Original Article Genetic Reconstitution of Autoimmune With Two Major Susceptibility in the Rat Norihide Yokoi,1,2 Chihiro Hayashi,1 Yuuka Fujiwara,1 He-Yao Wang,3 and Susumu Seino1

The Komeda diabetes-prone (KDP) rat is an animal model LETL, and KDP rats have the identical MHC haplotype, u u u u u of human autoimmune type 1 diabetes. We have previously RT1 (RT1.A B D C ), while LEW.1AR1-iddm rats have shown that two major susceptibility genes, the major his- a recombinant haplotype, RT1r2 (RT1.Aa Bu Du Cu). How- tocompatibility complex (MHC) RT1u haplotype and Cblb ever, the MHC class II haplotype (RT1.Bu Du)iscommon (Casitas B-lineage lymphoma b) mutation, are responsible to all of the rat models of type 1 diabetes, suggesting that for the development of diabetes in KDP rats, suggesting a the class IIu haplotype confers susceptibility to type 1 two- model for development of the disease. To confirm diabetes in rats (11). the two-gene model, we produced a congenic strain carry- To confirm the susceptibility genes in type 1 diabetes, ing mutated Cblb alleles of the KDP rat on a non-KDP genetic background harboring the RT1u haplotype on its several studies on the genetic reconstitution of the disease MHC. Despite the low incidence and delayed onset of on nondiabetic genetic backgrounds have been performed diabetes, the congenic strain did develop the disease, but have been unsuccessful to date (12–14). The congenic indicating that type 1 diabetes can be reconstituted on a rat strain carrying two major susceptibility alleles, the non-KDP genetic background with the RT1u haplotype and RT1u haplotype and Lyp/Ian4l1, of the BB rat on the F344 Cblb mutation. Similar to observations in KDP rats, the genetic background did not exhibit diabetes or insulitis congenic strain showed insulitis and thyroiditis, symptoms (12). Similarly, the congenic strain carrying the Lyp/Ian4l1 of autoimmunity. The low incidence and delayed onset of allele of the BB rat on the F344 background showed the the disease strongly suggest involvement of genetic modi- lymphopenia phenotype without insulitis and diabetes fiers; the congenic strain established in this study should be useful for the mapping and identification of such modi- (13). In addition, the congenic strains carrying susceptibil- fiers. Diabetes 56:506–512, 2007 ity alleles of the NOD mouse on the C57BL/6 (B6) genetic background did not develop diabetes, but insulitis was occasionally observed in some of the animals (14). In contrast, genetic reconstitution of another autoimmune disease, systemic lupus erythematosus, has successfully ype 1 diabetes is an autoimmune disease in been performed. In this model, the combination of three which both genetic and environmental factors susceptibility genes on the B6 genetic background resulted are involved. Only the major histocompatibility in development of the disease (15). Tcomplex (MHC) has been identified as a major Based on our previous studies (16,17), we proposed a genetic factor, but several other genes may be involved in two-gene model for the development of type 1 diabetes in INS the development of the disease, such as (insulin), KDP rats (18). In this model, two major susceptibility CTLA4, and PTPN22 (1,2). Several spontaneous animal genes, the RT1u haplotype and Cblb (Casitas B-lineage models of type 1 diabetes have been established, including lymphoma b) mutation determined tissue specificity to the nonobese diabetic (NOD) mouse (3), the BioBreeding pancreatic ␤-cells and autoimmune reaction, respectively. (BB) rat (4,5), the Long-Evans Tokushima Lean (LETL) rat However, two important questions remain to be answered. (6), the Komeda diabetes-prone (KDP) rat (7), and the First, can type 1 diabetes be reconstituted on non-KDP LEW.1AR1-iddm rat (8). Genetic analyses demonstrate genetic backgrounds with these two major susceptibility that MHC is the major genetic factor underlying the genes? And second, are