Monogenic Diabetes and Integrated Stress Response Genes Display Altered Gene Expression in Type 1 Diabetes

Abstract

Type 1 diabetes (T1D) has a multifactorial autoimmune etiology, involving environmental prompts and polygenic predisposition. We hypothesized that pancreata from individuals with and at risk for T1D would exhibit dysregulated expression of genes associated with monogenic forms of diabetes caused by nonredundant single-gene mutations. Using a "monogenetic transcriptomic strategy," we measured the expression of these genes in human T1D, autoantibody-positive (autoantibody+), and control pancreas tissues with real-time quantitative PCR in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Gene and protein expression was visualized in situ with use of immunofluorescence, RNAscope, and confocal microscopy. Two dozen monogenic diabetes genes showed altered expression in human pancreata from individuals with T1D versus unaffected control subjects. Six of these genes also saw dysregulation in pancreata from autoantibody+ individuals at increased risk for T1D. As a subset of these genes are related to cellular stress responses, we measured integrated stress response (ISR) genes and identified 20 with altered expression in T1D pancreata, including three of the four eIF2α-dependent kinases. Equally intriguing, we observed significant repression of the three arms of the ISR in autoantibody+ pancreata. Collectively, these efforts suggest monogenic diabetes and ISR genes are dysregulated early in the T1D disease process and likely contribute to the disorder's pathogenesis.

Figure 1

Monogenic diabetes genes were sorted into four physiological groups: immune, β-cell function, β-cell development, and ER function/stress (Supplementary Table 7). A: Scatter plot of gene expression data for STAT5B, a gene representing an immune monogenic diabetes gene, studied using RTqPCR in human organ donor pancreata. Cq values from the unaffected (control) and T1D cohorts were independently normalized using the geometric mean of three pancreas-specific RGs, and the FD is calculated based on the ratio of the means (Research Design and Methods). See Research Design and Methods for statistical analysis (P values and q values [estimation of false discovery rates]). N/D refers to number (N) of samples yielding no data out of the total (D). B: Widefield IF of STAT5B (green) and overlay with insulin (INS), glucagon (GCG), and somatostatin (SST) from a control and T1D pancreas. Final panel in each row shows combined STAT5B ISH (RNAscope [green dots]) coupled with IF for CD99 and KRT19. Magnification bars = 40 μm. C: Confocal imaging on a Nikon A1plus confocal microscope of STAT5B (green), insulin (INS), glucagon (GCG), and somatostatin (SST), showing an overlay of an islet from an unaffected and T1D pancreas (left panels). 3D Z-stacks were constructed with a 2.5-μm step size and compressed to a 2D maximum intensity projection for display. Individual channels in black and white identify cells positive only for STAT5B and negative for the other endocrine hormones, illustrated with yellow arrows (overlay) and with insets in the respective black and white channels.

Figure 2

A: Scatter plot of RTqPCR data depicting expression levels for GATA4, a monogenic diabetes gene in the category β-cell function (Supplementary Table 7). The FD is calculated based on the ratio of the means (Research Design and Methods). See Research Design and Methods for statistical analysis (P values and q values [estimation of false discovery rates]). N/D refers to number (N) of samples yielding no data out of the total (D). B: Widefield IF of GATA4 (green) and overlay with insulin (INS), glucagon (GCG), and somatostatin (SST) from a control and T1D pancreas. Final panel in each row shows combined GATA4 ISH (RNAscope [green dots]) coupled with IF for CD99 and KRT19. Magnification bars = 40 μm.

Figure 3

Scatter plot of RTqPCR data depicting expression levels for GLIS3, a monogenic diabetes gene in the category β-cell development (Supplementary Table 7). The FD is calculated based on the ratio of the means (Research Design and Methods). See Research Design and Methods for statistical analysis (P values and q values [estimation of false discovery rates]). N/D refers to number (N) of samples yielding no data out of the total (D). B: Widefield IF of GLIS3 (green) and overlay with insulin (INS), glucagon (GCG), and somatostatin (SST) from a control and T1D pancreas. Yellow outlines depict the islets to illustrate the significant reduction of GLIS3 in the T1D islet. Final panel in each row shows combined GLIS3 ISH (RNAscope [green dots]) coupled with IF for CD99 and KRT19. Magnification bars = 40 μm.

Figure 4

A: Scatter plot of RTqPCR data depicting expression levels for WFS1, a monogenic diabetes gene in the category ER function/stress (Supplementary Table 7). The FD is calculated based on the ratio of the means (Research Design and Methods). See Research Design and Methods for statistical analysis (P values and q values [estimation of false discovery rates]). N/D refers to number (N) of samples yielding no data out of the total (D). B: Widefield IF of WFS1 (green) and overlay with insulin (INS), glucagon (GCG), and somatostatin (SST) from a control and T1D pancreas. Final panel in each row shows combined WFS1 ISH (RNAscope [green dots]) coupled with IF for CD99 and KRT19. Magnification bars = 40 μm. C: Confocal imaging on a Nikon A1plus confocal microscope of WFS1 (green), insulin (INS), glucagon (GCG), and somatostatin (SST), showing an overlay of an islet from an unaffected and T1D pancreas (left panels). 3D Z-stacks were constructed with a 2.5-μm step size and compressed to a 2D maximum intensity projection for display. Individual channels in black and white identify cells positive only for WFS1 and negative for the other endocrine hormones, illustrated with yellow arrows (overlay) and with insets in the respective black and white channels.

Figure 5

A: Scatter plot of RTqPCR data depicting expression levels for EIF2AK3/PERK, a representative gene in arm 3 of the ISR. The FD is calculated based on the ratio of the means (Research Design and Methods). See Research Design and Methods for statistical analysis (P values and q values [estimation of false discovery rates]). N/D refers to number (N) of samples yielding no data out of the total (D). B: Widefield IF of PERK (green) and overlay with insulin (INS), glucagon (GCG), and somatostatin (SST) from a control and T1D pancreas. Final panel in each row shows combined PERK ISH (RNAscope [green dots]) coupled with IF for CD99 and KRT19. Magnification bars = 40 μm. C: Scatter plots of RTqPCR data depicting expression levels for ISR genes from the apex of each ISR arm 1–3 in T1D vs. unaffected control pancreata: ATF6, ERN1/IRE1α, EIF2AK2/PKR, EIF2AK3/PERK, and EIF2AK4/GCN2. The FD is calculated based on the ratio of the means. See Research Design and Methods for statistical analysis (P values and q values [estimation of false discovery rates]). D: Scatter plot of RTqPCR data depicting expression levels for genes significantly repressed in autoantibody+ (AAB+) pancreata in each of the three arms of the ISR: MBTPS1/S1P, MBTPS2/S2P, ERN1/IRE1α, and EIF2AK2/PKR.