While sharing the H2g7 MHC and many other important Type I diabetes susceptibility (Idd) genes with NOD mice, the NOR strain remains disease free due to resistance alleles within the ∼12% portion of their genome that is of C57BLKS/J origin. Previous F2 segregation analyses indicated multiple genes within the ‘Idd13’ locus on Chromosome 2 provide the primary component of NOR diabetes resistance. However, it was clear other genes also contribute to NOR diabetes resistance, but were difficult to detect in the original segregation analyses because they were relatively weak compared to the strong Idd13 protection component. To identify these further genetic components of diabetes resistance, we performed a new F2 segregation analyses in which NOD mice were outcrossed to a ‘genome-conditioned’ NOR stock in which a large component of Idd13-mediated resistance was replaced with NOD alleles. These F2 segregation studies combined with subsequent congenic analyses confirmed the presence of additional NOR resistance genes on Chr. 1 and Chr. 4, and also potentially on Chr. 11. These findings emphasize the value for diabetes gene discovery of stratifying not only MHC loci conferring the highest relative risk but also as many as possible of the non-MHC loci presumed to contribute significantly.
The NOD mouse is an inbred strain homozygous for a large collection of genes that collectively promotes loss of immunological tolerance to pancreatic β-cell products and the development of T-cell-mediated Type I diabetes. Contributors include some rare alleles, and also some common ones that acquire diabetogenic potential in the context of other susceptibility genes. This is exemplified by the complex of diabetogenic contributors contained within the gene-dense H2g7 MHC.1 Diabetes susceptibility loci are identified by outcross of NOD mice with diabetes-resistant inbred strains. In such outcrosses, inheritance of the H2g7 haplotype in homozygous state is almost always documented in progeny that develop diabetes. Additionally, a variable collection of other non-MHC genes must be inherited for diabetes development. The numbers and locations of the susceptibility-conferring non-MHC linkages identified in a given linkage study are contingent on the degree of relatedness to NOD of the inbred strain used in the outcross. Outcross of NOD to closely related, but diabetes-resistant strains segregate fewer resistance genes than outcrosses using unrelated strains.
NOR/Lt is an inbred and diabetes-resistant recombinant congenic strain that arose from an inadvertent genetic contamination in a research colony of NOD/Lt mice at The Jackson Laboratory. Genome-wide screening revealed an NOR/Lt genome approximately 88% derived from NOD and 12% from C57BLKS/J (BKS), the source of the contamination.2, 3 The NOR/Lt genomic intervals distinguishing these mice from NOD (originally reported polymorphisms on Chromosomes 1, 2, 4, 5, 7, 11, 12 and 18 plus new single-nucleotide polymorphisms (SNP) reported herein on Chr. 5, Chr. 7, Chr. 10, Chr. 12 and Chr. 14) are particularly interesting because BKS itself is the product of a genetic contamination of C57BL/6J (B6) with another strain, probably DBA/2J (DBA).4 Hence, the diabetes resistance in NOR represents the composite interactions of components from three distinct genomes. NOR/Lt diabetes resistance was particularly impressive because the strain shares identity with NOD/Lt not only across the diabetogenic H2g7 MHC (‘Idd1’) but also at strong non-MHC susceptibility regions on Chr. 3 (Idd3, 10, 17, 18) plus additional previously identified ‘Idd’ intervals on other chromosomes.5, 6 Perhaps because of the high degree of genomic preconditioning dictated by 88% identity with NOD/Lt, highly significant evidence for linkage of NOR/Lt resistance following NOD × NOR outcross could only be claimed for multiple contributions from one complex locus, provisionally designated ‘Idd13’ on Chr. 2.3 This protective Idd13 region in NOR derives from a region of BKS Chr. 2 that originated from B6 and includes both a common variant allele at β2-microglobulin (B2mb) as well as other alleles distinct from NOD extending distally through the IL-1 gene complex (Il1b, Il1a), and the gene encoding proliferating cell nuclear antigen (Pcna).3 Subsequent generation and analysis of NOD stocks carrying interval-specific regions of NOR/B6-derived Chr. 2 markers confirmed the genetic complexity of this linkage.7 Diabetes susceptibility was restored in a normally disease-free stock of NOD mice homozygous for a disrupted B2m allele following transgenic reintroduction of the NOD-type B2ma allele, but not another B2m variant.8 This indicated one of the susceptibility components of Idd13 indeed was the relatively common B2ma allele expressed by NOD. The single amino-acid difference distinguishing the two B2m isoforms produces differential conformations of the common class I H2-Kd and H2-Db gene products expressed by both NOD and NOR.7
