Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues

<div><p>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.</p></div

Overview of cutting sites for restriction enzymes.

<p>Overview of cutting sites for restriction enzymes.</p

Absolute values for global DNA methylation estimated by 4 different methods.

§<p>Data for measurements on DNA from colon biopsies are presented as mean % methylation for 10 paired samples of normal and tumor tissue (± standard error of the mean (SEM)). Data from analysis of DNA from the cells lines represent means of 3 technical replicates (± SEM).</p><p>NB: The nature of the measurements is different for each of the assays so direct comparison of the methylation percentages between assays cannot be made.</p>*<p><i>P</i><0.03 and **<i>P</i>≤0.009; non-parametric Wilcoxon matched-pair test was used for comparison of normal vs. tumor tissue for colon biopsies, and unpaired homoscedastic t-test was used to test for significance between 5-AzaC- treated and untreated cells (each cell line analyzed separately).</p

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

<p>As assessed by: A) the LINE1 assay (*<i>P</i> = 0.013), B) the Alu assay (**<i>P</i> = 0.005), C) the LUMA assay, and D) the HPLC method (*<i>P</i>≤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.</p

Overview of various assays to assess global DNA methylation, depicting their biological relevance.

§<p>Information taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079044#pone.0079044-Sellis1" target="_blank">[37]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079044#pone.0079044-Cordaux1" target="_blank">[38]</a>. ^# Information taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079044#pone.0079044-Fazzari1" target="_blank">[39]</a>.</p

Overview of primers and sequences for pyrosequencing assays.

<p>Overview of primers and sequences for pyrosequencing assays.</p

Linear regressions showing the relationships between assays.

<p>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.</p

Correction: Whole-Genome Saliva and Blood DNA Methylation Profiling in Individuals with a Respiratory Allergy

<p>Correction: Whole-Genome Saliva and Blood DNA Methylation Profiling in Individuals with a Respiratory Allergy</p

Comparison of methylation differences between RA cases and controls in PBMC and saliva as assessed by Illumina 450K bead chips and bisulfite pyrosequencing.

<p>Comparison of methylation differences between RA cases and controls in PBMC and saliva as assessed by Illumina 450K bead chips and bisulfite pyrosequencing.</p

Identification of differentially methylated probes between RA cases and healthy controls (485 DMS in saliva and 437 DMS in PBMC).

<p>216 probes were in common in PBMC and saliva. The common probes showed the same polarity of methylation levels (<i>hypermethylated</i>, <b>contramethylated</b>, <u>hypomethylated</u>).</p