Rapid Identification of Promoter Hypermethylation in Hepatocellular Carcinoma by Pyrosequencing of Etiologically Homogeneous Sample Pools

Abstract

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 CTNNB1) 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 ϳ1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material. (J Mol Diagn 2007, 9:510 -520;

Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer, Normal Colonic Mucosa, and Peripheral Blood Cells

“…17,32,[37][38][39][40][41][42][43][44][45] Genome-wide DNA hypomethylation plays an important role in genomic instability by reactivating transposable DNA sequences and, therefore, contributes to colorectal carcinogenesis. 1,3-6,35,39,46 -48 Previous studies have reported that most carcinomas of colon, stomach, breast, lung, prostate, liver, and esophagus show hypomethylation of LINE-1 compared with matched normal tissues.…”

DNA Methyltransferases 1 and 3b Expression in Huh-7 Cells Expressing HCV Core Protein of Different Genotypes

“…It has been already reported that DNMTs are dysregulated in HCC [17][18][19]. To elucidate the possible role of HCV core proteins on the development of HCV-induced HCC, we evaluated the mRNA and protein expression levels of DNMT1 and 3b by qRT-PCR and immunoblot detection in the human hepatoma cell line Huh-7 cells transiently expressing the HCV core protein of different genotypes.…”

Bisulfite-based epityping on pooled genomic DNA provides an accurate estimate of average group DNA methylation

“…To date, however, few studies have systematically analyzed the applicability of DNA pooling for the analysis of DNA methylation. Dejeux and colleagues successfully used pyrosequencing to screen DNA methylation across five loci in pooled DNA samples [ 15 ]. However, by pooling samples subsequent to sodium bisulfite treatment, their approach is potentially affected by differential bisulfite conversion biases, and requires relatively large amounts of starting material from each sample.…”