Expression-Based Genome-Wide Association Study Links Vitamin D-Binding Protein With Autoantigenicity in Type 1 Diabetes.

Type 1 diabetes (T1D) is caused by autoreactive T cells that recognize pancreatic islet antigens and destroy insulin-producing β-cells. This attack results from a breakdown in tolerance for self-antigens, which is controlled by ectopic antigen expression in the thymus and pancreatic lymph nodes (PLNs). The autoantigens known to be involved include a set of islet proteins, such as insulin, GAD65, IA-2, and ZnT8. In an attempt to identify additional antigenic proteins, we performed an expression-based genome-wide association study using microarray data from 118 arrays of the thymus and PLNs of T1D mice. We ranked all 16,089 protein-coding genes by the likelihood of finding repeated differential expression and the degree of tissue specificity for pancreatic islets. The top autoantigen candidate was vitamin D-binding protein (VDBP). T-cell proliferation assays showed stronger T-cell reactivity to VDBP compared with control stimulations. Higher levels and frequencies of serum anti-VDBP autoantibodies (VDBP-Abs) were identified in patients with T1D (n = 331) than in healthy control subjects (n = 77). Serum vitamin D levels were negatively correlated with VDBP-Ab levels in patients in whom T1D developed during the winter. Immunohistochemical localization revealed that VDBP was specifically expressed in α-cells of pancreatic islets. We propose that VDBP could be an autoantigen in T1D

Impact of blood collection and processing on peripheral blood gene expression profiling in type 1 diabetes

Abstract Background The natural history of type 1 diabetes (T1D) is challenging to investigate, especially as pre-diabetic individuals are difficult to identify. Numerous T1D consortia have been established to collect whole blood for gene expression analysis from individuals with or at risk to develop T1D. However, with no universally accepted protocol for their collection, differences in sample processing may lead to variances in the results. Here, we examined whether the choice of blood collection tube and RNA extraction kit leads to differences in the expression of genes that are changed during the progression of T1D, and if these differences could be minimized by measuring gene expression directly from the lysate of whole blood. Results Microarray analysis showed that the expression of 901 genes is highly influenced by sample processing using the PAXgene versus the Tempus system. These included a significant number of lymphocyte-specific genes and genes whose expression has been reported to differ in the peripheral blood of at-risk and T1D patients compared to controls. We showed that artificial changes in gene expression occur when control and T1D samples were processed differently. The sample processing-dependent differences in gene expression were largely due to loss of transcripts during the RNA extraction step using the PAXgene system. The majority of differences were not observed when gene expression was measured in whole blood lysates prepared from blood collected in PAXgene and Tempus tubes. Conclusion We showed that the gene expression profile of samples processed using the Tempus system is more accurate than that of samples processed using the PAXgene system. Variation in sample processing can result in misleading changes in gene expression. However, these differences can be minimized by measuring gene expression directly in whole blood lysates

Additional file 3: of Impact of blood collection and processing on peripheral blood gene expression profiling in type 1 diabetes

Correction Factor to convert expression in PAXgene-processed samples to expression in Tempus-processed samples. Correction factor (based on NanoString gene expression data) to convert expression of COMMD6, COX6C, COX7B, LSM3, RPS24, and SUB1 measured in PAXgene-processed RNA samples to expression measured in Tempus-processed RNA samples. (DOCX 46 kb

Additional file 1: of Impact of blood collection and processing on peripheral blood gene expression profiling in type 1 diabetes

Differentially expressed genes in peripheral blood of female vs. male subjects. List of genes that are differentially expressed in the peripheral blood of female vs. male subjects, gene descriptions, and fold-change data. (DOCX 89 kb

Additional file 2: of Impact of blood collection and processing on peripheral blood gene expression profiling in type 1 diabetes

Genes detected only using the PAXgene or the Tempus system. List of genes that are detected in the majority of samples collected in one type of tube (PAXgene or Tempus), but not detected in any samples collected with the other type of tube. Microarray probe IDs and gene descriptions are also included. (DOCX 84 kb