Circulating Unmethylated Insulin DNA As a Biomarker of Human Beta Cell Death: A Multi-laboratory Assay Comparison

Abstract

Context There is an unmet need for biomarkers of pancreatic beta-cell death to improve early diagnosis of type 1 diabetes, enroll subjects into clinical trials, and assess treatment response. To address this need, several groups developed assays measuring insulin deoxyribonucleic acid (DNA) with unmethylated CpG sites in cell-free DNA. Unmethylated insulin DNA should be derived predominantly from beta-cells and indicate ongoing beta-cell death. Objective To assess the performance of three unmethylated insulin DNA assays. Design and Participants Plasma or serum samples from 13 subjects undergoing total pancreatectomy and islet autotransplantation were coded and provided to investigators to measure unmethylated insulin DNA. Samples included a negative control taken post-pancreatectomy but pretransplant, and a positive control taken immediately following islet infusion. We assessed technical reproducibility, linearity, and persistence of detection of unmethylated insulin DNA for each assay. Results All assays discriminated between the negative sample and samples taken directly from the islet transplant bag; 2 of 3 discriminated negative samples from those taken immediately after islet infusion. When high levels of unmethylated insulin DNA were present, technical reproducibility was generally good for all assays. Conclusions The measurement of beta cell cell-free DNA, including insulin, is a promising approach, warranting further testing and development in those with or at-risk for type 1 diabetes, as well as in other settings where understanding the frequency or kinetics of beta cell death could be useful.

C-Peptide Levels in Subjects Followed Longitudinally Before and After Type 1 Diabetes Diagnosis in TrialNet

“…For example, the presence of low first-phase insulin response to intravenous glucose markedly increased the rate of disease progression and identified a cohort that responded to oral insulin (21), and we have recently reported that the insulin response to glucose-potentiated arginine stimulation is a reproducible measure that is thought to estimate function of the b-cell mass (22). Combined with other measures indicative of b-cell injury, such as impaired insulin (23) and islet amyloid polypetide (24) processing, and measures of b-cell death, such as demethylation of insulin DNA in circulating blood (25,26), such detailed and frequent analyses are likely to be informative during this period of time.…”

Cell-Free DNA Fragments as Biomarkers of Islet β-Cell Death in Obesity and Type 2 Diabetes

“…In immunocompetent mice, xenotransplantation of human islets results in the immediate release of unmethylated human INS DNA into the circulation, as measured by traditional quantitative PCR techniques [ 26 ]. Similarly, in humans, the levels of unmethylated INS DNA (or the ratio of unmethylated:methylated INS ) increased acutely following islet transplantation, consistent with death of β cells in the immediate post-transplant period [ 27 , 28 , 29 , 30 , 31 ]. Notably, this signal subsides within hours to days following transplantation, reflecting the balance between the half-life of the circulating DNA (likely hours to minutes) and the rate of ongoing β-cell lysis in any given individual.…”

“…A recent study evaluated subjects undergoing total pancreatectomy and islet autotransplantation using three different assays that measured unmethylated INS DNA. The study showed that during conditions when high levels of unmethylated INS DNA were present, the technical reproducibility was good amongst the three assays (two with dPCR and one with sequencing) [ 31 ].…”

Circulating Free DNA and Its Emerging Role in Autoimmune Diseases

“…Dying beta cells during the pathology of T1D release their DNA content into the circulation. The measurement of beta cell cfDNA, including insulin, using next-generation sequencing (NGS) or digital droplet PCR (ddPCR) is a promising approach to determine the methylation status in plasma or serum [ 98 ]. Another study suggests a correlation among the methylation patterns of cfDNA in recently diagnosed T1D patients, the rate of beta-cell death, and the risk of developing the disease [ 99 ].…”