Journal
JOURNAL OF DIABETES INVESTIGATION
Volume 13, Issue 7, Pages 1140-1148Publisher
WILEY
DOI: 10.1111/jdi.13806
Keywords
DNA methylation; Quantitative RT-PCR; Type 1 diabetes
Categories
Funding
- joint research program of the Institute for Molecular and Cellular Regulation, Gunma University [18020]
- [15K06910]
- [18K07448]
Ask authors/readers for more resources
Several research groups have reported methods for quantifying pancreatic beta cell injury by measuring CpG unmethylation of the insulin gene in circulation using digital droplet PCR or next-generation sequencing. The novel method developed in this study can detect four CpG unmethylations of the insulin gene, showing high linearity and the ability to detect a single copy of unmethylated insulin DNA.
Aims/Introduction Several research groups have reported methods for quantifying pancreatic beta cell (beta-cell) injury by measuring beta-cell-specific CpG unmethylation of the insulin gene in circulation using digital droplet PCR or next-generation sequencing. However, these methods have certain disadvantages, such as the need to consider the background signal owing to the small number of target CpG sites and the need for unique equipment. Materials and Methods We established a novel method for detecting four CpG unmethylations of the insulin gene using two-step amplification refractory mutation system PCR. We applied it to type 1 diabetes (T1D) patients with a wide range of disease durations and to healthy adults. Results The assay showed high linearity and could detect a single copy of unmethylated insulin DNA in experiments using methylated and unmethylated plasmid DNA. The unmethylated insulin DNA level in the type 1 diabetes group, whose beta-cell mass was considerably reduced, was similar to that of healthy adults. An inverse correlation was observed between copy number and disease duration in patients with unmethylated insulin DNA-positive type 1 diabetes. Conclusions We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real-time PCR system. This method would be useful for analyzing dynamic profiles of beta-cells in human disease such as type 1 diabetes.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available