Martin Trbušek

5-Methylcytosine as a marker for the monitoring of DNA methylation.

The extent of the DNA methylation of genomic DNA as well as the methylation pattern of many gene-regulatory areas are important aspects with regard to the state of genetic information, especially their expression. There is growing evidence that aberrant methylation is associated with many serious pathological consequences. As genetic research advances, many different approaches have been employed to determine the overall level of DNA methylation in a genome or to reveal the methylation state of particular nucleotide residues, starting from semiquantitative methods up to new and powerful techniques. In this paper, the currently employed techniques are reviewed both from the point of view of their relevance in genomic research and of their analytical application. The methods discussed include approaches based on chromatographic separation (thin-layer chromatography, high-performance liquid chromatography, affinity chromatography), separation in an electric field (capillary electrophoresis, gel electrophoresis in combination with methylation-sensitive restriction enzymes and/or specific sequencing protocols), and some other methodological procedures (mass spectrometry, methyl accepting capacity assay and immunoassays).

Detection and Functional Analysis of TP53 Mutations in CLL

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

613 Clonal Selection of TP53 Mutations in Chronic Lymphocytic Leukaemia Detected by Ultra-deep Pyrosequencing

patients with stage II, microsatellite stable colon cancer with clinical data and of colon mucosa samples from 50 healthy donors, obtained during routine colonoscopy using the newly developed 450,000 CpG site platform for DNA methylation studies (Illumina Infinium HumanMethylation450BeadChip). This array includes CpG and CNG sites, CpG islands/shores/shelves/isolated CpGs in the genome, non-coding RNA (microRNAs and long non-coding RNAs) and sites surrounding the transcription start sites (−200 bp to −1,500 bp, 5′-UTRs and exons 1) for coding genes, but also for the corresponding gene bodies and 3′-UTRs. This study has been developed in the context of the COLONOMICS project (www.colonomics.org) in which for those normal-tumor paired samples and controls we also have data on molecular expression, SNPs/CNVs and miRNAs. The biomarkers identified must be validated. Results and Discussion: The preliminary analysis of the methylation patterns among different groups show differences in the methylation patterns of tumor and normal mucosa. A total of 106,566 CpG sites were differentially methylated between tumor and normal tissue (at 5% significance level after Bonferroni correction). The analyses of principal component (PCA) clearly discriminate between tumor and normal tissue samples. Some tumors show a predominant tumor hypermethylation pattern while others show hypomethylation and these patterns depict tumor subgroups that are likely to have different phenotype and outcomes. Epigenetic events may contain prognostic information. Using supervised analyses we have been able to identify several potential candidates for diagnostic and prognostic biomarkers to discriminate between tumor and non-tumor tissues. Conclusion: These differences in the methylation patterns shown to be promising in predicting the diagnostic and prognosis of CRC patients based on epigenetic characteristics of the tumors and normal mucosa.