The diabetes-prone BioBreeding (BB) and Komeda diabetes-prone (KDP) rats are both spontaneous animal models of human autoimmune, T-cell-associated type 1 diabetes. Both resemble the human disease, and consequently, susceptibility genes for diabetes found in these two strains can be considered as potential candidate genes in humans. Recently, a frameshift deletion in Ian4, a member of the immune-associated nucleotide (Ian)-related gene family, has been shown to map to BB rat Iddm1. In the KDP rat, a nonsense mutation in the T-cell regulatory gene, Cblb, has been described as a major susceptibility locus. Following a strategy of examining the human orthologues of susceptibility genes identified in animal models for association with type 1 diabetes, we identified single nucleotide polymorphisms (SNPs) from each gene by resequencing PCR product from at least 32 type 1 diabetic patients. Haplotype tag SNPs (htSNPs) were selected and genotyped in 754 affected sib-pair families from the U.K. and U.S. Evaluation of disease association by a multilocus transmission/disequilibrium test (TDT) gave a P value of 0.484 for IAN4L1 and 0.692 for CBLB, suggesting that neither gene influences susceptibility to common alleles of human type 1 diabetes in these populations.

Development of diabetes in the BB rat involves at least three genes: Iddm1/lyp on chromosome 4, RT1u (at Iddm2) in the major histocompatibility complex (MHC) on chromosome 20, and a third unmapped gene (1,2). One unusual feature of this animal model is the severe lymphopenia that is essential for the development of the diabetic phenotype and that is inherited as a Mendelian trait (3). Life-long and profound T-cell lymphopenia is characterized by a reduction in peripheral CD4+ T-cells, an even greater reduction of CD8+ T-cells (4), and an almost total absence of RT6+ T-cells (5). The lymphopenia gene is involved in the regulation of apoptosis in the T-cell lineage and is, therefore, responsible for loss of critical T-cells, resulting in autoimmunity (6). Recently, two groups have independently shown, by positional cloning of Iddm1/lyp, that lymphopenia is due to a frameshift deletion in Ian4 (also called Ian5) of the immune-associated nucleotide (Ian)-related gene family (6,7), resulting in a truncated protein product. This deletion was only found in strains that have lymphopenia and diabetes (6). The human orthologue of Ian4 (IAN4L1) belongs to a family of at least 10 genes that encode GTP-binding proteins and are located in a 300-kb interval of human chromosome 7q36.

The KDP rat was derived as a substrain of the Long-Evans Tokushima lean (LETL) rat and shows 100% development of moderate to severe insulitis within 220 days of age (8,9). The LETL rat is characterized by sudden onset of polyuria, polyphagia, hyperglycemia, weight loss, and autoimmune destruction of pancreatic B-cells, while showing no significant T-cell lymphopenia and no sex-specific differences in rate of onset or severity (8). As with the BB rat, the KDP rat possesses the diabetogenic RT1u haplotype, adding to its relevance as a model of type 1 diabetes. In addition to the MHC, another unlinked locus, Iddm/kdp1, is essential in the development of moderate to severe insulitis and the onset of diabetes (10). Iddm/kdp1 has been mapped to a nonsense mutation in CBLB (Casitas B-lineage lymphoma b, or Cas-Br-M murine ecotropic retroviral transforming sequence b), a gene shown to have a role in the regulation of tyrosine kinase signaling pathways (1114). This mutation results in the removal of 484 amino acids, including the proline-rich and leucine zipper domains of the protein, and is specific to the KDP rat and the original LETL strain. It is not found in the nondiabetic KND (Komeda nondiabetic) or LETO (Long-Evans Tokushima Otsuka) strains (15). Homozygous mice generated to be deficient in Cblb develop spontaneous autoimmunity, characterized by T- and B-cell infiltration of multiple organs (16). Taken together, this evidence suggests that Cblb is probably the disease susceptibility gene at Iddm/Kdp1 and, consequently, a major susceptibility gene for diabetes in the rat.