other genetic factors involved in development of diabetes in all of these models. In addition, the development of this disease in KDP rats? To answer combinations of MHC and other susceptibility genes may these questions, we have produced and investigated a contribute to the development of the disease (9,10). BB, congenic strain carrying the mutated Cblb allele of the KDP rat on a non-KDP genetic background harboring the u From the 1Division of Cellular and Molecular Medicine, Kobe University RT1 haplotype on its MHC. Graduate School of Medicine, Chuo-ku, Kobe, Japan; the 2Clinical Genome Informatics Center, Kobe University Graduate School of Medicine, Chuo-ku, Kobe, Japan; and the 3Shanghai Institute of Materia Medica, Chinese Academy RESEARCH DESIGN AND METHODS of Sciences, Shanghai, China. Address correspondence and reprint requests to Susumu Seino, Division of Tester Moriyama (TM) rats were obtained from Dr. T. Serikawa at the Institute Cellular and Molecular Medicine, Kobe University Graduate School of Medi- of Laboratory Animals, Kyoto University. Both TM and KDP rats (19) were cine, Chuo-ku, Kobe 650-0017, Japan. E-mail: [email protected]. maintained under specific pathogen-free (SPF) conditions with a 12-h light/ Received for publication 24 July 2006 and accepted in revised form 23 dark cycle. A commercial diet (CE-2; CLEA Japan, Tokyo, Japan) and water September 2006. were available ad libitum at the Institute for Experimental Animals, Kobe MHC, major histocompatibility complex; SPF, specific pathogen free. University School of Medicine. The TM and KDP rats were crossed to produce DOI: 10.2337/db06-1027 the F1 progeny. Following this, six successive backcrosses (N7) to the TM rats © 2007 by the American Diabetes Association. were performed. An intercross between the N7 animals was conducted to The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance obtain the congenic strain (N7F1) carrying the mutated Cblb allele of the KDP with 18 U.S.C. Section 1734 solely to indicate this fact. rat on the TM genetic background. This congenic strain was maintained at the

506 DIABETES, VOL. 56, FEBRUARY 2007 N. YOKOI AND ASSOCIATES same facility. All animal experiments were approved by the Committee on developed the disease at ϳ120 days of age (156.3 Ϯ 9.4 Animal Experimentation, Kobe University School of Medicine (permission no. days, n ϭ 9). Despite the low incidence and the delay of P-031207) and carried out in accordance with the Guidelines for Animal Experimentation at Kobe University. The KDP rats are available from Japan onset, it is noteworthy that type 1 diabetes was reconsti- SLC (Shizuoka, Japan). The congenic strain (TM.KDP-Cblb) has been depos- tuted on the non-KDP genetic background with the two u ited (ref. no. 0354) and is available from the National Bio-Resource Project of major susceptibility genes (RT1 haplotype and Cblb Rat in Japan (www.anim.med.kyoto-u.ac.jp/nbr/). mutation). Phenotyping. Phenotyping was completed using a previously described We also compared body weight and changes in blood protocol (16). Diabetes was defined as glycosuria positivity and blood glucose glucose level in the congenic and KDP strains (Fig. 1B–D). levels Ն250 mg/dl under ad libitum dietary conditions. Data were obtained from five generations (N7F1 to N7F5) of the congenic strain and one At 210 days of age, both females and males in the congenic generation of the KDP strain. strain exhibited higher body weight than KDP rats (KDP Genotyping. Simple sequence-length polymorphism markers used in this vs. congenic: female 230 Ϯ 4.5 vs. 246.5 Ϯ 3.7 g, P ϭ 0.014; study have been described elsewhere (RATMAP, available at http://ratmap- male 377.8 Ϯ 5.7 vs. 450.5 Ϯ 10.2 g, P Ͻ 0.0001). In .gen.gu.se/; Rat Genome Database, available at http://rgd.mcw.edu/; 16,17,19). contrast, there was no difference in changes in blood Genotyping was performed as described in prior investigations (16,17,19). glucose