Diane Hopkins

A functional variant of SUMO4, a new I|[kappa]|B|[alpha]| modifier, is associated with type 1 diabetes

Previous studies have suggested more than 20 genetic intervals that are associated with susceptibility to type 1 diabetes (T1D), but identification of specific genes has been challenging and largely limited to known candidate genes. Here, we report evidence for an association between T1D and multiple single-nucleotide polymorphisms in 197 kb of genomic DNA in the IDDM5 interval. We cloned a new gene (SUMO4), encoding small ubiquitin-like modifier 4 protein, in the interval. A substitution (M55V) at an evolutionarily conserved residue of the crucial CUE domain of SUMO4 was strongly associated with T1D (P = 1.9 × 10−7). SUMO4 conjugates to IκBα and negatively regulates NFκB transcriptional activity. The M55V substitution resulted in 5.5 times greater NFκB transcriptional activity and ∼2 times greater expression of IL12B, an NFκB-dependent gene. These findings suggest a new pathway that may be implicated in the pathogenesis of T1D.

Association between type 1 diabetes and GWAS SNPs in the southeast US Caucasian population

The present study was conducted to assess genetic associations for type 1 diabetes (T1D) reported in previous genome-wide association studies (GWAS). A total of 21 previously reported single-nucleotide polymorphisms (SNPs) were genotyped by TaqMan assays in 1434 Caucasian T1D patients and 1864 normal controls from Georgia. Analysis of the samples identified 18 SNPs (PTPN22, INS, IFIH1, SH2B3, ERBB3, CTLA4, C14orf181, CTSH, CLEC16A, CD69, ITPR3, C6orf173, SKAP2, PRKCQ, RNLS, IL27, SIRPG and CTRB2) with putative association.

Lack of correlation between the levels of soluble cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and the CT-60 genotypes.

BackgroundCytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulation of antigen-activated immune response and polymorphisms at the CTLA-4 gene have been shown to be associated with several autoimmune diseases including type-1 diabetes (T1D). The etiological mutation was mapped to the CT60-A/G single nucleotide polymorphism (SNP) that is believed to control the processing and production of soluble CTLA-4 (sCTLA-4).MethodsWe therefore determined sCTLA-4 protein levels in the sera from 82 T1D patients and 19 autoantibody positive (AbP) subjects and 117 autoantibody negative (AbN) controls using ELISA. The CT-60 SNP was genotyped for these samples by using PCR and restriction enzyme digestion of a 268 bp DNA segment containing the SNP. Genotyping of CT-60 SNP was confirmed by dye terminating sequencing reaction.ResultsHigher levels of sCTLA-4 were observed in T1D (2.24 ng/ml) and AbP (mean = 2.17 ng/ml) subjects compared to AbN controls (mean = 1.69 ng/ml) with the differences between these subjects becoming significant with age (p = 0.02). However, we found no correlation between sCTLA-4 levels and the CTLA-4 CT-60 SNP genotypes.ConclusionConsistent with the higher serum sCTLA-4 levels observed in other autoimmune diseases, our results suggest that sCTLA-4 may be a risk factor for T1D. However, our results do not support the conclusion that the CT-60 SNP controls the expression of sCTLA-4.

Prospective assessment in newborns of diabetes autoimmunity (PANDA): Maternal understanding of infant diabetes risk

Purpose: To assess accuracy of mothers’ understanding of their newborns’ genetic risk for type 1 diabetes and to identify predictors of the comprehension and retention of genetic information. Methods: Mothers of 435 newborns genetically screened at birth were informed of the infant’s risk for type 1 diabetes using a standard script that provided both categorical and numerical risk information. The mothers’ comprehension and retention of this information were assessed by structured interview on two occasions, ∼3.6 weeks and ∼3.9 months postnotification. Results: At the initial interview, 73.1% of mothers gave a correct estimate of their child’s genetic risk, 3.2% overestimated risk, 13.3% underestimated risk, and 10.3% could not recall risk at all. At the follow-up interview, fewer mothers (61.9%) correctly estimated their child’s risk and more mothers (24.4%) underestimated their child’s risk. Maternal accuracy was associated with maternal education, ethnic minority status, infant risk status, maternal ability to spontaneously recall both categorical and numerical risk estimates, and length of time since risk notification. Underestimation of risk was associated with maternal education, family history of diabetes, time since risk notification, and maternal anxiety about the baby’s risk. Conclusion: The accuracy of mothers’ recall of infant risk declines over time, with an increasing number of mothers underestimating the infant’s risk. Effective risk communication strategies need to be developed and incorporated into genetic screening programs.

