Rapid identification of promoter hypermethylation in hepatocellular carcinoma by pyrosequencing of etiologically homogeneous sample pools

Aberrant DNA methylation patterns have been identified in a variety of human diseases, particularly cancer. Pyrosequencing has evolved in recent years as a sensitive and accurate method for the analysis and quantification of the degree of DNA methylation in specific target regions. However, the number of candidate genes that can be analyzed in clinical specimens is often restricted by the limited amount of sample available. Here, we present a novel screening approach that enables the rapid identification of differentially methylated regions such as promoters by pyrosequencing of etiologically homogeneous sample pools after bisulfite treatment. We exemplify its use by the analysis of five genes (CDKN2A, GSTP1, MLH1, IGF2, and C7NNB1) involved in the pathogenesis of human hepatocellular carcinoma using pools stratified for different parameters of clinical importance. Results were confirmed by the individual analysis of the samples. The screening identified all genes displaying differential methylation successfully, and no false positives occurred. Quantitative comparison of the pools and the samples in the pool analyzed individually showed a deviation of similar to 1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material.