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The Journal of Immunology, 2007, 179, 8341-8349
Copyright © 2007 by The American Association of Immunologists, Inc.
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Interactions between Idd5.1/Ctla4 and Other Type 1 Diabetes Genes1

Kara Hunter*, Dan Rainbow*, Vincent Plagnol*, John A. Todd*, Laurence B. Peterson{dagger} and Linda S. Wicker2,*

* Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; and {dagger} Department of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Two loci, Idd5.1 and Idd5.2, that determine susceptibility to type 1 diabetes (T1D) in the NOD mouse are on chromosome 1. Idd5.1 is likely accounted for by a synonymous single nucleotide polymorphism in exon 2 of Ctla4: the B10-derived T1D-resistant allele increases the expression of the ligand-independent isoform of CTLA-4 (liCTLA-4), a molecule that mediates negative signaling in T cells. Idd5.2 is probably Nramp1 (Slc11a1), which encodes a phagosomal membrane protein that is a metal efflux pump and is important for host defense and Ag presentation. In this study, two additional loci, Idd5.3 and Idd5.4, have been defined to 3.553 and 78 Mb regions, respectively, on linked regions of chromosome 1. The most striking findings, however, concern the evidence we have obtained for strong interactions between these four disease loci that help explain the association of human CTLA4 with T1D. In the presence of a susceptibility allele at Idd5.4, the CTLA-4 resistance allele causes an 80% reduction in T1D, whereas in the presence of a protective allele at Idd5.4, the effects of the resistance allele at Ctla4 are modest or, as in the case in which resistance alleles at Idd5.2 and Idd5.3 are present, completely masked. This masking of CTLA-4 alleles by different genetic backgrounds provides an explanation for our observation that the human CTLA-4 gene is only associated with T1D in the subgroup of human T1D patients with anti-thyroid autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Genes termed insulin-dependent diabetes (Idd)3 control the development of type 1 diabetes (T1D) in NOD mice. The Idd5 region from diabetes-resistant C57BL/10 (B10) or C57BL/6 (B6) mice provides protection from T1D when introgressed onto the NOD background (1, 2). Idd5 is located on mouse chromosome 1 and has been shown by congenic strain analysis to consist of at least two loci, Idd5.1 and Idd5.2, positioned at the proximal and distal ends, respectively, of an ~15 Mb interval (3). Idd5.1 was defined as a 2.0 Mb B10-derived resistance interval containing four genes including the candidate genes Ctla4 and Icos (3, 4), a remarkable finding since human T1D is associated with CTLA4 (5, 6). In addition to the human T1D association with CTLA4, functional studies support the candidacy of Ctla4 as the diabetes gene in the Idd5.1 interval because the B10 allele of Ctla4 produces more of the "ligand-independent" splice form of CTLA-4 (liCTLA-4) than does the NOD allele (6). The molecular basis for the splicing difference has been mapped to a single nucleotide polymorphism (SNP) in Ctla4 exon 2 that alters splicing in a similar manner to that mediated by a SNP in the human CD45 gene (7, 8, 9). The liCTLA-4 molecule mediates negative signaling in T cells thereby predicting that its higher expression in mice with the diabetes-protective B10 allele leads to reduced T cell activation and/or expansion (10). The Idd5.2 region was localized to a 1.5 Mb interval (3) in which Nramp1 is the most compelling candidate gene because there is a known functional missense polymorphism (Gly169) > (Asp169) distinguishing the NOD and B10 Nramp1 alleles. The NOD NRAMP1 protein is wild type and mediates protection from certain infectious diseases by contributing to the rapid acidification of the lysosome whereas the diabetes-resistant B10 NRAMP1 allotype is not functional (11). The likelihood of Nramp1 being Idd5.2 is very high because a knockdown of the gene mimics the biological effect of the natural knockout (12).

The current study was initiated to test the hypothesis that Idd5.1/Ctla4 and Idd5.2/Nramp1 alone are sufficient to account for the diabetes protection originally defined by the larger Idd5 locus containing both Idd5.1/Ctla4 and Idd5.2. The unexpectedly high frequency of diabetes observed when resistance alleles at Idd5.1/Ctla4 and Idd5.2/Nramp1 were combined revealed the existence of a third locus, Idd5.3, located between Idd5.1/Ctla4 and Idd5.2/Nramp1. This locus has been verified by the development of Idd5.3 congenic strains. In addition, the previously reported strong interaction of the Idd5 and Idd3 protective alleles causing nearly complete protection from diabetes and insulitis (1, 13) was shown to require resistance alleles at Idd5.2 and Idd5.3, because Idd3 and Idd5.1/Ctla4 did not recapitulate the protection observed in mice having resistance alleles at both Idd3 and Idd5.

