Background: Vascular complication of type 1 diabetes is associated with multiple factors. However, biomarkers which directly reflect vascular complication have not been elucidated. We have developed a method to detect unmethylated status of pancreatic β cell specific CpG sequences of insulin gene from cell-free DNA (cfDNA) in the circulation. Transmembrane protein ROBO4 is highly expressed in vascular endothelial cells, and it has been reported that the CpG sites in the promoter region are specifically unmethylated.
Aim: To develop a novel PCR method to specifically detect unmethylated CpG sites of ROBO4 gene promoter region in serum cfDNA and to evaluate this method in the supernatant of vascular endothelial cell culture and serum of patients with type 1 diabetes.
Material and Methods: After bisulfite conversion of cfDNA extracted from human umbilical vein endothelial cells (HUVEC) and patient serum, unmethylated ROBO4 gene was detected by the dual Amplifications Refractory Mutation Systems PCR, which amplifies only if 4 CpG sites (-185, -144, -118 and -103bp of transcription start sites of ROBO4) are all unmetylated. These methods were applied to 83 patients with type 1 diabetes (duration 16.1±13.9 years) and clinical characteristics were compared.
Results: In HUVEC culture supernatant under constant and intermittent high glucose condition, the detection of ROBO4 gene copy number increased depending on the number of dead cells and cfDNA concentration. A positive correlation (p <0.05) with the mean intima-media thickness (IMT) and MCV were observed for the ROBO4 copy numbers in patient serum cfDNA. According to the multiple regression analysis, unmethylated ROBO4 copy number (p <0.001) and age (p <0.001) were selected as the determinant factors of mean IMT.
Conclusion: Novel PCR method for ROBO4 could detect vascular endothelial cell injury and atherosclerosis quantitatively in patients with type 1 diabetes.
A. Kuroda: Speaker's Bureau; Self; Eli Lilly and Company, Novo Nordisk Inc., Sanofi. M.Y. Yamada: None. Y. Takashi: None. M. Ohishi: None. H. Mori: None. M. Ishizu: None. R. Suzuki: None. O. Yinhua: None. Y. Tominaga: None. M. Matsuhisa: Advisory Panel; Self; Eli Lilly and Company. Board Member; Self; Daiichi Sankyo Company, Limited. Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Mitsubishi Tanabe Pharma Corporation. Speaker's Bureau; Self; Astellas Pharma Inc., Eli Lilly and Company, Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Sanofi, Takeda Pharmaceutical Company Limited.
Japan Society for the Promotion of Science