We propose to develop a novel DNA chip specifically for high-throughput analysis of promoter hypermethylation in primary tumors. It is now clear that abnormal DNA methylation frequently occurs in multiple promoter CpG islands in cancer cells. Increased density of CpG methylation is known to alter local chromatin structure within a promoter , resulting in transcriptional silencing of the corresponding gene. At present, bisulfite DNA sequencing is considered to be the"""""""" gold standard"""""""" for marring methylated CpG sites within a gene promoter. This method, as well as other related techniques, has provided important insights into the functional relationship of promoter methylation and transcriptional silencing on a """"""""gene-by-gene"""""""" basis. Such approaches, however, have given limited pictures of complex epigenetic alterations in cancer and are restricted in throughput for routine clinical applications. In this proposal, we build on the approach of microarray techniques by developing a novel method, called Methylation-Specific Oligonucleotide (MSO) microarray, for high-throughput methylation analysis. Our goal aims at generating MSO chips in which thousands of short oligonucleotides are tethered to glass slide surfaces. These oligonucleotides specifically designed and tested are capable of differentiating methylated and unmethylated CpG sites at the specific locations of a promoter. Test (tumor) and reference samples are bisulfite-treated, PCR-modified products that may contain different pools of DNA fragments due to the hypermethylation status of the tumor genome. These DNA samples are co-hybridized to an MSO chip and quantitative differences in DNA methylation are determined by two-color fluorescence analysis. Distinct from the existing microarray technologies, the MSO approach allows simultaneous analysis of the anatomy of multiple promoter CpG islands in reference to the role of DNA methylation on gene silencing. In addition, this MSO chip can be directly applied to determine molecular signatures of different tumor subtypes. In the pilot R21 phase, we will apply a prototype MSO chip to analyze a small panel of tumor samples and determine its reliability in detecting DNA methylation. In the R33 phase, based on the experience we gain in the pilot study, we will design a full-fledged MSO chip for a comprehensive analysis of promoter methylation in multiple tumor types. An advanced computation system will be developed to handle a large set of methylation data and patients' clinicopathological information and to support heuristic queries. The MSO assay will furnish digital profiles of promoter hypermethylation for individual tumors and offers an alternative to cDNA microarray approaches for molecular classification of different cancer subtypes.
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