Genetic Loci Contributing to Spontaneous Autoimmune Diabetes

Abstract: Background and Aims: Spontaneous type 1 diabetes (T1D) in the BioBreeding (BB) rat mimics human T1D as the rats experience weight loss, polydipsia, polyuria, ketoacidosis, onset during puberty and insulin-dependency within a day after diagnosis. Because the DP rat develops T1D spontaneously, it is a prime laboratory animal for dissecting the genetics of T1D susceptibility without the need for external manipulation. The BB rat is comprised of two separate substrains; the diabetes prone (BBDP) and the diabetes resistant (BBDR). Failure to express the Gimap5 protein is associated with lymphopenia (lyp) and linked to T1D in the BBDP rat. In an intercross between F1(BBDP x F344) rats we identified a rat with a recombination event on rat chromosome (RNO) 4, allowing us to fix 34Mb of F344 between D4Rat253 and D4Rhw6 in the congenic DR.lyp rat line with two Mb of BBDP DNA, encompassing the Gimap5 mutation, introgressed on the DR genetic background. The aim of this thesis was to characterize the F344 DNA introgression, test the hypothesis that the introgression would result in 1) no effect on T1D development or 2) protection from T1D, generate congenic sublines and positionally clone and characterize the resulting candidate genes on rat RNO4. Material and Methods: The F344 fragment in the DRF.f/f rat line was fixed onto the DR.lyp background in a total of nine backcross and seven intercross matings. To generate DRF.f/f congenic sublines, DRF.f/f rats were crossed to inbred BBDR or DR.lyp/lyp rats and the offspring genotyped, phenotyped for lymphopenia and monitored for T1D. Positional candidate genes were then subjected to coding sequence analysis, cDNA sequencing and/or quantitative real-time (qRT) PCR expression analysis. Results: DRF.f/f rats, homozygous for the F344 allele, were lymphopenic but did not develop T1D while all (100%) DR.lyp/lyp rats developed T1D by 83 days of age. Generation of congenic sublines revealed that reduction of the DRF.f/f F344 DNA fragment by 26 Mb (42.52 Mb-68.51 Mb) retained complete T1D protection. Further dissection revealed that a 2 Mb interval of F344 DNA (67.41-70.17 Mb) (Iddm38) resulted in 47% protection while retaining <1 Mb of F344 DNA at the distal end (Iddm39) resulted in 28% protection, both of which significantly delayed onset. Comparative analysis of T1D frequency in the DRF.f/f congenic sublines refined the Iddm38 and Iddm39 intervals to approximately 670 Kb between SNP SS105325016 and D4Rat26 and 340 Kb proximal to Gimap5, respectively. Coding sequence analysis revealed TCR Vβ 8E, 12 and 13 as candidate genes in Iddm38 and Znf467 and Atp6v0e2 in Iddm39. Quantitative RT-PCR analysis of whole organ as well as in FACS sorted thymocytes and peripheral T-cells stained with CD4 and CD8 monoclonal antibodies showed a reduction in expression of four out of five Gimap genes located within the Iddm39 interval, in addition to Gimap5, in DR.lyp/lyp spleen and mesenteric lymph nodes (MLN) when compared to DR.+/+. Conclusions: Our data demonstrates that introgression of a 34 Mb region of the F344 genome, proximal to the mutated Gimap5 gene, renders the congenic DR.lyp/lyp rat T1D resistant despite being lymphopenic. Generation of congenic sublines revealed that spontaneous T1D in the BB rat is controlled, in part, by at least two genetic loci, Iddm38 and Iddm39, in addition to the Gimap5 mutation on RNO4. Coding sequence analysis revealed TCR Vβ 8E, 12 and 13 as candidate genes in Iddm38 and Znf467 and Atp6v0e2 as candidate genes in Iddm39. Quantitative RT-PCR expression analysis suggests that the lack of the Gimap5 protein in the DR.lyp/lyp congenic rat impairs expression of the entire Gimap gene family and regulates T-cell homeostasis in the peripheral lymphoid organs. The molecular identification and characterization of the genetic factors protecting from T1D in the DRF.f/f congenic rat line should prove critical to disclose the mechanisms by which T1D develops in the BB rat.