We, therefore, resequenced both IAN4L1 and CBLB as candidates for human type 1 diabetes susceptibility. For IAN4L1, we resequenced the entire gene, covering 12.2 kb, comprising three exons and introns and 3 kb 3′ and 5′ of the gene in 32 type 1 diabetic subjects, identifying 30 single nucleotide polymorphisms (SNPs), 19 of which were novel (Table 1). Of the 30 SNPs, 7 were exonic: 1 in exon 1, which contains the 5′ untranslated region, and 6 in exon 3. At CBLB, which extends over 230 kb (including three alternative, untranslated exon 1s), we resequenced 12.6 kb in 96 type 1 diabetic subjects, encompassing exons, intron/exon boundaries, and 2.5 kb 3′ and 5′ of the gene. From the CBLB sequence data, we identified 37 polymorphisms, of which 26 were novel (Table 2). These comprised 32 SNPs and five insertion/deletions. Of the 37 polymorphisms, 7 were exonic: 1 in each of exons 6, 9, 11, and 12 and 3 in exon 10. However, no nonsynonymous variants were observed in either gene, nor were there any other obvious candidates for variants that might change function or expression (Tables 1 and 2). For CBLB, we were unable to sequence exons 18, 1A, or 1B (although we covered 135 of 195 bp of exon 1C), and consequently, it was not possible to fully represent them directly with our haplotype tag SNP (htSNP) selection.

From the 21 polymorphisms in CBLB and 25 in IAN4L1 with allele frequencies >3%, we selected nine htSNPs for each, capturing the allelic variation within the genes with a minimum R2 of 0.8 (Tables 1 and 2), using the htSNP selection method described by Chapman et al. (17). To further reduce genotyping costs, we adopted a two-stage strategy, in which we only proceed to the second stage of genotyping if the results from the first stage offered some possibility of an overall significant result. In stage 1, a collection of 754 affected sib-pairs, comprising 472 U.K. and 282 U.S. multiplex type 1 diabetic families (equivalent to 1,400 trios; set 1), are genotyped and tested for association using the multilocus TDT, which tests for association between disease and htSNPs due to linkage disequilibrium (LD) with one or more causal variants (17). Transmissions of SNP alleles not genotyped in stage 1 can also be predicted using multiple regression equations computed in the course of htSNP selection from the initial sequencing data (17). Stage 2, genotyping in 1,708 additional families (set 2) only proceeds if the stage-1 multilocus TDT P value is <0.1. By setting a threshold P value relatively high at the first stage, in order to avoid rejecting true positives, little power is lost when compared with a single-stage approach. After genotyping of set 2, statistical analysis is performed on the entire dataset (2,462 families). Given the currently available sample collection and the two-stage strategy adopted, we have over 90% power to detect an association with P = 1 × 10−4, assuming a relative risk of 1.5 conferred by each copy of the causal allele and a population frequency of the causal allele of 0.1, regardless of whether genotyping proceeds to stage 2.

Approaches to the statistical analysis of htSNPs have been described by Chapman et al. (17). It was demonstrated that in regions of strong LD, simple models considering only the main effects of htSNP genotypes were optimal or near optimal for detecting disease association. Consequently, the multilocus TDT is considered the most appropriate test. In stage 1, the multilocus TDT P value for association between type 1 diabetes and IAN4L1 was 0.484 and for CBLB was 0.692. Therefore, we did not proceed to genotype the additional set 2 families in either gene. To illustrate the predictions of ungenotyped markers that are possible using this new approach, Tables 1 and 2 include single-locus tests for all the common polymorphisms in set 1 families.

