base quality from both ends (base quality > 30 represented 75%-85% of reads). The reads were aligned using 2 algorithms (Maq and Bowtie) and the methylation analysis was performed by Batman software (Bayesian Tool for Methylation Analysis). The creation of this genome-wide methylation map for AML permits the examination of the patterns for key genetic elements. Investigation of the 35,072 promoter regions identified 80 genes, which showed a significant differential methylation levels in leukemic cases in comparison to NBM; consistently high methylation levels in leukaemia were detected in the promoters of 70 genes e.g. DPP6, ID4, DCC, whereas high methylation levels in NBM, lost in leukaemia was observed in 10 genes e.g. ATF4. For each AML subtype, we also identified significant differentially methylated promoter regions e.g. PAX1 for t(8; 21), GRM7 for t(15; 17), NPM2 for NK. An analysis of gene body methylation identified 49 genes with significantly higher methylation in AML in comparison to NBM e.g. MYOD1 and 31 genes with a higher methylation in NBMs than AML e.g. GNG8. A similar analysis of 23,600 CpG islands identified 400 CpG islands with significant differential methylation levels between leukaemia and NBMs (212 CpG islands were found to have significantly increased methylation in leukaemia and 188 CpG islands had significantly higher methylation in NBMs). The pattern of methylation in CpG island “shores” (2 KB from either side of each CpG island) has been investigated and 312 CpG island shores showed a higher methylation in leukaemia and 88 CpG shores had a significant increase methylation levels in NBMs. This genome wide methylation map has been validated by using direct bisulfite sequencing of the regions identified above (Spearman r= 0.8, P <0.0001) and also by using Illumina Infinium assay (Spearman r= 0.7 P <0.0001) which interrogates regions at single representative CpGs. Comparison of previous array based gene expression data with this methylation map revealed a significant negative correlation between promoter methylation and gene expression (Pearson r= -0.9, P< 0.0001) while, gene body methylation showed a small negative correlation with gene expression, that was found in genes of CpG density >3% (Pearson r= -0.3, P< 0.0001). Conclusion: we have established a high-resolution (100bp) map of DNA methylation in AML and thus identified a novel list of genes, which have significantly differential methylation levels in AML. Disclosures: No relevant conflicts of interest to declare.
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Shi, Qi, Nana Feng, Qingyun Ma, et al. "ZNF354C Mediated by DNMT1 Ameliorates Lung Ischemia-Reperfusion Oxidative Stress Injury by Reducing TFPI Promoter Methylation to Upregulate TFPI." Oxidative Medicine and Cellular Longevity 2022 (July 19, 2022): 1–18. http://dx.doi.org/10.1155/2022/7288729.
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Background. Pulmonary ischemia reperfusion- (I/R-) induced dysfunction is a significant clinical problem after lung transplantation. In this study, we aim to explore the molecular mechanism of lung I/R injury (LIRI). Methods. Bioinformatic analysis of gene involved in oxidative stress. A HUVEC oxygen glucose deprivation/reoxygenation (OGD/R) model and I/R mouse model were first established via I/R. The cellular proliferation, migration, reactive oxygen species (ROS), and parameters of lung injury were assessed via CCK-8, EdU staining, Transwell, cellular ROS kit, and H&E staining. We also confirmed related gene expressions and protein levels and the interaction between the tissue factor pathway inhibitor (TFPI) promotor and ZNF354C. Results. Bioinformatic analysis results showed TFPI contributed to oxidative stress. OGD/R caused a reduction in cell viability and migration, hypermethylation of TFPI, increased ROS, and downregulation of ZNF354C, TFPI, and DNA methyltransferases (DNMTs) in HUVECs. Besides, ZNF354C could directly bind to the TFPI promoter, enhance proliferation and migration, and inhibit ROS in OGD/R-induced HUVECs by upregulating TFPI. More importantly, we discovered that 5-Aza could reduce TFPI methylation, upregulate TFPI, and enhance the binding of ZNF354C to the TFPI promoter in LIRI. Furthermore, DNMT1 silencing could induce proliferation and migration and prevent ROS in OGD/R-induced HUVECs by upregulating ZNF354C. Additionally, we verified that ZNF354C could alleviate LIRI by preventing DNA methylation in vivo. Conclusions. ZNF354C overexpression induced proliferation and migration, as well as suppressed ROS in OGD/R-induced HUVECs, and alleviated LIRI in mice by inhibiting TFPI promoter methylation to upregulate TFPI. Therefore, ZNF354C and TFPI methylation might be promising molecular markers for LIRI therapy.
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Isubakova, Daria S., Olga S. Tsymbal, Nikolai V. Litviakov, Ivan V. Milto, and Ravil M. Takhauov. "Relationship between methylation of promoters of apoptosis genes in blood lymphocytes with the frequency of chromosomal aberrations and the dose of radiation." Ecological genetics 20, no. 4 (2022): 315–23. http://dx.doi.org/10.17816/ecogen109119.
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BACKGROUND: Impaired apoptosis can have serious consequences: the accumulation of mutant cells, the development of teratogenic effects and malignant neoplasms. In this regard, the study of the mechanisms of changes in the activity of apoptosis due to methylation under the influence of long-term irradiation is urgent.
 AIM: The study of the degree of methylation of gene promoters involved in the induction of apoptosis in the personnel of the Siberian Chemical Plant, exposed to long-term technogenic irradiation of ionizing radiation in the course of their professional activities.
 MATERIALS AND METHODS: The study was performed on peripheral blood samples of employees of the Siberian Chemical Plant, with a total dose of external exposure from 100 to 300 mSv. Chromosomal aberrations were detected by standard karyotyping of cultured blood lymphocytes. The degree of gene promoters methylation was determined using MethylScreen technology.
 RESULTS: The degree of gene methylation BIRC2, CASP3, CASP9, CIDEB, CRADD, DAPK1, DFFA, FADD, GADD45A, LTBR, TNFRSF21, TNFRSF25 ranges from 0.31 to 41.75%. A strong negative correlation was found between the degree of methylation of GADD45A (r = 0.7364, р = 0.009) with an increased frequency of aberrant cells, moderate negative correlation GADD45A (r = 0.6347, р = 0.035) with an increased frequency of dicentric chromosomes, moderate negative correlation CASP9 (r = 0.6606, р = 0.026), and strong negative correlation CIDEB (r = 0.7982, р = 0.003) with an increased frequency of chromatid fragments. A moderate negative correlation of the methylation degree of CASP9 (r = 0.6636, р = 0.026), and CIDEB (r = 0.6636, р = 0.026) with the total dose of external exposure was shown.
 CONCLUSIONS: The decrease in the level of apoptosis at doses of 100300 mSv can be explained by the achievement of the demethylation threshold for the promoters of the proapoptotic genes GADD45A, CASP9, CIDEB. This once again testifies in favor of the threshold model of the dependence of the radiation effect on the radiation dose.
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Phipson, Belinda, Jovana Maksimovic, and Alicia Oshlack. "missMethyl: an R package for analyzing data from Illumina’s HumanMethylation450 platform." Bioinformatics 32, no. 2 (2015): 286–88. http://dx.doi.org/10.1093/bioinformatics/btv560.
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Abstract Summary: DNA methylation is one of the most commonly studied epigenetic modifications due to its role in both disease and development. The Illumina HumanMethylation450 BeadChip is a cost-effective way to profile &gt;450 000 CpGs across the human genome, making it a popular platform for profiling DNA methylation. Here we introduce missMethyl, an R package with a suite of tools for performing normalization, removal of unwanted variation in differential methylation analysis, differential variability testing and gene set analysis for the 450K array. Availability and implementation: missMethyl is an R package available from the Bioconductor project at www.bioconductor.org. Contact: alicia.oshlack@mcri.edu.au Supplementary information: Supplementary data are available at Bioinformatics online.
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Donoghue, M. J., B. L. Patton, J. R. Sanes, and J. P. Merlie. "An axial gradient of transgene methylation in murine skeletal muscle: genomic imprint of rostrocaudal position." Development 116, no. 4 (1992): 1101–12. http://dx.doi.org/10.1242/dev.116.4.1101.
