Mapping and Phenotype information for this QTL, its variants and associated markers
Recombinant congenic strains derived from NOD and C57BL/6J parentals resolved the Idd5 locus to two separate QTLs, Idd5a and Idd5b. Idd5a was localized to a region spanning 32.8 cM - 34.8 cM on mouse Chromosome 1 bounded by the markers D1Mit302 and D1Mit178. As a result, Cflar and Casp8 were excluded as candidate genes for Idd5a. Icos, Cd28, and Ctla4 remain candidate genes for Idd5a. C57BL/6J-derived alleles confer dominant resistance to diabetes at this locus. Idd5a is syntenic to human Chromosome 2q33 and may be the orthologue of IDDM12 in humans.
In this study the Idd5a interval was refined to 30.1 cM - 32.8 cM in subcongenic animals carrying C57BL/10J-derived DNA from D1Mit74 to D1Mit249 on an NOD genetic background. Parental strain NOD/MrkTac is susceptible to diabetes whereas parental strain C57BL/10J is resistant. Cd28, Casp8, and Cflar were eliminated as candidate genes as a result. Ctla4 (30.1 cM) and Icos (32 cM) remain strong candidate genes while 2810455B10Rik (Als2cr19) and Nrp2 are considered possible candidate genes. A recombination coldspot was observed between D1Mit249 and D1Mit303 as this distance is 2 Mb and only 3 recombination events occurred out of 22 expected.
A high amount of sequence polymorphism between C57BL/6J and NOD/MrkTac was observed within the reduced Idd5a interval. Several SNPs were observed in the intronic and 3' and 5' regulatory sequences of Ctla4 and Icos as well as 1 exonic SNP for each gene. Very few SNPs were observed for 2810455B10Rik. Differential expression of a Ctla4 splice isoform (liCTLA-4 mRNA) was observed for animals carrying the Idd5a<C57BL/10/J> allele vs animals carrying the Idd5a<NOD/MrkTac> allele. The Idd5a interval is syntenic to human Chromosome 2q33.3-q34.
The Idd5b interval was refined to a 1.52 Mb region between D1Mit180 (41 cM) and D1Mit46 (43.1 cM) using subcongenic lines designated R193 and R444s. 45 genes map to this interval. Scl11a1 (Nramp1) is a candidate gene for Idd5b and contains a functional polymorphism between C57BL/6J, C57BL/10J, and NOD/MrkTac. The Idd5b interval is syntenic to human Chromosome 2q35.
The presence of both Idd5a and Idd5b are required for full diabetes resistance. Each locus alone offers partial protection.
A series of subcongenic lines derived from C57BL/10J-derived donor DNA on a NOD/MrkTac genetic background was used to refine Idd5 (insulin dependent diabetes 5) on mouse Chromosome 1 into four separate QTLs- Idd5a, Idd5b, Idd5c and Idd5d. Donor strain C57BL/10J is resistant to type 1 diabetes mellitus compared to background strain NOD/MrkTac. The 4 QTLs that comprise the Idd5 locus display interactive effects. C57BL/10J alleles at Idd5a, Idd5b and Idd5c confer diabetes resistance while the same alleles at Idd5d confer diabetes susceptibility.
Idd5a (insulin dependent diabetes 5a) was localized to a 2 Mb region between D1Mit249 (32.8 cM; 60.8 Mb) and genetic sequence AL671560GA near Pard3b and Nrp2 (62.7 Mb). Ctla4 (30.1 cM; 60.9 cM) was named a likely candidate gene for Idd5a due to the presence of sequence variations and gene expression and splice isoform differences between C57BL/10J and NOD/MrkTac. C57BL/10J-derived diabetes resistance alleles at Idd5a have the strongest effect when C57BL/10J alleles are also present at Idd5b, Idd5c and Idd5d.
Idd5b (insulin dependent diabetes 5b) mapped to a 1.5 Mb interval where Slc11a1 (formerly Nramp1, 39.2 cM; 74.4 Mb) was named a likely candidate gene. Interaction between Idd5b and Idd3 on chromosome 4 was previously noted. C57BL/10J-derived alleles at Idd5b and Idd3 confer reduced insulitis, but do not confer diabetes resistance.
A novel diabetes resistance locus named Idd5c (insulin dependent diabetes 5c) was identified between rs30280887 (66.5 Mb) and rs30471359 (70.08 Mb). Eleven genes map to this 3.5 Mb interval of which Acadl (27.3 cM; 66.87 Mb) and Ikzf2 (69.58 Mb) are potential candidates for Idd5c. C57BL/10J-derived alleles at Idd5c are required for nearly complete protection from diabetes when resistance alleles are also present at Idd5a and Idd3 on mouse Chromosome 3 (19.2 cM; 60.2 Mb). It is unclear if the presence of Idd5c and Idd3 are sufficient to mediate diabetes resistance; however, congenic lines carrying both loci are now under construction to resolve this issue.
Idd5d (insulin dependent diabetes 5d) mapped to the distal portion of the Idd5 locus from approximately 76 Mb to 160 Mb. An intervening NOD/MrkTac-derived interval in Idd5d is located from approximately 140 Mb to 150 Mb due to a double recombination event in a progenitor congenic line. C57BL/10J-derived alleles at Idd5d confer diabetes susceptibility even when resistance alleles are present at Idd5b and Idd5c.
Idd5 on mouse Chromosome 1 was confirmed in a congenic strain carrying a C57BL/10J donor region spanning the entire Idd5 locus introgressed onto the NOD/MrkTac genetic background. Parental strain C57BL/10J is resistant to type 1 (insulin dependent diabetes) whereas parental strain NOD/MrkTac is susceptible. As expected, the congenic strain displayed resistance to type 1 diabetes.
Refinement of the Idd5 locus was accomplished by creation of several subcongenic lines. Idd5 was resolved to 2 distinct loci, Idd5a at 33.8 cM and Idd5b at 41 cM on mouse Chromosome 1. Idd5a spans 32.8 cM (D1Mit124) - 34.8 cM (D1Mit303) and includes candidate genes Cflar, Casp8, Cd28, and Ctla4. Sequence analysis of Casp8 revealed several polymorphisms between C57BL/10J and NOD/MrkTac. Idd5b spans 39.3 cM (D1Mcg5) - 43.1 cM (D1Mit46/D1Mit132) and includes candidate genes Slc11a1 (Nramp1) and Il8rb (Cmkar2). A functional polymorphism exists in the Slc11a1 sequence between C57BL/10J and NOD/MrkTac. C57BL/10J-derived alleles confer resistance to type 1 diabetes at Nidd5a and Nidd5b.
An interaction between Nidd5 and Nidd3 was also confirmed in this study. A double congenic carrying C57BL/10J donor DNA at both Nidd5 and Nidd3 on an NOD/MrkTac genetic background was constructed. The double congenic is extremely resistant to both type 1 diabetes and insulitis.