A novel ultra-sensitive assay does not detect beta-cell death based on cell-free DNA methylation in the plasma of patients with type 1 diabetes

There are currently no biomarkers for assessment of human pancreatic beta-cell death. It has been proposed that unmethylated insulin promoter fragments in plasma derive exclusively from beta-cells, reflect their recent demise, and hence can be used to assess beta-cell damage in type 1 diabetes (T1D) and in islet transplant recipients. We describe a method for simultaneous assessment of multiple DNA methylation markers in plasma cell-free DNA (cfDNA), and apply it to study beta-cell-derived cfDNA. We developed an ultra-sensitive assay based on 6 beta-cell-specific methylation markers, and demonstrate detection of beta-cell DNA in mixtures of DNA containing as little as 0.03% beta-cell DNA, or less than one beta-cell genome equivalent. The plasma of healthy individuals (n=170, age 4-78 years) contains on average just one beta-cell genome equivalent/ml. As expected, a strong signal is observed in the plasma of islet transplant recipients shortly after transplantation. We also detect a beta-cell cfDNA signal in a fraction of patients with islet cell tumors (not necessarily insulinomas), and in longitudinal samples from a patient with KATP congenital hyperinsulinism, consistent with substantial beta-cell turnover in this disease. Strikingly, in contrast to previous reports, we find no elevation of beta-cell-derived cfDNA in people at risk for T1D, in patients with recent onset T1D and in long-standing T1D patients, using 130 samples from 4 medical centers. We discuss the utility of sensitive beta-cell cfDNA analysis and potential explanations for the lack of a beta-cell cfDNA signal in T1D.