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We previously used mice bearing a myosin light chain-chloramphenicol acetyltransferase (MLC1-CAT) transgene to show that adult muscle cells bear a heritable, cell autonomous memory of their rostrocaudal position. CAT mRNA and protein are expressed in a &gt; 100-fold rostrocaudal gradient in skeletal muscles of developing and adult MLC1-CAT mice (Donoghue, M. J., Merlie, J. P., Rosenthal, N. and Sanes, J. R. (1991). Proc. Natl. Acad. Sci. USA 88, 5847–5851; Donoghue, M. J., Alvarez, J. D., Merlie, J. P. and Sanes, J. R. (1991). J. Cell Biol. 115, 423–434). Moreover, both in primary cultures and in myogenic cell lines prepared from individual muscles of these mice, CAT levels reflect the body position from which the myoblasts were derived (Donoghue, M.J., Morris-Valero, R., Johnson, Y.R., Merlie, J.P. and Sanes, J. R. (1992). Cell 69, 67–77). Here, we show that the methylation state of the MLC1-CAT transgene in skeletal muscles is also graded along the rostrocaudal axis: methylation levels decrease and expression levels increase in the order, jaw--&gt;neck--&gt;chest and forelimb--&gt;hindlimb. Methylation levels are also approx. 10-fold higher in rostrally derived than in caudally derived myogenic cell lines, which express low and high levels of CAT, respectively. Within each cell line, undifferentiated cells (myoblasts), which do not express the transgene, and differentiated cells (myotubes), which do, are indistinguishable in methylation state. Thus, differentiation-related changes in transgene expression do not affect position-related levels of transgene methylation. On the other hand, treatment of rostrally derived lines with the demethylating agent, 5-azacytidine, decreases methylation and increases expression of the transgene. Thus, perturbation of methylation affects expression. Taken together, these results suggest that methylation provides a genomic imprint of rostrocaudal body position that may serve as a component of the positional memory that mammalian cells retain into adulthood.
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Knödlseder, Nastassia, Guillermo Nevot, Maria-José Fábrega, et al. "Engineering selectivity of Cutibacterium acnes phages by epigenetic imprinting." PLOS Pathogens 18, no. 3 (2022): e1010420. http://dx.doi.org/10.1371/journal.ppat.1010420.
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Cutibacterium acnes (C. acnes) is a gram-positive bacterium and a member of the human skin microbiome. Despite being the most abundant skin commensal, certain members have been associated with common inflammatory disorders such as acne vulgaris. The availability of the complete genome sequences from various C. acnes clades have enabled the identification of putative methyltransferases, some of them potentially belonging to restriction-modification (R-M) systems which protect the host of invading DNA. However, little is known on whether these systems are functional in the different C. acnes strains. To investigate the activity of these putative R-M and their relevance in host protective mechanisms, we analyzed the methylome of six representative C. acnes strains by Oxford Nanopore Technologies (ONT) sequencing. We detected the presence of a 6-methyladenine modification at a defined DNA consensus sequence in strain KPA171202 and recombinant expression of this R-M system confirmed its methylation activity. Additionally, a R-M knockout mutant verified the loss of methylation properties of the strain. We studied the potential of one C. acnes bacteriophage (PAD20) in killing various C. acnes strains and linked an increase in its specificity to phage DNA methylation acquired upon infection of a methylation competent strain. We demonstrate a therapeutic application of this mechanism where phages propagated in R-M deficient strains selectively kill R-M deficient acne-prone clades while probiotic ones remain resistant to phage infection.
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Patel, Manishkumar S., Ellen K. Kendall, Sarah Ondrejka, et al. "Gene Expression and Epigenetic Analysis in Relapsed/Refractory Diffuse Large B Cell Lymphoma Provides Insights into Evolution of Treatment Resistance to R-CHOP." Blood 136, Supplement 1 (2020): 26. http://dx.doi.org/10.1182/blood-2020-138645.
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Background Diffuse large B cell lymphoma (DLBCL) is curable in ~60-70% of patients using standard chemoimmunotherapy, but the prognosis is poor for relapsed/refractory (R/R) DLBCL. Therefore, understanding the underlying molecular mechanisms will facilitate early prediction and effective management of resistance to therapy. Recent studies of paired diagnostic-relapse biopsies from patients have relied on a single "omics" approach, examining either gene expression or epigenetic evolution. Here we present a combined analysis of gene expression and DNA methylation profiles of paired diagnostic-relapse DLBCL biopsies to identify changes responsible for relapse after R-CHOP. Methods Biopsies from 23 DLBCL patients were obtained at the time of diagnosis and relapse following frontline R-CHOP chemoimmunotherapy. The cohort had 18 (78.3%) male patients with median age of 62 (range, 35-86) years and median IPI of 2.5 (range, 1-5). The median time from diagnosis to relapse was 7 (range, 0-57) months. DNA and RNA were extracted simultaneously from formalin-fixed paraffin embedded (FFPE) biopsy samples. DNA methylation levels were measured through Illumina 850k Methylation Array for 22 pairs of diagnostic-relapse biopsies. RNA from diagnostic-relapse paired biopsies from 6 patients was sequenced using Illumina HiSeq4000. Differentially methylated probes were identified using the DMRcate package, and differentially expressed genes were identified using the DESeq2 package. Gene set enrichment analysis was performed using canonical pathway gene sets from MSigDB. Pearson's correlation with a Bonferroni correction to the p-value was used to calculate the correlation between regularized log transformed gene expression counts and methylation beta values. Results In a pairwise comparison of gene expression between diagnostic and R/R biopsy pairs, we found 14 differentially expressed genes (FDR&lt;0.1 & Log2FC&gt;|1|) consistent across all pairs. Compared to gene expression at diagnosis, five genes (CYP1B1, LGR4, ATXN1, CTSC, ZMAT3) were downregulated, and eight genes (ERBB3, CD19, CARD11, MT-RNR2, IGHG3, CCDC88C, ATP2A3, CENPE, and PCNT) were up-regulated in the R/R samples. Many of these genes have been previously implicated in oncogenesis, such as ERBB3, a member of the epidermal growth receptor family. Importantly, some of these genes have known roles in DLBCL biology, such as CD19, a member of the B-cell receptor complex, and CARD11, a gene in which several oncogenic mutations have been identified in DLBCL as a mediator of NF-KB activation. Gene set enrichment analysis revealed overexpression of immune signatures such as cytokine-cytokine receptor interaction, chemokine receptor-chemokine binding, and the IL-12-STAT4 pathway at diagnosis. At relapse, cell cycle, B-cell receptor, and NOTCH signaling pathways were overexpressed. Interestingly, in a pairwise comparison of methylation between diagnostic and R/R biopsy pairs, there were no differentially methylated probes (FDR&lt;0.05), suggesting no coordinated epigenetic evolution between diagnostic and R/R pairs. For biopsy pairs that had both gene expression and methylation data (5 pairs), we correlated gene expression and methylation values. We found that none of the differentially expressed genes between the diagnostic and R/R biopsies were significantly correlated with methylation status (adjusted p-value&lt;0.05). Conclusions By analyzing paired diagnostic and relapse DLBCL biopsies, we found that at the time of relapse, there are significant transcriptomic changes but no significant epigenetic changes when compared to diagnostic biopsies. Activation of B-cell receptor and NOTCH signaling, as well as the loss of immune signaling at relapse, cannot be attributed to coordinated epigenetic changes in methylation. As the epigenetic profile of the biopsies did not consistently evolve, these data emphasize the need for better understanding of the baseline methylation profiles at the time of diagnosis, as well as acquired somatic mutations that may contribute to the emergence of therapeutic resistance. Future studies are needed to focus on how activation of signaling pathways triggered by genomic alterations can be targeted in relapsed/refractory DLBCL. Disclosures Hsi: Seattle Genetics: Consultancy, Honoraria; Miltenyi: Consultancy, Honoraria; Abbvie: Research Funding; Eli Lilly: Research Funding; CytomX: Consultancy, Honoraria. Hill:Takeda: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding.
