Congenic mapping of the type 1 diabetes locus, ldd3, to a 780-kb region of mouse chromosome 3:: Identification of a candidate segment of ancestral DNA by haplotype mapping

Type I diabetes in the nonobese diabetic (NOD) mouse arises as a consequence of T cell-mediated destruction of the insulin-producing beta cells of the pancreas. Although little is known of the events that initiate and subsequently drive beta-cell destruction it is clear that the entire process is under complex genetic control. Ar present 19 loci have been mapped that influence the development of diabetes either at the level of initiation of insulitis or at the level of progression from insulitis to overt diabetes, or both. Previously, we have mapped one of these loci, 1dd3, to a 0.35-cM interval on proximal mouse chromosome 3. In the present study we have narrowed the map position of this locus to an interval of 0.15 cM by a combination of novel congenic strains and an ancestral haplotype analysis approach. We have constructed a physical contig in bacterial artificial chromosome (BAC) clones across the minimal interval. Restriction mapping of the BAC contig placed the maximum size of the 1dd3 interval at 780 kb between the markers D3Nds36 and D3Nds76. To refine further the 1dd3 interval we developed a series of novel single nucleotide polymorphisms (SNPs) and carried our haplotype analysis on DNA From mouse strains known to carry either 1dd3 susceptibility or protective alleles. This haplotype analysis identified a 145-kb segment of ancestral DNA between the microsatellite marker D3Nds6 and the SNP 81.3. One haplotype of this ancestral segment of DNA is Found in mouse strains carrying an 1dd3 susceptibility allele and another is found in mouse strains carrying an Idd3 protective allelle. Within the 780-kb congenically defined interval this 145-kb segment represents the most likely location for 1dd3. The 112 gene, which encodes the cyrokine interleukin 2 [IL2], maps to this interval and is a strong candidate for 1dd3. To investigate whether sequence variation exists in the promoter region of the 112 gene, which might alter its expression, we sequenced the promoter region of the 112 gene from mouse strains carrying either an 1dd3 susceptibility or resistance allele. Two sequence variants were identified, neither of which Fell In known regulatory elements within the 112 promoter. In agreement with this observation steady-state 112 mRNA levels showed no variation between susceptible and resistant mouse strains. These data suggest that the profound protection from diabetes seen in congenic mice carrying an 1dd3 protective allele is unlikely to be due to differences in the level of expression of the 112 gene. Instead, all of the current data support our hypothesis that Idd3 corresponds to amino acid variation at the amino terminus of 112.