These results suggest that common alleles of IAN4L1 and CBLB do not contribute significantly to the familial clustering of human type 1 diabetes in the two populations analyzed. We cannot exclude the possibility that a common variant exists in either gene with an effect that is too small to be detected in a study of this size or that there is an unidentified polymorphism that is in much weaker LD with the htSNPs we analyzed. Had we genotyped all identified markers, our probability of detecting disease association would not have been substantially increased. Large introns and more extensive flanking DNA regions can be analyzed for association in the future by using the genome-wide SNP map that is under construction (18). By adopting an htSNP and a two-stage strategy, these candidate genes were quickly and economically evaluated for association with type 1 diabetes. This approach has allowed us to significantly reduce the genotyping burden (by ∼84% for CBLB and ∼87% for IAN4L1) and decrease turnaround time 1) by avoiding redundant genotyping of markers that can be imputed easily from the genotyping data of other markers and the patterns of LD across the gene and 2) by refraining from genotyping additional families in which there is limited possibility of obtaining an overall significant result. Although, in these data, common allelic variation in neither the IAN4L1 nor CBLB coding regions is associated with type 1 diabetes, genetic susceptibility data obtained from animal models can be directly applicable to humans, as has been found with the MHC (19) and CTLA4 (20). In addition, in our study, we have not excluded the possibility that alleles with frequencies <3% affect susceptibility to type 1 diabetes, and this remains a possibility. Whether or not exactly the same disease susceptibility genes in animal models are contributors to the familial clustering of disease in humans depends on the frequencies of causal alleles of the gene orthologues in human populations, a parameter that is subject to wide random variation. Nevertheless, even if a direct genetic susceptibility concordance is not found, the pathways that emerge from genetic studies of representative models and humans improve our understanding of disease mechanisms and how these might be modulated to reduce the risk of disease.

The 754 type 1 diabetic families were white European or of Caucasian European descent, with two parents and at least one affected child (472 Diabetes U.K. Warren 1 multiplex [21] and 282 multiplex ascertained in the U.S., obtained from the Human Biological Data Interchange [22]).

SNP identification and genotyping.

Direct sequencing of nested PCR products from 96 type 1 diabetic individuals for CBLB and 32 for IAN4L1 was performed using an Applied Biosystems (ABI) 3700 capillary sequencer (Foster City, CA). Polymorphisms were identified using the Staden Package (http://www.mrc-lmb.cam.ac.uk/pubseq/) and mapped to the golden path sequence (NCBI build 33). htSNPs were selected from the polymorphisms with >3% minor allele frequency in our sequencing panel using Stata (http://www.stata.com) and the htSNP package available from http://www-gene.cimr.cam.ac.uk/clayton/software/stata/.

Genotyping was performed using either Taqman MGB chemistry (Applied Biosystems) (23) or the Invader biplex assay (Third Wave Technologies, Madison, WI) (24). All genotyping data were double scored to minimize error. All SNP sequences are in dbSNP; sequencing and genotyping data can be obtained upon request (http://www-gene.cimr.cam.ac.uk/todd/human_data.shtml).

Annotation.

CBLB (European Molecular Biology Laboratory [EMBL] accession nos. U26710, full-length human CBLB mRNA; U26711, truncated form 1, human CBLB, lacking leucine zipper mRNA; amd U26712, truncated form 2, human CBLB, lacking leucine zipper mRNA) and IAN4L1 (EMBL accession no. AK002158) were annotated locally, importing Ensembl information into a temporary ACeDB database. Here, the gene structure was verified following a more thorough Blast analysis and then reextracted from ACeDB in GFF format and submitted to a local Gbrowse database (National Center for Biotechnology Information build 33) (DIL annotations viewable at http://dil-gbrowse.cimr.cam.ac.uk).

Statistical analysis.

All statistical analyses were performed within Stata making specific use of the Genassoc package (http://www-gene.cimr.cam.ac.uk/clayton/software/stata). All genotyping data were assessed for, and found to be in, Hardy-Weinberg equilibrium (P > 0.05).