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Caspers, Maarten, Sara Blocquiaux, Ruben Charlier, et al. "Intensity-Specific Differential Leukocyte DNA Methylation in Physical (In)Activity: An Exploratory Approach." Twin Research and Human Genetics 21, no. 2 (2018): 101–11. http://dx.doi.org/10.1017/thg.2018.10.
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The aim of this exploratory study was to investigate how sedentary behavior (SB) and physical activity (PA) influence DNA methylation at a global, gene-specific, and health-related pathway level. SB, light PA (LPA), and moderate-to-vigorous PA (MVPA) were assessed objectively for 41 Flemish men using the SenseWear Pro 3 Armband. CpG site-specific methylation in leukocytes was determined using the Illumina HumanMethylation 450 BeadChip. Correlations were calculated between time spent on the three PA intensity levels and global DNA methylation, using a z-score-based method to determine global DNA methylation levels. To determine whether CpG site-specific methylation can be predicted by these three PA intensity levels, linear regression analyses were performed. Based on the significantly associated CpG sites at α = 0.005, lists were created including all genes with a promoter region overlapping these CpG sites. A biological pathway analysis determined to what extent these genes are overrepresented within several pathways. No significant associations were observed between global DNA methylation and SB (r = 0.084), LPA (r = -0.168), or MVPA (r = -0.125), although the direction of the correlation coefficients is opposite to what is generally reported in literature. SB has a different impact on global and gene-specific methylation than PA, but also LPA and MVPA affect separate genes and pathways. Furthermore, the function of a pathway seems to determine its association with SB, LPA, or MVPA. Multiple PA intensity levels, including SB, should be taken into account in future studies investigating the effect of physical (in)activity on human health through epigenetic mechanisms.
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Ribeiro, Andre M., Hiruni Wijesena, Daniel C. Ciobanu, Steve Horvath, and Matthew L. Spangler. "PSIII-7 Relationship of Age and Genetics with the Methylation Profile of Beef Cattle." Journal of Animal Science 99, Supplement_1 (2021): 159. http://dx.doi.org/10.1093/jas/skab054.272.
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Abstract This study aimed to compare models for the prediction of cow age from DNA methylation profiles and estimate the heritability of the proportion of methylated sites (PM) and methylation status at each site (MS). Methylation data from blood samples of cows (n=136) were generated from the HorvathMammalMethylChip40 array that consists of 34,324 CpG sites that mapped to the bovine genome. Methylation status was determined by the distribution of the methylation values, with values above, within and below 2 standard deviations classified as methylated (2), intermediately methylated (1) and unmethylated (0), respectively. Principal component analysis (PCA) was applied to a (co)variance methylation status matrix. The first and second PC accounted for 25.65% and 9% of the total variance, respectively. Five Bayesian models (Bayesian ridge regression, BayesA, BayesB, BayesCπ and Bayesian LASSO) were implemented with the BGLR package in R. Bootstrapping validation (n=400) was used to evaluate the tested models, with 102 and 34 individuals in the training and validation sets, respectively. The correlation between the predicted and true age was high (r = 0.97 to 0.99). A BayesA model performed the best (r = 0.99, MSE = 0.11 and slope = 0.93), while Bayesian LASSO was the least accurate (r = 0.97, MSE = 0.26 and slope = 0.88). Heritability was estimated using GBLUP implemented in the BGLR package. The mean (SD) heritability estimate for PM was 0.46 ± 0.10 and the heritability of MS ranged from 0.18 to 0.73 (mean = 0.33). The 10% of sites with the highest heritability (343 sites; mean = 0.62) were located in exon (91), intron (84), intergenic (152), and promoter (16) regions. The largest number of these top sites (31) were located on chromosome 3 in genetic or intergenic regions close to transcription factor binding sites (i.e., FOXO6, ELAV4 and LMO4).
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Tawbi, H. A., S. Buch, P. Pancoska, et al. "Prediction of response to alkylator-based chemotherapy in metastatic melanoma (MM) using gene expression and promoter methylation signatures." Journal of Clinical Oncology 27, no. 15_suppl (2009): 9009. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.9009.
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9009 Background: Temozolomide and dacarbazine (TMZ and DTIC) remain the mainstay of alkylator-based chemotherapy for MM, despite response rates of 10–15% and the absence of any impact on survival. Classification of patients according to responsiveness can guide the individualization of therapy and inform approaches to abrogate mechanisms of chemotherapy resistance. Epigenetic mechanisms play an important role in regulation of genes associated with resistance and were evaluated in tandem with gene expression profiling in biological samples from MM patients (pts) to refine our understanding of the epigenomic-genomic-phenotypic interplay. Methods: We examined promoter methylation and gene expression in tumor tissues of 21 pts with MM treated with TMZ or DTIC, using high throughput technologies (Illumina Inc). The cases were divided into responder (R) and non-responder (NR) groups based on clinical response. The data were analyzed using Prediction Analysis of Microarrays (PAM) from BRB array tools. Results: Differential promoter methylation analysis revealed that 63.6% of promoter sites were hypomethylated in tumors obtained from R pts (p<0.0001). PAM analysis of gene expression data revealed that a classifier set consisting of 82 genes was able to predict NRs from Rs with 83% sensitivity and 89% specificity. Promoter methylation profiling did not independently correlate with R status. A simultaneous analysis of the promoter methylation and gene expression values first stratified into 3 data-driven categories and then combined into a 3 by 3 matrix allowed us to identify a common gene expression/methylation signature of 15 genes that classified both NR and R groups accurately 100% of the time. Conclusions: Gene expression signatures independently predict response to chemotherapy in MM, however promoter methylation profiling alone does not. Analysis of combined gene expression and promoter methylation in a well- annotated clinical data set dichotomized according to response identified a highly predictive signature. The findings from this study are qualified by the relatively small sample size and are currently being validated in an expanded sample set. Supported in part by the ECOG Paul Carbone, MD, Fellowship Award. No significant financial relationships to disclose.
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Hulme, Bethany, Altug Didikoglu, Steven Bradburn, et al. "Epigenetic Regulation of BMAL1 with Sleep Disturbances and Alzheimer’s Disease." Journal of Alzheimer's Disease 77, no. 4 (2020): 1783–92. http://dx.doi.org/10.3233/jad-200634.
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Background: An early symptom of Alzheimer’s disease (AD) is a disturbance of the circadian rhythm that is associated with disrupted sleep/wake cycles. Objective: To investigate if BMAL1, a key gene that drives the circadian cycle, is epigenetically regulated in brains in relation to longitudinal changes in cognition, sleep quality, and AD neuropathology. Methods: Frontal cortex tissues were acquired from the Manchester Brain Bank (N = 96). DNA methylation at six CpG sites at the promoter of BMAL1, determined using bisulfite pyrosequencing, was tested for associations with Braak stage, CERAD score and Thal phase, longitudinal changes in cognition, sleep measurements and cross-section measures of depressive symptoms (BDI score). Results: Methylation across all the CpGs strongly correlated with each other. We found increased CpG2 methylation with higher Braak (t(92), p = 0.015) and CERAD (t(94), p = 0.044) stages. No significance was found between longitudinal fluid intelligence, processing speed and memory tests, but methylation at CpG1 (r = 0.20, p = 0.05) and CpG4 (r = 0.20, p = 0.05) positively correlated with vocabulary. CpG2 positively correlated with cross-sectional fluid intelligence (r = 0.20 p = 0.05) and vocabulary (r = 0.22 p = 0.03). Though longitudinal analysis revealed no significance between sleep duration, midsleep and efficiency for any of the CpG sites, CpG3 (B = 0.03, 95% CI, p = 0.03) and CpG5 (B = 0.04, 95% CI, p = 0.01) significantly correlated with night wake. CpG4 correlated with depressive symptoms (B = –0.27, 95% CI, p = 0.02). Conclusion: Methylation of BMAL1 associated with tau pathology, changes in cognitive measures, a measure of sleep and depressive symptoms, suggesting an involvement of the circadian cycle.
