Detection of promoter hypermethylation of genes in serum dna

Promoter hypermethylation has been identified as a mechanism in which genes, such as putative tumor suppressor genes, can be transcriptionally inactivated. These epigenetic changes can occur early in lung tumourigenesis. Thus hypermethylation may prove useful as a diagnostic tool. This study is aimed at analysing whether aberrant methylation can be determined in the serum of patients with smoking histories. Methods: Methylation specific PCR (MSP), a technique designed to identify methylated CPG sites within promoter regions, was utilised. Briefly this technique requires bisulfite modification of DNA. Following treatment, genes with methylated cytosines (C) will remain unchanged whereas unmethylated cytosines will change to thymines (T). PCR Primers designed to distinguish between unmodified and modified regions can then identify the methylated promoter regions in certain DNA samples. The bisulfite treatment and the MSP procedure were performed on DNA isolated from serum using the QIAamp blood midi kit (QIAGEN). Results: In this study, we report the detection of methylated gene promoter regions in DNA isolated from serum of smoking patients with and without lung cancer. From the 9 sera tested so far, DNA was detected in all specimens using primers designed to anneal to unmethylated sequences, indicating that serum DNA is reliably obtained and extracted. Of the serum DNA, MSP detected I case with aberrant hypermethylation of the/ 16'-fl promoter. Conclusions: MSP detection of aberrant promoter hypermethylation in serum is feasible. We are now prospectively testing the diagnostic utility of this method in patients with lung cancer