levels between the strains, except for age of onset Histological analysis. Histological analysis was performed as described previously (7,16), with some modifications. Briefly, tissues were fixed in 10% of diabetes: blood glucose levels elevated rapidly from formalin, and paraffin sections were stained with hematoxylin and eosin. ϳ100 to ϳ400 mg/dl within a week in both the congenic Serial sections were viewed via light microscopy by an examiner blind to the and KDP strains. experimental conditions of the animals. Each animal was rated on the degree Degree of insulitis of the congenic and KDP strains. of insulitis, which ranged from none to severe (none [0%], slight [Ͼ0 and Յ5%], We then investigated the degree of insulitis shortly after mild [Ͼ5 and Յ20%], moderate [Ͼ20 and Յ70%], and severe [Ͼ70%]) based the onset of diabetes in diabetic animals and at 210 days of mainly on the percentage of moderately and severely infiltrated islets. The term “slight insulitis” refers to at least one infiltrated islet across the sections age in nondiabetic animals (Fig. 2). In the congenic strain, and a percentage of infiltrated islets of Յ5%. Animals were also rated on the the Cblb homozygous mutants (n ϭ 35) showed slight to degree of thyroiditis, which ranged from none to severe (none [0%], slight to severe insulitis, while heterozygous (n ϭ 22) and wild-type mild [Ͼ0% and Յ20%], and moderate to severe [Ͼ20%]) based on the (n ϭ 14) animals exhibited none or only slight insulitis percentage of infiltrated regions in thyroid sections. Data were obtained from (TM.KDP-Cblb homozygotes vs. heterozygotes, ␹2 [df ϭ 4] five generations (N7F1 to N7F5) of the congenic strain and one generation of ϭ 43.7, P Ͻ 0.0001; TM.KDP-Cblb homozygotes vs. wild- the KDP strain, except that the degree of insulitis at 90 days of age was ␹2 ϭ ϭ Ͻ evaluated in the N7F9 generation of the congenic strain. types, [df 4] 37.2, P 0.0001). In contrast, all of the Statistical analysis. Differences in the incidence of diabetes and in the Cblb homozygous mutants (n ϭ 18) of KDP rats showed degree of insulitis or thyroiditis were assessed using ␹2 tests. Differences in severe insulitis, which is consistent with 100% incidence of body weights were assessed using two-tailed Student’s t tests. Nominal P diabetes (KDP homozygotes vs. TM.KDP-Cblb homozy- values are listed for all analyses. gotes, ␹2 [df ϭ 3] ϭ 26.2, P Ͻ 0.0001). Although the degree of insulitis was relatively mild in the congenic strain compared with that in KDP rats, these results indicate that RESULTS insulitis was reconstituted on the non-KDP genetic back- Establishment of a TM.KDP-Cblb congenic strain. By ground with the two major susceptibility genes (RT1u crossing the TM and KDP rats followed by six successive haplotype and Cblb mutation). backcrosses with the speed congenic approach (20), we To clarify the onset of insulitis in the congenic strain, we successfully produced a congenic strain carrying the mu- further investigated the degree of insulitis at 90 days of age tated Cblb allele of the KDP rat on the TM genetic (Fig. 2). At that age, KDP rats showed 70% incidence of background harboring the RT1u haplotype, TM.KDP-Cblb. diabetes, whereas none of the animals in the congenic Genetic profiling revealed that the ϳ14-cM (ϳ15-Mb) strain developed diabetes. The Cblb homozygous mutants region between D11Rat68 and D11Mgh4 on (n ϭ 10) of the congenic strain showed none to moderate 11 harboring the mutated Cblb allele of the KDP rat had insulitis, which is milder than at 210 days of age. These been introgressed into the TM genetic background and results indicate that the onset and progression of insulitis that the KDP genome was not detected on other chromo- were delayed in the congenic strain, thus resulting in the somes (Table 1). Since the Cblb homozygous mutants have delayed onset of diabetes. poor reproductive ability (17,19), we have been maintain- Degree of thyroiditis of the congenic and KDP ing the congenic strain that has the Cblb region in the strains. We also investigated the degree of thyroiditis of heterozygous state. the congenic and KDP strains at the same ages as those for Incidence of diabetes in the congenic and KDP insulitis (Fig. 3). Regarding the congenic strain, 23% (8 of strains. We compared the incidence of diabetes in the 35) and 20% (7 of 35) of the Cblb homozygous mutants congenic strain with that in KDP rats under the same SPF showed slight to mild and moderate to severe thyroiditis, condition (Fig. 1A). At 210 days of age, 26% (9 of 35) of the respectively, while heterozygous (n ϭ 22) and wild-type Cblb homozygous mutants of the congenic strain devel- (n ϭ 14) animals exhibited none or only slight thyroiditis oped diabetes, while none of the heterozygous (0 of 22) or (TM.KDP-Cblb homozygotes vs. heterozygotes, ␹2 [df ϭ 2] wild-type (0 of 14) animals developed the disease ϭ 10.0, P ϭ 0.0067; TM.KDP-Cblb homozygotes vs. wild- (TM.KDP-Cblb homozygotes vs. heterozygotes, ␹2 [df ϭ 1] types, ␹2 [df ϭ 2] ϭ 8.65, P ϭ 0.0133). In KDP rats, 11% (2 ϭ 6.72, P ϭ 0.0095; TM.KDP-Cblb homozygotes vs. wild- of 18) of the Cblb homozygous mutants showed moderate types, ␹2 [df ϭ 1] ϭ 4.41, P ϭ 0.0357). In contrast, all (18 to severe thyroiditis. In both strains, mild to severe thy- of 18) of the Cblb homozygous mutants of KDP rats roiditis was observed in either the nondiabetic animals or developed diabetes at 210 days of age (KDP homozygotes those with delayed onset of diabetes. In contrast to the vs. TM.KDP-Cblb homozygotes, ␹2 [df ϭ 1] ϭ 26.3, P Ͻ results for insulitis and diabetes, the congenic strain 0.0001). In addition to the low incidence, the onset of showed a higher incidence of thyroiditis than that seen in diabetes was delayed in the congenic strain. KDP rats KDP rats (KDP homozygotes vs. TM.KDP-Cblb homozy- developed diabetes as early as 60 days of age (96.3 Ϯ 8.8 gotes, ␹2 [df ϭ 2] ϭ 6.43, P ϭ 0.0401). Because thyroiditis days [means Ϯ SE], n ϭ 18), while the congenic strain is one of the autoimmune symptoms observed in KDP rats

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TABLE 1 TABLE 1 Genetic profile of the TM.KDP-Cblb congenic strain Continued

*Distances (in cM) were based on the genetic linkage map (19) for chromosome (Chr) 11 or on the Genome-Wide Rat Screening Set (Invitrogen, Carlsbad, CA) for other . †Distances (in Mb) were obtained from the NCBI MapViewer (RGSC v3.4, available at www.ncbi.nlm.nih.gov/). ‡TM, homozygous for the TM allele; KDP, continued homozygous for the KDP allele. ND, not determined.

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FIG. 1. Cumulative incidence of diabetes (A), body weight (B), and changes in blood glucose level (C and D) in the congenic and KDP strains. A: Under the same SPF condition, cumulative incidence of diabetes was observed in the congenic and KDP strains. In the KDP strain, data were In the TM.KDP-Cblb congenic strain, data were obtained from the animals .(18 ؍ obtained from the animals homozygous for the Cblb mutation (n for the Cblb mutation. Note that the incidence patterns of the TM.KDP-Cblb (14 ؍ and wild type (n ,(22 ؍ heterozygous (n ,(35 ؍ homozygous (n heterozygous and wild-type animals overlap. B: Body weights at 210 days of age were compared in animals wild type for the Cblb mutation in the ,male ;10 ؍ and those homozygous for the Cblb mutation in the TM.KDP-Cblb congenic strain (female, n (9 ؍ male, n ;6 ؍ KDP strain (female, n ;P < 0.0001 vs. KDP. C and D: Representative examples of changes in blood glucose level are shown for the KDP strain (C** ;0.014 ؍ P* .(11 ؍ n .Nonfasting blood glucose levels were checked once a week until the onset of diabetes .(5 ؍ and TM.KDP-Cblb congenic strain (D; n (4 ؍ n

