Detection of Pancreatic Beta-Cell DNA in the Circulation Using the Dual Amplification Refractory Mutation System PCR

Background: CpG cytosine in the human insulin gene (INS) is uniquely unmethylated in pancreatic β-cells. It has been reported that a few unmethylated CpG specific PCR in circulating cell-free DNA could detect injury of pancreatic β-cells. However, the same CpG methylation pattern with these CpG sites are also existing in non-β-cells, thus the false positive results might be involved in these reports.

Aim: To develop a precise method for detecting pancreatic β-cell DNA in the circulation.

Methods: We have developed the dual Amplification Refractory Mutation System (ARMS) PCR, which amplifies only if 4 CpG sites were all unmethylated. The first ARMS PCR amplifies if 2 CpG sites (+331, 404bp of transcription start sites (TSS) of INS) are unmethylated simultaneously, and followed by the second ARMS PCR, which amplifies if 2 CpG sites (+367, 374bp of TSS of INS) are unmethylated simultaneously. This method was applied to 52 patients with type 1 diabetes (T1D) (duration 12.6 +/- 10.1 years). We confirmed the positive samples using DNA sequence analysis.

Results: Pancreatic β-cell DNA were detected in 3 T1D (11.8, 2.5, 912.7 copies in 0.1mL of serum) who are diagnosed as slowly progressive T1D, and 4 among 16 healthy control subjects (1.2, 2.2, 1.9, 3.7 copies in 0.1mL of serum).

Conclusion: We have developed the dual ARMS PCR which is a precise method to detect circulating pancreatic β-cell DNA. Almost no β-cell DNA was circulating in the long standing T1D, however, β-cell DNA was detectable in slowly progressive T1D. Also, there were not so many, but a few β-cell DNA in the circulation of healthy control subjects. Therefore, this method is useful to evaluate pathogenesis of type 1 diabetes.