Another novel bicongenic strain consisting of Idd5.2/Nramp1 and Idd5.3 had an unexpectedly low frequency of diabetes, leading to the discovery of a fourth Idd region on chromosome 1, Idd5.4. In the case of Idd5.4, it is the B10 allele, rather than the NOD allele, that confers susceptibility to T1D. We demonstrate that an interaction between the B10 Idd5.1/Ctla4 resistance allele and the B10 Idd5.4 susceptibility allele exists and that the B10 Idd5.1/Ctla4 allele can, when present in different genetic contexts, provide potent, moderate, or undetectable protection from diabetes. Our study also has implications for the ongoing search and characterization of human T1D susceptibility genes, one of which is CTLA4 (5, 6).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Congenic mouse strains and assessment of diabetes, insulitis, and insulin autoantibodies

The breeding and genotyping strategies for the development of the congenic mouse strains protected from T1D have been reviewed (14). The current study extends the fine mapping of the Idd5 region that has been previously published (1, 3) and, for clarity, all of the strains used or referred to are summarized in Fig. 1. An asterisk in Fig. 1 indicates the new NOD.B10 chromosome 1 congenic lines developed specifically for the current study. The origins of all of the Idd5 strains in Fig. 1 can be traced to lines R8 or R2. R8 and R2 were used to develop the R974 and R444 lines and the R2s line, respectively, by backcrossing to the NOD parental strain and identifying recombination events in progeny that were subsequently selectively bred and selected to be homozygous for the congenic interval. All congenic strains have been backcrossed to NOD 10 to 20 times. The centromeric segment of R2s was originally produced as a congenic strain from R8 to localize Idd5.1 and it was shown to have a frequency identical to the NOD strain (L. Wicker, L. Peterson, unpublished observations). To perform a direct comparison with R444, the distal segment of R2s was developed from the R2 strain and combined with the centromeric segment described above and is shown in Fig. 1. Three lines were subsequently developed from R2s: lines 3700, 6359, and 6360. The remaining Idd5 congenic strains in Fig. 1 were developed from R444. The NOD.B10 Idd5R426+R52 (R426+R52) bi-congenic strain was developed by intercrossing the R426 and R52 strains, backcrossing the resulting F1 mice to the NOD/MrkTac (Taconic Farms) parental strain and selecting for mice heterozygous at both regions, indicating that a recombination event had occurred between the two Idd intervals. Bicongenic heterozygous mice were intercrossed and homozygous mice selected as founders of the new strain.


Figure 1
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  		FIGURE 1. Genetic intervals present in the Idd5 congenic strains referred to in this study. Filled regions are B10-derived or B6-derived segments of DNA for chromosomes 1 and 3, respectively, defined by the most centromeric and telomeric non-NOD allelic markers. Open regions represent the region between the last non-NOD allelic marker and the first NOD allelic marker at each boundary. Lines represent NOD-derived DNA. Vertical arrows designate the Idd5.1, Idd5.2, Idd5.3, and Idd5.4 regions. The Idd5.4 interval contains a segment that is NOD-derived due to a double recombination event (initially undetected) that occurred during the development of the R8 strain. The diagram is to scale.

 
To confirm the purity of the genetic background of the congenic strains, DNA from lines R2, R974, R444 and R2s (R8 DNA was not available), was tested by genotyping using a 5K mouse SNP chip and was performed by ParAllele Biosciences. For all of the strains tested, no non-NOD SNPs were identified outside of the defined congenic regions.

Three congenic strains having disease resistant Idd alleles on two chromosomes, Idd3 on chromosome 3, and different portions of Idd5 on chromosome 1, were used in the current study. The previously published line 1591 has the R444 and Idd3 genetic segments, line 1573 has the R467 Idd5 segment together with Idd3, and line 2402 has the R46 segment together with Idd3. The Idd3 congenic strain used to make lines 1591, 1573, and 2402 is NOD.B6 Idd3 R450 (15). The NOD.B6 Idd3 R450 was also shown to be free of non-NOD SNPs outside of the congenic region using the 5K mouse SNP chip. The R974, R46, R193, R2, R2s, R444, R467+Idd3, and Idd3 congenic strains are available from Taconic Farms through the Emerging Models Program (Lines 974, 2193, 2574, 1092, 1595, 1094, 1573, and 1098, respectively). Line 1591 is available as line 6109. Lines 6360 and 6359 are also available.

Elevated urinary glucose was detected using Diastix (Miles). Animals were considered diabetic when urinary glucose was over 500 mg/dl. Diabetic mice also exhibited polydipsia, polyuria, and weight loss. The assessment of insulitis has been described previously (13). In brief, each pancreas received a single score for the degree of insulitis observed: 0 no mononuclear cell infiltrates in the islets, 1 mild insulitis, <20% of the islets have infiltrates; 2 moderate insulitis, 20–60% of the islets have infiltrates; 3 severe insulitis, most islets (>60%) are infiltrated; and 4 extensive insulitis, nearly all islets are either completely infiltrated or appear as a residual islet. All procedures were conducted according to approved protocols of the Institutional Animal Care and Use Committee of Merck Research Laboratories.

Statistical analyses

Diabetes frequencies were analyzed with survival curves generated with the GraphPad Prism 4 (GraphPad Software) software package. Survival curves were created using the product limit method of Kaplan and Meier and strain comparisons were performed using the log rank test. The difference in the degree of insulitis between strains was analyzed by 2 x 3 contingency tables created using GraphPad Prism 4. Comparisons between strains were performed using {chi}-square test. The two-tailed unpaired t test (GraphPad Prism 4) was used to compare "mean age of diabetes onset" between strains.

