GLAD-PCR Assay of DNA Methylation Markers Associated with Colorectal Cancer

Abstract

Hypermethylation of the gene regulatory regions is documented for many cancer diseases. Such an aberrant DNA methylation in cancer cells is catalyzed by DNA methyltransferases Dnmt3a and Dnmt3b, which predominantly recog- nize and methylate RCGY sequences with formation of R(5mC)GY sites. Recently, based on a new methyl-directed DNA endonuclease GlaI, we developed a GLAD-PCR assay, which allows determining R(5mC)GY site in a defined posi- tion of the genomic DNA. In this work we applied GLAD-PCR assay for identification of the methylated RCGY sites in the regulatory regions of some downregulated genes associated with colorectal cancer (CRC). This list includes ADHFE1, ALX4, CNRIP1, EID3, ELMO1, ESR1, FBN1, HLTF, LAMA1, NEUROG1, NGFR, RARB, RXRG, RYR2, SDC2, SEPT9, SFRP2, SOCS3, SOX17, THBD, TMEFF2, UCHL1, and VIM genes. GLAD-PCR analysis of selected RCGY sites within the regulatory regions of some of these genes demonstrates a good prognostic potential with relatively high sensitivity and specificity of CRC detection in tumor DNA.