development of a quantitative methylation-specific polymerase chain reaction method for monitoring beta cell death in type 1 diabetes定量methylation-specific聚合酶链反应方法的发展在1型糖尿病监测β细胞死亡.pdfVIP

Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes 1 2 1 1 1 1 Mohamed I. Husseiny , Akio Kuroda , Alexander N. Kaye , Indu Nair , Fouad Kandeel , Kevin Ferreri * 1 Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of City of Hope, Duarte, California, United States of America, 2 Diabetes Therapeutics and Research Center, The University of Tokushima, Tokushima, Japan Abstract DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (Ins2) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined Ins2 exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse Ins2 gene was methylated in vitro and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic