Abstract
The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.
Highlights
It is estimated that there are more than 347 million diabetics worldwide [1] of whom 5%–10% have type 1 diabetes (T1D)
These results demonstrate that even in a spontaneous diabetic model that the methylation-specific PCR (MSP) methodology is capable of detecting beta cell death at the onset of insulitis and six weeks prior to the rise in blood sugar
Methylation pattern of the human insulin gene We previously reported that the promoter of the human insulin gene (INS) contains nine potential methylation (CpG dinucleotide) sites that are located at positions 2357, 2345, 2234, 2206, 2 180, 2135, 2102, 269 and 219 bp relative to the transcription start site (TSS) and that these sites are predominantly unmethylated in pancreatic beta cells yet methylated in other pancreatic cells [19]
Summary
It is estimated that there are more than 347 million diabetics worldwide [1] of whom 5%–10% have type 1 diabetes (T1D). We and others have recently developed methods for monitoring beta cell death by specific detection of beta cell-derived unmethylated DNA in circulation before the onset of diabetes [16,17,18]. This approach is based on the observation that the DNA encoding portions of the insulin gene in mice (Ins2) and humans (INS) is tissue- unmethylated in insulin-producing beta cells [19], and that this DNA is released into circulation when beta cells are lost during the progression of the disease. To monitor beta cell death, we developed a highly sensitive quantitative methylation-specific PCR (qMSP) assay to detect the circulating beta cell DNA and demonstrated that this signal appears prior to hyperglycemia in a chemically-induced diabetic mouse model [16]
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