Identification of cancer-associated DNA methylation markers by the methylated CpG island recovery assay (MIRA)

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Lung cancer accounts for 30% of all deaths from cancer and for 1.5 million annual deaths worldwide. The relevance of a screening method is emphasized by the fact that the five-year survival rate for lung cancer is much lower than that of breast or prostate cancers for which proper screening tests exist. In normal tissues, CpG islands are associated with the regulatory regions of many cancer-relevant genes are usually unmethylated, but a subset of islands becomes methylated during tumor development. Cancer specific changes in the methylation status of CpG islands can be used as early markers for tumorigenesis. We recently developed an in vitro method to investigate the methylation status of CpG islands. The methyl-CpG island recovery assay (MIRA) is based on the fact that the MBD2b protein can specifically recognize methylated CpG dinucleotides and this interaction is strongly enhanced by the MBD3L1 protein. To identify early markers for human cancers, we combined the MIRA technique with commercially available human CpG island microarrays. Isolated methylated CpG islands from normal and cancer cells were specifically labeled with fluorescent dyes and hybridized to human CpG island microarrays. In a second approach, the identity of the MIRA-enriched methylated CpG islands is determined by DNA sequencing. Verification of the methylation status of the isolated CpG islands is in progress and this information will be used to develop a MIRA-based screening method.