Evidence that the complete Type I diabetes resistance of NOR/Lt mice of both sexes had a more complex genetic basis than simply the B6 contributions on Chr. 2 was provided by the finding of a 29% disease frequency in F2 female segregants homozygous for NOR/B6 alleles across the entire Idd13 interval.3 Similarly, a stock of NOD mice congenic for this NOR-Idd13 interval developed diabetes at a 16% frequency rather than showing complete resistance.7 The original (NOD × NOR)F2 analysis indicated suggestive (weak) evidence for resistance loci linked to NOR polymorphic markers on Chr. 4 and Chr. 1.3 NOR and NOD mice differ with regard to the leukocytic infiltrates in the pancreas. In both strains, lymphoaccumulation is observed around the pancreatic vasculature and ducts, but in aging NOR/Lt mice, leukocytes remain primarily associated with the islet perimeters (‘peri-insulitis’). Conversely, in NOD mice, an initial peri-insulitis invariably transits to widespread intraislet infiltrates with β-cell destruction (‘invasive insulitis’).9, 10 Scoring insulitis severity as a quantitative trait in (NOD × NOR)F2 progeny, Fox et al10 reported protective NOR/Lt contributions regulating this histopathologic shift through an interaction between the Idd13 region on Chr. 2 and a B6-derived NOR genomic segment on Chr. 1 in the Idd5 region.
In order to detect additional NOR linkages contributing to this strain's resistance to development of both invasive insulitis and spontaneous development of Type I diabetes we established a new (NOD × NOR)F2 cross wherein the NOR genome was further ‘conditioned’ by replacing some of the protective NOR/B6 alleles within the Idd13 interval with NOD variants. We reasoned this would favor detection of resistance loci on other chromosomes whose contributions were masked in the presence of the intact NOR/B6-derived Idd13 resistance complex. The chromosome-substituted stock developed for this purpose proved to retain the NOR/B6 B2mb allele, but carried NOD alleles distally throughout the rest of the Idd13 region. We demonstrate that this genome conditioning now permits detection of additional NOR resistance contributions on Chr. 1, Chr. 4 and Chr. 11. We confirm the linkages on Chr. 1 and Chr. 4 by development and analysis of NOD stocks congenic for each individual resistance interval.
While the original intention was to completely replace the BKS/B6-derived Idd13 region on NOR Chr. 2 with NOD genome, the stock eventually produced retained a small residuum of B6 alleles immediately proximal to Il1a that includes the B6-derived B2mb allele. Data in Figure 1a show the results of this congenic replacement in the NOR.NOD-Il1 stock. Despite the reversion to NOD type of a significant number of NOR Idd13 region alleles marking peak resistance in the original (NOD × NOR)F2 study,3 the NOR.NOD-Ill congenic stock showed no diabetes development in 20 females and 20 males aged 40 weeks. Thus, the strong NOR resistance to spontaneous diabetes has an oligogenic basis. In the original (NOD × NOR) outcross, 33% (10/30) of F1 females developed clinical diabetes by 1 year of age.3 In contrast, 90% (9/10) of (NOD × NOR.NOD-Il1)F1 females in the present study were diabetic by 40 weeks (Figure 2). This onset was significantly delayed compared to NOD controls, with first appearance of disease in the F1 progeny at 28 weeks when 5/10 were diagnosed. This compares to a 95% incidence in standard NOD/Lt females by the 28 week time point (Figure 2). Hence, replacement of some Idd13 resistance subcomponents clearly conditioned the genome to greater susceptibility. This was apparent when comparing the original F2 cross3 to the Idd13-conditioned F2 cross in the present study. In the previously published (NOD × NOR)F2 study, 29.3% of 123 females followed to a year of age-developed clinical diabetes. Of the 185 (NOD × NOR.NOD-Il1)F2 females followed to 40 weeks in the present study, 86 (46.5%) were diabetic, with first onset at 12 weeks (Figure 2). Insulitis Index (II) of the 99 diabetes-free survivors showed the full range of scores from incipient diabetic (II=1.00) to virtually no insulitis (II=0.02). Equal weight was given to each range of data, time-of-diabetes onset and II, and thus the full group of 185 animals can be used as a single QTL analysis (Figure 3).