TABLE 1

SNPs identified in IAN4L1 and single-locus test results

Variant nameMap positiondbSNPMinor allele frequencyAllelic R2TransmittedNot transmittedP valueLocation
DIL4283 149746862 rs3757411 0.355 99.35 1612 1602 0.78 5′ 
DIL4284 149747929 ss14452109 0.012 — — — — 5′ 
DIL4285 149748019 ss13452110 0.078 85.47 255 248 0.72 5′ 
DIL4287* 149748426 ss13452112 0.094 htSNP 343 333 0.67 5′ 
DIL4286* 149748431 ss13452111 0.281 htSNP 810 811 0.97 5′ 
DIL4344 149749043 rs3807383 0.281 100.00 1986 1985 0.97 5′ 
DIL4343* 149749122 rs917805 0.047 htSNP 49 66 0.07 5′ 
DIL4348 149749357 ss13452128 0.161 100.00 419 424 0.79 5′ 
DIL4347 149749574 ss13452127 0.016 — — — — Exon 1 
DIL4345* 149750328 ss13452125 0.266 htSNP 838 859 0.54 Intron 1 
DIL4346 149750358 ss13452126 0.375 100.00 1182 1192 0.78 Intron 1 
DIL4288 149750835 ss13452113 0.016 — — — — Intron 1 
DIL4289 149751082 ss13452114 0.172 100.00 2338 2323 0.60 Intron 1 
DIL4349 149752812 ss13452129 0.266 100.00 838 859 0.54 Intron 1 
DIL4350 149752813 ss13452130 0.016 — — — — Intron 1 
DIL4351 149752974 rs4725936 0.391 100.00 1615 1611 0.92 Intron 2 
DIL4352* 149753023 rs4725359 0.125 htSNP 2453 2470 0.49 Intron 2 
DIL4353* 149753046 ss13452133 0.438 htSNP 838 861 0.50 Intron 2 
DIL4354 149753102 ss13452134 0.016 — — — — Intron 2 
DIL4338 149753443 ss13452121 0.300 97.40 845 866 0.54 Intron 2 
DIL4355 149753684 ss13452135 0.276 100.00 838 859 0.54 Intron 2 
DIL4342 149754385 rs759011 0.321 86.19 846 867 0.54 Exon 3 
DIL4290 149754928 rs1046355 0.224 91.85 757 775 0.55 Exon 3 
DIL4291* 149755077 ss13452116 0.172 htSNP 2681 2655 0.11 Exon 3 
DIL4293 149755128 rs10361 0.269 88.14 775 796 0.51 Exon 3 
DIL4292 149755534 rs6598 0.261 80.68 699 717 0.53 Exon 3 
DIL4294* 149755599 rs2286899 0.097 htSNP 376 378 0.93 Exon 3 
DIL4295 149755861 rs2286898 0.274 97.32 838 857 0.58 3′UTR 
DIL4357* 149756048 ss13452136 0.160 htSNP 343 327 0.53 3′UTR 
DIL4358 149757510 ss13452137 0.267 98.39 2001 1981 0.54 3′ 
Variant nameMap positiondbSNPMinor allele frequencyAllelic R2TransmittedNot transmittedP valueLocation
DIL4283 149746862 rs3757411 0.355 99.35 1612 1602 0.78 5′ 
DIL4284 149747929 ss14452109 0.012 — — — — 5′ 
DIL4285 149748019 ss13452110 0.078 85.47 255 248 0.72 5′ 
DIL4287* 149748426 ss13452112 0.094 htSNP 343 333 0.67 5′ 
DIL4286* 149748431 ss13452111 0.281 htSNP 810 811 0.97 5′ 
DIL4344 149749043 rs3807383 0.281 100.00 1986 1985 0.97 5′ 
DIL4343* 149749122 rs917805 0.047 htSNP 49 66 0.07 5′ 
DIL4348 149749357 ss13452128 0.161 100.00 419 424 0.79 5′ 
DIL4347 149749574 ss13452127 0.016 — — — — Exon 1 
DIL4345* 149750328 ss13452125 0.266 htSNP 838 859 0.54 Intron 1 
DIL4346 149750358 ss13452126 0.375 100.00 1182 1192 0.78 Intron 1 
DIL4288 149750835 ss13452113 0.016 — — — — Intron 1 
DIL4289 149751082 ss13452114 0.172 100.00 2338 2323 0.60 Intron 1 
DIL4349 149752812 ss13452129 0.266 100.00 838 859 0.54 Intron 1 
DIL4350 149752813 ss13452130 0.016 — — — — Intron 1 
DIL4351 149752974 rs4725936 0.391 100.00 1615 1611 0.92 Intron 2 
DIL4352* 149753023 rs4725359 0.125 htSNP 2453 2470 0.49 Intron 2 
DIL4353* 149753046 ss13452133 0.438 htSNP 838 861 0.50 Intron 2 
DIL4354 149753102 ss13452134 0.016 — — — — Intron 2 
DIL4338 149753443 ss13452121 0.300 97.40 845 866 0.54 Intron 2 
DIL4355 149753684 ss13452135 0.276 100.00 838 859 0.54 Intron 2 
DIL4342 149754385 rs759011 0.321 86.19 846 867 0.54 Exon 3 
DIL4290 149754928 rs1046355 0.224 91.85 757 775 0.55 Exon 3 
DIL4291* 149755077 ss13452116 0.172 htSNP 2681 2655 0.11 Exon 3 
DIL4293 149755128 rs10361 0.269 88.14 775 796 0.51 Exon 3 
DIL4292 149755534 rs6598 0.261 80.68 699 717 0.53 Exon 3 
DIL4294* 149755599 rs2286899 0.097 htSNP 376 378 0.93 Exon 3 
DIL4295 149755861 rs2286898 0.274 97.32 838 857 0.58 3′UTR 
DIL4357* 149756048 ss13452136 0.160 htSNP 343 327 0.53 3′UTR 
DIL4358 149757510 ss13452137 0.267 98.39 2001 1981 0.54 3′ 

