Project description:Recent advances in the understanding of the genetics of type 2 diabetes (T2D) susceptibility have focused attention on the regulation of transcriptional activity within the pancreatic beta-cell. MicroRNAs (miRNAs) represent an important component of regulatory control, and have proven roles in the development of human disease and control of glucose homeostasis. We set out to establish the miRNA profile of human pancreatic islets and of enriched beta-cell populations, and to explore their potential involvement in T2D susceptibility. We used Illumina small RNA sequencing to profile the miRNA fraction in three preparations each of primary human islets and of enriched beta-cells generated by fluorescence-activated cell sorting. In total, 366 miRNAs were found to be expressed (i.e. >100 cumulative reads) in islets and 346 in beta-cells; of the total of 384 unique miRNAs, 328 were shared. A comparison of the islet-cell miRNA profile with those of 15 other human tissues identified 40 miRNAs predominantly expressed (i.e. >50% of all reads seen across the tissues) in islets. Several highly-expressed islet miRNAs, such as miR-375, have established roles in the regulation of islet function, but others (e.g. miR-27b-3p, miR-192-5p) have not previously been described in the context of islet biology. As a first step towards exploring the role of islet-expressed miRNAs and their predicted mRNA targets in T2D pathogenesis, we looked at published T2D association signals across these sites. We found evidence that predicted mRNA targets of islet-expressed miRNAs were globally enriched for signals of T2D association (p-values <0.01, q-values <0.1). At six loci with genome-wide evidence for T2D association (AP3S2, KCNK16, NOTCH2, SCL30A8, VPS26A, and WFS1) predicted mRNA target sites for islet-expressed miRNAs overlapped potentially causal variants. In conclusion, we have described the miRNA profile of human islets and beta-cells and provide evidence linking islet miRNAs to T2D pathogenesis. Examination of the miRNA profiles in 3 preparations of isolated pancreatic islets and 3 preparations of FACS-enriched pancreatic beta-cells

Project description:Recent advances in the understanding of the genetics of type 2 diabetes (T2D) susceptibility have focused attention on the regulation of transcriptional activity within the pancreatic beta-cell. MicroRNAs (miRNAs) represent an important component of regulatory control, and have proven roles in the development of human disease and control of glucose homeostasis. We set out to establish the miRNA profile of human pancreatic islets and of enriched beta-cell populations, and to explore their potential involvement in T2D susceptibility. We used Illumina small RNA sequencing to profile the miRNA fraction in three preparations each of primary human islets and of enriched beta-cells generated by fluorescence-activated cell sorting. In total, 366 miRNAs were found to be expressed (i.e. >100 cumulative reads) in islets and 346 in beta-cells; of the total of 384 unique miRNAs, 328 were shared. A comparison of the islet-cell miRNA profile with those of 15 other human tissues identified 40 miRNAs predominantly expressed (i.e. >50% of all reads seen across the tissues) in islets. Several highly-expressed islet miRNAs, such as miR-375, have established roles in the regulation of islet function, but others (e.g. miR-27b-3p, miR-192-5p) have not previously been described in the context of islet biology. As a first step towards exploring the role of islet-expressed miRNAs and their predicted mRNA targets in T2D pathogenesis, we looked at published T2D association signals across these sites. We found evidence that predicted mRNA targets of islet-expressed miRNAs were globally enriched for signals of T2D association (p-values <0.01, q-values <0.1). At six loci with genome-wide evidence for T2D association (AP3S2, KCNK16, NOTCH2, SCL30A8, VPS26A, and WFS1) predicted mRNA target sites for islet-expressed miRNAs overlapped potentially causal variants. In conclusion, we have described the miRNA profile of human islets and beta-cells and provide evidence linking islet miRNAs to T2D pathogenesis.

Project description: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.

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Single-cell chromatin accessibility identifies pancreatic islet cell type- and state-specific regulatory programs of diabetes risk

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