Table 1 shows the logarithm of odds ratio (LOD) scores for each marker typed and Figure 4a shows the genome-wide interval mapping of the first Principal component (PC1) analyzed for all 185 animals. Resistance in F2 females was most strongly conveyed by NOR genome on Chr. 4. Interval mapping computed peak linkage at D4Mit71 (61.9 cM, MGI; 132.2 Mb, Ensembl) with an LOD score of 5.87 (Table 1 and Figure 4a). This locus was only suggestively linked to resistance in a previous cross between NOD and NOR.3 ANOVA indicated that this B6 resistance contribution was additive with heterozygotes showing an intermediate resistance compared to females homozygous for the NOR marker alleles. The support interval for this NOR-derived diabetes resistance QTL overlaps that of the Idd11 locus originally identified in an outcross of NOD to B6,11 and subsequently confirmed by subcongenic analyses.12 A C57BL/10 (B10)-derived diabetes resistance locus on Chr. 4 designated Idd9 has been found to contain at least three disease protective genes.13 The support interval for the NOR-derived diabetes resistance QTL revealed in the current study, as well as the previously identified Idd11 protective locus from B6, both overlap the most proximal Idd9.1 subcomponent of the multigenic Idd9 locus.13 It should be noted that the nature of the recombination zones of B6 and DBA/2-like alleles on NOR Chr. 4 is more complex than we originally reported. Original typing suggested the region of NOR Chr. 4 spanning the flanking markers D4Mit31 (51.3 cM; 105.5 Mb) and D4Mit16 (59.1 cM; 126.5 Mb) was of DBA origin. However, SNP typing revealed that in addition to DBA-derived genome, this region also contains three small chromosomal segments of B6 and one of NOD origin (Table 1 and Figure 1c; SNP data from Petkov et al14). Initial typing also indicated the region of NOR Chr. 4 delineated by the flanking markers D4Mit203 (60.0 cM; 127.9 Mb) and D4Mit310 (71.0 cM; 146.6 Mb) was of B6 origin. Additional typing revealed this segment also includes a DBA-derived region distal to the linkage markers for the NOR diabetes resistance QTL on Chr. 4 (Table 1, Figure 1c).
A somewhat weaker but still significant NOR resistance contribution was mapped to the proximal BKS region on Chr. 1 that contains both B6 and DBA-like derived genome. The LOD scores were flat over the region, 3.08 at D1Mit3 (11.0 cM, MGI; 20.0 Mb, Ensembl) and 3.04 at D1Mit232 (20.8 cM, MGI; 35.1 Mb, Ensembl) (Table 1 and Figure 4a), although a posterior probability density (PPD) plot calculated for this region suggests that a peak may lie closer to the DBA-derived genome marked by D1Mit232 (data not shown). However, in the original ‘unconditioned’ cross, suggestive evidence for resistance was indicated in the more distal zone of B6 alleles at the marker D1Mit46 (43.1 cM, MGI; 76.0 Mb, Ensembl). In the present ‘conditioned’ cross, interval mapping over the whole chromosome computed a peak linkage at ∼24–35 cM. However, within this interval, NOD, NOR and NOR.NOD-Il1a congenic mice all proved to share allelic identity for the microsatellites D1Mit322 and D1Mit178 as well as the Ctla4 SNP variant likely to be the NOD Idd5.1 susceptibility gene.15 In contrast, the BKS Ctla4 SNP was identical to that reported for B10.15 In a previously published segregation analyses of NOD Idd loci, a region of peak linkage that was identified on Chr. 3 in an initial interval mapping study16 was ultimately shown to reflect effects mediated by both a more proximal and several more distal contributors.17, 18 This suggests that the computer-generated peak at Ctla4 is an artifact, and in reality most likely marks two flanking resistance loci with at least one gene in each region. That the more distal NOR/B6-derived linkage on Chr. 1 contributes resistance was independently confirmed in an NOD.NOR-Chr. 1 congenic stock described below.
