Identification of the inflammasome Nlrp1b as the candidate gene conferring diabetes risk at the Idd4.1 locus in the nonobese diabetic mouse.

Type 1 diabetes in the NOD mouse model has been linked to >30 insulin-dependent diabetes (Idd) susceptibility loci. Idd4 on chromosome 11 consists of two subloci, Idd4.1 and Idd4.2. Using congenic analysis of alleles in NOD and NOD-resistant (NOR) mice, we previously defined Idd4.1 as an interval containing >50 genes that controlled expression of genes in the type 1 IFN pathway. In this study, we report refined mapping of Idd4.1 to a 1.1-Mb chromosomal region and provide genomic sequence analysis and mechanistic evidence supporting its role in innate immune regulation of islet-directed autoimmunity. Genetic variation at Idd4.1 was mediated by radiation-sensitive hematopoietic cells, and type 1 diabetes protection conferred by the NOR allele was abrogated in mice treated with exogenous type 1 IFN-ß. Next generation sequence analysis of the full Idd4.1 genomic interval in NOD and NOR strains supported Nlrp1b as a strong candidate gene for Idd4.1. Nlrp1b belongs to the Nod-like receptor (NLR) gene family and contributes to inflammasome assembly, caspase-1 recruitment, and release of IL-1ß. The Nlrp1b of NOR was expressed as an alternative spliced isoform that skips exon 9, resulting in a premature stop codon predicted to encode a truncated protein. Functional analysis of the truncated NOR Nlrp1b protein demonstrated that it was unable to recruit caspase-1 and process IL-1ß. Our data suggest that Idd4.1-dependent protection from islet autoimmunity is mediated by differences in type 1 IFN- and IL-1ß-dependent immune responses resulting from genetic variation in Nlrp1b.

Polymorphism in the innate immune receptor SIRPα controls CD47 binding and autoimmunity in the nonobese diabetic mouse.

The signal regulatory protein (SIRP) locus encodes a family of paired receptors that mediate both activating and inhibitory signals and is associated with type 1 diabetes (T1D) risk. The NOD mouse model recapitulates multiple features of human T1D and enables mechanistic analysis of the impact of genetic variations on disease. In this study, we identify Sirpa encoding an inhibitory receptor on myeloid cells as a gene in the insulin-dependent diabetes locus 13.2 (Idd13.2) that drives islet inflammation and T1D. Compared to T1D-resistant strains, the NOD variant of SIRPα displayed greater binding to its ligand CD47, as well as enhanced T cell proliferation and diabetogenic potency. Myeloid cell-restricted expression of a Sirpa transgene accelerated disease in a dose-dependent manner and displayed genetic and functional interaction with the Idd5 locus to potentiate insulitis progression. Our study demonstrates that variations in both SIRPα sequence and expression level modulate T1D immunopathogenesis. Thus, we identify Sirpa as a T1D risk gene and provide insight into the complex mechanisms by which disease-associated variants act in concert to drive defined stages in disease progression.

Genome-wide end-sequenced BAC resources for the NOD/MrkTac() and NOD/ShiLtJ() mouse genomes.

Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive loss of insulin-secreting beta-cells by an autoimmune driven process. NOD mice represent a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe the development and characterization by end-sequencing of bacterial artificial chromosome (BAC) libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library, estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly (NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the CHORI-29 library has an average depth over the autosomes of 5.0-fold and 2.8-fold coverage of the X chromosome, the reduced X chromosome coverage being due to the use of a male donor for this library. Clones from this library have an average insert size of 205 kb and map to 93.9% of the reference mouse genome assembly, covering 95.7% of Ensembl genes. We have identified and validated 191,841 single nucleotide polymorphisms (SNPs) for DIL NOD and 114,380 SNPs for CHORI-29. In total we generated 229,736,133 bp of sequence for the DIL NOD and 121,963,211 bp for the CHORI-29. These BAC libraries represent a powerful resource for functional studies, such as gene targeting in NOD embryonic stem (ES) cell lines, and for sequencing and mapping experiments.

The idd4 locus displays sex-specific epistatic effects on type 1 diabetes susceptibility in nonobese diabetic mice.

The nonobese diabetic (NOD) mouse recapitulates many aspects of the pathogenesis of type 1 diabetes in humans, including inheritance as a complex trait. More than 20 Idd loci have been linked to type 1 diabetes susceptibility in NOD mice. Previously, we used linkage analysis of NOD crossed to the nonobese diabetes-resistant (NOR) strain and NOD congenic strains to map susceptibility to both spontaneous and cyclophosphamide-accelerated type 1 diabetes to the Idd4 locus on chromosome 11 that displayed a sex-specific effect on diabetes susceptibility. Here, we elucidate the complex genetic architecture of Idd4 by analysis of congenic strains on the NOD and NOR backgrounds. We previously refined Idd4.1 to 1.4 Mb and demonstrated an impact of this interval on type 1 interferon pathways in antigen-presenting cells. Here, we identify a second subregion, the 0.92 Mb Idd4.2 locus located telomeric to Idd4.1. Strikingly, Idd4.2 displayed a sex-specific, epistatic interaction with Idd4.1 in NOR.NOD congenic females that was not observed in syngenic males. Idd4.2 contains 29 genes, and promising candidates for the Idd4.2 effect on type 1 diabetes are described. These data demonstrate sex-dependent interaction effects on type 1 diabetes susceptibility and provide a framework for functional analysis of Idd4.2 candidate genes.

Molecular genetic analysis of the Idd4 locus implicates the IFN response in type 1 diabetes susceptibility in nonobese diabetic mice.

High-resolution mapping and identification of the genes responsible for type 1 diabetes (T1D) has proved difficult because of the multigenic etiology and low penetrance of the disease phenotype in linkage studies. Mouse congenic strains have been useful in refining Idd susceptibility loci in the NOD mouse model and providing a framework for identification of genes underlying complex autoimmune syndromes. Previously, we used NOD and a nonobese diabetes-resistant strain to map the susceptibility to T1D to the Idd4 locus on chromosome 11. Here, we report high-resolution mapping of this locus to 1.4 megabases. The NOD Idd4 locus was fully sequenced, permitting a detailed comparison with C57BL/6 and DBA/2J strains, the progenitors of T1D resistance alleles found in the nonobese diabetes-resistant strain. Gene expression arrays and quantitative real-time PCR were used to prioritize Idd4 candidate genes by comparing macrophages/dendritic cells from congenic strains where allelic variation was confined to the Idd4 interval. The differentially expressed genes either were mapped to Idd4 or were components of the IFN response pathway regulated in trans by Idd4. Reflecting central roles of Idd4 genes in Ag presentation, arachidonic acid metabolism and inflammation, phagocytosis, and lymphocyte trafficking, our combined analyses identified Alox15, Alox12e, Psmb6, Pld2, and Cxcl16 as excellent candidate genes for the effects of the Idd4 locus.