Variant name based on local naming scheme for polymorphisms. Map positions on human chromosome 7 from NCBI build 33. Minor allele frequencies shown are based on the sequencing panel of 32 type 1 diabetic subjects. Transmitted and not transmitted were estimated for the ungenotyped SNPs with allele frequencies >0.03 from the regression equations computed in the preliminary sequencing study.

*

Denotes htSNP. dbSNP numbers for all novel polymorphisms in boldface. Single-locus tests at SNPs other than htSNPs were calculated from imputed data based on htSNP genotypes and LD information obtained from the sequencing panel. Single-locus P values were obtained using paired t tests. Data were not imputed and tests not performed for SNPs with allele frequencies <0.03. UTR, untranslated region.

TABLE 2

Polymorphisms identified in CBLB and single-locus test results

Variant nameMap positiondbSNPMinor allele frequencyAllelic R2TransmittedNot transmittedP valueLocation
DIL4620 106873087 rs1503921 0.244 97.07 2220 2225 0.88 5′ 
DIL4621 106872769 ss13452139 0.023 — — — — 5′ 
DIL4622 106872519 ss13452140 0.005 — — — — 5′ 
DIL4623 106871969 rs1503922 0.005 — — — — 5′ 
DIL4624* 106867943 ss13452142 0.229 htSNP 599 594 0.89 5′ 
DIL4625 106776408 ss13452143 0.028 — — — — Intron 4 
DIL4649 106776404-5 ss13452167 0.128 87.18 323 321 0.94 Intron 4 
DIL4650 106776371-2 ss13452168 0.021 — — — — Intron 4 
DIL4651 106776332-6 ss13452169 0.261 88.48 663 655 0.79 Intron 4 
DIL4626* 106776303 rs3772512 0.109 htSNP 261 259 0.92 Intron 4 
DIL4627 106745995 ss13452145 0.016 — — — — Exon 6 
DIL4628* 106740946 rs3213928 0.027 htSNP 2727 2731 0.74 Intron 6 
DIL4629 106740829 ss13452147 0.005 — — — — Intron 6 
DIL4652 106740755 ss13452170 0.006 — — — — Intron 6 
DIL4630* 106737184 rs2289746 0.413 htSNP 1861 1879 0.62 Intron 8 
DIL5960 106734298 ss13452173 0.026 — — — — Intron 8 
DIL5961 106734124 ss13452174 0.005 — — — — Exon 9 
DIL4634 106720318 ss13452152 0.005 — — — — Exon 10 
DIL4633 106720255 rs2305035 0.258 93.40 624 609 0.61 Exon 10 
DIL4632 106720186 rs2305036 0.258 93.40 624 609 0.61 Exon 10 
DIL4631 106720058 ss13452149 0.012 — — — — Intron 10 
DIL4636* 106704073 rs2305037 0.286 htSNP 781 771 0.76 Exon 11 
DIL4635 106703765 ss13452153 0.286 100.00 781 771 0.76 Intron 11 
DIL4637 106702263 rs3772534 0.043 99.58 2761 2745 0.19 Exon 12 
DIL4663 106681919 ss13452172 0.005 — — — — Intron 14 
DIL4638 106658744 rs1042852 0.290 97.59 2059 2070 0.75 3′UTR 
DIL4639* 106658224 ss13452157 0.096 htSNP 199 199 0.99 3′ 
DIL4640 106657549 ss13452158 0.016 — — — — 3′ 
DIL4641* 106656495 ss13452159 0.042 htSNP 2762 2745 0.18 3′ 
