Novel method utilizing bisulfite conversion with dual amplification-refractory mutation system polymerase chain reaction to detect circulating pancreatic beta cell cfDNA.

2020 Fiscal Year Final Research Report

• Project/Area Number: 18K07448
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 52010:General internal medicine-related
• Research Institution: The University of Tokushima
• Principal Investigator: KURODA Akio 徳島大学, 先端酵素学研究所, 准教授 (70571412)
• Co-Investigator(Kenkyū-buntansha): 山下 美鈴 (山田美鈴) 徳島大学, 先端酵素学研究所(糖尿病), 徳島大学専門研究員 (90451690) ; 松久 宗英 徳島大学, 先端酵素学研究所(糖尿病), 教授 (60362737)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Keywords: 1型糖尿病 / 細胞死 / PCR

Outline of Final Research Achievements

Pancreatic beta cell death plays a key role in type 1 diabetes (T1D) progression. CpG cytosines in the insulin gene are uniquely unmethylated in pancreatic beta cells in mice and humans. In this report, we used specific and quantitative demethylation-specific amplification refractory mutation system (ARMS) PCR in streptozotocin (STZ)-induced diabetic mice and patients with T1D. Murine beta cell-derived cfDNA was detected after STZ treatment. Thirty-two of 114 T1D patients were positive for beta cell-derived cfDNA. Although 105 copies of methylated insulin DNA were not detected by demethylation-specific ARMS PCR, one copy of unmethylated insulin DNA was detected. The patient age and duration of T1D were negatively correlated with the beta cell cfDNA copy number significantly. The detection of bisulfite-converted cfDNA by ARMS PCR is a novel method of detecting circulating CpG methylation-specific cfDNA and has good sensitivity and specificity with low costs.

Free Research Field

糖尿病臨床

Academic Significance and Societal Importance of the Research Achievements

膵β細胞の自己免疫による破壊により1型糖尿病が発症するが、その直接的な破壊を定量的に評価する方法はなかった。今回我々は膵β細胞のインスリン遺伝子の特徴を利用した極めて精度の高い検出法を開発して膵β細胞の破壊を定量的に評価する方法を開発した。同様な方法を用いることであらゆる細胞の破壊を検出することが可能であり医学の発展に大変大きな意義がある。