Discovery and Validation of Serum Protein Changes in Type 1 Diabetes Patients Using High Throughput Two Dimensional Liquid Chromatography-Mass Spectrometry and Immunoassays

Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10−27), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10−20), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10−16); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10−5) and myeloperoxidase (OR = 0.51, p < 10−41). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10−37), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10−46), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10−16) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10−11) and myeloperoxidase (OR = 0.10, p < 10−43) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.

Type 1 diabetes patients have significantly lower frequency of plasmacytoid dendritic cells in the peripheral blood

Dendritic cells uniquely orchestrate the delicate balance between T cell immunity and regulation and an imbalance favoring immunogenic rather than tolerogenic DC is believed to contribute to the development of autoimmune diseases such as type 1 diabetes (T1D). In this study, we determined the frequencies of three blood DC subsets (pDC, mDC1 and mDC2) in 72 T1D patients and 75 normal controls using the Miltenyi blood DC enumeration kit. The frequency of blood pDC was found to be negatively correlated with subject age in both normal controls and T1D patients (p=0.0007), while the frequency of mDC1 and mDC2 do not change significantly with subject age. More importantly, the mean frequency of pDC in blood was, after adjusting for age, significantly lower in T1D (mean=0.127%) than controls (mean=0.188%) (p<6.0 x 10(-5)), whereas no difference was observed for mDC1 and mDC2 between T1D and controls. Furthermore, T1D patients have a lower proportion of pDC and higher proportion of mDC1 among the total blood DC population than normal controls. These results indicate that the frequency of blood pDC and the pDC/mDC1 ratio are negatively associated with T1D.

Genetically Dependent ERBB3 Expression Modulates Antigen Presenting Cell Function and Type 1 Diabetes Risk

Type 1 diabetes (T1D) is an autoimmune disease resulting from the complex interaction between multiple susceptibility genes, environmental factors and the immune system. Over 40 T1D susceptibility regions have been suggested by recent genome-wide association studies; however, the specific genes and their role in the disease remain elusive. The objective of this study is to identify the susceptibility gene(s) in the 12q13 region and investigate the functional link to the disease pathogenesis. A total of 19 SNPs in the 12q13 region were analyzed by the TaqMan assay for 1,434 T1D patients and 1,865 controls. Thirteen of the SNPs are associated with T1D (best p = 4×10−11), thus providing confirmatory evidence for at least one susceptibility gene in this region. To identify candidate genes, expression of six genes in the region was analyzed by real-time RT-PCR for PBMCs from 192 T1D patients and 192 controls. SNP genotypes in the 12q13 region are the main factors that determine ERBB3 mRNA levels in PBMCs. The protective genotypes for T1D are associated with higher ERBB3 mRNA level (p<10−10). Furthermore, ERBB3 protein is expressed on the surface of CD11c+ cells (dendritic cells and monocytes) in peripheral blood after stimulation with LPS, polyI:C or CpG. Subjects with protective genotypes have significantly higher percentages of ERBB3+ monocytes and dendritic cells (p = 1.1×10−9); and the percentages of ERBB3+ cells positively correlate with the ability of APC to stimulate T cell proliferation (R2 = 0.90, p<0.0001). Our results indicate that ERBB3 plays a critical role in determining APC function and potentially T1D pathogenesis.

