Human pancreatic islets methylation array

Circulating cell-free unmethylated DNA fragments arising from the human INS gene have been proposed as biomarkers of β-cell death for the presymptomatic detection of diabetes. However, given the variability of CpG methylation in the INS gene in different cell types, this gene alone may not yield sufficiently specific information to unambiguously report β-cell damage. We employed an unbiased approach using data from a human DNA methylation gene array to identify the CHTOP gene as a candidate biomarker whose CpGs show a greater frequency of unmethylation in human islets. When tested across an array of non-islet human tissues by digital PCR, both INS and CHTOP contain unmethylated CpG sites in several of these tissues, but in a non-overlapping pattern: INS showed a slightly higher frequency of unmethylation in adipose tissue, whereas CHTOP appeared to be unmethylated in pancreas, brain, and skeletal muscle. Notably, INS and CHTOP genes are both unmethylated in human β and α cells, suggesting that each species at best would represent markers of islet cell death in general, and together might distinguish death arising from islets vs. other tissues. To validate unmethylated CHTOP as a biomarker for islet cell damage, we used digital PCR to measure cell-free circulating DNA in human populations. Compared to healthy controls, we observed that levels of differentially methylated CHTOP and INS were higher in youth with new onset type 1 diabetes and in healthy youth who have first-degree relatives with T1D. When tested in youth across a spectrum of metabolic dysfunction, increased levels of unmethylated INS and CHTOP were observed in obese individuals compared to lean controls. Together, these data suggest that simultaneous measurement of both circulating differentially methylated INS and CHTOP is likely to detect islet death in T1D, and raise new questions about β-cell health in populations at risk for both T1D and T2D. Importantly, our data support the use of multiple parameters to increase the accuracy of biomarkers of β-cell health in youth with diabetes. Bisulphite-converted DNA from 64 samples of human pancreatic islets were hybridised to the Illumina Infinium 450K Human Methylation BeadChip.