Epigenetic Analyses / Genotyping

Changes in genomic DNA methylation patterns are one of the earliest and most consistent features of tumourigenesis. It has been demonstrated that aberrant DNA methylation profiles can be used as a valuable marker for clinical tumour characterisation. However, very little is known about the nature of these changes and their role in cellular transformation. Progress in the field has been hampered by the unavailability of methods for genomic epigenetic profiling. We have established a method for the detection of methylation variations on oligonucleotide microarrays, using bisulfite treatment to uncover the methylation status. Sodium bisulfite induces methylation-dependent single nucleotide polymorphisms by converting unmethylated cytosine to uracil and, upon PCR amplification, to thymine. 5-methylcytosine is not affected by sodium bisulfite treatment and thus becomes amplified as cytosine.

Conversion of unmethylated cytosine to uracil by a treatment with bisulfite.


We utilise this technology toward a genome-wide, gene-specific analysis of DNA methylation. This approach will result in a detailed characterisation of genomic methylation patterns. The data will be evaluated together with the available clinical data and results from transcriptional profiling. Our data will allow fundamental insights into the role of DNA methylation during tumourigenesis and provide the foundation for an epigenetic classification of tumours.

As part of the National German Research Network (NGFN), we utilise microarray technology toward a genome-wide, gene-specific analysis of DNA methylation patterns. The platform is based on two projects funded earlier by the Deutsche Forschungsgemeinschaft and within the DHGP (see below).

The group assembled in this platform has established the means for (1) the sodium bisulfite modification of DNA samples for the generation of methylation-dependent polymorphisms, (2) the detection of the resulting single-base polymorphisms on complex oligonucleotide microarrays and (3) bioinformatics platforms for subsequent data analysis, with a special focus on combining the epigenetic information with transcript profiles and clinical information. Also, for the analyses performed within the SMP, clinical material with corresponding clinicopathological information is available.

Infrastructure will be provided that will be used in collaborations within the NGFN-2 network and beyond for a global analysis of epigenetic variations by means of microarray-based analyses. Different systems, which meet the respective requirements, will be provided. By comparison between them, standards will be defined that will allow comparability of data across platforms and across various areas of analysis. Also for this purpose, the existing close connection and interrelation of this partnership with international efforts is crucial. The platform combines groups with complementary but nevertheless sufficiently overlapping expertise, competence and capability to provide the proposed platform. Parallel to application, technical developments will be performed in order to improve quality and reliability.

The work plan can be divided into three parts: standardisation, evaluation, production. Initially, the array systems will be compared. Also, the preparative steps will be harmonised. Objective of this work portion is a standardisation of protocols, handling steps, analysis tools, algorithms and processes that allow the comparison of the data irrespective of their origin.

During the evaluation phase, the SMP will establish across the platform the entire process of high-throughput screening, transfer of the gained knowledge into appropriate chip formats and their subsequent use in actual analyses. In terms of biology, there will be a focus on chronic lymphocytic leukemia, melanoma, pancreatic and prostate cancer, since well-defined material for these tissues is available within the SMP.

After these two phases, the whole platform will be operational with defined standards, established protocols and communication processes within the SMP and to the partners in NGFN (and beyond). Depending on the requirements of the individual study, the platform will then offer a portfolio of systems, from which the combination can be select that will be best suited to achieve the respective objective.

Platform objectives:

• High-throughput screening based on pre-selected methylation sites.
• High-throughput screening based on comprehensive CpG-island array.
• Chip design and evaluation.
• Research-type medium-throughput analysis on complex arrays
• Routine analysis on microarrays of low complexity.
• High-throughput analyses on complex arrays.
• Methodical analysis of the methylation sites in chromosome 21.

   

The basis for the above mentioned NGFN platform was laid in collaborative projects with Frank Lyko (Division of Epigenetics, DKFZ) as well as the company Epigenomics in Berlin. Because of the bisulfite conversion, methylation patterns can be presented as chemically introduced single nucleotide polymorphisms (SNPs) and analysed accordingly using a number of different microarray-based approaches.

To date, we performed the analysis mainly by direct hybridisation to complementary oligonucleotides synthesised in situ on the Geniom system of febit. Due to the density of CpG sites in CpG islands, however, frequently there is more than one CpG contained in a single oligonucleotide. For an analysis, all possibke permutations need to be representedon the microarray. Nevertheless, accuracy is effected negatively because of this.

In a pilot study, the approach was used to study the CpG island in the promoter region of the tumour suppressor gene p16. In total, 876 oligonucleotide probes of 21 nucleotides in length were used to inspect the methylation status of 53 CpG dinucleotides, producing correct signals in colorectal cancer cell lines as well as control samples with a defined methylation status. The information was validated by established alternative methods. The overall methylation pattern was consistent for each cell line, while different between them. At the level of individual cytosines, however, significant variations between individual cells of the same type were found, but also consistencies across the panel of cancer cell lines. The probe sequences used for the analysis of p16 (Mund et al., Nucleic Acids Res. 33, e73) are available here.

Alternatively, an on-chip primer extension reaction can be performed. This is reducing the number of oligonucleotide significantly and concomitantly increases the accuracy of base calling, since the process combines binding to a complementary oligonucleotide with the specificity of the polymerase. We use the APEX system established in the group of Andres Metspaly (Tartu University, Estonia). The chip-bound primers hybridise to the genomic sequence directly adjacent to the base of interest. Upon an incubation with labelled dideoxynucleotides, the fitting nucleotide is incorporated by the polymerase and can be detected by means of the specific fluorophor.