Identification of new microsatellite markers and genotyping

Mice were initially genotyped by PCR using primers to the previously published microsatellite markers. To map recombination points more precisely, additional microsatellites and SNPs were identified and characterized as described previously (3). PCR were optimized using B10 and NOD DNA templates. Markers that were polymorphic between these two strains were used in further genotyping. All PCR were performed using Amplitaq Gold with buffer II (Applied Biosystems). When PCR product sizes were not distinguishable between B10 and NOD samples by 4% agarose gel electrophoresis, genotyping was performed using an ABI Prism 3100 Genetic analyzer (Applied Biosystems). All primers were ordered from Sigma-Genosys. Forward primers were labeled at the 5' end with the fluorescent dye HEX for analysis on the ABI genetic analyzer. SNPs were analyzed following restriction enzyme digestion of the PCR product and visualized on agarose gels. Primer sequences for novel markers used in this study are shown in Table I.


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  		Table I. Novel markers developed for this study

 
Gene identification

Inspection of mouse EnsEMBL and the orthologous region in human EnsEMBL was used to determine the gene content of Idd5.3.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Discovery of a novel Idd locus on chromosome 1, Idd5.3

From the analysis of congenic strains of mice, the Idd5 interval on chromosome 1 consists of two loci, Idd5.1 and Idd5.2 (1, 3). As summarized in Fig. 1, the centromeric boundary of Idd5.1 is defined by the R974 strain and is between the markers D1Mit249 and Cd28distal3. The distal Idd5.1 boundary is between AL671560TATG and AL671560GA, which is defined by the R46 strain (3). The gene for CTLA-4 is encoded within the 2.0 Mb Idd5.1 region at 61.3 Mb; it has a sequence variation between the NOD and B10 alleles that alters production of the liCTLA-4 splice form and, consequently, the level of negative signaling in T cells (3, 6, 10). Thus, Ctla4 is the prime candidate gene for Idd5.1. The Idd5.2 region has its centromeric and distal boundaries at ~75 Mb on chromosome 1 defined by the R193 and R444s strains, respectively (Fig. 1). Nramp1, which is within the 1.5 Mb Idd5.2 region, is functionally polymorphic between NOD and B10 mice, and knockdown experiments strongly support the hypothesis that Nramp1 is Idd5.2 (12).

To test the hypothesis that the Idd5.1/Ctla4 and Idd5.2/Nramp1 regions as defined above account for the protection conferred by strains such as R444 or R444s, in which the region between Idd5.1/Ctla4 and Idd5.2/Nramp1 is also derived from B10, the R52 (Idd5.2/Nramp1 from B10) and R426+R52 (Idd5.1/Ctla4 combined with Idd5.2/Nramp1 from B10) congenic strains were developed (Fig. 1). The frequency of T1D in these two novel strains was compared with the NOD parental strain as well as the R426 (Idd5.1/Ctla4 from B10) and R444s (Idd5.1/Ctla4 and Idd5.2/Nramp1 and the region between from B10) congenic strains (Figs. 1 and 2). As reported previously (1, 3), the presence of the B10 resistance allele at Idd5.1/Ctla4 alone confers modest but highly significant protection from T1D as compared with NOD mice (R426 vs NOD, p = 4.6 x10–7). A similar level of protection was observed when the Idd5.2/Nramp1 region alone was derived from B10, as in the R52 strain (R52 vs NOD, p = 1.7 x 10–9). Surprisingly, when the Idd5.1/Ctla4 and Idd5.2/Nramp1 regions were combined in the bicongenic strain, R426+R52, no additional protection was observed above that with either region alone (p = 0.47 vs R52, p = 0.24 vs R426). The frequency of diabetes observed in the R426+R52 strain was also higher than that in the R444s strain (p = 0.02). Furthermore, the delay of diabetes observed in R444s as compared with NOD (mean days of onset for R444s and NOD are 179 ± 6 and 130 ± 3, respectively, p = 1.4 x 10–8, t test) was also present when R444s was compared with R426 (Idd5.1/Ctla4), R52 (Idd5.2/Nramp1), and R426+R52 (mean day of onset for R426, R52, and R426+R52, are 155 ± 5, 143 ± 7, and 146 ± 6, respectively, p values vs R444s are all ≤8.0 x10–4, t test). We, therefore, hypothesize that a B10-derived resistance allele is located in a region designated in this study as Idd5.3, which is defined by the difference between the distal boundary of the R426 congenic strain and the proximal boundary of the R52 strain, a 7.27 Mb interval.


Figure 2
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  		FIGURE 2. Discovery of Idd5.3. Frequency of diabetes in female Idd5 congenic mice reveals the existence of a third Idd5 subregion, Idd5.3. The p values were obtained by comparing the survival curves of the two indicated strains using the log-rank test. NS, Not significant.