Conditioning for Idd13 components in the (NOD × NOR.NOD-Il1)F2 cross also provided suggestive evidence for linkage on Chr. 11 where none was previously detected in the ‘unconditioned’ F2 analysis. As with Chr. 4, the previously reported complexity of NOR Chr. 113 was also further refined by SNP typing in the present study (Table 1). Linkage analysis generated a broad plateau of LOD scores of 2.30–2.34 between D11Mit87 (27.5 cM, 52.0 Mb) and SNP 11-064063416-N (63.2 Mb). Interval mapping computed peak PPD linkage at 26 cM close to D11Mit87, an area the present analyses indicated to be characterized by a series of heretofore undetected recombination events between genomic elements of B6 and DBA origin on NOR Chr. 11 (Table 1). Interestingly, this computationally generated peak is considerably more proximal than both the B6-derived Idd4.1 and Idd4.2 resistance loci identified by Grattan et al19 in a region from 43 to 49 cM. The fact that NOR does not carry B6 alleles, but rather DBA-like alleles across the previously-defined Idd4 complex would explain why linkage at the B6-defined Idd4.1 and Idd4.2 loci was not found in this cross. The NOR resistance peak on Chr. 11 was closer to the IL-12p40-encoding Il12b locus (19 cM) whose overexpression in NOD macrophages was previously associated with an NOD allele linked to the Idd4 complex.20 Given that evidence for linkage was borderline (Figure 4a), it is quite likely that, like most other non-MHC Idd complex regions, multiple genes each contribute a small part of the phenotypic variance. Again, the NOR resistance locus on Chr. 11 was dominant. This region approximates a previous report of a potential C57L-contributed resistance locus in an NOD tricongenic stock designated NOD.DR2.21
Figure 4a shows the QTL analysis for the PC1 (Prin1) as a combined trait (age at onset and insulitis severity/extent). This principal component captures the effect that contrasts the least severe insulitis with the late onset of diabetes on an equally weighted basis. A principal components summary of multiple traits is most effective when the traits are being driven by a common genetic mechanism, in this case, insulitis as a measurement of disease progression. In practice, it is impossible to know that this is the case in advance, and thus a QTL analysis of each trait was performed individually (Figure 4b and c) to assess the extent to which they share common features. The Chr. 1, Chr. 4 and Chr. 11 linkages were each significantly associated with the age of onset, with Chr. 1 and Chr. 4 showing stronger linkage than Chr. 11 (Figure 4b). However, only the Chr. 4 linkage was significantly associated with both II and age at onset (Figure 4b and c). Thus, as reported by others, the linkage on Chr. 1 and Chr. 11 contribute to insulitis severity as measured by the rate at which clinical disease is diagnosed (eg ‘timing genes’).10, 17 The contributions of the Chr. 4 linkage are more complex, indicating that genes on this chromosome not only control the rate at which insulitis destruction progresses but also whether a destructive insulitis will or will not develop. No evidence for interactions among any of these three loci was detected. Also, the newly discovered polymorphic SNPs distinguishing discrete regions of NOR Chr. 5 (∼115 Mb), Chr. 7 (∼77–83 Mb), Chr. 10 (∼18–48 Mb), Chr. 12 (∼6–20 Mb) and Chr. 14 (∼76–89 Mb) failed to show any evidence for linkage (Table 1).
It was anticipated that the small residual NOR genomic segment remaining on Chr. 2 of the NOR.NOD-Il1 congenic stock that included a B6-derived B2mb allele would be a significant resistance contributor in this conditioned F2 cross. As shown in Table 1 and Figure 4a, no contribution from this locus was detected. Currently, the most centromeric B6-derived Chr. 2 allele reported in NOR/Lt is D2Mit395 at 66.9 cM.10 In physical distance, the conserved B6 segment could not exceed the 7.6 Mb interval between D2Mit62 and D2Mit398 in the NOR.NOD-Il1 congenic stock (Figure 1a). Hence, an NOR-derived gene(s) from the distal component of the Idd13 complex is seemingly required to detect a diabetes protective effect mediated by the more proximal region containing the B2mb variant. We previously found that transgenic expression of the B2ma, but not the B2mb variant, reversed the diabetes resistance of an NOD stock homozygous for a disrupted B2m gene contained within a Chr. 2 congenic interval derived from a donor strain with a mixed 129 and B6 genetic background.8 In this NOD transgene recipient stock, the 129/B6-derived congenic interval containing the inactivated