Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

Mathias Ehrich, Matthew R Nelson, Patrick Stanssens, Marc Zabeau, Triantafillos Liloglou, George Xinarianos, Charles R Cantor, John K Field, Dirk van den Boom

Research output: Contribution to journalArticle (journal)peer-review

712 Citations (Scopus)

Abstract

Methylation is one of the major epigenetic processes pivotal to our understanding of carcinogenesis. It is now widely accepted that there is a relationship between DNA methylation, chromatin structure, and human malignancies. DNA methylation is potentially an important clinical marker in cancer molecular diagnostics. Understanding epigenetic modifications in their biological context involves several aspects of DNA methylation analysis. These aspects include the de novo discovery of differentially methylated genes, the analysis of methylation patterns, and the determination of differences in the degree of methylation. Here we present a previously uncharacterized method for high-throughput DNA methylation analysis that utilizes MALDI-TOF mass spectrometry (MS) analysis of base-specifically cleaved amplification products. We use the IGF2/H19 region to show that a single base-specific cleavage reaction is sufficient to discover methylation sites and to determine methylation ratios within a selected target region. A combination of cleavage reactions enables the complete evaluation of all relevant aspects of DNA methylation, with most CpGs represented in multiple reactions. We successfully applied this technology under high-throughput conditions to quantitatively assess methylation differences between normal and neoplastic lung cancer tissue samples from 48 patients in 47 genes and demonstrate that the quantitative methylation results allow accurate classification of samples according to their histopathology.

Original languageEnglish
Pages (from-to)15785-90
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number44
DOIs
Publication statusPublished - 1 Nov 2005

Keywords

  • Binding Sites
  • Cluster Analysis
  • DNA Methylation
  • Diagnostic Techniques and Procedures
  • Genomics/methods
  • Humans
  • Lung Neoplasms/classification
  • Neoplasms/diagnosis
  • Polymerase Chain Reaction/methods
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Sulfites

Research Institutes

  • Health Research Institute

Research Centres

  • Cardio-Respiratory Research Centre

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