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Originally published online as doi:10.2353/jmoldx.2007.060209 on August 9, 2007

Published online before print August 9, 2007
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Journal of Molecular Diagnostics 2007, Vol. 9, No. 4
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2007.060209

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

Emelyne Dejeux*, Virginie Audard{dagger}, Catherine Cavard{dagger}, Ivo Glynne Gut*, Benoit Terris{dagger}{ddagger} and Jörg Tost*

From the Laboratory for Epigenetics, * Centre National de Génotypage, Evry; the Department of Endocrinology, Metabolism and Cancer, {dagger} Institut Cochin, INSERM U567, Centre National de la Recherche Scientifique UMR-S 8104 and University Paris 5, Faculté de Médecine René Descartes, UM3, Paris; and the Service d’Anatomopathologie, {ddagger} Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France

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






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Copyright © 2007 by the American Society for Investigative Pathology and the Association for Molecular Pathology.