 
To verify the existence of Idd5.3 and to further map the region, we developed additional congenic strains: lines 6359, 6360, and 3700 (Fig. 1). The frequency of diabetes was assessed in female mice from lines 3700, 6359, and 6360 and compared with that of NOD mice, which revealed that lines 3700 and 6360 had a significant protection from diabetes (p = 1.08 x 10–2 and p = 1.2 x 10–3 respectively). Line 6359 had a diabetes frequency indistinguishable from NOD (p = 5.57 x 10–1) (Fig. 3). These new congenic lines now define Idd5.3 to a 3.553 Mb region, with the proximal boundary defined by line 6359, and the distal boundary defined by lines 3700 and 6360 (Fig. 4).


Figure 3
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  		FIGURE 3. Fine mapping of Idd5.3. Frequency of diabetes in females from lines 3700, 6360, and 6359 congenic mice are compared with NOD females. Line 6359 is not significantly protected from diabetes compared with the NOD strain. Lines 3700 and 6360 have indistinguishable diabetes frequencies and are protected from diabetes compared with NOD females (p values 1.08 x 10–2 and 1.2 x 10–3, respectively) and are also significantly protected from diabetes compared with line 6359 (p values 2.54 x 10–2 and 4.1 x 10–3, respectively). A 1.43 x 10–4 p value is obtained when a meta analysis is done after combining the frequency data of the two strains having resistant alleles at Idd5.3 (lines 3700 and 6360) and the two strains having susceptible alleles at Idd5.3 (line 6359 and the NOD parental strain). The p values were obtained by comparing the survival curves of the indicated strains using the log-rank test.

 

Figure 4
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  		FIGURE 4. Genes in the Idd5.3 region. From the T1D frequencies observed in Fig. 3, the proximal and distal boundaries of the Idd5.3 region are defined by strain 6359 and 6360, respectively. Genes present in the orthologous human genomic region are also indicated. Open regions represent the region between the last non-NOD allelic marker and the first NOD allelic marker at each boundary. Lines represent NOD-derived DNA.

 
Gene content of Idd5.3

As detailed in Fig. 4, there are 11 genes annotated within the 3.553 Mb region of Idd5.3, including the functional candidate Ikzf2, which encodes the Ikaros family zinc finger 2 protein, a T cell restricted transcription factor (16). By virtue of its differential expression in CD4 T cells following activation (17), Acadl, which encodes long chain acyl CoA dehydrogenase (18), is also a candidate gene for Idd5.3.

Idd5.3 is essential for the nearly complete protection from T1D caused by resistance alleles at Idd5 and Idd3

The combination of protective alleles at Idd5 and Idd3 provides nearly complete protection from diabetes, insulitis, and the occurrence of insulin autoantibodies (1, 13). To assess the individual contributions of the Idd5 subregions to the interaction of Idd3 and Idd5 (19), two congenic strains combining Idd3 with various portions of the Idd5 region were developed, lines 2402 and 1573 (Fig. 5A). Line 2402 has its Idd5 interval from R46 (B10 alleles at Idd5.1/Ctla4) and line 1573 has its Idd5 interval from R467 (B10 alleles at Idd5.1/Ctla4 and Idd5.3). Both lines 2402 and 1573 were selected to have resistance alleles at Idd3, which were derived from line 1098. The diabetes frequencies of these two novel strains were determined along with those of the parental NOD strain (data not shown for clarity in the figure), line 1098 (Idd3 resistance alleles only), and line 1591 (13), a Idd3/5 congenic strain having its Idd5 interval derived from the R444 strain and, therefore, having resistance alleles at Idd5.1/Ctla4, Idd5.2/Nramp1, and Idd5.3. The results (Fig. 5A) showed equivalent (p = 5.9 x 10–1, 1591 vs 1573), nearly complete, protection from T1D in lines 1591 (p = 2.6 x 10–5 vs 1098) and 1573 (p = 3.1 x 10–3 vs 1098); however, line 2402 was not protected from T1D as completely as lines 1591 and 1573 because it had a disease frequency equivalent to line 1098 (p = 0.76, 2402 vs 1098). These results not only confirm the existence of Idd5.3 (line 1573 is more protected from T1D than line 2402) but also show that a resistance allele at Idd5.3 is essential for the nearly complete protection from diabetes observed when resistance alleles at Idd5 and Idd3 are both present. It is also notable that resistance alleles at Idd5.1/Ctla4 (R46) and Idd3 together did not increase protection from diabetes as compared with Idd3 resistance alleles alone. In contrast to Idd5.3, resistance alleles at Idd5.2/Nramp1 are not required for Idd3/5-mediated disease protection because lines 1573 and 1591 were equally protected from T1D. Although resistance alleles at Idd5.1/Ctla4 are not sufficient for the interaction with resistance alleles at Idd3, further analysis of the isolated Idd5.3 congenic strain, line 6360, in conjunction with Idd3 will be needed to determine whether the Idd5.3 locus is sufficient on its own to mediate the interaction. It is possible that resistance alleles at both Idd5.1/Ctla4 and Idd5.3 are essential, in combination, for the genetic interaction with resistance alleles at Idd3 to occur.


