Identification of genomic regions controlling experimental autoimmune uveoretinitis in rats

Shu-Hui Sun, Phyllis B. Silver, Rachel R. Caspi, Ying Du1, Chi-Chao Chan, Ronald L. Wilder1 and Elaine F. Remmers1

Laboratory of Immunology, National Eye Institute, and
1 Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, MD 20892, USA

Correspondence to: R. R. Caspi, Laboratory of Immunology, National Eye Institute, NIH, Building 10, Room 10N222, 10 Center Drive MSC 1857, Bethesda, MD 20892, USA


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The present study attempts to identify specific genetic loci contributing to experimental autoimmune uveoretinitis (EAU) susceptibility in F2 progeny of resistant Fischer (F344/N) and susceptible Lewis (LEW/N) inbred rats. F2 progeny of F344/N x LEW/N inbred rats were immunized with the R16 peptide of interphotoreceptor retinoid-binding protein (IRBP). A genome-wide scan was conducted using 125 simple sequence length polymorphism markers in selected F2 animals that developed severe eye disease or remained unaffected to identify phenotype:genotype co-segregation. The F2 population (n = 1287) demonstrated a wide range of histologically assessed EAU scores (assessed on a scale of 0–4). The disease incidence and severity were not consistent with a simple Mendelian inheritance model. Of the F2 hybrid rats, 60% developed EAU, implying the existence of a potent susceptibility locus with incomplete penetrance associated with the LEW genome or a more complex polygenic model of inheritance. Two genomic regions, on chromosomes 4 and 12, showed strong genetic linkage to the EAU phenotype (P < 0.0016), suggesting the presence of susceptibility loci in these chromosomal regions. In conclusion, we have identified two genomic candidate intervals from D4Arb8 to D4Mit17 on chromosome 4 and from the chromosome end to D12Arb8 on chromosome 12, that appear to influence EAU susceptibility in LEW/F344 rats. Further analysis of these genomic regions may lead to identification of the susceptibility genes and to characterization of their function.

Keywords: experimental autoimmune uveoretinitis, rat, QTL


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Endogenous uveitis is a major cause of visual handicap in the US and has been estimated to account for up to 10% of the cases of legal blindness. Although autoimmune mechanisms have been implicated in pathogenesis, the etiology of endogenous uveitis and the hereditary predisposing factors are largely unknown. There is considerable evidence that the development of uveitis has a strong genetic basis. For example, strong HLA associations have been noted in a number of human uveitic diseases such as Behcet's disease, Vogt–Koyanagi–Harada syndrome, sympathetic ophthalmia and birdshot retinochoroidopathy (1,2). However, systematic genetic analysis of human uveitis is limited by the size of pedigrees and its etiologic heterogeneity.

Experimental autoimmune uveoretinitis (EAU) in animals serves as a laboratory model for human ocular inflammatory diseases of a presumed autoimmune nature. EAU is a T cell-mediated autoimmune disease that targets the neural retina, and is induced in susceptible rodent strains by immunization with retinal proteins and their fragments. EAU susceptibility in rats, mice and guinea pigs is strongly strain dependent, indicating an important genetic influence on disease development. In previous studies, both MHC and non-MHC genes were suggested to play a role in regulating the expression of EAU (14). While MHC restriction of susceptibility is likely to be connected to recognition of the appropriate antigenic epitopes, control by loci outside the MHC is complex and is less well understood. Currently, directed breeding of backcross or intercross animals allows the generation of large numbers of affected offspring that can be used to map contributing loci or genes even in multigenic diseases (5) by correlating co-segregation of phenotype with genotype. We have, therefore, employed the EAU model to identify specific genetic factors that may determine susceptibility to uveitic disease.

Our previous study tentatively mapped MHC control of susceptibility to EAU to class II genes (4). In the present study, we performed a genome-wide analysis to identify specific genetic loci outside of MHC class II that contribute to EAU susceptibility. For this purpose we used EAU-susceptible LEW/N and EAU-resistant F344 rat strains, which share the same MHC class II (RT1l) but differ in multiple non-MHC genes. Their F2 progeny (n = 1287) were immunized with a pathogenic peptide derived from the interphotoreceptor-retinoid binding protein (IRBP) and EAU severity was measured by histopathology. Polymorphic (F334 versus LEW) PCR-typable markers for microsatellites (simple sequence repeat sequences) throughout the rat genome were analyzed in selected F2 progeny and the data were analyzed for phenotype:genotype co-segregation. We found that genomic intervals on chromosome 4 and on chromosome 12 were likely to be linked to EAU susceptibility loci. The existence of additional susceptibility loci cannot be excluded.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals and breeding
Specific pathogen-free Fischer 344 (F344/N) and Lewis (LEW/N) rats were obtained from Harlan-Sprague-Dawley (Indianapolis, IN). Animal care was in accordance with the National Institutes of Health guidelines and the ARVO Resolution on Use of Animals in Research. Using the convention of (femalexmale) to indicate strain parentage, F1 progeny were generated from four mating pairs: two (F344xLEW) and two (LEWxF344). Brother–sister mating of F1 animals yielded F2 progeny.

Antigen
Peptide R16 of bovine IRBP (sequence ADGSSWEGVGVVPDV, residues 1177–1191) was synthesized using conventional solid-phase chemistry as described previously (6). Peptide R16 encodes a major uveitogenic epitope recognized by the RT1l haplotype (6).

