Comparison of methods for quantification of global DNA methylation in human cells and tissues

Abstract

DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

Figure 1. Methylation levels cell lines before and after treatment with the demethylating agent 5-AzaC.

As assessed by: A) the LINE1 assay (**P≤0.001), B) the Alu assay (*P = 0.009), C) the LUMA assay (*P = 0.019), and D) the HPLC method (**P≤0.001). Data are presented as the mean % of methylation (n = 3) relative to the control (i.e. untreated cells). Error bars represent standard deviations.

Figure 2. Methylation levels in tumor samples and in matched biopsies of normal colorectal mucosa.

As assessed by: A) the LINE1 assay (*P = 0.013), B) the Alu assay (**P = 0.005), C) the LUMA assay, and D) the HPLC method (*P≤0.029). Data are presented as the mean % of methylation (n = 10) relative to the control (i.e. normal colon biopsies). Error bars represent standard deviations.

Figure 3. Linear regressions showing the relationships between assays.

Using both data from the cell lines (⧫ 5-AzaC treated or ▴ untreated) and the colon tissues (•), significant linear associations were observed A) between the HPLC method and the LINE1 assay and B) HPLC method versus the Alu assay. Absolute values were used for these analyses.

Figure 4. Correlation coefficients for the estimates of global DNA methylation as quantified by the various methods.

A) Pearson correlations for all data combined (n = 32), B) Pearson correlation coefficients when using the data from the cells (n = 6/cell type), and C) Pearson correlations for the data from the colon biopsies (n = 20).

Figure 5. Bland-Altman plots representing the agreement of each of the surrogate assays.

A) LINE1, B) Alu, and C) LUMA, with the gold standard HPLC method. The differences between the two methods (HPLC – second assay) were plotted against the averages of the two measurements, using both data from the cell lines (⧫ 5-AzaC treated or ▴ untreated) and the colon tissues (•). Horizontal lines were drawn at the mean difference, and at the limits of agreement, which are defined as the mean difference plus or minus two times the standard deviation of the differences. For these comparative analyses, absolute values were converted to percentages relative to the overall mean of each method (i.e. mean set at 100%), in order to acquire values of similar magnitude for all of the methods.