Figure 5
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  		FIGURE 5. Fine mapping of Idd5.3 by virtue of its ability to increase protection from diabetes in conjunction with protective alleles at Idd3. A, The frequency of diabetes was monitored in strains having resistance alleles at Idd3 and various subregions of Idd5. p values were obtained by comparing the survival curves of the two indicated strains using the log-rank test. Note that the y-axis is truncated due to the low frequency of T1D in the strains examined. B, Degree of insulitis in strains having combinations of Idd5 and Idd3 protective alleles. N = Number of pancreata examined. Each pancreas was examined using two noncontiguous longitudinal sections. Data from strains 1591, 1573, and 2402 are from 7-mo-old females. As a positive insulitis control, 3-mo-old NOD males were included. The p values were obtained using a 2 x 3 contingency table comparing the indicated strains.

 
Similar to the diabetes phenotype, we have previously shown that as compared with mice having resistance alleles at Idd3 or Idd5 alone, the frequency of insulitis is significantly reduced when resistance alleles at Idd3 and Idd5 are both present (1, 13). Therefore, we assessed the occurrence and severity of insulitis at 7 mo of age, or following the development of diabetes, in females from lines 1591, 1573, and 2402. In contrast to diabetes, there was a hierarchy in the occurrence and severity of insulitis with line 2402 having more mice with insulitis than 1573 (p = 0.05, Fisher’s exact test) and 1573 having more insulitis than 1591 (p = 0.01) (Fig. 5B). This latter finding is of particular note because the Idd5.2/Nramp1 resistance alleles were not required for the interaction with Idd3 when diabetes was the phenotype assessed (Fig. 5A) but are required to provide the genetic interaction causing a marked reduction in insulitis.

B10-derived susceptibility allele distal to Idd5.2/Nramp1 on chromosome 1

Previously, we had observed that the R2 strain, which has B10 alleles at Idd5.3 and Idd5.2/Nramp1 (Fig. 1), was not protected from T1D (1). This is at odds with the protection observed in this study for the R52 strain, which has a resistance allele at Idd5.2/Nramp1 alone (Figs. 1 and 2). Because the R52 interval is completely contained within the boundaries of the R2 strain, this result suggested that a B10-derived susceptibility allele present in the R2 strain masks the effects of the protective Idd5.2 allele defined by the R52 strain. This hypothesis was substantiated by the analysis (Fig. 6) of a newly derived strain, R2s, which has resistant alleles at Idd5.2 and Idd5.3, but like R2, has a NOD allele at Idd5.1/Ctla4 (Fig. 1). As found for the R52 congenic strain, the R2s strain is protected from T1D (Fig. 6, p = 2 x 10–11, R2 vs R2s). Hence, we proposed the existence of a fourth T1D locus, distal to Idd5.2, designated Idd5.4.


Figure 6
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  		FIGURE 6. Discovery of Idd5.4. The frequency of diabetes in the R2 and R2s strains reveals a B10-derived susceptibility allele at the Idd5.4 locus. The p values were obtained by comparing the survival curves of the two indicated strains using the log-rank test.

 
We sought to confirm the existence of the Idd5.4 B10-derived T1D susceptibility allele by hypothesizing that if one dose of the B10 susceptibility allele was detectable in the context of one dose each of B10-derived resistance alleles at Idd5.1/Ctla4, Idd5.2/Nramp1, and Idd5.3, that the frequency of diabetes in Idd5.1/Idd5.2/Idd5.3/Idd5.4 heterozygotes should be greater than in Idd5.1/Idd5.2/Idd5.3 heterozygotes even though the Idd5.1/Idd5.2/Idd5.3/Idd5.4 and Idd5.1/Idd5.2/Idd5.3 homozygous strains, R974 and R444, respectively, are protected equivalently from T1D (3). Thus, Idd5.1/Idd5.2/Idd5.3/Idd5.4 (R974 x NOD)F1 and Idd5.1/Idd5.2/Idd5.3 (R444 x NOD)F1 heterozygous mice and the three parental strains were tested for their frequencies of T1D. As seen in Fig. 7A, the frequency of T1D was marginally higher in the (R974 x NOD)F1 mice than in the (R444 x NOD)F1 animals ({square} vs {circ}, p = 0.04) even though the R974 and R444 parental strains have identical disease frequencies. These data are consistent with the existence of a B10-derived susceptibility and that the effect of one dose of this allele is only partially silenced by one dose of a B10-derived resistance allele at Idd5.1/Ctla4.


Figure 7
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  		FIGURE 7. Confirmation of a B10 susceptibility allele at Idd5.4. A, The higher diabetes frequency in R974 x NOD F1 vs R444 x NOD F1 mice confirms the influence of Idd5.4. B, Homozygosity for the resistance allele at Ctla4/Idd5.1 counters the T1D-susceptibility mediated by the B10 allele at Idd5.4. The p values were obtained by comparing the survival curves of the two indicated strains using the log-rank test.

