Published online before print May 24, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: jmoldx.2007.060167v1 9/3/368    most recent Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mikeska, T. Articles by Waha, A. Search for Related Content PubMed PubMed Citation Articles by Mikeska, T. Articles by Waha, A.

Journal of Molecular Diagnostics 2007, Vol. 9, No. 3
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2007.060167

Optimization of Quantitative MGMT Promoter Methylation Analysis Using Pyrosequencing and Combined Bisulfite Restriction Analysis

Thomas Mikeska^*, Christoph Bock, Osman El-Maarri, Anika Hübner^*, Denise Ehrentraut^*, Johannes Schramm, Jörg Felsberg^¶, Philip Kahl^||, Reinhard Büttner^||, Torsten Pietsch^* and Andreas Waha^*

From the Departments of Neuropathology, * Experimental Hematology and Transfusion Medicine, Neurosurgery, and Pathology, ^|| University of Bonn, Bonn; the Max-Planck-Institut für Informatik, Saarbrücken; and the Department of Neuropathology, ^¶ University Düsseldorf, Düsseldorf, Germany

Resistance to chemotherapy is a major complication during treatment of cancer patients. Hypermethylation of the MGMT gene alters DNA repair and is associated with longer survival of glioblastoma patients treated with alkylating agents. Therefore, MGMT promoter methylation plays an important role as a predictive biomarker for chemotherapy resistance. To adopt this established correlation into a molecular diagnosis procedure, we compared and optimized three experimental techniques [combined bisulfite restriction analysis, a primer extension- and denaturing high-performance liquid chromatography-based method named SIRPH (SNuPE ion pair-reverse phase high-performance liquid chromatography), and pyrosequencing] with regard to their accuracy of detecting MGMT promoter methylation. Initially, bisulfite sequencing was used to obtain a comprehensive methylation profile of the MGMT promoter region in 22 glioblastoma samples and in three normal brain controls. Next, we statistically identified CpG sites that best discriminate between methylated and unmethylated MGMT promoters. These results were then used to design optimal combined bisulfite restriction analysis, SIRPH, and pyrosequencing assays for accurate and cost-efficient assessment of MGMT promoter methylation. We compared all three techniques with regard to their reliability and reproducibility on well-characterized tumor samples. The optimized pyrosequencing assay performed best and provides a sensitive, robust, and easy-to-use method for quantitative assessment of MGMT methylation, for both snap-frozen and paraffin-embedded specimens.

This article has been cited by other articles:

				A. J. Iafrate and D. N. Louis "MGMT for pt mgmt": Is Methylguanine-DNA Methyltransferase Testing Ready for Patient Management? J. Mol. Diagn., July 1, 2008; 10(4): 308 - 310. [Abstract] [Full Text] [PDF]

				C. Bock, J. Walter, M. Paulsen, and T. Lengauer Inter-individual variation of DNA methylation and its implications for large-scale epigenome mapping Nucleic Acids Res., June 1, 2008; 36(10): e55 - e55. [Abstract] [Full Text] [PDF]

				L. S. Kristensen, T. Mikeska, M. Krypuy, and A. Dobrovic Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection Nucleic Acids Res., April 1, 2008; 36(7): e42 - e42. [Abstract] [Full Text] [PDF]

				C. Bock and T. Lengauer Computational epigenetics Bioinformatics, January 1, 2008; 24(1): 1 - 10. [Abstract] [Full Text] [PDF]