DIL4642 106656481 ss13452160 0.307 93.01 2012 2024 0.70 3′ 
DIL4653 106656362-3 ss13452171 0.135 81.28 378 373 0.83 3′ 
DIL4643 106656361 ss13452161 0.297 94.25 2050 2056 0.89 3′ 
DIL4644 106656043 ss13452162 0.286 100.00 2051 2061 0.76 3′ 
DIL4645* 106656038 ss13452163 0.130 htSNP 2547 2583 0.09 3′ 
DIL4646 106656037 ss13452164 0.005 — — — — 3′ 
DIL4647* 106655931 rs2293148 0.443 htSNP 1626 1640 0.70 3′ 
DIL4648 106655861 ss13452166 0.005 — — — — 3′ 
Variant nameMap positiondbSNPMinor allele frequencyAllelic R2TransmittedNot transmittedP valueLocation
DIL4620 106873087 rs1503921 0.244 97.07 2220 2225 0.88 5′ 
DIL4621 106872769 ss13452139 0.023 — — — — 5′ 
DIL4622 106872519 ss13452140 0.005 — — — — 5′ 
DIL4623 106871969 rs1503922 0.005 — — — — 5′ 
DIL4624* 106867943 ss13452142 0.229 htSNP 599 594 0.89 5′ 
DIL4625 106776408 ss13452143 0.028 — — — — Intron 4 
DIL4649 106776404-5 ss13452167 0.128 87.18 323 321 0.94 Intron 4 
DIL4650 106776371-2 ss13452168 0.021 — — — — Intron 4 
DIL4651 106776332-6 ss13452169 0.261 88.48 663 655 0.79 Intron 4 
DIL4626* 106776303 rs3772512 0.109 htSNP 261 259 0.92 Intron 4 
DIL4627 106745995 ss13452145 0.016 — — — — Exon 6 
DIL4628* 106740946 rs3213928 0.027 htSNP 2727 2731 0.74 Intron 6 
DIL4629 106740829 ss13452147 0.005 — — — — Intron 6 
DIL4652 106740755 ss13452170 0.006 — — — — Intron 6 
DIL4630* 106737184 rs2289746 0.413 htSNP 1861 1879 0.62 Intron 8 
DIL5960 106734298 ss13452173 0.026 — — — — Intron 8 
DIL5961 106734124 ss13452174 0.005 — — — — Exon 9 
DIL4634 106720318 ss13452152 0.005 — — — — Exon 10 
DIL4633 106720255 rs2305035 0.258 93.40 624 609 0.61 Exon 10 
DIL4632 106720186 rs2305036 0.258 93.40 624 609 0.61 Exon 10 
DIL4631 106720058 ss13452149 0.012 — — — — Intron 10 
DIL4636* 106704073 rs2305037 0.286 htSNP 781 771 0.76 Exon 11 
DIL4635 106703765 ss13452153 0.286 100.00 781 771 0.76 Intron 11 
DIL4637 106702263 rs3772534 0.043 99.58 2761 2745 0.19 Exon 12 
DIL4663 106681919 ss13452172 0.005 — — — — Intron 14 
DIL4638 106658744 rs1042852 0.290 97.59 2059 2070 0.75 3′UTR 
DIL4639* 106658224 ss13452157 0.096 htSNP 199 199 0.99 3′ 
DIL4640 106657549 ss13452158 0.016 — — — — 3′ 
DIL4641* 106656495 ss13452159 0.042 htSNP 2762 2745 0.18 3′ 
DIL4642 106656481 ss13452160 0.307 93.01 2012 2024 0.70 3′ 
DIL4653 106656362-3 ss13452171 0.135 81.28 378 373 0.83 3′ 
DIL4643 106656361 ss13452161 0.297 94.25 2050 2056 0.89 3′ 
DIL4644 106656043 ss13452162 0.286 100.00 2051 2061 0.76 3′ 
DIL4645* 106656038 ss13452163 0.130 htSNP 2547 2583 0.09 3′ 
DIL4646 106656037 ss13452164 0.005 — — — — 3′ 
DIL4647* 106655931 rs2293148 0.443 htSNP 1626 1640 0.70 3′ 
DIL4648 106655861 ss13452166 0.005 — — — — 3′ 