 
Gene dosage effects at Idd5.1/Ctla4

The finding that one dose of a protective allele at Idd5.1/Ctla4 was insufficient to provide a high degree of protection from T1D in the presence of one dose of Idd5.4 led us to test the hypothesis that two doses of an Idd5.1/Ctla4 resistance allele could more effectively counter a single dose of a susceptibility allele at Idd5.4. We, therefore, compared T1D frequencies in (R974 x NOD)F1 and (R46 x R974)F1 mice, the latter strain having two doses of the B10 resistance allele at Idd5.1/Ctla4. As seen in Fig. 7B, significantly (p = 8.8 x 10–8) fewer (R46 x R974) F1 mice ({diamond}) developed diabetes than (R974 x NOD)F1 mice ({square}). These results strongly confirm the existence of Idd5.4 and support the hypothesis that resistance alleles at Idd5.1/Ctla4 can, in a dose-dependent manner, mask the increase of diabetes that is mediated by a B10 susceptibility allele at Idd5.4.

Genetic control by the Idd5.1/Ctla4 region is altered in different genetic contexts

The discovery of Idd5.4 has caused us to the reconsider the genetic control mediated by the extended Idd5 region that now contains at least four loci: Idd5.1/Ctla4, Idd5.2/Nramp1, Idd5.3, and Idd5.4. As shown in Fig. 8, depending on the genetic context, the protective B10 alleles at Idd5.1/Ctla4 provide vastly different levels of protection from T1D. When present on the NOD background alone, the B10 allele at Idd5.1/Ctla4 provides a small, but significant (p = 3 x 10–4), level of protection from T1D (Fig. 8A, see also Fig. 2). In contrast, in the presence of B10 protective alleles at Idd5.2/Nramp1 and Idd5.3 (Figs. 6 and 8B) there is a small (Fig. 6, p = 4.5 x10–2, R2s vs R444s) or no (Fig. 8B, p = 0.33, R2s vs R444s) effect of the B10 resistant alleles at the Idd5.1/Ctla4 region. Similarly, the combination of resistance alleles at Idd5.1/Ctla4 and Idd5.2/Nramp1 (Fig. 2) or Idd5.1/Ctla4 and Idd5.3 (1, 3) does not reduce the frequency of diabetes below that seen with resistance alleles at Idd5.1/Ctla4 alone. Only when B10-derived alleles are present in the Idd5.4, Idd5.3 and Idd5.2/Nramp1 regions is highly significant protection provided by the addition of Idd5.1/Ctla4 resistance alleles, as seen in the comparison of the R974 and R2 strains (4) (Fig. 8C, p = 1.8 x 10–5 for R974 vs R2).


Figure 8
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  		FIGURE 8. Idd5.1/Ctla4 is sensitive to genetic background. The level of protection conferred by B10 alleles at Idd5.1/Ctla4 is dependent on which alleles are present at other Idd5 subregions. Examples of moderate (A), small or nonexistent (B), and large effects of a protective B10 allele (C) at Idd5.1/Ctla4 are shown. The p values were obtained by comparing the survival curves of the two indicated strains using the log-rank test. NS = not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Gene interactions in type 1 diabetes

This study was initiated to determine whether the Idd5.1/Ctla4 and Idd5.2/Nramp1 T1D resistance alleles derived from the B10 strain at the centromeric and distal regions, respectively, of the Idd5 region on chromosome 1 are sufficient to explain the disease resistance produced when the intervening region is also derived from the B10 parent, such as in the R444 strain (Fig. 1) (3). Unexpectedly, this was found not to be the case, and because of these results we hypothesized that additional T1D genes exist in the Idd5 region. We designed a series of experiments to test this hypothesis and defined two new Idd loci on chromosome 1, Idd5.3, which is in the intervening segment between Idd5.1/Ctla4 and Idd5.2/Nramp1, and Idd5.4, which is distal of Idd5.2/Nramp1. The most unexpected finding in this study was the evidence for strong and complex interactions between the four disease loci: protective alleles at Idd5.1/Ctla4 could completely mask potent susceptibility alleles at Idd5.4; in the absence of protective alleles at Idd5.1/Ctla4, susceptibility alleles at Idd5.4 can, in turn, completely mask the effect of protective alleles at Idd5.3 and Idd5.2/Nramp1 as seen in the T1D susceptible R2 strain (Fig. 6); disease protection mediated by B10-derived, T1D-resistant alleles at Idd5.1/Ctla4 varied depending on the genetic background in which they function. In the gene combinations examined in the current study, Idd5.1/Ctla4 is a very potent disease susceptibility gene only when the Idd5.3, Idd5.2/Nramp1, and Idd5.4 alleles are all derived from the B10 genome (Figs. 7B and 8C). Indeed the effect of variation at Ctla4 is almost completely masked when B10 alleles are present at Idd5.3 and Idd5.2/Nramp1 but not Idd5.4 (Fig. 8B).

