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Background: The 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.
Results: Three 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.
Conclusions: Although 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.
Aim: Exposure to opioids has been associated with epigenetic effects. Studies in rodents suggested a role of varying degrees of DNA methylation in the differential regulation of μ-opioid receptor expression across the brain.
Methods: In a translational investigation, using tissue acquired postmortem from 21 brain regions of former opiate addicts, representing a human cohort with chronic opioid exposure, μ-opioid receptor expression was analyzed at the level of DNA methylation, mRNA and protein.
Results & conclusion: While high or low μ-opioid receptor expression significantly correlated with local OPRM1 mRNA levels, there was no corresponding association with OPRM1 methylation status. Additional experiments in human cell lines showed that changes in DNA methylation associated with changes in μ-opioid expression were an order of magnitude greater than differences in brain. Hence, different degrees of DNA methylation associated with chronic opioid exposure are unlikely to exert a major role in the region-specificity of μ-opioid receptor expression in the human brain.