Disagreement between two common biomarkers of global DNA methylation

Abstract

BackgroundThe quantification of global DNA methylation has been established in epigenetic screening. As more practicable alternatives to the HPLC-based gold standard, the methylation analysis of CpG islands in repeatable elements (LINE-1) and the luminometric methylation assay (LUMA) of overall 5-methylcytosine content in “CCGG” recognition sites are most widely used. Both methods are applied as virtually equivalent, despite the hints that their results only partly agree. This triggered the present agreement assessments.ResultsThree different human cell types (cultured MCF7 and SHSY5Y cell lines treated with different chemical modulators of DNA methylation and whole blood drawn from pain patients and healthy volunteers) were submitted to the global DNA methylation assays employing LINE-1 or LUMA-based pyrosequencing measurements. The agreement between the two bioassays was assessed using generally accepted approaches to the statistics for laboratory method comparison studies. Although global DNA methylation levels measured by the two methods correlated, five different lines of statistical evidence consistently rejected the assumption of complete agreement. Specifically, a bias was observed between the two methods. In addition, both the magnitude and direction of bias were tissue-dependent. Interassay differences could be grouped based on Bayesian statistics, and these groups allowed in turn to re-identify the originating tissue.ConclusionsAlthough providing partly correlated measurements of DNA methylation, interchangeability of the quantitative results obtained with LINE-1 and LUMA was jeopardized by a consistent bias between the results. Moreover, the present analyses strongly indicate a tissue specificity of the differences between the two methods.

Global DNA methylation levels are altered by modifiable clinical manipulations in assisted reproductive technologies

“…The reasons for this difference in discriminatory power between surrogate global measures of DNA methylation are unclear. CCGG sites are enriched in hypomethylated CpG islands [ 39 , 40 ], which are found near the promoters of approximately half of all genes, while LINE1 elements are dispersed in repeated sequences that are generally hypermethylated [ 40 ]. ART is associated with an increase in methylation in the usually hypomethylated CCGG site (Table 2 ) and a decrease in methylation in the usually hypermethylated LINE1 sites (Table 3 ).…”

Evaluating LINE-1 Methylation in Cleft Lip Tissues and its Association with Early Pregnancy Exposures

“…Infants with non-syndromic cleft lip with or without palate were identified in the context of the ongoing PENTACLEFT project [44]. The PENTACLEFT project protocol includes the recruitment of non-syndromic CL/P cases, their parents and maternal grandparents, and the collection of genomic DNA from peripheral blood or buccal swab samples.…”

Genomic 5-mC contents in peripheral blood leukocytes were independent protective factors for coronary artery disease with a specific profile in different leukocyte subtypes

“…Since a large portion of 5-mC is found in repeat sequences and transposable elements, such as long interspersed nuclear element (LINE-1) and ALU [ 12 – 14 ], methylation of these repetitive elements has thus been used as a surrogate for the global methylation of the genome [ 15 ]. It could be directly detected using bisulfite sequencing or other methods primarily based on the digestion of genomic DNA by restriction enzymes, like Luminometric Methylation Assay, LUMA, and the [ 3 H]-methyl acceptance-based method [ 16 , 17 ].…”