High-resolution methylome analysis of renal cell carcinoma and identification of aberrant epigenetic changes in gene bodies.

Abstract

e15000 Background: Metastatic renal cell carcinoma (RCC) has a very poor prognosis with limited therapeutic options. Despite this, there have been very few global genetic and epigenetic studies of RCC. DNA methylation is an epigenetic change that has been extensively studied in cancer, but most studies have focused on changes in promoters and CpG islands. We utilized a novel high resolution, high throughput assay, the HELP (HpaII tiny fragment enrichment by ligation-mediated PCR) assay, to determine DNA methylation in RCC. The HELP assay examines CpG methylation at 1.3 million loci in the genome, including promoters, gene bodies and intergenic regions, making it the most comprehensive study of the RCC methylome available. Methods: We performed the HELP assay on 13 clear cell RCC and 13 control samples. The HELP assay compares restriction enzyme digestion with a methylation-sensitive enzyme (HpaII) to a methylation-insensitive enzyme (MspI) at CCGG sequences in the genome. These digestion fragments are amplified by PCR, fluorescently labeled, and co-hybridized onto a custom microarray. Methylation can be determined at each loci by comparison of the HpaII to MspI signal. Results: Unsupervised clustering showed that the RCC samples were epigenetically distinct and clustered completely separately from controls. Next, we determined common epigenetic alterations in all samples of RCC and observed that these tumors are characterized by a dramatic hypermethylation of a large number of loci in the gene bodies when compared to controls. Ingenuity pathway analysis of genes that were methylated and underexpressed in RCC revealed the TGFβ1 pathway to be the dominant pathway affected by epigenetic silencing, while cell death and cancer were two major biological functions influenced by epigenetic methylation. Conclusions: The HELP assay revealed widespread novel epigenetic changes and identified differentially regulated genes in RCC. This is the first study of DNA methylation changes in gene bodies in RCC. We hope to correlate methylation profiles with clinical response to uncover novel signatures of prognosis in RCC and identify new therapeutic targets within its epigenome.