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. Disclosure A. Kuroda: None. M.Y. Yamada: None. Y. Tominaga: None. R. Suzuki: None. M. Tamaki: None. Y. Akehi: None. Y. Takashi: None. D. Koga: Employee; Self; Otsuka Pharmaceutical Co.,Ltd.. E. Shimokita: None. F. Tanihara: None. K. Kurahashi: None. S. Yoshida: None. I. Endo: None. K. Aihara: None. M. Abe: None. K. Ferreri: None. M. Matsuhisa: None.