The iddm4 locus segregates with diabetes susceptibility in congenic WF.iddm4 rats

Abstract

Viral antibody-free BBDR and WF rats never develop spontaneous diabetes. BBDR rats, however, develop autoimmune diabetes after perturbation of the immune system, e.g., by viral infection. We previously identified a disease-susceptibility locus in the BBDR rat, iddm4, which is associated with the development of autoimmune diabetes after treatment with polyinosinic:polycytidylic acid and an antibody that depletes ART2(+) regulatory cells. We have now developed lines of congenic WF.iddm4 rats and report that in an intercross of N5 generation WF.iddm4 rats, approximately 70% of animals either homozygous or heterozygous for the BBDR origin allele of iddm4 became hyperglycemic after treatment to induce diabetes. Fewer than 20% of rats expressing the WF origin allele of iddm4 became diabetic. Testing the progeny of various recombinant N5 WF.iddm4 congenic rats for susceptibility to diabetes suggests that iddm4 is centered on a small segment of chromosome 4 bounded by the proximal marker D4Rat135 and the distal marker D4Got51, an interval of <2.8 cM. The allele at iddm4 has 79% sensitivity and 80% specificity in prediction of diabetes in rats that are segregating for this locus. These characteristics suggest that iddm4 is one of the most powerful non-major histocompatibility complex determinants of susceptibility to autoimmune diabetes described to date.

FIG. 1

Kaplan Meier analysis of diabetes in WF.iddm4 N5F1 rats. From among N5 WF.iddm4 progeny scored for markers linked to iddm4, suitable rats were selected to generate intercross progeny as described in RESEARCH DESIGN AND METHODS. In this figure, progeny were typed as homozygous or heterozygous for the presence WF or BBDR origin alleles of the microsatellite marker D4Arb9. A total of 58 of the progeny expressed the ART2.1 regulatory T-cell alloantigen and could therefore be tested for susceptibility to the induction of diabetes by administration of poly I:C and anti-ART2.1 mAb. Among WF.iddm4^d/d rats, 67% became diabetic, with a median latency of 25 days. Among WF.iddm4^d/w animals, 70% became diabetic, with a median latency of 27 days. The cumulative frequency of diabetes in iddm4^w/w progeny was statistically significantly less (18%, P = 0.007 vs. iddm4^d/d and P =0.003 vs. iddm4^d/w). The cumulative frequency of diabetes in homozygous iddm4^d/d rats and heterozygous iddm4^d/w rats was statistically similar (P = 0.57). Significance levels were determined by log-rank statistic with adjustment for ART2 genotype (see RESULTS).

FIG. 2

Map of diabetes susceptibility in heterozygous WF.iddm4 congenic rats. The left-most bar represents the entire length of rat chromosome 4 (RN04). The solid region of the bar indicates the iddm4 interval present in the N5 parents of the N5F1 rats shown in Fig. 1 and the N6 congenic progeny analyzed in Table 1. A magnified view of the iddm4 interval with the relevant microsatellite markers is shown in the adjacent bar, with the intermarker distances indicated in centiMorgans. The remaining bars represent the segment of this interval present in the parent of each of the seven subcongenic types, with the type number indicated on the top of each bar. At the bottom of each bar is the percentage of progeny of these animals that developed diabetes after treatment, as explained in Table 1 and RESULTS. Areas of the bars with no fill indicate WF origin alleles, and areas with solid fill indicate BBDR-derived alleles. Diagonal fill indicates transition intervals for which the strain of origin could not be determined with available markers. Note that these chromosomal segments are those of the parents and do not take into account recombinations that may be present in their progeny. Considering only the definitively typed regions (solid fill) and omitting from consideration type 11, which is indeterminate in its susceptibility to diabetes, the data from progeny testing define a 2.8-cM interval for iddm4 bounded by D4Rat135 and D4Got51.

FIG. 3

Kaplan Meier analysis of diabetes in WF.iddm4^d (D4Arb9^d/w) and WF.iddm4^w (D4Arb9^w/w) N6 congenic rats. Congenic N6 generation WF.idddm4 rats were bred and treated with poly I:C and anti-ART2.1 mAb as described in RESEARCH DESIGN AND METHODS. All animals were genotyped and grouped according to the presence of the WF and BBDR alleles of the microsatellite marker D4Arb9, as described in RESULTS and in the legend to Table 1. One WF.iddm4^d rat that died on day 17 with unknown glycemic status has been omitted. The cumulative frequency of diabetes was 49% in the WF.iddm4^d rats and 6% in the WF.iddm4^w group (P < 0.0001, log-rank statistic after adjustment for ART2 genotype).

FIG. 4

Kaplan Meier analysis of diabetes in adoptive recipients of thymocytes. Thymocytes were obtained from the N5F1 WF.iddm4 rats described in Fig. 1 and prepared for adoptive transfer to WAG rnu/rnu rats, as described in RESEARCH DESIGN AND METHODS. Thymi were obtained from diabetic donors within 1 day of diagnosis; additional thymi were obtained from animals still nondiabetic at the conclusion of the protocol on day 40. Recipients were monitored for the development of diabetes for 90 days after transfer. Overall life table analysis, after adjustment for the ART2 genotype of the thymocyte donor, confirmed a statistically significant difference in the behavior of the three groups at the P < 0.005 level. Thymocytes from WF.iddm4^d/d and WF.iddm4^d/w were equally effective in transferring diabetes (P = 0.5), and both of these groups differed significantly from the WF.iddm4^w/w thymocyte donors (P < 0.01). Recipients of thymocytes from donors bearing the WF allele of D4Arb9 became diabetic, with a median latency to onset of 56 days, whereas recipients of thymocytes from donors bearing a BBDR origin allele of D4Arb9 became diabetic, with a median latency of 33 days. There was no statistically significant independent effect attributable to the presence or absence of diabetes in the thymocyte donor. N indicates the number of rats entered into the study, and the small vertical bars indicate censored data, i.e., rats that either died during the study (n = 4) or were still normoglycemic at the end of the period of observation.