Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes

<div><p>DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (<em>Ins2</em>) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined <em>Ins2</em> exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse <em>Ins2</em> gene was methylated <em>in vitro</em> and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic DNA background. We also compared different methods for data analysis and found that the Relative Expression Ratio method is the most robust method since it incorporates both a reference value to normalize day-to-day variability as well as PCR reaction efficiencies to normalize between the methylation-specific and bisulfite-specific components of the calculations. The assay was applied in the streptozotocin-treated diabetic mouse model and detected a significant increase in circulating beta cell DNA before the rise in blood glucose level. These results demonstrate that this qMSP assay can be used for monitoring circulating DNA from insulin-producing cells, which will provide the basis for development of assays to detect beta cell destruction in early T1DM.</p>