Rezultaty
The results from this study demonstrate that a significant proportion of the analysed loci within the MHC show tissue-specific methylation profiles and inter-individual methylation differences are common. Tissue specific differences in DNA methylation suggest that epigenetic mechanisms are involved in the use of alternative transcriptional start sites. The generated methylation data allow the identification of MVPs that can be typed with high quantitative resolution and sensitivity using MALDI-MS, providing a tool for large population-based studies and diagnosing diseases in the future. The study reported here lays the foundation for the HEP, which aims to analyze the methylation state of the regulatory regions of all annotated genes in most major cell types and their diseased variants.
The main objective of this project was, as a pilot study towards the Human Epigenome Project, to establish a methylation analysis pipeline and to apply it to determine methylation patterns within the Major Histocompatibility Complex (MHC), a region essential to our immune system and associated with many common diseases. Data from the study add strong support that based on methylation it is possible to distinguish between cell types in general. About 250 loci (two per gene on average) encompassing most of the expressed genes of the MHC were analysed in 64 samples from 9 different tissues. In total, the methylation status of about 150.000 CpG sites has been determined revealing major differences in the methylation pattern between the analysed tissues. The generated data have been integrated with existing genome annotation and have been publicly released at http://www.epigenome.org.
A prototype for a robotic facility for high-throughput epigenotyping was developed and a device for sample preparation, bisulfite treatment and amplification has been developed (Epigenomics AG). MHC-specific amplification protocols for an integrated pipetting robot/amplification system enabled the generation of a sufficient amount of template material for the project (Epigenomics AG). The development of specific amplification protocols made the generation of amplicons for 255 gene fragments from over 100 genes in the MHC feasible. From 64 samples, these gene fragments were amplified and sequenced (Wellcome Trust Sanger Institute).
A subset of those fragments that turned out to be differentially methylated in the sequencing reactions were analysed by an independent high-throughput epigenotyping method, the GOOD assay which provides more accurate quantitation (Centre National de Génotypage). Using the completed human MHC genomic sequence as a reference, Epigenomics AG and the Wellcome Trust Sanger Institute initially selected 150 MHC genes that had a high CpG content and were therefore likely to contain methylation variable positions (MVPs). At least two PCR fragments had been generated per gene, from the promoter and early exon regions. Genomic DNA was subjected to bisulfite treatment and the PCR fragments have been amplified from this source by Epigenomics AG. The Wellcome Trust Sanger Institute sequenced these PCR fragments and the methylation levels were analysed with a software program (developed by Epigenomics AG).
In total, primers from 255 fragments have been used for sequencing DNA generated from 64 samples, comprising of 9 different tissues. Sequences that passed the strict quality criteria (e.g. 21,081 reads) were analysed with the ESME software (developed by Epigenomics AG) to calculate the % methylation level for all CpG sites. An assay based on multiplexed primer extension for allele-discrimination and quantitative matrix assisted laser desorption/ionisation time of flight (MALDI TOF) mass spectrometric detection was developed (Centre National de Génotypage) using bisulfite treated genomic DNA as starting material. All parameters concerning the performance of the molecular biology reactions and on the instrument were thoroughly investigated. The assay was implemented with an extensive degree of automation making it amenable to high-throughput processing. Quantitative analysis by MALDI mass spectrometry was performed using a statistical approach to compensate for systematic fluctuation. Amplificates with differential methylation between nine different tissues types identified by sequencing were translated into epigenotyping assays for high-resolution locus specific quantitation of methylation by MALDI mass spectrometry.
Assays were designed for 231 methylation variable positions in 77 amplificates and validated on bisulphite treated genomic DNA. Calibration with mixtures of known methylation degree were shown to be essential for accurate quantification as a bias towards either methylated or non-methylated allele was observed for some methylation variable positions and was included in the subsequent analysis. 355.000 data points are generated which results in 15.000 high-resolution calibrated epigenotypes for the 64 DNA samples of nine different tissue types. Special software to interpret the accumulated data was developed and integrated into the process. Based on the haplotype analysis, differences in disease expression, by comparing DNA from two different tissues, skin biopsies and lymphocytes have been investigated. Therefore skin biopsies had to be organized. The MHC linked disease, Psoriasis, was investigated at the CNG by HLA-haplotyping of 188 individuals and genotyping of 1017 individuals using microsatellites.
All results from the study were made publicly available. Links to the ENSEMBL database and further associated data are provided via the project's web site at http://www.epigenome.org/.
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