(17), these results indicate that the autoimmune pheno- confirm the susceptibility loci and identify the responsible type was reconstituted on the non-KDP genetic back- genes in animal models of type 1 diabetes, many congenic ground with the two major susceptibility genes (RT1u strains have been produced in NOD mice and BB rats. haplotype and Cblb mutation). These studies have shown that most congenic strains DISCUSSION carrying susceptibility allele(s) on nondiabetic genetic backgrounds do not develop diabetes (12–14). In addition, In the present study, we successfully reconstituted auto- immune type 1 diabetes with two major susceptibility most congenic strains carrying resistant allele(s) on dia- genes, the RT1u haplotype and Cblb mutation, on a non- betes-prone genetic backgrounds suppressed the develop- KDP genetic background using the congenic approach. ment of the disease. The BB congenic strains carrying Genetic analysis of type 1 diabetes in the MHC-matched segments of chromosome 6 or 18 of the spontaneously backcrosses, (TM ϫ KDP)F1 ϫ KDP and (LETO [Long- hypertensive rat on the BB/OK background showed a Evans Tokushima Otsuka] ϫ KDP)F1 ϫ KDP, revealed reduction in diabetes frequency compared with BB/OK that the genetic predisposition to diabetes is accounted for rats (86% vs. 14 or 34%, respectively) (21). Moreover, NOD by a recessively acting , Iddm/kdp1 (16). This raised congenic strains carrying resistant alleles (Idd3, Idd5, the possibility that introgression of Iddm/kdp1 (Cblb) onto Idd10,orIdd18) suppressed the development of both the TM or LETO genetic backgrounds might confer suffi- insulitis and diabetes (22). In contrast, the NOD congenic cient genetic susceptibility for type 1 diabetes. Because strain carrying B6-derived alleles on chromosome 13 ac- the LETO strain was derived from the same outbred celerated diabetes, suggesting that B6 mice harbor more colony as LETL (the original strain of KDP) (6), a consid- diabetogenic alleles than NOD mice for this locus (23). erable part of the genetic background may be common These findings indicate that restricted combinations of among LETO, LETL, and KDP rats. Therefore, we selected susceptibility alleles are not sufficient for the development the TM strain as the recipient strain for backcrossing. To of type 1 diabetes on nondiabetic genetic backgrounds.

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FIG. 3. Degree of thyroiditis of the congenic and KDP strains. The degree of thyroiditis in each animal was evaluated shortly after the onset of diabetes or at 210 days of age for nondiabetic animals. In the KDP strain, data were obtained from animals homozygous for the Cblb In the TM.KDP-Cblb congenic strain, data were .(18 ؍ mutation (n ؍ heterozygous (n ,(35 ؍ obtained from the animals homozygous (n .for the Cblb mutation (14 ؍ and wild type (n ,(22

study. However, as reported previously (19), the incidence of diabetes in each generation fluctuates from 70 to 100%. In addition, the present KDP colony at Japan SLC exhib- FIG. 2. Degree of insulitis of the congenic and KDP strains. The degree ϳ of insulitis in each animal was evaluated shortly after the onset of ited 80% incidence of diabetes (N. Masui, H. Asai, S. diabetes or at 210 days of age for nondiabetic animals. All of the Yokose, personal communication). Therefore, the inci- diabetic animals showed severe insulitis. In the KDP strain, data were dence of diabetes in the present KDP colony is similar to .(18 ؍ obtained from the animals homozygous for the Cblb mutation (n In the TM.KDP-Cblb congenic strain, data were obtained from the that of the previous reports (16,19). The degree of insulitis and wild type in the congenic strain supports the previous findings that ,(22 ؍ heterozygous (n ,(35 ؍ animals homozygous (n for the Cblb mutation. In addition, the degree of insulitis in homozygosity for the KDP allele (Cblb mutation) at the (14 ؍ n) the animals homozygous for the Cblb mutation in the congenic strain Iddm/kdp1 (Cblb) locus is strongly associated with the .