B2m gene is ∼10 cM in length. This fact also supports the possibility that B2mb can only contribute to diabetes resistance when coexpressed with a linked distal gene(s) derived from a strain other than NOD. Fox et al10 previously reported that T-cell recruitment to the insulitis lesion in (NOD × NOR)F2 pancreata entailed an interaction between D2Mit395 marking the proximal end of the Idd13 complex and D1Mit305 marking the distal end of the Idd5 region on Chr. 1. No such pair-wise interaction was observed in the conditioned (NOD × NOR.NOD-Il1)F2 population, indicating that the interaction entailed contributions from the more distal components of the Idd13 resistance complex. Further, the region of NOR Chr. 1 identified as conferring resistance in the present cross was considerably more proximal than the region previously shown to control insulitis development through interaction with Chr. 2.10
The diabetes-retardant effect of both the NOR-derived Chr. 4 and Chr. 1 linkages indicated by F2 segregation analysis was confirmed by analysis of diabetes resistance in NOD stocks congenic for NOR intervals on each chromosome (Figure 5). The NOR-derived intervals introgressed into NOD are depicted in Figure 1b (Chr. 1) and 1c (Chr. 4). Consistent with the F2 analysis, the female incidence of diabetes in N5F1 NOD.NOR-Chr. 4 homozygous segregants was significantly less than in those that were either homozygous (P<0.0001) or heterozygous (P=0.0013) for NOD-derived alleles across the congenic segment (Figure 5a). Although Chr. 4 heterozygotes (D/R) developed diabetes at a more protracted rate than NOD homozygotes (D/D), the difference was not significant by survival analysis. Comparative analysis of insulitis scores for the three genotypic classes confirmed a QTL contribution to this phenotype. A mean insulitis score of 1.0 (‘end-stage insulitis’) was assigned to all overtly diabetic mice necropsied prior to the 30 weeks end point of the study. A mean score of 1.0±0 was recorded for D/D homozygotes (n=20), 0.90±0.07 for D/R heterozygotes (n=12) and 0.58±0.08 for R/R homozygotes (n=19). Interestingly, the R/R segregants showed approximately the same degree of protection from clinical disease development as had previously been observed in NOD.NOR-Chr. 2 homozygous females.7 Brodnicki et al12 showed comparable protection in an NOD congenic stock carrying B6-derived alleles across this region designated as Idd11. Similarly, comparable diabetes resistance was also reported by Lyons et al13 in an NOD congenic stock (Idd9R28) carrying B10 alleles across a longer, and more distally extended region of Chr. 4 that encompassed the minimum of three protective Idd9 region genes. Finally, a diabetes-resistant tricongenic stock of NOD mice (NOD.DR2) carrying C57L alleles at the Idd11 locus has also been described.21 The NOD.NOR-Chr. 4 congenic reported herein presumably spans the B6-defined Idd11 region and the overlapping portion of the B10-defined Idd9.1 interval and possibly a portion of Idd9.2 including B6 alleles at Cd30 and Tnfr2, but not Cd137. However, in NOR, it should be noted that most of the Idd9.1 region originally defined by B10-mediated resistance is instead a complex mix of B6, DBA-like and NOD genome. The B6-derived portion of NOR Chr. 4 from ∼128 to ∼135 Mb contained the diabetes resistance QTL peak in our conditioned F2 cross (at ∼132 Mb, 61.9 cM). Hence, we infer that the major resistance contribution is probably B6 derived. It should be noted that the length of the NOR-derived Chr. 4 segment in our congenic stock is slightly shorter than in the donor parental strain as evidenced by the distal recombination between D4Mit234 and D4Mit310. This segment is B6-derived in NOR and NOR.NOD-Il1, but NOD-derived in NOD.NOR-Chr. 4 (Table 1, Figure 1c).
Data in Figure 5b confirm a significant contribution of NOR alleles on Chr. 1 to the strain's diabetes resistance. The resistance alleles in our NOD.NOR-Chr. 1 stock overlap the protective Idd5.2, but not the more proximal Idd5.1 region, both previously identified by truncation of a B10-derived congenic interval.22 The NOD.NOR-Chr. 1 stock also does not carry the even more proximal NOR block shown in Table 1 that contains a mixture of B6- and DBA-derived genome that segregated with diabetes resistance in the F2 progeny. Again, the survival difference between the NOD/NOR heterozygotes and the NOD homozygotes was not significant, but the diabetes-free survival advantage of females homozygous for the NOR alleles depicted in Figure 1 differed significantly from both groups combined (P=0.0053).