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Toussirot, E., S. Pasquereau, C. Vauchy, et al. "POS0329 ABERRANT GLOBAL DNA METHYLATION IN PERIPHERAL BLOOD CELL SUBPOPULATIONS OF PATIENTS WITH AXIAL SPONDYLOARTHRITIS." Annals of the Rheumatic Diseases 81, Suppl 1 (2022): 416–17. http://dx.doi.org/10.1136/annrheumdis-2022-eular.1876.
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BackgroundAxial spondyloarthritis (axSpA) corresponds to a group of chronic inflammatory diseases mainly affecting the axial skeleton. TNFα and IL-17A have been identified as key inflammatory mediators driving the inflammatory process of axSpA. Epigenetics refers to different mechanisms that alter gene expression without involving changes in DNA sequence. DNA methylation is an important epigenetic mechanism, playing a role in gene expression regulation. It is recognized that aberrant DNA methylation can result in immune cell autoreactivity.Objectivesepigenetic features have been rarely evaluated in axSpA. Prior studies have identified DNA methylation changes at both the gene-specific and genowe-wide levels in radiographic ax-SpA (r-axSpA). In this study, we aimed to evaluate the global DNA methylation of patients with axSpA.Methodscase-control study (NCT03092583). Patients with r-axSpA or non radiographic (nr) axSpA (ASAS criteria) and healthy controls (HC) were evaluated. All the patients were biologic naïve and under NSAIDs. Disease activity was evaluated by BASDAI and ASDAS. CD4+T cells and CD14+ monocytes were isolated from peripheral blood and then DNA was extracted. Global DNA methylation (5-mC) was determined using MethylAmp global DNA methylation quantification kit (Epigentek) using 150 ng of total DNA.Results104 patients with axSpA including 59 with r-axSpA (45 M; age [mean ± SD]: 47.1 ± 15 y; disease duration: 15.2 ± 13 y; B27: 86.4%) and 45 nr-axSpA (21 M, age: 39.6 ± 13.2; disease duration: 7 ± 7.8; B27: 65.1%) and 79 healthy controls (HC) (51 M; age: 43.4 ± 12.2 y) were evaluated. Patients had active disease (BASDAI and ASDAS in r-axSpA and nr-axSpA: 5.1 ± 1.8 and 3.04 ± 1.1; 5.04 ± 1.1 and 2.8 ± 1.0, respectively). In CD4+ T lymphocytes, global DNA methylation was higher in the whole SpA group compared to HC (1.45 ± 3.6 vs 0.44 ± 0.9 of 5-mC (p= 0.0092). Similarly, DNA methylation was higher in monocytes from patients with ax-SpA compared to HC (1.94 ± 2.3 vs 0.62 ± 0.9 of 5-mC)(p= 0.0004). When analysing the results between ax-SpA subgroups, DNA methylation remains higher in both CD4 T lymphocytes and monocytes of each patient subgroup compared to HC (p< 0.05 for all comparisons) (Figure 1). The levels of DNA methylation did not correlate with laboratory (ESR, CRP) or clinical (BASDAI) measures of disease activity, excepting ASDAS which was weakly correlated with DNA methylation in CD4+ T lymphocytes from the whole group of axSpA (r = 0.18, p = 0.08). History of uveitis was associated with higher DNA methylation in monocytes (p= 0.017). Previous smoker patients had higher DNA methylation in their monocytes compared to current or never smoker patients (p = 0.06). In a limited number of patients (N = 15) who started a TNFi, DNA methylation decreased in both CD4 T lymphocytes and monocytes after 3 months of treatment.Figure 1.global DNA methylation of CD4+ T lymphocytes and monocytes from patients with ankylosing spondylitis (AS or radiographic axial spondyloarthritis) and non radiographic axial spondyloarthritis (nr-axSpA) and healthy controls (HC) (* p <0.05; *** p <0.001)Conclusiona global DNA hypermethylation was observed in patients with axSpA, both in T CD4 lymphocytes and monocytes. These modifications involved both the radiographic and non radiographic forms. These results were weakly correlated with disease activity and only in monocytes. Collectively, these changes in DNA methylation could alter recruitment of methyl binding proteins (MBP) that regulate chromatin structure and/or impair binding of transcription factors, resulting in down regulation of gene expression relevant to the pathogenesis of axSpA. We currently evaluated the level of expression of DNA methyltransferase (DNMT) and MBP proteins and specific DNA methylation status of the promoters of gene involved in inflammation such as TNFa.AcknowledgementsThis work was supported by a grant (APICHU) from CHU de BesançonDisclosure of InterestsNone declared
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Wang, Yue, Jennifer M. Franks, Michael L. Whitfield, and Chao Cheng. "BioMethyl: an R package for biological interpretation of DNA methylation data." Bioinformatics 35, no. 19 (2019): 3635–41. http://dx.doi.org/10.1093/bioinformatics/btz137.
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AbstractMotivationThe accumulation of publicly available DNA methylation datasets has resulted in the need for tools to interpret the specific cellular phenotypes in bulk tissue data. Current approaches use either single differentially methylated CpG sites or differentially methylated regions that map to genes. However, these approaches may introduce biases in downstream analyses of biological interpretation, because of the variability in gene length. There is a lack of approaches to interpret DNA methylation effectively. Therefore, we have developed computational models to provide biological interpretation of relevant gene sets using DNA methylation data in the context of The Cancer Genome Atlas.ResultsWe illustrate that Biological interpretation of DNA Methylation (BioMethyl) utilizes the complete DNA methylation data for a given cancer type to reflect corresponding gene expression profiles and performs pathway enrichment analyses, providing unique biological insight. Using breast cancer as an example, BioMethyl shows high consistency in the identification of enriched biological pathways from DNA methylation data compared to the results calculated from RNA sequencing data. We find that 12 out of 14 pathways identified by BioMethyl are shared with those by using RNA-seq data, with a Jaccard score 0.8 for estrogen receptor (ER) positive samples. For ER negative samples, three pathways are shared in the two enrichments with a slight lower similarity (Jaccard score = 0.6). Using BioMethyl, we can successfully identify those hidden biological pathways in DNA methylation data when gene expression profile is lacking.Availability and implementationBioMethyl R package is freely available in the GitHub repository (https://github.com/yuewangpanda/BioMethyl).Supplementary informationSupplementary data are available at Bioinformatics online.
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Andreopoulos, Bill, and Dimitris Anastassiou. "Integrated Analysis Reveals hsa-miR-142 as a Representative of a Lymphocyte-Specific Gene Expression and Methylation Signature." Cancer Informatics 11 (January 2012): CIN.S9037. http://dx.doi.org/10.4137/cin.s9037.
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Gene expression profiling has provided insights into different cancer types and revealed tissue-specific expression signatures. Alterations in microRNA expression contribute to the pathogenesis of many types of human diseases. Few studies have integrated all levels of gene expression, miRNA and methylation to uncover correlations between these data types. We performed an integrated profiling to discover instances of miRNAs associated with a gene expression and DNA methylation signature across multiple cancer types. Using data from The Cancer Genome Atlas (TCGA), we revealed a concordant gene expression and methylation signature associated with the microRNA hsa-miR-142 across the same samples. In all cancer types examined, we found a signature of co-expression of a gene set R and methylated sites M, which correlate positively (M+) or negatively (M–) with the expression of hsa-miR-142. The set R consistently contains many genes, such as TRAF3IP3, NCKAP1L, CD53, LAPTM5, PTPRC, EVI2B, DOCK2, LCP2, CYBB and FYB. The signature is preserved across glioblastoma, ovarian, breast, colon, kidney, lung, uterine and rectum cancer. There is 28% overlap of methylation sites in M between glioblastoma (GBM) and ovarian cancer. There is 60% overlap of genes in R between GBM and ovarian ( P = 1.3e−-11). Most of the genes in R are known to be expressed in lymphocytes and haematopoietic stem cells, while M reflects membrane proteins involved in cell-cell adhesion functions. We speculate that the hsa-miR-142 associated signature may signal haematopoietic-specific processes and an accumulation of methylation events triggering a progressive loss of cell-cell adhesion. We also observed that GBM samples belonging to the proneural subtype tend to have underexpressed hsa-miR-142 and R genes, hypomethylated M+ and hypermethylated M–, while the mesenchymal samples have the opposite profile.