(10 ؍ was also evaluated at 90 days of age (n development of moderate to severe insulitis and that Iddm/kdp1 (Cblb) acts in a recessive manner (16). How- Therefore, to our knowledge, the present study is the first ever, in addition to the observation in (TM ϫ KDP)F1 demonstration of genetic reconstitution of autoimmune animals (16), slight insulitis was observed in 68% (15 of 22) type 1 diabetes on a nondiabetic genetic background with of Cblb heterozygous and 43% (6 of 14) of wild-type a combination of susceptibility genes. animals in the congenic strain (Fig. 2). These data indicate The KDP rats showed 100% incidence of diabetes in this that slight insulitis may occur in the KDP, TM, or (TM ϫ

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KDP)F1 genetic backgrounds, irrespective of Cblb muta- disease. The responsible genes for these modifiers and tion, and that the Cblb mutation acts in a semidominant their physiological roles remain to be investigated. The manner in the development of slight insulitis. congenic strain established in this study should be useful The congenic strain showed a higher incidence of for mapping and identifying such modifiers and for the thyroiditis than KDP rats. Mild to severe thyroiditis was elucidation of mechanisms underlying the development of rarely found in animals with early onset of diabetes but autoimmune diseases. was found in those with delayed onset of diabetes in both the KDP and congenic strains. In addition, mild to severe ACKNOWLEDGMENTS thyroiditis was found in nondiabetic congenic rats. These This study was supported by Grants-in-Aid for Scientific results suggest that the onset and progression of thyroid- Research (15700313) and Specially Promoted Research itis are delayed compared with those of insulitis and are (15002002) from the Ministry of Education, Culture, independent of the development of insulitis. Similar to the Sports, Science and Technology of Japan. case of insulitis, homozygosity for the Cblb mutation is We thank the late Professor K. Komeda (Tokyo Medical necessary for the development of mild to severe thyroid- u University) and Drs. T. Serikawa (Kyoto University) and K. itis. However, it is not clear whether RT1 is involved in Yasuda (International Medical Center of Japan) for their the development of thyroiditis. The study of BB rats helpful advice during the course of this study. We also suggested that the susceptibility of RT1 haplotypes to thank A.C. Kentner (University of Ottawa) and T. Hirata thyroiditis is distinct from that of insulitis and that RT1a u for critical reading of the manuscript and technical assis- confers greater susceptibility to thyroiditis than RT1 (24). tance, respectively. Although type 1 diabetes was reconstituted in the con- Part of this study was conducted at the Department of genic strain, the incidence of diabetes was low and pro- Cellular and Molecular Medicine, Graduate School of gression of the disease was delayed compared with KDP Medicine, Chiba University, with which N.Y., H.-Y.W., and rats. Since breeding and phenotyping of both the congenic S.S. were formerly affiliated. and KDP strains were performed in the same room at the same facility, environmental factors are likely to be similar for both strains, except for the microenvironment, such as REFERENCES cages or mothers. Thus, the primary cause of the differ- 1. Maier LM, Wicker LS: Genetic susceptibility to type 1 diabetes. Curr Opin Immunol 17:601–608, 2005 ence in incidence of diabetes between the two strains is 2. Barker JM: Clinical review: type 1 diabetes-associated autoimmunity: most likely due to genetic rather than environmental natural history, genetic associations, and screening. J Clin Endocrinol factors. These genetic factors (modifiers) may well inter- Metab 91:1210–1217, 2006 act with environmental factors to promote or suppress the 3. Makino S, Kunimoto K, Muraoka Y, Mizushima Y, Katagiri K, Tochino Y: development of insulitis and type 1 diabetes. There are Breeding of a non-obese, diabetic strain of mice. Exp Anim (Jikken diabetes-promoting modifiers in the KDP genetic back- Dobutsu) 29:1–13, 1980 4. Like AA, Butler L, Williams RM, Appel MC, Weringer EJ, Rossini AA: ground, whereas there are diabetes-suppressing modifiers Spontaneous autoimmune diabetes mellitus in the BB rat. Diabetes 31 in the TM genetic background. Differences in the pheno- (Suppl. 