Variant name based on local naming scheme for polymorphisms. Map positions on human chromosome 3 from NCBI build 33. Minor allele frequencies shown are based on the sequencing panel of 96 type 1 diabetic subjects. Transmitted and not transmitted were estimated for the ungenotyped SNPs with allele frequencies >0.03 from the regression equations computed in the preliminary sequencing study.

*

Denotes htSNP. dbSNP numbers for all novel polymorphisms in boldface. Single-locus tests at SNPs other than htSNPs were calculated from imputed data based on htSNP genotypes and LD information obtained from the sequencing panel. The single-locus P values were obtained using paired t tests. Data were not imputed and tests not performed for SNPs with allele frequencies <0.03. UTR, untranslated region.

F.P. and D.J.S. contributed equally to this study.

This work was funded by the Wellcome Trust and the Juvenile Diabetes Research Foundation International. We thank the Human Biological Data Interchange and Diabetes U.K. for U.S. and U.K. multiplex families, respectively.

1.
Colle E, Guttmann RD, Seemayer T: Spontaneous diabetes mellitus syndrome in the rat. I. Association with the major histocompatibility complex.
J Exp Med
154
:
1237
–1242,
1981
2.
Jacob HJ, Pettersson A, Wilson D, Mao Y, Lernmark A, Lander ES: Genetic dissection of autoimmune type I diabetes in the BB rat.
Nat Genet
2
:
56
–60,
1992
3.
Bieg S, Koike G, Jiang J, Klaff L, Pettersson A, MacMurray AJ, Jacob HJ, Lander ES, Lernmark A: Genetic isolation of iddm 1 on chromosome 4 in the biobreeding (BB) rat.
Mamm Genome
9
:
324
–326,
1998
4.
Ramanathan S, Poussier P: BB rat lyp mutation and type 1 diabetes.
Immunol Rev
184
:
161
–171,
2001
5.
Greiner DL, Handler ES, Nakano K, Mordes JP, Rossini AA: Absence of the RT-6 T cell subset in diabetes-prone BB/W rats.
J Immunol
136
:
148
–151,
1986
6.
MacMurray AJ, Moralejo DH, Kwitek AE, Rutledge EA, Van Yserloo B, Gohlke P, Speros SJ, Snyder B, Schaefer J, Bieg S, Jiang J, Ettinger RA, Fuller J, Daniels TL, Pettersson A, Orlebeke K, Birren B, Jacob HJ, Lander ES, Lernmark A: Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene.
Genome Res
12
:
1029
–1039,
2002
7.
Hornum L, Romer J, Markholst H: The diabetes-prone BB rat carries a frameshift mutation in Ian4, a positional candidate of Iddm1.
Diabetes
51
:
1972
–1979,
2002
8.
Kawano K, Hirashima T, Mori S, Saitoh Y, Kurosumi M, Natori T: New inbred strain of Long-Evans Tokushima lean rats with IDDM without lymphopenia.
Diabetes
40
:
1375
–1381,
1991
9.
Komeda K, Noda M, Terao K, Kuzuya N, Kanazawa M, Kanazawa Y: Establishment of two substrains, diabetes-prone and non-diabetic, from Long-Evans Tokushima Lean (LETL) rats.
Endocr J
45
:
737
–744,
1998
10.
Yokoi N, Kanazawa M, Kitada K, Tanaka A, Kanazawa Y, Suda S, Ito H, Serikawa T, Komeda K: A non-MHC locus essential for autoimmune type I diabetes in the Komeda Diabetes-Prone rat.
J Clin Invest
100
:
2015
–2021,
1997
11.
Bustelo XR, Crespo P, Lopez-Barahona M, Gutkind JS, Barbacid M: Cbl-b, a member of the Sli-1/c-Cbl protein family, inhibits Vav-mediated c-Jun N-terminal kinase activation.
