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PMID: 17690210 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

The Journal of molecular diagnostics : JMD ·Vol. 9 ·No. 4 ·2007-09-00 ·Pages 510-20

Dejeux E, Audard V, Cavard C, Gut IG, Terris B, Tost J

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 approximately 1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material.

MeSH Terms
Carcinoma, Hepatocellular/genetics CpG Islands DNA Methylation DNA, Neoplasm/genetics Humans Promoter Regions, Genetic/genetics Sample Size Sequence Analysis, DNA/methods
Chemicals
DNA, Neoplasm
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Dejeux Emelyne
Laboratory for Epigenetics, Centre National de Génotypage, Bâtiment G2, 2 rue Gaston Crémieux, CP 5721, 91057 Evry Cedex, France.
Audard Virginie
Cavard Catherine
Gut Ivo Glynne
Terris Benoit
Tost Jörg
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Article Info
Journal
The Journal of molecular diagnostics : JMD
Abbr.
J Mol Diagn
ISSN
1525-1578
DOI
10.2353/jmoldx.2007.060209
Published
2007-09-00
Epub
2007-00-09
Pages
510-20
Language
English
Region
United States
NLM ID
100893612
PMCID
PMC1975099
Subset
IM
External Links
PubMed source DOI: 10.2353/jmoldx.2007.060209 PMC Full Text View journal details
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