Risk of Type 1 Diabetes Progression in Islet Autoantibody-Positive Children Can Be Further Stratified Using Expression Patterns of Multiple Genes Implicated in Peripheral Blood Lymphocyte Activation and Function

There is tremendous scientific and clinical value to further improving the predictive power of autoantibodies because autoantibody-positive (AbP) children have heterogeneous rates of progression to clinical diabetes. This study explored the potential of gene expression profiles as biomarkers for risk stratification among 104 AbP subjects from the Diabetes Autoimmunity Study in the Young (DAISY) using a discovery data set based on microarray and a validation data set based on real-time RT-PCR. The microarray data identified 454 candidate genes with expression levels associated with various type 1 diabetes (T1D) progression rates. RT-PCR analyses of the top-27 candidate genes confirmed 5 genes (BACH2, IGLL3, EIF3A, CDC20, and TXNDC5) associated with differential progression and implicated in lymphocyte activation and function. Multivariate analyses of these five genes in the discovery and validation data sets identified and confirmed four multigene models (BI, ICE, BICE, and BITE, with each letter representing a gene) that consistently stratify high- and low-risk subsets of AbP subjects with hazard ratios >6 (P < 0.01). The results suggest that these genes may be involved in T1D pathogenesis and potentially serve as excellent gene expression biomarkers to predict the risk of progression to clinical diabetes for AbP subjects.

The Expression of Inflammatory Genes Is Upregulated in Peripheral Blood of Patients With Type 1 Diabetes

OBJECTIVE Our previous gene expression microarray studies identified a number of genes differentially expressed in patients with type 1 diabetes (T1D) and islet autoantibody-positive subjects. This study was designed to validate these gene expression changes in T1D patients and to identify gene expression changes in diabetes complications. RESEARCH DESIGH AND METHODS We performed high-throughput real-time RT-PCR to validate gene expression changes in peripheral blood mononuclear cells (PBMCs) from a large sample set of 928 T1D patients and 922 control subjects. RESULTS Of the 18 genes analyzed here, eight genes (S100A8, S100A9, MNDA, SELL, TGFB1, PSMB3, CD74, and IL12A) had higher expression and three genes (GNLY, PSMA4, and SMAD7) had lower expression in T1D patients compared with control subjects, indicating that genes involved in inflammation, immune regulation, and antigen processing and presentation are significantly altered in PBMCs from T1D patients. Furthermore, one adhesion molecule (SELL) and three inflammatory genes mainly expressed by myeloid cells (S100A8, S100A9, and MNDA) were significantly higher in T1D patients with complications (odds ratio [OR] 1.3–2.6, adjusted P value = 0.005–10−8), especially those patients with neuropathy (OR 4.8–7.9, adjusted P value <0.005). CONCLUSIONS These findings suggest that inflammatory mediators secreted mainly by myeloid cells are implicated in T1D and its complications.

Chemokine (C-C motif) ligand 2 (CCL2) in sera of patients with type 1 diabetes and diabetic complications.

Background Chemokine (C-C motif) ligand 2 (CCL2), commonly known as monocyte chemoattractant protein-1 (MCP-1), has been implicated in the pathogenesis of many diseases characterized by monocytic infiltration. However, limited data have been reported on MCP-1 in type 1 diabetes (T1D) and the findings are inconclusive and inconsistent. Methods In this study, MCP-1 was measured in the sera from 2,472 T1D patients and 2,654 healthy controls using a Luminex assay. The rs1024611 SNP in the promoter region of MCP-1 was genotyped for a subset of subjects (1764 T1D patients and 1323 controls) using the TaqMan-assay. Results Subject age, sex or genotypes of MCP-1 rs1024611SNP did not have a major impact on serum MCP-1 levels in either healthy controls or patients. While hemoglobin A1c levels did not have a major influence on serum MCP-1 levels, the mean serum MCP-1 levels are significantly higher in patients with multiple complications (mean = 242 ng/ml) compared to patients without any complications (mean = 201 ng/ml) (p = 3.5×10−6). Furthermore, mean serum MCP-1 is higher in controls (mean = 261 ng/ml) than T1D patients (mean = 208 ng/ml) (p<10−23). More importantly, the frequency of subjects with extremely high levels (>99th percentile of patients or 955 ng/ml) of serum MCP-1 is significantly lower in the T1D group compared to the control group (odds ratio = 0.11, p<10−33). Conclusion MCP-1 may have a dual role in T1D and its complications. While very high levels of serum MCP-1 may be protective against the development of T1D, complications are associated with higher serum MCP-1 levels within the T1D group.