title: 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 be affected by environmental factors. As an indirect measure of β-cell death, we developed an assay 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 the β-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 < .0001). More than one-third of T1D patients and one-half of at-risk subjects had levels that were more than 2 SD than the mean of nondiabetic control subjects. We conclude that droplet digital PCR is a useful method to detect β-cell death and is more specific and feasible than other methods, such as nested real-time PCR. This new method may be a valuable tool for analyzing pathogenic mechanisms and the effects of treatments in all forms of diabetes.
authors.affiliation: L2 Diagnostics (S.U.-B., M.L.), New Haven, Connecticut 06511; Departments of Immunobiology and Internal Medicine (J.L., K.C.H.), Yale University School of Medicine, New Haven, Connecticut 06520; Barbara Davis Center for Childhood Diabetes (A.K.S.), University of Colorado Denver, Aurora, Colorado 80045; and Department of Pediatrics (C.B.), University of South Florida, Tampa, Florida 33606.
authors.forename: Sahar
authors.initials: S
authors.lastname: Usmani-Brown
authors.forename: Jasmin
authors.initials: J
authors.lastname: Lebastchi
authors.forename: Andrea K
authors.initials: AK
authors.lastname: Steck
authors.forename: Craig
authors.initials: C
authors.lastname: Beam
authors.forename: Kevan C
authors.initials: KC
authors.lastname: Herold
authors.forename: Michel
authors.initials: M
authors.lastname: Ledizet
mesh_terms: D000293:Adolescent; D016923:Cell Death; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D005260:Female; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D055815:Young Adult
chemical_list: D004247:DNA