Conditioning the genome of diabetes-resistant strains used in outcross to NOD by first substituting their MHC with the diabetes-permissive H2g7 haplotype of NOD has proven an essential first step prior for segregation analysis to identify non-MHC Idd linkages.5, 17 As NOD and NOR are H2g7 identical, genomic conditioning of this strain required reversion to NOD genome of Idd13, the original non-MHC diabetes resistance locus identified in NOR. Multiple studies indicated that the genetic basis for NOR resistance to recruitment/activation of autoimmune T-effector cells into the islets was more genetically complex. Diabetes resistance in NOR mice congenic for NOD-derived TCR transgenes from the diabetogenic NY8.3 CD8+ clone was associated with deletion of that clonotype.23 This NOR protection appeared to act recessively. Reciprocally, diabetes protection in NOR mice congenic for NOD-derived TCR transgenes from the diabetogenic NY4.1 CD4+ clone was not associated with clonotype-specific deletion, but rather activation of more normal immunoregulatory pathways in a dominant manner.23 That the NOR immune repertoire contains a variety of potentially pathogenic effectors was confirmed by tetramer staining showing that islet-reactive BDC2.5 CD4+ T cells were also present in the periphery.24 One regulatory cell type capable of blunting pathogenic activation of diabetogenic T cells is the NKT cell. NKT numbers are decreased in NOD mice and their functions impaired.25 NOD genes contributing to this immunodeficiency have been reported in the Idd4, Idd5, Idd9.1 and Idd13 regions, regions distinguishing NOR from NOD.26, 27 Certain of the immune anomalies associated with NOD antigen-presenting cells (APC) also are more normal in NOR, including NF-κB regulation that promotes more normal control of IL-12p70, TNFα and IL-1α cytokine secretion.28 Thus, a combination of NOR genes serve to suppress autoimmune diabetes development at both the T-cell and APC levels.
The present study underscores the difficulty of identifying Idd loci even in a model cross of reduced genetic complexity wherein both the NOD mouse and the outcross partner NOR strain share extensive susceptibility modifiers, including the MHC and large complement of other genes important for disease development. In a previous outcross of NOD with NOR, insulitis developing in 80-day-old F2 females entailed an epistatic interaction between NOR/B6-derived alleles in the Idd5.2 region and the Idd13 complex of genes on Chr. 2.9 In this previous study, the more proximal ∼11–20 Mb region of NOR Chr. 1 that was shown in our current ‘conditioned’ cross to possibly contain a gene(s) contributing to diabetes resistance did not segregate with suppressed insulitis development. This could be explained if a gene(s) residing within the ∼11–20 Mb region of NOR Chr. 1 does contribute to diabetes resistance, but in a weakly acting manner that can only be unmasked if a potentially more strongly acting protective effect(s) elicited by interactions between Idd5.2 and distal Idd13 region genes are eliminated. Such an unmasking effect could be detected in our ‘conditioned’ cross. Furthermore, analyses of F2 segregants from the ‘conditioned’ cross continued to provide suggestive evidence that even in the absence of distal Idd13 region contributions, a Idd5.2 region gene(s) provides a degree of diabetes resistance in the NOR strain. The diabetes protective effect of this NOR Idd5.2 region gene(s) was confirmed by congenic strain analyses. Curiously, interval mapping software assigned peak linkage in the F2 segregants for an NOR diabetes resistance locus on Chr. 1 to a region previously defined as Idd5.1 using NOD stocks bearing various truncations of B10- or B6-derived congenic segment.29, 30 However, within the peak linkage interval assigned by the software, all NOR markers typed proved to share allelic identity with NOD including a Ctla4 SNP that is likely to be the actual Idd5.1 susceptibility variant.15 Thus, it is possible that the computer assigned peak linkage to the Idd5.1 region is an artifact, and in reality is due to contributions from two flanking loci each containing at least one diabetes resistance gene. This was previously shown to be the case for an Idd gene linkage on Chr. 3 that subsequent analyses demonstrated was actually the result of contributions from both proximal and distal resistance variants.17, 18
In conclusion, removal of components of a major resistance locus, Idd13, from NOR/Lt, an MHC-identical but diabetes-resistant relative of NOD/Lt, uncovered a hierarchy of additional linkages whose significant contributions were difficult to demonstrate in the presence of this locus. This study emphasizes the value for diabetes gene discovery of stratifying not only MHC loci conferring the highest relative risk but also as many as possible of the non-MHC loci presumed to contribute significantly.