Interactions between Idd5.1/Ctla4 and Idd5.4

Because disease resistance by the protective B10 allele at Idd5.1/Ctla4 is mediated by increased production of the negative-signaling molecule liCTLA-4 (10), a logical hypothesis is that the B10-derived susceptibility allele at Idd5.4 causes an event that can be reversed or prevented by more negative signaling via liCTLA-4 in one or more cell types. The activity of Idd5.1/Ctla4 is dose-dependent when there is one copy of the susceptibility allele at Idd5.4 (Fig. 7). This suggests that a homeostatic, quantitative interaction exists between the molecular and/or cellular events caused by Idd5.1/Ctla4 and Idd5.4. Thus far, we have only studied the activity of a B10 allele at Idd5.4 in the context of protective alleles at Idd5.3 and Idd5.2/Nramp1 on chromosome 1. It is possible that if NOD.B10 Idd5.4 congenic mice were developed, they would have a higher frequency of diabetes than that of the NOD parental strain or that T1D would occur at a younger age. However, it is also possible that Idd5.4-mediated disease susceptibility is dependent on molecular or cellular events caused by resistance alleles at Idd5.3 and/or Idd5.2/Nramp1. It would be informative to determine whether susceptibility at Idd5.4 overcomes the protection mediated by other resistance alleles such as Idd3 or Idd9.3 (20). Especially as the identity of more Idd loci becomes known, the consequences of combining particular resistance and susceptibility alleles should contribute to an increased understanding of the pleiotropic effects of the loci on T1D pathogenesis.

Combining protective alleles does not always lead to increased protection from T1D

Another unexpected result of this study was that no, or only a slight, increase in diabetes resistance was observed when protective alleles at particular loci are combined: Idd5.1/Ctla4 and Idd5.2/Nramp1 (Fig. 2) or Idd5.1/Ctla4 with Idd5.2/Nramp1 and Idd5.3 (Figs. 6 and 8B). We also now realize that when protective alleles at Idd5.1/Ctla4 and Idd5.3 are both present, as they unknowingly were in several strains studied in a previous report (3), no additional protection was observed above that seen with T1D-resistance alleles at Idd5.1/Ctla4 alone (R46, R426, R467, and R193 all had the same T1D frequencies in Ref. 3). These results imply that the mechanisms of protection by Idd5.1/Ctla4 are either redundant or masked when protective alleles are also present at either Idd5.2/Nramp1 or Idd5.3 or that the distinct mechanisms provided by Idd5.1/Ctla4 and Idd5.2/Nramp1 or Idd5.1/Ctla4 and Idd5.3 do not result in the increased protection observed in, for example, the combination of T1D-resistant alleles at Idd3 and Idd5.1/Idd5.3 (Fig. 5).

Genetic control of Idd3/Idd5-mediated protection from T1D

The discovery of Idd5.3 is particularly important when considering the nearly complete T1D protection provided by a combination of resistance alleles at Idd3 and the loci on chromosome 1 (18). Resistance alleles at Idd5.3 and Idd5.1/Ctla4 together with Idd3 provided as much protection from T1D as all three Idd5 loci and Idd3 (Fig. 5A), although the loss of the protective allele at Idd5.2/Nramp1 was observed in the insulitis score (Fig. 5B). In contrast, protective alleles at Idd5.1/Ctla4 only in conjunction with resistance alleles at Idd3 were not sufficient to provide this nearly complete protection. Because of these findings, we have developed an Idd5.3 congenic strain and are in the process of developing an Idd5.3/Idd3 double congenic line. It is also important to consider that NOD mice with T1D-resistance alleles at Idd3 (but not T1D-resistance alleles at Idd5.1/Ctla4) have increased expression of CTLA-4 on the surface of activated CD4+ and CD8+ T cells as compared with activated NOD T cells (20). It is possible that the increased negative signaling provided by higher levels of liCTLA-4 in mice with T1D-resistant alleles at Idd5.1/Ctla4 (10) is somewhat equivalent to the increase in negative signaling that would be mediated by higher levels of full-length CTLA-4 caused by protective alleles at Idd3. This potential overlap in the mechanism of protection provided by T1D-resistant alleles at Idd3 and Idd5.1/Ctla4 could provide an explanation for our observation that protective alleles at Idd5.1/Ctla4 in conjunction with resistance alleles at Idd3 did not provide more protection from T1D than protective alleles at the Idd3 region alone (Fig. 5A).

Idd5.1/Ctla4 is sensitive to genetic background

Another lesson learned from the current study is that the mapping of Idd5.1/Ctla4 may not have been accomplished without the existence of the Idd5.4 region, even though we did not know of the existence and effect of Idd5.4 at the time (1). Localization of Idd5.1/Ctla4 via sequential truncations of the proximal end of a strain such as R444 would have failed to reveal the Idd5.1/Ctla4 region because there is very little or no difference in the frequency of diabetes between strains having T1D resistance alleles at Idd5.1/Ctla4, Idd5.3, and Idd5.2/Nramp1 and those having protective alleles at Idd5.3 and Idd5.2/Nramp1. Thus, in the absence of the B10-derived susceptibility allele at Idd5.4, we most likely would have missed the differential effects of resistant and susceptible alleles at Idd5.1/Ctla4.

Candidate genes

The development of Idd5.3 congenic strains has allowed the fine mapping of the Idd5.3 region to an interval of 3.553 Mb which contains 11 genes. It is not practical to reduce this region by further congenic strain development and, consequently, we are now taking a candidate gene approach. Because we have observed that Acadl is differentially expressed in CD4 T cells obtained from NOD and NOD.Idd3/5 mice (17), it is the primary candidate gene for Idd5.3. Future studies will address the expression of other polymorphic genes within the Idd5.3 interval as well as functional consequences of the differential expression of Acadl.

