Usmani-Brown Sahar, Lebastchi Jasmin, Steck Andrea K, Beam Craig, Herold Kevan C, Ledizet Michel
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.
Endocrinology. 2014 Sep;155(9):3694-8. doi: 10.1210/en.2014-1150. Epub 2014 Jul 8.
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.
1型糖尿病（T1D）和其他形式的糖尿病是由于β细胞被破坏所致。然而，β细胞的损失仅通过液体餐或葡萄糖的功能研究来评估，而这些研究可能会受到环境因素的影响。作为β细胞死亡的间接测量方法，我们开发了一种使用新型液滴数字PCR的检测方法，该方法可检测源自β细胞的胰岛素（INS）DNA。带有表观遗传修饰（未甲基化的CpG）的INS DNA的释放可识别DNA的β细胞来源。该检测方法可检测大约600拷贝/微升至0.7拷贝/微升范围内的未甲基化DNA，对数转换拷贝数的回归系数为0.99。该检测方法对与甲基化INS DNA混合的未甲基化INS DNA具有特异性。我们分析了近期发病的T1D患者和疾病高危正常血糖受试者中未甲基化INS DNA的水平，发现与非糖尿病对照受试者相比，未甲基化INS DNA水平升高（P <.0001）。超过三分之一的T1D患者和一半的高危受试者的水平比非糖尿病对照受试者的平均值高出2个标准差以上。我们得出结论，液滴数字PCR是检测β细胞死亡的一种有用方法，比其他方法（如巢式实时PCR）更具特异性和可行性。这种新方法可能是分析所有形式糖尿病的致病机制和治疗效果的宝贵工具。