1):S7–S13, 1982 types of Cblb-deficient mice also support the involvement 5. Chappel CI, Chappel WR: The discovery and development of the BB rat of modifiers: Bachmaier et al. (25) did, whereas Chiang et colony: an animal model of spontaneous diabetes mellitus. Metabolism 32 al. (26) did not, observe spontaneous development of (Suppl. 1):S8–S10, 1983 several autoimmune phenotypes in mice. Based on genetic 6. Kawano K, Hirashima T, Mori S, Saitoh Y, Kurosumi M, Natori T: New inbred strain of Long-Evans Tokushima lean rats with IDDM without analyses of backcrosses between the KDP rat and three lymphopenia. Diabetes 40:1375–1381, 1991 strains (TM, LETO, and BN rats), we previously identified 7. Komeda K, Noda M, Terao K, Kuzuya N, Kanazawa M, Kanazawa Y: u the two recessively acting genes, the RT1 haplotype and Establishment of two substrains, diabetes-prone and non-diabetic, from Cblb mutation, and found that most of the genetic predis- Long-Evans Tokushima Lean (LETL) rats. Endocr J 45:737–744, 1998 position to diabetes in these crosses can be accounted for 8. Lenzen S, Tiedge M, Elsner M, Lortz S, Weiss H, Jorns A, Kloppel G, by these two major genes (16,17). However, in these Wedekind D, Prokop CM, Hedrich HJ: The LEW. 1AR1/Ztm-iddm rat: a new model of spontaneous insulin-dependent diabetes mellitus. Diabetologia analyses using backcrosses, dominantly acting genes 44:1189–1196, 2001 could not be identified. The results in this study, together 9. Mordes JP, Bortell R, Blankenhorn EP, Rossini AA, Greiner DL: Rat models with the previous genetic studies, strongly suggest that of type 1 diabetes: genetics, environment, and autoimmunity. ILAR J there are dominantly acting diabetes-promoting modifiers 45:278–291, 2004 in the KDP genetic background. The two recessively acting 10. Mathews CE: Utility of murine models for the study of spontaneous genes, the RT1u haplotype and Cblb mutation, are neces- autoimmune type 1 diabetes. Pediatr Diabetes 6:165–177, 2005 11. Ellerman KE, Like AA: Susceptibility to diabetes is widely distributed in sary for the development of type 1 diabetes, while domi- normal class IIu haplotype rats. Diabetologia 43:890–898, 2000 nantly acting diabetes-promoting modifiers are not 12. Hornum L, Lundsgaard D, Markholst H: An F344 rat congenic for BB/DP essential but influence the development of the disease. rat-derived diabetes susceptibility loci Iddm1 and Iddm2. Mamm Genome Although both KDP and TM rats have the same RT1u 12:867–868, 2001 haplotype, it is possible that subtle differences in coding or 13. Moralejo DH, Park HA, Speros SJ, MacMurray AJ, Kwitek AE, Jacob HJ, regulatory sequences of RT1.Bu and RT1.Du between KDP Lander ES, Lernmark A: Genetic dissection of lymphopenia from autoim- munity by introgression of mutated Ian5 gene onto the F344 rat. J and TM rats may influence the incidence and age of onset Autoimmun 21:315–324, 2003 of diabetes. In addition, accumulating evidence in both 14. Yui MA, Muralidharan K, Moreno-Altamirano B, Perrin G, Chestnut K, humans and NOD mice (27–29) indicates that genes out- Wakeland EK: Production of congenic mouse strains carrying NOD- side the class II MHC may contribute to susceptibility to derived diabetogenic genetic intervals: an approach for the genetic dissec- type 1 diabetes. tion of complex traits. Mamm Genome 7:331–334, 1996 Genetic variations in the human CBLB gene do not seem 15. Morel L, Croker BP, Blenman KR, Mohan C, Huang G, Gilkeson G, Wakeland EK: Genetic reconstitution of systemic lupus erythematosus to be a major cause of type 1 diabetes (30–32). However, immunopathology with polycongenic murine strains. Proc Natl Acad Sci U human orthologues of diabetes-promoting modifiers of the SA97:6670–6675, 2000 KDP rat may be involved in the development of the 16. Yokoi N, Kanazawa M, Kitada K, Tanaka A, Kanazawa Y, Suda S, Ito H,

