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? cell death and dysfunction during type 1 diabetes development in at-risk individuals.

ABSTRACT: UNLABELLED:Role of the funding source: Funding from the NIH was used for support of the participating clinical centers and the coordinating center. The funding source did not participate in the collection or the analysis of the data. BACKGROUND:The ? cell killing that characterizes type 1 diabetes (T1D) is thought to begin years before patients present clinically with metabolic decompensation; however, this primary pathologic process of the disease has not been measured. METHODS:Here, we measured ? cell death with an assay that detects ? cell-derived unmethylated insulin (INS) DNA. Using this assay, we performed an observational study of 50 participants from 2 cohorts at risk for developing T1D from the TrialNet Pathway to Prevention study and of 4 subjects who received islet autotransplants. RESULTS:In at-risk subjects, those who progressed to T1D had average levels of unmethylated INS DNA that were elevated modestly compared with those of healthy control subjects. In at-risk individuals that progressed to T1D, the observed increases in unmethylated INS DNA were associated with decreases in insulin secretion, indicating that the changes in unmethylated INS DNA are indicative of ? cell killing. Subjects at high risk for T1D had levels of unmethylated INS DNA that were higher than those of healthy controls and higher than the levels of unmethylated INS DNA in the at-risk progressor and at-risk nonprogressor groups followed for 4 years. Evaluation of insulin secretory kinetics also distinguished high-risk subjects who progressed to overt disease from those who did not. CONCLUSION:We conclude that a blood test that measures unmethylated INS DNA serves as a marker of active ? cell killing as the result of T1D-associated autoimmunity. Together, the data support the concept that ? cell killing occurs sporadically during the years prior to diagnosis of T1D and is more intense in the peridiagnosis period. TRIAL REGISTRATION:Clinicaltrials.gov NCT00097292. FUNDING:Funding was from the NIH, the Juvenile Diabetes Research Foundation, and the American Diabetes Association.

SUBMITTER: Herold KC

PROVIDER: S-EPMC4362259 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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β cell death and dysfunction during type 1 diabetes development in at-risk individuals.

Herold Kevan C KC Usmani-Brown Sahar S Ghazi Tara T Lebastchi Jasmin J Beam Craig A CA Bellin Melena D MD Ledizet Michel M Sosenko Jay M JM Krischer Jeffrey P JP Palmer Jerry P JP

The Journal of clinical investigation 20150202 3

<h4>Unlabelled</h4>Role of the funding source: Funding from the NIH was used for support of the participating clinical centers and the coordinating center. The funding source did not participate in the collection or the analysis of the data.<h4>Background</h4>The β cell killing that characterizes type 1 diabetes (T1D) is thought to begin years before patients present clinically with metabolic decompensation; however, this primary pathologic process of the disease has not been measured.<h4>Method ...[more]

PMID: 25642774

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Project description:DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (Ins2) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined Ins2 exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse Ins2 gene was methylated in vitro and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic DNA background. We also compared different methods for data analysis and found that the Relative Expression Ratio method is the most robust method since it incorporates both a reference value to normalize day-to-day variability as well as PCR reaction efficiencies to normalize between the methylation-specific and bisulfite-specific components of the calculations. The assay was applied in the streptozotocin-treated diabetic mouse model and detected a significant increase in circulating beta cell DNA before the rise in blood glucose level. These results demonstrate that this qMSP assay can be used for monitoring circulating DNA from insulin-producing cells, which will provide the basis for development of assays to detect beta cell destruction in early T1DM.

Dataset Information

? cell death and dysfunction during type 1 diabetes development in at-risk individuals.

Publications

β cell death and dysfunction during type 1 diabetes development in at-risk individuals.

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