Oncogene
15
:
2511
–2520,
1997
12.
Ettenberg SA, Magnifico A, Cuello M, Nau MM, Rubinstein YR, Yarden Y, Weissman AM, Lipkowitz S: Cbl-b-dependent coordinated degradation of the epidermal growth factor receptor signaling complex.
J Biol Chem
276
:
27677
–27684,
2001
13.
Lavagna-Sevenier C, Marchetto S, Birnbaum D, Rosnet O: The CBL-related protein CBLB participates in FLT3 and interleukin-7 receptor signal transduction in pro-B cells.
J Biol Chem
273
:
14962
–14967,
1998
14.
Zhang Z, Elly C, Qiu L, Altman A, Liu YC: A direct interaction between the adaptor protein Cbl-b and the kinase zap-70 induces a positive signal in T cells.
Curr Biol
9
:
203
–206,
1999
15.
Yokoi N, Komeda K, Wang HY, Yano H, Kitada K, Saitoh Y, Seino Y, Yasuda K, Serikawa T, Seino S: Cblb is a major susceptibility gene for rat type 1 diabetes mellitus.
Nat Genet
31
:
391
–394,
2002
16.
Bachmaier K, Krawczyk C, Kozieradzki I, Kong YY, Sasaki T, Oliveira-dos-Santos A, Mariathasan S, Bouchard D, Wakeham A, Itie A, Le J, Ohashi PS, Sarosi I, Nishina H, Lipkowitz S, Penninger JM: Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b.
Nature
403
:
211
–216,
2000
17.
Chapman JM, Cooper JD, Todd JA, Clayton DG: Detecting disease associations due to linkage disequilibrium using haplotype tags: a class of tests and the determinants of statistical power.
Hum Hered
56
:
18
–31,
2003
18.
Couzin J: Human genome: HapMap launched with pledges of $100 million.
Science
298
:
941
–942,
2002
19.
Todd JA, Bell JI, McDevitt HO: HLA-DQ beta gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus.
Nature
329
:
599
–604,
1987
20.
Ueda H, Howson JM, Esposito L, Heward J, Snook H, Chamberlain G, Rainbow DB, Hunter KM, Smith AN, Di Genova G, Herr MH, Dahlman I, Payne F, Smyth D, Lowe C, Twells RC, Howlett S, Healy B, Nutland S, Rance HE, Everett V, Smink LJ, Lam AC, Cordell HJ, Walker NM, Bordin C, Hulme J, Motzo C, Cucca F, Hess JF, Metzker ML, Rogers J, Gregory S, Allahabadia A, Nithiyananthan R, Tuomilehto-Wolf E, Tuomilehto J, Bingley P, Gillespie KM, Undlien DE, Ronningen KS, Guja C, Ionescu-Tirgoviste C, Savage DA, Maxwell AP, Carson DJ, Patterson CC, Franklyn JA, Clayton DG, Peterson LB, Wicker LS, Todd JA, Gough SC: Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease.
Nature
423
:
506
–511,
2003
21.
Bain SC, Todd JA, Barnett AH: The British Diabetic Association: Warren repository.
Autoimmunity
7
:
83
–85,
1990
22.
Lernmark A, Ducat L, Eisenbarth G, Ott J, Permutt MA, Rubenstein P, Spielman R: Family cell lines available for research.
Am J Hum Genet
47
:
1028
–1030,
1990
23.
Ranade K, Chang MS, Ting CT, Pei D, Hsiao CF, Olivier M, Pesich R, Hebert J, Chen YD, Dzau VJ, Curb D, Olshen R, Risch N, Cox DR, Botstein D: High-throughput genotyping with single nucleotide polymorphisms.
Genome Res
11
:
1262
–1268,
2001
24.
Olivier M, Chuang LM, Chang MS, Chen YT, Pei D, Ranade K, de Witte A, Allen J, Tran N, Curb D, Pratt R, Neefs H, de Arruda Indig M, Law S, Neri B, Wang L, Cox DR: High-throughput genotyping of single nucleotide polymorphisms using new biplex invader technology.
Nucleic Acid Res
30
:
e53
,
2002