Materials and methods
The NOR.NOD-(Il1-D2Mit144)/Lt (hereafter NOR.NOD-Il1) congenic stock (JAX MICE stock no. 2347) was produced by mating NOR/Lt to NOD/Lt, and backcrossing 20 times (N21) to NOR with selection only for NOD genotype at Il1a by typing segregants at each backcross cycle for a restriction fragment length polymorphism identified by Southern blot.2 At N21, flanking polymorphic microsatellite markers on Chr. 2 were typed by PCR3 to define the length of the introgressed NOD congenic interval. Additionally, a genome-wide scan was performed to verify NOR homozygosity at all other chromosomal regions where NOD and NOR were known to differ. This scan indeed confirmed NOR homozygosity at all non-Chr. 2 genomic regions. No microsatellite or SNP polymorphisms have been found that distinguish NOD and NOR alleles on Chr. 2 between the centromere and the marker D2Mit62 (119.7 Mb, Ensembl). As depicted in Figure 1a, this congenic stock, designated NOR.NOD-Il1, exhibits replacement of NOR/B6 alleles by NOD alleles on Chr. 2 between the selected Il1a/Il1b complex at ∼129 Mb and extending distally through Pcna to D2Mit48 at 156.2 Mb (NCBI Ensembl, Build 33). Between the D2Mit62 microsatellite at 117.9 Mb demarcating the boundary of NOD genome resident on the proximal end NOR Chr. 2 and D2Mit398 at 125.5 Mb, proximal to Il1a, the selected NOD allele, a recombination occurred. This recombination resulted in an NOR/B6 genomic residium at D2Mit277 at 123.2 Mb continuing up through the B2m gene at 121.9 Mb to D2Mit395 at 119.3 Mb. The retention of the NOR/B6 B2mb allele was detected as described previously7 and confirmed by flow cytometry using a B2mb-specific monoclonal antibody (Lym11b, Pharmingen, San Diego, CA, USA). In all, 20 females and 20 males were aged for diabetes incidence as well as 10 F1 females generated by mating NOD/Lt females to NOR.NOD-Il1 males. Other F1's were intercrossed to produce the F2 generation. A total of 185 F2 females were aged for incidence of diabetes, with survivors killed at 40 weeks of age and tissue collected for DNA isolation and histology. The onset of diabetes was measured every 2 weeks, beginning at 10 weeks of age, by urinalysis of glucose using Diastix (kindly provided by Bayer). Those animals testing positive over two consecutive weeks were killed and tissue collected for DNA isolation.
Development of additional congenic stocks to confirm Chr. 1 and Chr. 4 linkages
Two congenic stocks were constructed on the NOD/LtDvs background: one containing the distal portion of Chr.1 from NOR/Lt containing alleles of B6 origin (Figure 1b), and the second containing both the B6 and the DBA/2-like alleles contained on NOR Chr. 4 (Figure 1c). NOR/Lt was crossed with NOD/Lt with backcrossing to NOD/Lt with selection for NOR genotype at D1Mit532 (73.7 Mb) and D1Mit8 (83.5 Mb) for the Chr. 1 congenic, and D4Mit31 (105.5 Mb), D4Mit11 (121.6 Mb) and D4Mit160 (142.8 Mb) for the Chr. 4 congenic. At N3 and N4, mice were typed for replacement of the known BKS-derived alleles at sites other than Chr. 1 or Chr. 4 by the respective NOD alleles. As NOR is already 88% NOD derived, fewer backcrosses than usual are required; thus, the congenics were intercrossed at N5. Mice homozygous NOR/NOR and NOD/NOD, as well as heterozygous NOR/NOD for each congenic region at N5F1 and N5F2 were aged for diabetes incidence.
The pancreas from diabetes-free F2 survivors was fixed in Bouin's solution. The pancreas was sectioned at three different levels and granulated β cells stained with aldehyde fuchsin and counterstained with hematoxylin and eosin. Islets were scored for level of insulitis on the following scale: 0, no lymphocytes in or around the islet: (1) peri-insulitis without intraislet infiltration; (2) peri-insulitis with intraislet infiltration reducing β-cell mass by <25%; (3) heavy insulitis with ∼50% reduction in β-cell mass; and (4) complete destruction of the islet. An II was calculated by multiplying the number of islets in each score category by the category score, summing them and then dividing by the total number of islets times 4.0, the maximal total score.31 Thus, the resulting II ranged from a value of 0 (no lesions) to 1 (maximal destruction). Chronically diabetic females were arbitrarily assigned an II score of 1.0.