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Spadotto, Valeria, Roberto Giambruno, Enrico Massignani, et al. "PRMT1-mediated methylation of the microprocessor-associated proteins regulates microRNA biogenesis." Nucleic Acids Research 48, no. 1 (2019): 96–115. http://dx.doi.org/10.1093/nar/gkz1051.
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Abstract MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17–92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.
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Zhong, Xingming, Fenpin Jin, Chuican Huang, Mengxuan Du, Mengge Gao, and Xiangcai Wei. "DNA methylation of AMHRII and INSR gene is associated with the pathogenesis of Polycystic Ovary Syndrome (PCOS)." Technology and Health Care 29 (March 25, 2021): 11–25. http://dx.doi.org/10.3233/thc-218002.
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BACKGROUND: Polycystic ovary syndrome (PCOS) is a common gynecologic endocrinopathy, characterized by menstrual disorders, ovulation disorders, polycystic ovary, hyperandrogen syndrome and insulin resistance. At present, the etiology and exact pathogenesis of PCOS are still unclear. Anti-Müllerian hormone is a local regulator secreted by ovarian granulosa cells, and participates in regulating the occurrence and development of PCOS. Insulin resistance is another important pathophysiological feature of PCOS. Although the expression of anti-müllerian hormone receptor (AMHR) and insulin receptor (INSR) in PCOS have been previously reported, the DNA methylation of the genes have not been well characterized. OBJECTIVE: To study AMHR II/INSR and its role in gene methylation in Ovarian and endometrial pathology of PCOS subjects. METHODS: We recruited seventy-five women with PCOS as cases and twenty healthy women as controls, using immunohistochemical method, study localization, distribution and expression of MHRII/INSR in ovary and endometrium and then discover the correlation of AMHRII/INSR gene methylation. RESULTS: Different clinical features in PCOS group AMHRII gene methylation level and insulin resistance relations have significant differences (r= 0.532, P= 0.000); INSR gene methylation level and insulin resistance relations have significant differences (r= 0.281, P= 0.03). CONCLUSIONS: The analysis of DNA methylation suggested that methylation of AMHRII and INSR genes was associated with basic clinical characteristics and insulin resistance of PCOS. These results provide evidence for AMHRII and INSR genes, and their methylation levels are intimately associated with the pathogenesis of PCOS.
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Iwagami, Shiro, Yoshifumi Baba, Masayuki Watanabe, et al. "The association between smoking and LINE-1 hypomethylation (global DNA hypomethylation) in normal esophageal epithelium of patients with esophageal squamous cell carcinoma." Journal of Clinical Oncology 30, no. 4_suppl (2012): 41. http://dx.doi.org/10.1200/jco.2012.30.4_suppl.41.
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41 Background: DNA methylation is a major epigenetic mechanism in X-chromosome inactivation, imprinting and repression of transposable elements and endogenous retroviral sequences. Global DNA hypomethylation appears to play an important role in genomic instability, leading to cancer development. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. Smoking and alcohol is extremely important as the etiology of esophageal squamous cell carcinoma. Nonetheless, whether or not smoking and alcohol affect LINE-1 methylation level in normal esophageal epithelium of esophageal cancer patients remains uncertain. Methods: We quantified LINE-1 methylation of normal esophageal mucosa using pyrosequencing technology in 118 resected esophageal squamous cell carcinomas. The data on smoking (Brinkman index, absence or presence) and alcohol amount are available in all cases. We excluded preoperatively treated cases. Results: LINE-1 methylation in normal esophageal epithelium of esophageal cancer patients ranged from 57.1 to 92.8 of 0-100 scale (N=118; mean 81.2; median 80.0; standard deviation 7.2). LINE-1 methylation level (continuous variable) was significantly associated with Brickman index (continuous variable) (r=0.12, p=0.0002); heavy smoker had lower LINE-1 methylation level of normal esophageal mucosa. LINE-1 methylation level was lower in patients with smoking history (mean 79.7) than in patients without smoking history (mean 83.2) (p=0.034). Alcohol assumption was not associated with LINE-1 methylation level (r=0.003, p=0.58). Conclusions: Smoking was associated with LINE-1 hypomethylation in esophageal normal epithelium, suggesting the possibility of epigenetic field effects caused by cigarette in esophageal tumorigenesis. Considering that DNA methylation alterations are reversible and can thus be targets for chemoprevention, our findings may have clinical implication.
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Kostadinov, Rumen, Robert Scharpf, Sarven Sabunciyan, Daniel Magoon, Rafael Irizarry, and Patrick Brown. "Identifying Methylation Changes Driving Evolution of Relapse in MLL-Rearranged Acute Lymphoblastic Leukemias." Blood 124, no. 21 (2014): 3797. http://dx.doi.org/10.1182/blood.v124.21.3797.3797.
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Abstract Introduction The MLL gene is rearranged (MLL-r) in 80% of infants with B-lymphoblastic leukemia (B-ALL). MLL-r infant B-ALL has a poor prognosis, with 4-year event-free survival less than 45%. The typical pattern of failure in MLL-r infant ALL is successful remission induction followed by early relapse, suggesting rapid emergence and/or selection of one or more chemoresistant subclones. The markedly lower observed remission induction rates at relapse (<40%) vs. diagnosis (>90%) are consistent with this hypothesis. Genomic studies of MLL-r B-ALL have revealed a striking paucity of cooperating genomic abnormalities compared to other subsets of B-ALL, which suggests heritable epigenetic changes may drive leukemogenesis, chemoresistance and evolution of relapse in MLL-r B-ALL. MLL, its fusion partners and various components of its large complexes have functional domains with known or suspected epigenetic activity. Thus, MLL rearrangement in B-ALL may trigger chromatin modifications and DNA CpG methylation changes that interplay to disrupt normal gene transcription and expression. We hypothesized that infant MLL-r B-ALL cells dynamically acquire heritable DNA CpG methylation changes, some of which may contribute to chemoresistance and evolution of relapse. To test this hypothesis we performed whole-genome bisulfite sequencing (WGBS) using paired diagnosis-relapse (DX-RL) samples to identify methylation changes that may drive relapse. Methods We evaluated paired DX-RL specimens with >95% leukemic blasts from two infants with B-ALL harboring MLL-ENL fusions: A 4 month-old female who relapsed after 1 year (case a), and an 8 month-old male who relapsed after 5 months (case b). We prepared WGBS libraries and ran paired-end sequencing (2x100bp) using Illumina HiSeq at average 25X coverage. We used Bismark for aligning reads and calling CpG methylation states. We developed an analysis pipeline to convert CpG states into CpG haplotypes, detect methylation changes at the haplotype level between DX and RL, annotate methylation changes to genes and promoters, and perform gene set enrichment analysis (GSEA) on these genes. Results First, we filtered reads falling into repeated DNA. We then obtained methylation calls at 15.6/14.3 (DX/RL) million CpGs in case (a) and 15.5/15.6 (DX/RL) million CpGs in case (b). We extracted uniquely aligned reads covering 4 contiguous CpGs, which defined a “4 CpG-site”, or “site”. We required that each site in both DX and RL is covered by at least 4 reads, which yielded 26,747 and 85,174 sites for (a) and (b). We compared the methylation level at each site in DX versus RL (e.g. a “1-0-1-1” 4 CpG-site has a methylation level of 0.75). For (a), 165 sites showed a 50% increase in methylation level at relapse (e.g., from 0.2 to 0.7), and 468 showed a 50% decrease in methylation level at relapse. For (b) there were 605 increased and 57 decreased. The 633 methylation changes in (a) mapped to 175 genes (2kb upstream to transcription end site) and the 662 changes in (b) mapped to 135 genes. Some of these genes are known to be involved in MLL-r ALL [e.g., IKZF1, in (a) and TLX2 in (b)]. GSEA was performed independently using the 175 and 135 genes in (a) and (b), respectively. Interestingly, there was substantial overlap: The top 3 gene sets for (b) (FDR q-value ≤ 2.9E-8) were also within the top 9 sets for (a) (FDR ≤ 1.1E-5). Strikingly, all 3 of these sets involved components of the polycomb repressive complex 2 (PRC2) (1. EED target genes, 2. H3K27me3 bound genes and 3. Genes increased after EZH2 knockdown). Also in common was the KEGG gene set for antigen processing and presentation [FDR 3.4E-6 in (a) and 1.7E-4 in (b)]. Conclusion WGBS allows an in-depth look at the epigenetic state of leukemic cell populations. While >98% of the methylome remained relatively stable between diagnosis and relapse (< 50% change), the hundreds of differentially methylated sites may be sufficient to influence expression of key genes and drive selection during the evolution of relapse. In addition, the ~15 million CpG methylation states are stably inherited yet variable, and comprise a rich source of information that can be used to identify evolving subclones, particularily in MLL-r ALL given its silent genomic landscape. Validation of the functional significance of the methylation changes using RNA-seq and identification of functionally important epigenetically-defined subclones is underway. Disclosures No relevant conflicts of interest to declare.