The identification of the gene or genes accounting for the Idd5.4 genetic effect will require the development of additional strains of congenic mice. Even though global differential expression analyses have highlighted Daf1 as a compelling candidate gene (17), the Idd5.4 region is currently quite large (78 Mb) and we have demonstrated previously how compelling functional candidate genes for Idd regions can be eliminated by refined genetic analyses (22, 23). Therefore, to test the candidacy of Daf1, a NOD.B10 Daf1 congenic strain is currently being developed and will be tested for its ability to alter the frequency of type 1 diabetes.

Subclassification of autoimmune diabetes

The T1D frequency of the R2 strain having B10 alleles at Idd5.3, Idd5.2/Nramp1, and Idd5.4 is equivalent to that observed in the NOD parental strain (Fig. 6). However, it is likely that some of the cellular pathways causing autoimmune diabetes are altered by the introgression of the B10-derived region on chromosome 1 that contains at least two B10-derived protective alleles and one B10-derived susceptibility allele. The resulting disease processes in the NOD vs R2 strains could be described as two subtypes of type 1 diabetes, a situation that we suggest is analogous to the one in human families in which different segregating combinations of susceptibility and resistance alleles cause the development of what is normally assumed to be clinically identical type 1 diabetes. For example, the gene combination present in R2 mice could render this strain more or less resistant to an immune modulation protocol that is effective in the NOD strain, a possibility that can be tested. It is therefore possible that multiple strains of diabetes-susceptible NOD-related mice could be developed for the purpose of mimicking a portion of the genetic variation present in humans, especially in light of criticisms that rodent models such as the NOD are only representative of one case report of human diabetes (24). Such a congenic strain panel should be more effective than the NOD model alone for evaluating and prioritizing new therapeutic agents in the future.

Human gene discovery and disease subclassification

Implications from this study for mapping and understanding the effects of genetic variants on human autoimmune disease are significant. The human CTLA-4 gene is associated with a number of autoimmune diseases; in particular, noncoding SNPs near the CTLA-4 structural gene are strongly associated with autoimmune thyroid disease (AITD) (6). These same SNPs are also associated with T1D but much more weakly than in AITD (6). Recently, we (5) and others (25) have discovered that the CTLA4 SNPs are strongly associated with T1D but in only those T1D cases with AITD autoantibodies or diagnosed AITD. Our interpretation of the results from these human studies on CTLA4 is based on our current and previous observations in the NOD mouse model: the strength of the effect of allelic variation of Ctla4 is very dependent on different combinations of other susceptibility loci, including complete masking of the effect (that is no association with disease), and different combinations of Idd loci give rise to quite different autoimmune phenotypes within the general NOD genetic background, including AITD and an autoimmune liver disease (26, 27). It is likely that the genetic basis of human T1D and related diseases that occur more frequently together in T1D cases and families than expected, such as AITD and rheumatoid arthritis, is similar: different sets of alleles from multiple loci give rise to different but related diseases. In these sets, there will be susceptibility loci in common (e.g., HLA class II alleles and haplotypes, and PTPN22) (28, 29, 30, 31, 32, 33), and other loci specific to certain diseases or phenotypes (e.g., the gene encoding insulin for T1D) (34). For the sets of susceptibility genes in humans that can give rise to isolated T1D, not complicated with AITD disease or related autoantibodies, the CTLA4 SNPs are not associated, or only very weakly, with this subclass of T1D, and we proposed that this is due to the presence and function of human genes similar to Idd5.3 and Idd5.2/Nramp1 that can mask the effect of variation in the CTLA-4 gene. In the minor subgroup of T1D that is complicated with AITD or AITD autoantibodies, ~10–15% of T1D cases, CTLA-4 genetic variation has a strong effect, presumably via its role in regulation of peripheral tolerance, in which the disease-associated CTLA4 haplotype is predisposing to a failure in tolerance to multiple organs or tissues (5). Our current results highlight the complexities of studying autoimmunity genes even in a well-controlled experimental model, and promote further caution in the design, execution, and interpretation of susceptibility gene mapping in humans.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by National Institutes of Health Grant NIH P01 AI039671. The availability of NOD congenic mice through the Taconic Farms Emerging Models Program was supported by grants from the Merck Genome Research Institute, National Institute of Allergy and Infectious Diseases, and the Juvenile Diabetes Research Foundation. L.S.W. and J.A.T. were supported by grants from the Juvenile Diabetes Research Foundation and the Wellcome Trust, and L.S.W. was a Juvenile Diabetes Research Foundation/Wellcome Trust Principal Research Fellow. Back

2 Address correspondence and reprint requests to Dr. Linda S. Wicker, Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome Trust/Medical Research Council Building, Addenbrooke’s Hospital, Cambridge, U.K. E-mail address: linda.wicker{at}cimr.cam.ac.uk Back

3 Abbreviations used in this paper: Idd, insulin dependent diabetes; T1D, type 1 diabetes; liCTLA-4, "ligand-independent" splice form of CTLA-4; SNP, single nucleotide polymorphism; AITD, autoimmune thyroid disease. Back

Received for publication September 11, 2006. Accepted for publication August 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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