Analysis of β-cell death in type 1 diabetes by droplet digital PCR

Abstract

Type 1 diabetes (T1D) and other forms of diabetes are due to the killing of β-cells. However, the loss of β-cells has only been assessed by functional studies with a liquid meal or glucose that can beaffected by environmental factors. Asanindirectmeasureofβ-cell death,wedevelopedanassay using a novel droplet digital PCR that detects INS DNA derived from β-cells. The release of INS DNA with epigenetic modifications (unmethylated CpG) identifies theβ-cellular source of the DNA. The assay can detect unmethylated DNA between a range of approximately 600 copies/μL and 0.7 copies/μL, with a regression coefficient for the log transformed copy number of 0.99. The assay was specific for unmethylated INS DNA in mixtures with methylated INS DNA. We analyzed the levels of unmethylated INS DNA in patients with recent onset T1D and normoglycemia subjects at high risk for disease and found increased levels of unmethylated INS DNA compared with nondiabetic control subjects (P