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Ross, Jason P., Isao Suetake, Shoji Tajima, and Peter L. Molloy. "Recombinant mammalian DNA methyltransferase activity on model transcriptional gene silencing short RNA–DNA heteroduplex substrates." Biochemical Journal 432, no. 2 (2010): 323–32. http://dx.doi.org/10.1042/bj20100579.
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The biochemical mechanism of short RNA-induced TGS (transcriptional gene silencing) in mammals is unknown. Two competing models exist; one suggesting that the short RNA interacts with a nascent transcribed RNA strand (RNA–RNA model) and the other implying that short RNA forms a heteroduplex with DNA from the unwound double helix, an R-loop structure (RNA–DNA model). Likewise, the requirement for DNA methylation to enact TGS is still controversial. In vitro assays using purified recombinant murine Dnmt (DNA methyltransferase) 1-dN (where dN indicates an N-terminal truncation), 3a and 3b enzymes and annealed oligonucleotides were designed to question whether Dnmts methylate DNA in a RNA–DNA heteroduplex context and whether a RNA–DNA heteroduplex R-loop is a good substrate for Dnmts. Specifically, model synthetic oligonucleotides were used to examine methylation of single-stranded oligonucleotides, annealed oligonucleotide duplexes, RNA–DNA heteroduplexes, DNA bubbles and R-loops. Dnmt methylation activity on the model substrates was quantified with initial velocity assays, novel ARORA (annealed RNA and DNA oligonucleotide-based methylation-sensitive restriction enzyme analysis), tBS (tagged-bisulfite sequencing) and the quantitative PCR-based method MethylQuant. We found that RNA–DNA heteroduplexes and R-loops are poor substrates for methylation by both the maintenance (Dnmt1) and de novo (Dnmt3a and Dnmt3b) Dnmts. These results suggest the proposed RNA/DNA model of TGS in mammals is unlikely. Analysis of tagged-bisulfite genomic sequencing led to the unexpected observation that Dnmt1-dN can methylate cytosines in a non-CpG context in DNA bubbles. This may have relevance in DNA replication and silencing of transcriptionally active loci in vivo.
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Zhu, Yinghui, Xin He, Haojie Dong, et al. "Inhibition of PRMT1 Mediated FLT3 Arginine Methylation As a Potent Therapeutic Strategy for MLL-r ALL." Blood 132, Supplement 1 (2018): 892. http://dx.doi.org/10.1182/blood-2018-99-115139.
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Abstract Mixed-lineage leukemia-rearranged (MLL-r) ALL, seen in 70% of infant ALL, has a dismal prognosis compared to those with wild type MLL1 gene. Transcriptional profiling has identified Fms-like receptor tyrosine kinase 3 (FLT3) as one of the most significantly upregulated genes in MLL-r ALL. The highly expressed FLT3 protein is activated by the autocrine ligand, making the kinase a therapeutic target. FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412, although effective in kinase inhibition, partially impair survival of MLL-r ALL cells and clinical trial results are not promising, promoting us to ask whether FLT3 regulates the ALL cells survival also through a kinase-independent mechanism. Herein, we report the finding of dimethylated arginines on FLT3, detected through mass spectrometry analysis of a MLL-r ALL specimen and a MLL-r ALL line SEM. The most conserved and enriched of dimethylated arginines are residues R972/R973. Using home-made arginine methylation (R-Me) antibody, we found that PRMT1, which is responsible for most type I arginine methyltransferases activity, catalyzes FLT3 methylation. Immunoblot (IB) analysis validated the expression of FLT3 R-Me in MLL-r ALL samples (6 out of 6) and MLL-r ALL lines (4 out of 4). Analysis of the GEO dataset (GSE13204) revealed that PRMT1 mRNA levels are increased in MLL-r ALL relative to normal cells (MLL-r, n=70 vs. normal, n=73, p<0.0001). We studied FLT3 R-Me biological function using two approaches that specifically blocked FLT3 methylation levels: cells expressing FLT3 methylation deficient construct (R972/973K, arginine [R] to lysine [K]) exhibited reduced survival (BaF3: FLT3-WT 98.5±0.11% vs. R972/973K 71.5±0.53%, p=0.0004); knockdown of PRMT1 in SEM cells also had an inhibitory effect (siCtrl 95.1±0.1% vs. siPRMT1 74.7±0.5%, p=0.0007). Moreover, the type I arginine methyltransferase inhibitor MS023 (5 µM) treatment markedly induced apoptosis of primary ALL cells but spared normal counterparts from healthy donors (ALL: vehicle 10.4±0.4% vs. MS023 23.7±0.8%, n=4; p<0.0001; normal CD19+: 8.3±0.3% vs. 8.2±0.1%, n=3, p=0.86). Interestingly, inhibition of FLT3 methylation decreased FLT3 phosphorylation at tyrosine 969 (Y969) but not Y589/591 or Y842. Expression of R972/973K decreased FLT3 downstream signaling like phospho-STAT5 and -AKT to a greater extent than that of Y969F mutant (Y to phenylalanine [F] substitution, mimics loss of Y phosphorylation). Next, FLT3 WT, R972/973K or Y969F transduced primary MLL-r ALL cells were transplanted into NSGS mice for analysis of leukemia development (n=6/group). Mice transplanted with FLT3 Y969F MLL-r ALL had longer survival relative to FLT-WT injected animals (p=0.0031), and the median survival was further extended in mice injected with R972/973K mutant compared with FLT3 Y969F MLL-r ALL (p=0.0007). Additionally, PKC412 treatment alone did not alter FLT3 R-Me, and high FLT3 methylation level in SEM cells was not affected by FLT3 ligand stimulation, confirming that the function of R-Me is independent of FLT3 phosphorylation. Importantly, we observed that the combination of MS023 with PKC412 significantly induced a higher rate of apoptosis in primary MLL-r ALL cells compared with each drug alone (control, 10±0.43%, MS023, 21.1±1.2%, PKC412, 21.5±0.11%, combination, 39.8±2.9%, PKC412 vs combination, p<0.01, n=4). We further tested the effects of in vivo administration of MS023 plus PKC412 on primary MLL-r ALL cells xenografted in NSGS mice. Following engraftment >1% in peripheral blood, mice were subdivided into four groups and treated with vehicle, PKC412 (100 mg/kg, i.g.), MS023 (80 mg/kg, i.p, bid), or the combination (n=7/group) for 4 weeks. The BM tumor burden of CD45+ CD19+ cells was reduced in single drug-treated mice cohorts, with further reduction after combination treatment (vehicle, 94.4±0.5%, PKC412, 50.2±6.3%, MS023, 55.6±4.5%, combination, 30.7±4.9%, PKC412 vs. combination, p<0.001). Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced BM engraftment at 16 weeks compared to PKC412 treatment alone (PKC412, 62.2±4.9%, combination, 8.4±5.1%, n=5, p<0.0001), indicating reduced leukemia initiating capacity. Our results support further exploring the molecular function of FLT3 R-Me. We will determine whether PRMT1 and FLT3 methylation are potential druggable targets in MLL-r ALL. Disclosures Konopleva: Stemline Therapeutics: Research Funding.
