Abstract 2299: An epigenetic study of DNA methylation and histone modification of brain cancer Glioblastoma multiforme

Abstract

Abstract Epigenetic modifications to the genome affect gene expression, causing increased risk for cancers and other diseases. DNA methylation and modification of histone proteins are ways in which this occurs. The specific aim of this study was to identify DNA methylation percentages in Glioblastoma multiforme (GBM) samples compared against normal astrocyte genomic DNA(gDNA). Within the context of DNA methylation, a global genome methylation analysis was performed to determine gDNA methylation differences between GBM and a normal astrocyte samples using an Imprint® Methylated DNA Quantification Kit (Catalog # MDQ1, Sigma®, Saint Louis USA). . Methyl-sensitive cut counting analysis (MSCC) technique was used further estimate and compare methylation of cancer and normal gDNA. DNA samples were digested using the restriction endonuclease HpaII, which is methyl-sensitive and will cut DNA at unmethylated CCGG sites. It is estimated that generally 90% of CpG islands in genomic DNA have at least one HpaII site . HpaII sites must be greater than 40 bases from another HpaII site to be designated an individual HpaII site. In this case, the identified gene sequence is considered to be a single site. DNA (2 μg) was digested using 20 units of HpaII.An adaptor with the recognition site for the MmeI restriction enzyme was added using T4 DNA ligase. The DNA was then ethanol precipitated and nicks were repaired using an 8 unit Bst DNA polymerase. MmeI (2 units) was added to the DNA, which served to cleave at 18 bases adjacent to the HpaII sites. These segments were then ligated to another adapter for PCR amplification and sequencing. Tag sizes were purified using a 10% PAGE gel and amplified with quantitative PCR and Bio-Rad iProof high-fidelity polymerase. An Illumina HiSeq 2000 Genome Analyzer at the University of Nebraska Medical Center Sequencing Core Facility was used for tag sequencing. Tag sequences were paired against the human genome (h19) library using Bowtie, a short sequence aligner . Sequence tags, representing an unmethylated HpaII site, were counted in GBM and normal astrocyte gDNA. The change in tag counts between the two samples represented a change in the methylation status of the HpaII site. The results showed GBM genomic DNA(gDNA) possessed lower gDNA methylation percentages compared to normal samples. Methyl-sensitive cut counting analysis (MSCC) showed fold decreases in GBM gDNA methylation sites for genes PBK, KIF23, COL6A3 and LOX.Results could be used as prognostic indicators for glioblastoma therapeutic strategy. ACKNOWLEDGEMENT:Supported by a NIH-RISE GRANT to ELIZABETH CITY STATE UNIVERSITY and a TMCF-DOE award to Dr.Hirendra Banerjee. Citation Format: Hirendra N. Banerjee, GWYN HYMAN, Jeffrey Rousch, VINOD MANGALIK, Deidre Vann, Christopher Krauss, sabrina sharpe, DAVID KLINKEBIEL, SANTOSH MANDAL, MUKESH VERMA. An epigenetic study of DNA methylation and histone modification of brain cancer Glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2299. doi:10.1158/1538-7445.AM2014-2299