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.

The effect of morphine upon DNA methylation in ten regions of the rat brain

“…Global methylation levels were estimated using two assays to interrogate Line1 methylation (L1-utr and L1-orf) that have been widely utilized for this purpose since their first development 59 and which correlate well with measures of global 5-mc content by high-performance liquid chromatography 60 . Gene-specific assays were designed to interrogate promoter regions for the Bdnf, Comt, Il1b, Il6, Nr3c1 and Tnf genes.…”

Assessment of DNA methylation patterns in breast tumours and apparently healthy subjects in Anambra state, Nigeria

“…DNA methylation changes in cancer cells include the loss of methylation at normally methylated sequences (hypomethylation) and the gain of methylated sequences at sites usually unmethylated (hypermethylation) [22]. In this study, presentation of normal DNA methylation is low in subjects with breast tumours, while DNA methylation shift such as hypomethylation, unmethylation and hypermethylation were significantly high in subjects with malignant and benign breast tumours.…”

“…DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations [22]. DNA demethylation has an important role in cancer by turning on the expression of pro-metastatic genes, such as the heparanase gene, MMP2 (which encodes matrix metalloproteinase-2) and uPA (which encodes urokinase plasminogen activator) [23].…”

Repeats as global DNA methylation marker in bovine preimplantation embryos

“…For example, the Alu bisulfite primers used in a recent study could amplify only 16 different copies, with length differing from 46 up to 1017 bp (Lisanti et al 2013), making it dubious as marker for global methylation status. In the present study, we tried to amplify as many as possible copies of the repeat families to represent the whole genome.…”