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Cedoz, Pierre-Louis, Marcos Prunello, Kevin Brennan, and Olivier Gevaert. "MethylMix 2.0: an R package for identifying DNA methylation genes." Bioinformatics 34, no. 17 (2018): 3044–46. http://dx.doi.org/10.1093/bioinformatics/bty156.
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Gómez-González, Belén, and Andrés Aguilera. "Looping the (R) Loop in DSB Repair via RNA Methylation." Molecular Cell 79, no. 3 (2020): 361–62. http://dx.doi.org/10.1016/j.molcel.2020.07.015.
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Salcini, Anna Elisabetta. "Dangerous R loops form in the absence of H3K9 methylation." Nature Genetics 48, no. 11 (2016): 1299–300. http://dx.doi.org/10.1038/ng.3705.
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Gevaert, O. "MethylMix: an R package for identifying DNA methylation-driven genes." Bioinformatics 31, no. 11 (2015): 1839–41. http://dx.doi.org/10.1093/bioinformatics/btv020.
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Abramycheva, Nataliya Yu, Ekaterina Yu Fedotova, Evgenii P. Nuzhnyi, et al. "Epigenetics of Friedreich’s Disease: Methylation of the (GAA)n-Repeats Region in FXN Gene." Annals of the Russian academy of medical sciences 74, no. 2 (2019): 80–87. http://dx.doi.org/10.15690/vramn1099.
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Background: Friedreich’s disease (FD) is the most common hereditary ataxia. It is associated, most frequently, with homozygous GAA repeats expansion in intron 1 of the FXN gene. Methylation of the FXN gene can play an important role in the pathogenesis of FD. Aims: to study methylation pattern in CpG sites flanking GAA-expansion in intron 1 of the FXN gene in patients with FD and their heterozygous relatives as well as its relationship with clinical features. Materials and methods: We studied DNA samples from patients with FD (n=18), their relatives carrying heterozygous GAA expansion (n=12), and control group (n=15). Pattern of methylation was studied by direct sequencing of DNA regions after bisulphide processing. Results: We analyzed 18 CpG sites in the UP-GAA region of the gene (before GAA-repeats) and 12 CpG sites in the DOWN-GAA region (after GAA-repeats). In the UP-region, the mean methylation level of CpG sites in FD patients was higher compared to controls (n=15) (р0.05), while in the DOWN-region there was a decrease of mean methylation level in FD compared to controls (р0.05). Analysis of methylation level in different CpG sites in the UP-GAA region revealed hypermethylation for 15 of 18 CpG-sites as compared to controls (р0.05). The most significant differences in methylation level in the UP-GAA region were seen for CpG sites 50−54, 57 and 58. In contrast, in the DOWN-GAA region almost all CpG sites were fully methylated in the control group, while in FD patients methylation was significantly lower (р0.05). We revealed positive correlation of mean methylation level and more expanded allele length for the UP-GAA region in FD (r=0.63; p=0.03), and no correlations for the DOWN-GAA region. In heterozygous carriers we observed an analogous positive correlations in the UP-GAA region for CpG site 50 (r=0.77; p=0.04), while in the DOWN-GAA region there was inverse correlation of methylation with GAA repeat number in the expanded allele (r=-0.83, p=0.02). Negative correlation was found between the hypermethylation of some CpG-sites in the UP-GAA region and age of the disease onset (p0.05). Conclusion: We revealed hypermethylation in the UP-GAA region and hypomethylation in the DOWN-GAA region in patients with FD compared to controls and correlations of methylation level with the GAA expansion length and age of disease onset.
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Kendall, Ellen K., Manishkumar S. Patel, Sarah Ondrejka, et al. "Integrative DNA Methylation and Gene Expression Analysis Reveals Candidate Biomarkers Associated with Dichotomized Response to Chemoimmunotherapy in Diffuse Large B-Cell Lymphoma." Blood 136, Supplement 1 (2020): 22. http://dx.doi.org/10.1182/blood-2020-137180.
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Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. While 60% of DLBCL patients achieve complete remission with frontline therapy, relapsed/refractory (R/R) DLBCL patients have a poor prognosis with median overall survival below one year, necessitating investigation into the biological principles that distinguish cured from R/R DLBCL. Recent analyses have identified unfavorable molecular signatures when accounting for gene expression, copy number alterations and mutational profiles in R/R DLBCL. However, an integrative analysis of the relationship between epigenetic and transcriptomic changes has yet to be described. In this study, we compared baseline methylation and gene expression profiles of DLBCL patients with dichotomized clinical outcomes. Methods: Diagnostic DLBCL biopsies were obtained from two patient cohorts: patients who relapsed or were refractory following chemoimmunotherapy ("R/R"), and patients who entered durable clinical remission following therapy ("cured"). The median age for R/R and cured cohorts were 62 (range 35-86) years vs. 64 (range 28-83) years (P= 0.27). High-intermediate or high IPI scores were present in 14 vs. 6 patients (P= 0.08) in the R/R and cured cohorts, respectively. All patients were treated with frontline R-CHOP or R-EPOCH. DNA and RNA were extracted simultaneously from formalin-fixed, paraffin embedded biopsy samples. An Illumina 850k Methylation Array was used to identify DNA methylation levels in 29 R/R patients and 20 cured patients. RNA sequencing was performed on 9 R/R patients and 7 cured patients at diagnosis using Illumina HiSeq4000. Differentially methylated probes were identified using the DMRcate package, and differentially expressed genes were identified using the DESeq2 package. Gene set enrichment analysis was performed using canonical pathway gene sets from MSigDB. Results: At the time of diagnosis, we found significant epigenetic and transcriptomic differences between cured and R/R patients. Comparing cured to R/R samples, there were 8,159 differentially methylated probes (FDR&lt;0.05). Differentially methylated regions between R/R and cured cohorts overlap with genes previously identified as mutation hotspots in DLBCL. Upon comparing transcriptomic profiles between R/R and cured, 267 genes were found to be differentially expressed (Log2FC&gt;|1| and FDR&lt;0.05). Gene set enrichment analysis revealed gene sets related to cell cycle, membrane trafficking, Rho and Rab family GTPase function, and transcriptional regulation were upregulated in the R/R samples. Gene sets related to innate immune signaling, Type I and II interferon signaling, fatty acid and carbohydrate metabolism were upregulated in the cured samples. To identify genes likely to be regulated by specific changes in methylation, we selected genes that were both differentially expressed and differentially methylated between the R/R and cured cohorts. In the R/R samples, 13 genes (ARMC5, ARRDC1, C12orf57, CCSER1, D2HGDH, DUOX2, FAM189B, FKBP2, KLF5, MFSD10, NEK8, NT5C, and WDR18) were significantly hypermethylated and underexpressed when compared to cured specimens, suggesting that epigenetic silencing of these genes is associated with lack of response to chemoimmunotherapy. In contrast, 12 genes (ATP2B1, C15orf41, FAM102B, FAM3C, FHOD3, FYTTD1, GPR180, KIAA1841, LRMP, MEF2A, RRAS2, and TPD52) were significantly hypermethylated and underexpressed in cured patients, suggesting that epigenetic silencing of these genes is favorable for treatment response. Many of these epigenetically modified genes have been previously implicated in cancer biology, including roles in NOTCH signaling, chromosomal instability, and biomarkers of prognosis. Conclusions: This is the first integrative epigenetic and transcriptomic analysis of diagnostic biopsies from cured and R/R DLBCL patients following chemoimmunotherapy. At the time of diagnosis, both the methylation and gene expression profiles significantly differ between patients that enter durable remission as opposed to those who are R/R to therapy. Soon, the hypomethylating agent CC-486 (i.e. oral azacitidine) will be explored in combination with mini-R-CHOP for older DLBCL patients in whom DNA methylation is likely increased. These data support the use of hypomethylating agents to potentially restore sensitivity of DLBCL to chemoimmunotherapy. Disclosures Hsi: Eli Lilly: Research Funding; Abbvie: Research Funding; Miltenyi: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; CytomX: Consultancy, Honoraria. Hill:Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Takeda: Research Funding; Beigene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding.
