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, 102 (44), 15785-90

Quantitative High-Throughput Analysis of DNA Methylation Patterns by Base-Specific Cleavage and Mass Spectrometry

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Quantitative High-Throughput Analysis of DNA Methylation Patterns by Base-Specific Cleavage and Mass Spectrometry

Mathias Ehrich et al. Proc Natl Acad Sci U S A.

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.

Figures

Fig. 1.
Fig. 1.
Analysis of methylation by base-specific cleavage and MALDI-TOF MS. Treated bisulphite is PCR amplified by using primers located outside of the CpG islands with one primer tagged with a T7 promoter sequence. The PCR product is transcribed into a RNA transcript and cleaved base specifically. The cleavage products are analyzed by MALDI-TOF MS, and a characteristic mass signal pattern is obtained. In the case shown here, the PCR product is transcribed from the reverse strand and cleaved U specifically. A methylated template (here indicated in yellow) carries a conserved cytosine, and, hence, the reverse transcript of the PCR product contains CG sequences. In an unmethylated template (indicated in red), the cytosine is converted to uracil. The reverse transcript of the PCR product therefore contains adenosines in the respective positions. The sequence changes from G to A yield 16-Da mass shifts. Cleavage product 1 has two methylation sites. Mass signals of the cleavage product will differ by 32 Da when both CpG sites are either methylated or nonmethylated. For cleavage products 2 and 3, mass shifts of 16 Da will be observed, because each contains only one methylation site. The spectrum can be analyzed for the presence/absence of mass signals to determine which CpGs in the template sequence are methylated, and the ratio of the peak areas of corresponding mass signals can be used to estimate the relative methylation. This assay enables the analysis of mixtures without cloning the PCR products.
Fig. 2.
Fig. 2.
Differences in mass signal patterns between a methylated and a nonmethylated sequence. (a) C-specific cleavage results for the IGF2/H19 region. Spectra were generated by base-specific cleavage of clones representing either a fully methylated or a fully nonmethylated template. In the nonmethylated DNA, all cytosines have been converted to uracil during bisulfite treatment. The RNA transcript derived after PCR amplification does not contain any cytosine positions and, except for an engineered start and end tag, no cleavage can occur in the C-specific cleavage reaction. On the contrary, in a fully methylated template, each methylated CpG site will carry a cytosine after bisulphite treatment and cleavage can occur. A methylated template leads to multiple mass signals representing the 25 methylated CpGs in the selected IGF2/H19 region. Each mass signal represents methylation at two CpG sites (see text for further details). (b) Spectra generated by C-specific cleavage of the forward transcript derived from a mixing experiment. Mixtures of methylated and nonmethylated DNA were generated from cloned DNA and were used for PCR amplification, in vitro transcription, and base-specific cleavage. The CpG units are annotated according to their order in the amplified IGF2/H19 region. Presence of a mass signal at the marked positions reflects the occurrence of methylation at the indicated CpG. The mass signal marked S_01 represents the first cleavage product of the transcript (5′-GGGAGAAGGCU-3′). This cleavage product does not contain a CpG site. Methylation-specific mass signals, and, hence, the presence of methylated DNA in a sample, can be detected in mixtures containing as little as 5% methylated DNA without methylation-specific PCR.
Fig. 3.
Fig. 3.
Mixing experiments to determine the limits of quantitation and the precision of methylation analysis in mixtures. (a) An overlay of four representative mass spectra generated by U-specific cleavage of the reverse transcript amplified from mixtures of methylated and nonmethylated DNA. The mass window shown spans one pair of peaks representing the methylation status of CpG 2 of the IGF2/H19 amplicon. The mass signals labeled NM (3,207 Da) and M (3,223 Da) represent nonmethylated and methylated cleavage products, respectively. (b) Relationship between the estimated relative methylation [calculated as intensity of the methylated cleavage product peak (IM) divided by the sum of the intensities of the methylated and nonmethylated cleavage peaks (IM + INM)] and the relative amount of methylated DNA spiked into the mixture.
Fig. 4.
Fig. 4.
A two-way hierarchical cluster analysis of the relative methylation of 68 CpG units (columns) measured on 96 tissue samples from 48 lung cancer cases (rows). Tissue samples are identified on the left vertical axis as white boxes (normal tissue) and black boxes (tumor tissue). CpG units are identified at the bottom horizontal axis as the gene and the fragment number within the gene. The relative methylation of each fragment within each sample is presented in the central image plot with values ranging from zero (red) to one (yellow; see color key). Missing values are represented as gray.

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