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Serikawa T, Komeda K: A non-MHC locus essential for autoimmune type I Santos A, Mariathasan S, Bouchard D, Wakeham A, Itie A, Le J, Ohashi PS, diabetes in the Komeda diabetes-prone rat. J Clin Invest 100:2015–2021, Sarosi I, Nishina H, Lipkowitz S, Penninger JM: Negative regulation of 1997 lymphocyte activation and autoimmunity by the molecular adaptor Cblb. 17. Yokoi N, Komeda K, Wang H-Y, Yano H, Kitada K, Saitoh Y, Seino Y, Yasuda Nature 403:211–216, 2000 K, Serikawa T, Seino S: Cblb is a major susceptibility gene for rat type 1 26. Chiang YJ, Kole HK, Brown K, Naramura M, Fukuhara S, Hu R-J, Jang IK, diabetes mellitus. Nat Genet 31:391–394, 2002 Gutkind JS, Shevach E, Gu H: Cblb regulates the CD28 dependence of 18. Yokoi N: Identification of a major gene responsible for type 1 diabetes in T-cell activation. Nature 403:216–220, 2000 the Komeda diabetes-prone rat. Exp Anim 54:111–115, 2005 27. Nejentsev S, Gombos Z, Laine AP, Veijola R, Knip M, Simell O, Vaarala O, 19. Yokoi N, Namae M, Fuse M, Wang HY, Hirata T, Seino S, Komeda K: Akerblom HK, Ilonen J: Non-class II HLA gene associated with type 1 Establishment and characterization of the Komeda diabetes-prone rat as a diabetes maps to the 240-kb region near HLA-B. Diabetes 49:2217–2221, segregating inbred strain. Exp Anim 52:295–301, 2003 2000 20. Markel P, Shu P, Ebeling C, Carlson GA, Nagle DL, Smutko JS, Moore KJ: 28. Johansson S, Lie BA, Todd JA, Pociot F, Nerup J, Cambon-Thomsen A, Theoretical and empirical issues for marker-assisted breeding of congenic Kockum I, Akselsen HE, Thorsby E, Undlien DE: Evidence of at least two mouse strains. Nat Genet 17:280–284, 1997 type 1 diabetes susceptibility genes in the HLA complex distinct from 21. Klo¨ ting I, van den Brandt J, Kuttler B: Genes of SHR rats protect HLA-DQB1, -DQA1 and -DRB1. Genes Immun 4:46–53, 2003 spontaneously diabetic BB/OK rats from diabetes: lessons from congenic 29. Ikegami H, Makino S, Yamato E, Kawaguchi Y, Ueda H, Sakamoto T, BB.SHR rat strains. Biochem Biophys Res Commun 283:399–405, 2001 Takekawa K, Ogihara T: Identification of a new susceptibility locus for 22. Robles DT, Eisenbarth GS, Dailey NJ, Peterson LB, Wicker LS: Insulin insulin-dependent diabetes mellitus by ancestral haplotype congenic map- autoantibodies are associated with islet inflammation but not always ping. J Clin Invest 96:1936–1942, 1995 related to diabetes progression in NOD congenic mice. Diabetes 52:882– 30. Kosoy R, Yokoi N, Seino S, Concannon P: Polymorphic variation in the 886, 2003 CBLB gene in human type 1 diabetes. Genes Immun 5:232–235, 2004 23. Brodnicki TC, Quirk F, Morahan G: A susceptibility allele from a non- 31. Payne F, Smyth DJ, Pask R, Barratt BJ, Cooper JD, Twells RC, Walker NM, diabetes-prone mouse strain accelerates diabetes in NOD congenic mice. Lam AC, Smink LJ, Nutland S, Rance HE, Todd JA: Haplotype tag single Diabetes 52:218–222, 2003 nucleotide polymorphism analysis of the human orthologues of the rat 24. Awata T, Guberski DL, Like AA: Genetics of the BB rat: association of type 1 diabetes genes Ian4 (Lyp/Iddm1) and Cblb. Diabetes 53:505–509, autoimmune disorders (diabetes, insulitis, and thyroiditis) with lymphope- 2004 nia and major histocompatibility complex class II. Endocrinology 136: 32. Bergholdt R, Taxvig C, Eising S, Nerup J, Pociot F: CBLB variants in type 5731–5735, 1995 1 diabetes and their genetic interaction with CTLA4. J Leukoc Biol 25. Bachmaier K, Krawczyk C, Kozieradzki I, Kong Y-Y, Sasaki T, Oliveira-dos- 77:579–585, 2005

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