Experiment
Development of type 1 diabetes in NOD/Jsd mice can be accelerated with cyclophosphamide treatment, but not in NOR/LtJ diabetes resistant mice. This phenotype was mapped using linkage analysis of 66 (NOD/Jsd x NOR/LtJ)F1 x NOD/Jsd backcross animals. NOR/LtJ is a recombinant inbred strain derived from a NOD genetic background (88%) with C57BLKS/J donor regions over 10 different chromosomes. Some of these regions contain Idd susceptibility loci. NOD/Jsd is susceptible to spontaneous and cyclophosphamide-accelerated type 1 diabetes whereas NOR/LtJ is resistant.
Significant linkage to severe insulitis in cyclophosphamide-treated backcross animals mapped to 43 cM on mouse chromosome 11 near D11Mit219 (LOD=4.9) and to 60 cM on mouse Chromosome 4 near D4Mit338 (LOD=7.4). The chromosome 11 locus accounts for 30% of the variance and corresponds to previously identified diabetes QTL Idd4. The Idd4 1-LOD confidence interval spans a 7.8 cM interval on chromosome 11 between 37 cM - 44.8 cM. The chromosome 4 locus accounts for 43% of the variance and corresponds to previously identified diabetes QTL Idd9. The Idd9 1-LOD confidence interval spans a 12.4 cM interval on chromosome 4 between 53.6 cM -66 cM. All animals with progression to severe insulitis following cyclophosphamide treatment are homozygous for NOD/Jsd-derived alleles at both Idd4 and Idd9.
Analysis of reciprocal congenic strains derived from NOD/Jsd and NOR/LtJ confirmed the involvement of Idd4 and Idd9 in cylcophosphamide-accelerated insulitis. Furthermore, Idd4 was localized to a 6.9 cM interval between D11Mit30 (40 cM) and D11Mit33 (46.9 cM). NOR/LtJ-derived alleles at Idd4 confer protection from cyclophospamide-induced insulitis in female animals, but not in male animals. NOR/LtJ-derived alleles at Idd9 confer protection from cyclophosphamide-induced insulitis in both male and female animals. The NOR/LtJ allele at Idd4 also protects against spontaneous development of type 1 diabetes. The NOD/Jsd allele at Idd4 confers susceptibility to type 1 diabetes with a stronger effect observed in male animals.
Potential candidate genes mapping near the Idd4 interval are Nos2 (45.6 cM), Tnfaip1 (45.1 cM), Evi2a (46.1 cM), Tcf2 (44 cM), Cryba1 (44.7 cM), and the Scya cluster (47 cM). Previously identified QTLs Eae7 (48 cM) and Orch3 (44 cM) also map to this region.
Potential candidate genes for Idd9 include Csf3r (57.5 cM), Iapls1-19 (57.2 cM), sno (58.3 cM), and Lck (59 cM). Previously identified diabetes QTL Idd11 colocalizes with Idd9 at 65 cM on mouse Chromosome 4. Idd11 confers a protective effect against cyclophosphamide-accelerated type 1 diabetes, which corroborates with the findings from this study.
Animals doubly congenic for NOD/Jsd-derived alleles both Idd4 and Idd9 exhibit significantly greater susceptibility to cyclophosphamide-accelerated type 1 diabetes compared to NOR/LtJ controls, but less so than NOD/Jsd parentals. This finding suggested the presence of other protective loci in the NOR/LtJ background, possibly contributed by Idd5 (40 cM)onmouse Chromosome 1 or Idd13 (71 cM) on mouse Chromosome 2. Reciprocal congenic lines for Idd5 and Idd13 were derived from NOD/Jsd and NOR/LtJ to evaluate this hypothesis. NOD.NOR-Idd5 congenic animals exhibited protection from cyclophosphamide-accelerated type 1 diabetes whereas NOD.NOR-Idd13 congenics did not. However, the reciprocal congenic NOR.NOD-Idd5 did not exhibit increased susceptibility to type 1 diabetes after cyclophosphamide treatment, indicating that Idd5 only confers a protective effectwith the NOR/LtJ-derived allele. Ctla4 was previously identified as a potential candidate gene for Idd5 but since the resistant Idd5 congenic in this study carried NOD/Jsd-derived Ctla4 DNA its candidacy was excluded. Potential Idd5 candidate genes include Slc11a1 (formerly Nramp1, 39.2 cM), Chrng (52.3 cM), Col6a3 (53.9 cM), Hdlbp (55.3 cM), and Ramp1 (56 cM).
NOD/Jsd and NOR/LtJ share the same Idd1 alleles at 19.5 cM on mouse Chromosome 17. After cyclophosphamide treatment both strains show signs of insulitis, although it is mild in NOR/LtJ and severe in NOD/Jsd, progressing to diabetes after 18 days of continued treatment. Controls strains C57BL/6J and BALB/cJ do not exhibit any signs of insulitis after cyclophosphamide treatment. This finding suggests that NOD-derived alleles at Idd1 confer susceptibility to cyclophosphamide-induced insulitis.
Analysis Tools