Abstract
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 (Ins2) 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 Ins2 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 Ins2 gene was methylated in vitro 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.
Highlights
Type 1 diabetes mellitus (T1DM) results from the autoimmune destruction [1,2] of the insulin-producing beta cells by localized inflammation around the pancreatic islets involving cytotoxic T cells [3,4] anti-islet antibodies [5], and antigen presenting cells [6]
Our previous methylation mapping results of the mouse insulin-2 gene (Ins2) promoter revealed three CpG dinucleotide sites that are located at positions 2414, 2182, and 2171 bp relative to the transcription start site (TSS) and these CpG sites have a specific methylation pattern in insulin-producing pancreatic beta cells and NIT-1 mouse insulinoma cells compared to other tissues [21]
The CpG site at +190 was unmethylated in 13 of 17 clones (76%) from the enriched beta cell fraction versus 2 of 23 non-beta cells (9%), CpG at +310 was unmethylated in 11 of 17 beta (65%) versus 0 of 23 non-beta (0%), CpG at +337 was unmethylated in 11 of 17 beta (65%) versus 2 of 23 non-beta (9%), and CpG at +340 was unmethylated in 14 of 17 beta (82%) versus 1 of 23 nonbeta (4%). These results demonstrate that exon 2 of the mouse Ins2 gene exhibits a tissue-specific pattern of DNA methylation similar to what we previously reported for the mouse Ins2 promoter
Summary
Type 1 diabetes mellitus (T1DM) results from the autoimmune destruction [1,2] of the insulin-producing beta cells by localized inflammation around the pancreatic islets involving cytotoxic T cells [3,4] anti-islet antibodies [5], and antigen presenting cells [6]. Several studies have identified biomarkers for disease risk, such as anti-islet antibodies [9] and HLA genotyping [10], but a method of monitoring the underlying basis of the disease, namely the destruction of the beta cells, might allow early detection of the disease and provide mechanistic assessment of new therapeutic interventions. Several clinical assays have been developed to monitor cell death in vivo based on the detection of nucleic acids that are released into the circulation by dying cells [11,12]. These molecules can be detected by specific PCR-based assays [13,14] providing a new approach for noninvasive assessment of the patient’s status. The critical factor in these tests is the identification of a cell-specific nucleic acid biomarker for the disease
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