Abstract 3014: Quantitative accuracy of Illumina HumanMethylation27 Infinium BeadChip data assessed by pyrosequencing

Purpose. Aberrant DNA methylation can lead to changes in gene expression, and this alteration is highly relevant in the context of cancer. The ability to accurately assess DNA methylation status is therefore of great importance in health and disease. The Illumina HumanMethylation27 Infinium BeadChip allows for analysis of whole genome DNA methylation by interrogating the methylation status of 27,578 CpG sites from the promoter regions of 14,475 annotated genes. However, one potential limitation of this platform as specified by the manufacturer is the inability to detect a β value difference between specimens of <0.2 (∼20% methylation). The present study assessed the quantitative nature of the Infinium platform through independent validation using a pyrosequencing approach.Procedures. Genomic DNA from 46 ovarian cell lines and 4 cultured normal cells, including ovarian surface epithelium and endometriotic cells, was modified with sodium bisulfite using the EZ DNA methylation kit from Zymo Research. Bisulfite conversion efficiency was confirmed by pyrosequencing at two chromosomal loci. Comparisons between normal and cancer cells identified differentially methylated genes using criteria of p0.3. Ten candidate differentially methylated genes (C14orf105, HNF1 β, HNF1α, KIF12, MIA2, PAX8, SERPINA6, SGK2, SRC, and TM4SF4) were selected for validation. Pyrosequencing assays were designed to allow for direct comparison of the same CpG site(s). The relationship between pyrosequencing and Infinium data was evaluated using correlation analyses.Results. Bisulfite conversion was >97% at BRCA1 (chr17) and IGF2 (chr11). Hypermethylation of 1,162 genes (1,452 CpG sites) and hypomethylation of 63 genes (74 CpG sites) was found in ovarian cancer cell lines as compared with cultured normal cells. The β values for 10 genes from Infinium analysis and percent methylation by pyrosequencing of the same CpG site(s) were highly correlated (R>0.98, p0.91, p<0.001). There was a significant positive correlation between platforms when data for all ten genes were grouped into 0-20%, 21-40%, 41-60% 61-80% and 81-100% bins (R value range, 0.32-0.73; p< 0.00002).Conclusions. Ovarian cancer cell lines exhibit significantly increased methylation as compared with normal cultured cells using the HumanMethylation27 Infinium BeadChip assay. The strong correlations between the Infinium and pyrosequencing data at the same CpG sites suggest that the Infinium platform is quantitative, lending support for use of this technology for assessment of methylation differences among specimens analyzed.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 3014. doi:10.1158/1538-7445.AM2011-3014

Duke Scholars

Author Andrew Berchuck Obstetrics and Gynecology, Gynecologic Oncology

Author Susan Kay Murphy Obstetrics and Gynecology, Reproductive Sciences