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Wang, Yanli, Sijun Diao, Maoqing Hu, and Lin Zhang. "Methylation of Hypothalamic Tsc1-mTOR Signaling in Regulation of Obesity and Obesity Resistance." BioMed Research International 2020 (December 30, 2020): 1–6. http://dx.doi.org/10.1155/2020/8723869.
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The Tsc1-mTOR signaling pathway is often related to obesity, and epigenetic modification may lead to expression changes of obesity-related gene. Therefore, we aim to investigate the methylation of the Tsc1-mTOR signaling pathway in regulation of obesity susceptibility. Wistar rats were fed a normal diet or a high-fat diet to develop animal models. Protein and mRNA expression levels of Tsc1-mTOR signaling in the hypothalamus were determined by Western blot and quantitative real-time PCR. Methylation of Tsc1 gene promoter was detected by bisulfite genomic sequence. Both mRNA and protein expression levels of Tsc1 in DIO group hypothalamus were lower; mTOR and its downstream targets S6K1, 4EBP1, and S6 protein expression levels were higher than those of the DIO-R group and the chow group. The Tsc1 gene promoter methylation rate in the hypothalamus was 92.05 ± 3.07 % in the DIO group, 87.27 ± 1.91 % in the DIO-R group, and 88.18 % ± 3.20 % in the chow group, respectively, with significantly higher levels in the DIO group. Both the expression levels of Tsc1 gene promoter methylation and Tsc1-mTOR signaling pathway in the hypothalamus of DIO rats and DIO-R rats are different. These findings may shed light on the potential mechanism for the differentiation of obesity susceptibility.
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Aczel, Dora, Ferenc Torma, Matyas Jokai, et al. "The Circulating Level of Klotho Is Not Dependent upon Physical Fitness and Age-Associated Methylation Increases at the Promoter Region of the Klotho Gene." Genes 14, no. 2 (2023): 525. http://dx.doi.org/10.3390/genes14020525.
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(1) Background: Higher levels of physical fitness are believed to increase the physiological quality of life and impact the aging process with a wide range of adaptive mechanisms, including the regulation of the expression of the age-associated klotho (KL) gene and protein levels. (2) Methods: Here, we tested the relationship between the DNA methylation-based epigenetic biomarkers PhenoAge and GrimAge and methylation of the promoter region of the KL gene, the circulating level of KL, and the stage of physical fitness and grip force in two groups of volunteer subjects, trained (TRND) and sedentary (SED), aged between 37 and 85 years old. (3) Results: The circulating KL level is negatively associated with chronological age in the TRND group (r = −0.19; p = 0.0295) but not in the SED group (r = −0.065; p = 0.5925). The age-associated decrease in circulating KL is partly due to the increased methylation of the KL gene. In addition, higher plasma KL is significantly related to epigenetic age-deceleration in the TRND group, assessed by the biomarker of PhenoAge (r = −0.21; p = 0.0192). (4) Conclusions: The level of physical fitness, on the other hand, does not relate to circulating KL levels, nor to the rate of the methylation of the promoter region of the KL gene, only in males.
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Bacolod, Manny D., and Francis Barany. "MGMT Epigenetics: The Influence of Gene Body Methylation and Other Insights Derived from Integrated Methylomic, Transcriptomic, and Chromatin Analyses in Various Cancer Types." Current Cancer Drug Targets 21, no. 4 (2021): 360–74. http://dx.doi.org/10.2174/1568009621666210203111620.
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Background: MGMT (O6-methylguanine-DNA methyltransferase) is primarily responsible for limiting the activity of some widely used chemotherapeutic agents, including temozolomide (TMZ) and carmustine (BCNU). The gene encoding this protein is epigenetically regulated, and assessment of methylation at its promoter region is used to predict glioma patients’ response to TMZ. Methods: In this report, we employed a bioinformatic approach to elucidate MGMT’s epigenetic regulation. Integrated for the analysis were genome-wide methylation and transcription datasets for > 8,600 human tissue (representing 31 distinct cancer types) and 500 human cancer cell line samples. Also crucial to the interpretation of results were publicly available data from the ENCODE Project: tracks for histone modifications (via ChIP-seq) and DNase I hypersensitivity (via DNaseseq), as well as methylation and transcription data for representative cell lines (HeLa-S3, HMEC, K562). Results and Discussion: We were able to validate (perhaps more comprehensively) the contrasting influences of CpG methylation at promoter region and at gene body on MGMT transcription. While the MGMT promoter is populated by CpG sites whose methylation levels displayed high negative correlation (R) with MGMT mRNA counts, the gene body harbors CpG sites exhibiting high positive R values. The promoter CpG sites with very high negative R’s across cancer types include cg12981137, cg12434587, and cg00618725. Among the notable gene body CpG sites (high positive R’s across cancer types) are cg00198994 (Intron 1), cg04473030 (Intron 2), and cg07367735 (Intron 4). For certain cancer types, such as melanoma, gene body methylation appears to be a better predictor of MGMT transcription (compared to promoter methylation). In general, the CpG methylation v. MGMT expression R values are higher in cell lines relative to tissues. Also, these correlations are noticeably more prominent in certain cancer types such as colorectal, adrenocortical, esophageal, skin, and head and neck cancers, as well as glioblastoma. As expected, hypomethylation at the promoter region is associated with more open chromatin, and enrichment of histone marks H3K4m1, H3K4m2, H3K4m3, and H3K9ac. Conclusion: Overall, our analysis illustrated the contrasting influence of promoter and gene body methylation on MGMT expression. These observations may help improve diagnostic assays for MGMT.
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Lin, Hsiang-Yu, Chung-Lin Lee, Sisca Fran, et al. "Quantitative DNA Methylation Analysis and Epigenotype-Phenotype Correlations in Taiwanese Patients with Beckwith-Wiedemann Syndrome." Journal of Personalized Medicine 11, no. 11 (2021): 1066. http://dx.doi.org/10.3390/jpm11111066.
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Background: Beckwith-Wiedemann syndrome (BWS; OMIM 130650) is a rare overgrowth syndrome with tumor predisposition resulting from the abnormal expression or function of imprinted genes of the chromosome 11p15.5 imprinting gene cluster. The aim of this study was to identify the epigenotype-phenotype correlations of these patients using quantitative DNA methylation analysis. Methods: One hundred and four subjects with clinically suspected BWS were enrolled in this study. All of the subjects had been referred for diagnostic testing which was conducted using methylation profiling of H19-associated imprinting center (IC) 1 and KCNQ1OT1-associated IC2 in high-resolution melting analysis and methylation quantification with the MassARRAY assay. Correlations between the quantitative DNA methylation status and clinical manifestations of the enrolled subjects were analyzed. Results: Among the 104 subjects, 19 had IC2 hypomethylation, 2 had IC1 hypermethylation, and 10 had paternal uniparental disomy (pUPD). The subjects with IC2 hypomethylation were characterized by significantly more macroglossia but less hemihypertrophy compared to the subjects with pUPD (p < 0.05). For 19 subjects with IC2 hypomethylation, the IC2 methylation level was significantly different (p < 0.05) between the subjects with and without features including macroglossia (IC2 methylation level: 11.1% vs. 30.0%) and prenatal or postnatal overgrowth (8.5% vs. 16.9%). The IC2 methylation level was negatively correlated with birth weight z score (p < 0.01, n = 19) and birth height z score (p < 0.05, n = 13). For 36 subjects with clinically diagnosed BWS, the IC2 methylation level was negatively correlated with the BWS score (r = −0.592, p < 0.01). The IC1 methylation level showed the tendency of positive correlation with the BWS score without statistical significance (r = 0.137, p > 0.05). Conclusions: Lower IC2 methylation and higher IC1 methylation levels were associated with greater disease severity in the subjects with clinically diagnosed BWS. Quantitative DNA methylation analysis using the MassARRAY assay could improve the detection of epigenotype-phenotype correlations, which could further promote better genetic counseling and medical care for these patients.