Abstract LB-181: Genome-wide DNA methylation analysis reveals novel hypermethylated genes in astrocytic tumors
Authors: Shani A. Mulholland, Rifat A. Hamoudi, Deborah S. Malley, V. Peter Collins, Koichi Ichimura
Journal: Cancer Research (2011)

Abstract Introduction: Astrocytomas are the most common subtype of glioma, accounting for about 70% of all malignant tumors found in the central nervous system. Genetic alterations in gliomas have been extensively researched, but epigenetic changes are less well investigated. The aim of this study is to analyze genome-wide patterns of DNA methylation in a set of astrocytic tumors to identify aberrantly methylated genes, which could serve as candidates for new diagnostic markers and/or therapeutic targets. Material &amp; Methods: DNA methylation was investigated genome-wide by methyl-DNA immunoprecipitation (MeDIP) and hybridization to customized Agilent Human CpG island (250K) oligonucleotide arrays. In total, 18 glioblastoma cell lines, 59 astrocytic tumor samples (4 diffuse astrocytomas WHO grade II, 17 anaplastic astrocytomas grade III and 38 glioblastomas grade IV) and 5 normal whole brain samples were analyzed. Data quality control, normalization and analysis was performed through the arrayQuality and LIMMA packages of R. Statistical comparisons were made between tumors grouped according to various parameters including tumor vs normal, malignancy grades and known genotypes such as IDH mutations using an empirical Bayes t-test. Methylation levels were validated by pyrosequencing and compared with mRNA expression measured by real-time-qPCR (RT-QPCR). Results: A number of differentially methylated genes (Log2 fold change &amp;gt; 1.5, adjusted p value &amp;lt; 0.001 after Benjamini &amp; Hochberg multiple test correction) were identified in tumors as compared to normal brain samples. A selection of hypermethylated candidates were confirmed by pyrosequencing. The normalized array data significantly (p&amp;lt;0.0001) correlated to methylation (Pearson's correlation test) for SIM1 (r = 0.84), OTX2 (r = 0.78), NEFM (r = 0.88) and CDYL (r = 0.82). RT-QPCR was performed for NEFM and confirmed correlation between DNA methylation and mRNA expression (r = −0.76, p &amp;lt; 0.0001). SIM1 and OTX2 are transcription factors that play a key role in neurogenesis. NEFM encodes for a neurofilament and is expressed during neuronal differentiation. CDYL has recently been shown to be a REST/NRSF corepressor and key in the repression of the proto-oncogene TrkC. Functional annotation of the candidate gene lists indicated the significant (p&amp;lt;0.0001) enrichment of binding sites for the following transcription factors: E2F, PAX4, PAX5 and STAT5A. Conclusion: We have demonstrated the effectiveness of using MeDIP and the Agilent array platform to profile DNA methylation genome-wide in astrocytic tumors and identified a number of aberrantly hypermethylated genes in astrocytic tumors. Some of these genes are involved in transcriptional control of neuronal differentiation, suggesting that suppression of this pathway may be involved in the pathogenesis of these tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-181. doi:10.1158/1538-7445.AM2011-LB-181