DNA was prepared by standard methods from spleen or kidney. F2 intercross animals were genotyped using microsatellite markers known to be polymorphic between NOD and NOR in all the C57BLKS/J-derived regions as well as SNP. Most of the microsatellite typing for genome-wide screening was carried out in a TETRAD cycler (MJ Research) with products analyzed on an ABI 3700 instrument (Applied Biosystems). Some fill-in typing was carried out in a PTC-100 (MJ Research) and run in 4% Metaphor (Cambrex Bio Science, Rockland, ME, USA) agarose gels. SNP typing was carried out by KBioscience (Hoddesdon, UK). Additional new SNP polymorphisms distinguishing NOD/LtJ from NOR/LtJ have recently been described.14 The data are accessible at http://www.genome.org/cgi/content/full/14/9/1806/DC1. Additional SNPs distinguishing NOD, NOR, B6 and DBA were obtained from a database generated by the Welcome Trust (http://www.well.ox.ac.uk/mouse/INBREDS). A condensed summary of SNPs distinguishing NOR from NOD, BKS, B6, DBA/2 and NZB (a potential source of contamination) has been posted at the Leiter laboratory website: http://www.jax.org/staff/leiter/labsite/type1_genomics.html. The Ctla4 allele in the Idd5.1 region was assessed by sequence analysis of the polymorphic SNP in exon 2 reported by Ueda et al15 to produce an altered splice variant in NOD (NOD=G; B10=A).
Database organization and ANOVA analysis for dominant/recessive traits were carried out in StatView (Abacus Concepts, Berkeley, CA, USA). Kaplan–Meier analysis for diabetes-free survival was performed using JMP® software (SAS Institute Inc., Cary, NC, USA). Genome-wide scans for QTL were performed using the method of Sen and Churchill.32 This method is similar to the interval mapping procedure of Lander and Botstein,33 but uses a different algorithm to compute model likelihoods. First, we carried out one-dimensional genome scans on a single QTL basis to detect QTL with main effects. LOD scores were computed at 2 cM intervals across the genome and significance was determined by permutation testing.34 Significant and suggestive QTL meet or exceed the 95 and 90% genome-wide thresholds, respectively. Then, simultaneous genome scans for all pairs of markers were implemented to detect epistatic interactions. The search strategy has been described by Sen and Churchill32 and Sugiyama et al.35 Briefly, the genome scan searches through all pairs of loci by fitting a two-way ANOVA model with an interaction item. An LOD score contrasting the full model to a null model (with no genomic effects) is computed and genome-wide significance is established by permutation analysis. A secondary test for the significance of the interaction term is computed only for those pairs that pass the genome-wide screening. A stringent nominal significance level (0.001) is used for interaction test and only those locus pairs passing both tests are deemed to be interacting. Finally, all the detected main effect and interacting QTLs were used to fit multiple regression models. The type III sum of square of each marker or marker pair to the total sum square is the percentage of variance explained by this marker or marker pair. The software package used in this study, Pseudomarker release version 1.01, is available at http://www.jax.org/research/churchill.
Principle components analysis was used in this study to combine the information content of the two quantifiable phenotypes (a discrete variable, age at diabetes onset and a continuous variable, severity/extent of insulitis in nondiabetic survivors). We found that the PC1 captured 72% of the phenotypic variance. Our assumption was that each of the measures is capturing some aspect of an underlying susceptibility to diabetes. II has a numerical score from 0.0 to 1.0. Onset of diabetes was converted to a similar scale covering 15 biweekly sampling intervals where earliest onset time (12 weeks) was assigned a value of 2.0 and latest onset time (40 weeks) a value of 1.07. Both phenotypes (severity/extent of insulitis and week of clinical diabetes onset) were equally weighted. In essence, the scale from 0.0 to 2.0 is a measurement of diabetes progression over time (Figure 3).
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We gratefully acknowledge the assistance provided by Pamela Stanley, Bruce Regimbal and Steve Langley for animal breeding, and by Gunjan Wagner for assistance with SNP genotyping. This work was supported by NIH Grants DK27722 and DK36175. Institutional shared services were supported by National Cancer Institute Center Support Grant CA-34196.
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Reifsnyder, P., Li, R., Silveira, P. et al. Conditioning the genome identifies additional diabetes resistance loci in Type I diabetes resistant NOR/Lt mice. Genes Immun 6, 528–538 (2005). https://doi.org/10.1038/sj.gene.6364241
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