Analysis of conditional gene deletion using probe based Real-Time PCR

Following publication of this article [1] the authors noticed that an incorrect probe reference was cited on page 3, 4, 5 and 6 ("UP #69, Roche Applied Science"). The correct probe that was used for the 1lox/2lox allele ratio analysis in the paper is as follow

Epigenetics and proteomics join transcriptomics in the quest for tuberculosis biomarkers

CITATION: Esterhuyse, M. M. et al. 2015. Epigenetics and proteomics join transcriptomics in the quest for tuberculosis biomarkers. mBio, 6(5):e01187-15, doi:10.1128/mBio.01187-15.The original publication is available at http://mbio.asm.orgAn estimated one-third of the world’s population is currently latently infected with Mycobacterium tuberculosis. Latent M. tuberculosis infection (LTBI) progresses into active tuberculosis (TB) disease in ~5 to 10% of infected individuals. Diagnostic and prognostic biomarkers to monitor disease progression are urgently needed to ensure better care for TB patients and to decrease the spread of TB. Biomarker development is primarily based on transcriptomics. Our understanding of biology combined with evolving technical advances in high-throughput techniques led us to investigate the possibility of additional platforms (epigenetics and proteomics) in the quest to (i) understand the biology of the TB host response and (ii) search for multiplatform biosignatures in TB. We engaged in a pilot study to interrogate the DNA methylome, transcriptome, and proteome in selected monocytes and granulocytes from TB patients and healthy LTBI participants. Our study provides first insights into the levels and sources of diversity in the epigenome and proteome among TB patients and LTBI controls, despite limitations due to small sample size. Functionally the differences between the infection phenotypes (LTBI versus active TB) observed in the different platforms were congruent, thereby suggesting regulation of function not only at the transcriptional level but also by DNA methylation and microRNA. Thus, our data argue for the development of a large-scale study of the DNA methylome, with particular attention to study design in accounting for variation based on gender, age, and cell type.http://mbio.asm.org/content/6/5/e01187-15.abstract?sid=fe0ea1c7-6da2-4e53-b4a4-5cd8233777c7Publisher's versio

withdrawn 2017 hrs ehra ecas aphrs solaece expert consensus statement on catheter and surgical ablation of atrial fibrillation

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Abstract 4350: Cell-of-origin differentiation stages define methylation-based subtypes of human colorectal cancer

Abstract Introduction: Colorectal cancer accounts for a significant fraction of cancer-related mortalities, but has proven to be surprisingly refractory to consensus pathological and molecular subclassification. Similarly, little is known about the molecular profiles of colorectal cancer cells-of-origin. DNA methylation is an important epigenomic marker of cellular identity and can therefore be used to infer cancer cells-of-origin. Methods: Infinium 450k data from colorectal cancer samples provided by “The Cancer Genome Atlas” was applied to a consensus clustering algorithm. The identified epigenetic subgroups were tested for features including well established clinical parameters or subgroup specific gene expression. Additionally, the subgroups were compared with Infinium 450k data of non-malignant colorectal adenomas. Finally, clinical significance was addressed by testing for subgroup specific overall survival rates. Results: Analysis of the TCGA dataset defined 5 distinct epigenetic subtypes of human colorectal cancer. These subgroups showed an overlap to the microsatellite instability phenotype and the CpG island methylator phenotype. Gene expression based analysis revealed that the subtypes also form a continuum of epigenetic programs reflecting various intestinal crypt cell differentiation stages. Patient survival correlated with the differentiation stage, with a particular poor prognosis for patients with stem cell-related signatures. Interestingly, its prognostic potential outperformed a recently established gene expression-based classifier. Finally, non-malignant colorectal adenomas could be classified into the same methylation subtypes, reflecting their shared cell-types of origin with carcinomas Conclusion: Our results establish a novel and clinically relevant approach for colorectal cancer classification and illustrate how differences in the cell-type of origin shape the tumor methylome. Citation Format: Felix Bormann, Manuel Rodríguez-Paredes, Yehudit Bergman, Heinz G. Linhart, Frank Lyko. Cell-of-origin differentiation stages define methylation-based subtypes of human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4350. doi:10.1158/1538-7445.AM2017-4350</jats:p

Genes methylated by DNA methyltransferase 3b are similar in mouse intestine and human colon cancer

Human cancer cells frequently have regions of their DNA hypermethylated, which results in transcriptional silencing of affected genes and promotion of tumor formation. However, it is still unknown whether cancer-associated aberrant DNA methylation is targeted to specific genomic regions, whether this methylation also occurs in noncancerous cells, and whether these epigenetic events are maintained in the absence of the initiating cause. Here we have addressed some of these issues by demonstrating that transgenic expression of DNA methyltransferase 3b (Dnmt3b) in the mouse colon initiates de novo DNA methylation of genes that are similar to genes that become methylated in human colon cancer. This is consistent with the notion that aberrant methylation in cancer may be attributable to targeting of specific sequences by Dnmt3b rather than to random methylation followed by clonal selection. We also showed that Dnmt3b-induced aberrant DNA methylation was maintained in regenerating tissue, even in the absence of continuous Dnmt3b expression. This supports the concept that transient stressors can cause permanent epigenetic changes in somatic stem cells and that these accumulate over the lifetime of an organism in analogy to DNA mutations

Folate Deficiency Induces Genomic Uracil Misincorporation and Hypomethylation But Does Not Increase DNA Point Mutations

BACKGROUND AND AIMS: Epidemiologic studies have linked nutritional folate deficiency to an increased risk of cancer, but recent trials suggest that folate supplementation does not protect against tumor formation. Our aim was to analyze the genetic and epigenetic consequences of folate deficiency and to investigate whether impairment of the uracil base excision repair pathway can enhance its effects. METHODS: Wild-type mice and those deficient in uracil DNA glycosylase (Ung(−/−)) were placed on a folate-deficient diet for 8 months. We measured tumor incidence in major organs, DNA mutation rates, DNA mutation spectra, local DNA methylation, and global DNA methylation in colon epithelial cells. RESULTS: The experimental diet increased plasma homocysteine (60%, P < .001) and DNA uracil content (24%, P < .05) but not tumor formation. Global DNA methyllation was slightly decreased in splenocytes (9.1%) and small intestinal epithelial cells (4.2%), and significantly reduced in colon epithelial cells (7.2%, P < .04). No gene-specific changes in methylation were detected at the mouse B1 element, the H19 DMR, or the Oct4 gene. By λ CII assay and sequencing analysis of 730 mutants, we found that Ung(−/−) mice had a higher frequency of point mutations and increased C:G to T:A transitions at non-CpG sites. However, folate deficiency had no additional effect on the DNA mutation frequency or spectrum in Ung(−/−) or wild-type mice. CONCLUSIONS: Contradicting current concepts, these findings indicate that the effects of a low-folate diet on DNA methylation and point mutations are insufficient to promote tumor development, even in the presence of Ung deficiency