Immunization
R16 peptide was emulsified in complete Freund's adjuvant containing 2.5 mg/ml Mycobacterium tuberculosis H37RA (Difco, Detroit, MI). Rats were injected with 30 µg R16 in 0.1 ml emulsion into one hind foot-pad.

Phenotype analysis by EAU scoring
Clinical onset of EAU was recorded as anterior chamber inflammation. Experiments were terminated when EAUsusceptible animals had been positive for 5–7 days, typically 16–18 days after immunization. Quantitation of disease was done by histopathology. Eyes were graded on a scale of 0–4 in a masked fashion by one of us who is an ophthalmic pathologist (C.-C. C.). Eyes were assigned disease scores (0–4) according to the extent of inflammation and tissue damage, using previously described criteria (7).

Disease severity for each individual animal was calculated as the average of both eyes.

Genotype analysis
DNA was extracted from 500 mg pulverized frozen liver by a standard protocol (8). Genotypes were determined by PCR amplification of DNA fragments containing simple sequence length polymorphisms as described (8,9), except that volumes were reduced to 10 µl. The amplification conditions are also described at the Arthritis and Rheumatism Branch Rat Genetic Database website (http://www.nih.gov/niams/scientific/ratgbase/index.htm). Genetic maps were constructed with the computer program MAPMAKER/EXP version 3.0b (10). Genotypes resulting in apparent double crossovers (detected with the MAPMAKER error checking feature) were re-read on the original films and then the error-checked data were used for {chi}2 analysis. All marker information is available on the Arthritis and Rheumatism Branch Rat Genetic Database website.

Genetic analysis
A non-parametric approach was used to detect loci that co-segregate with EAU susceptibility. A genome-wide scan was performed using 125 polymorphic microsatellite markers that distinguish LEW and F344 alleles. Non-random allele segregation was evaluated using a {chi}2 analysis of the observed allele frequency compared to an expected random distribution of LEW and F344 alleles. Genomic regions encompassing markers with non-random allele frequencies (P < 1.6x10–3, {chi}2 test statistic >10.16) were considered suggestive for linkage to a susceptibility locus in a genome-wide analysis, unless segregation distortion was detected by genotyping unaffected progeny.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
EAU score distribution in LEW, F344, F1 and F2 hybrid rats
Our previous work has demonstrated that LEW rats are susceptible to EAU and F344 rats are resistant (3). To confirm the EAU susceptibility pattern in the parental stock used in the present study (LEW and F344) and to determine susceptibility in their F1 generation, 13 animals in each group were immunized with peptide R16 of IRBP, containing a major pathogenic epitope for the RT1l haplotype, and their EAU scores were evaluated by histopathology (Fig. 1aGo). The parental strains exhibited the expected susceptibility phenotypes, with LEW rats developing EAU by day 10 and F344 remaining disease-free. Eleven of the 13 F1 progeny developed EAU between day 10 and 12. The median score of the F1 progeny was intermediate between those of LEW and F344 parents.



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Fig. 1. EAU scores in LEW/N, F344/N and F1 and F2 hybrid rats immunized with the R16 peptide of IRBP. EAU scores were evaluated by histopathology as described in Methods. (a) EAU scores in LEW and F344 parental strains, and their F1 generation (n = 13). Horizontal bars represent the medians of each group. (b) EAU score distribution in (LEWxF344 ) F2 hybrid rats (n = 1287).

 
To identify specific genetic regions contributing to the development of EAU in this animal model, 1287 F2 (LEWxF344) rats were immunized with R16 peptide and their EAU scores were evaluated by histopathology. The F2 population demonstrated the complete range of EAU scores from 0 to 4 (Fig. 1bGo). Only 60% of F2 animals developed EAU, which suggested the existence of either (i) an incompletely penetrant dominant susceptibility locus in the LEW genome, (ii) two interacting dominant susceptibility loci or (iii) a more complex polygenic model.

Microsatellite-based genome-wide scanning for EAU loci
To detect genetic loci that may co-segregate with disease susceptibility, a genome-wide analysis using 125 microsatellite markers throughout the rat genome was first performed on 40 F2 rats with EAU scores >=1.0 (Table 1Go). As a result, 25 markers on ten chromosomes (2, 4, 5, 6, 7, 10, 12, 14, 15 and 20) were found to exhibit nominally significant differences ({chi}2 test P < 0.05) from a random distribution (Table 1Go). Because a genome-wide analysis involves multiple hypothesis testing, a significant portion of these genomic regions were likely to be false positives. Therefore, for a genome-wide analysis in an F2 population, P < 0.0016 is suggestive of linkage to a disease locus (11). Among these 22 markers, four groups (12 markers) on four different chromosomes 12) included at least one marker with a nominal P < 0.01 (Table 1Go). These 12 markers were further genotyped (17) on DNA samples of the remaining 77 F2 rats with EAU scores >=1.0. To eliminate the possibility that the observed non-random distributions were caused by segregation distortion, these 12 markers were also analyzed in 40 F2 rats that failed to develop EAU.


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Table 1. Genome scan of Chromosomes 1–20 in 40 F2 EAU-affected rats
 
Three markers on three chromosomes 2, 4 and 12 demonstrated significant deviation (P < 0.0016) from expected allele frequencies in the 117 affected animals, suggesting linkage to genes controlling EAU (Table 2Go). Two of these, however, D2Mgh15 (chromosome 2) and D4Arb7 (Npy) (chromosome 4), had a lower than expected number of F344 alleles in the 40 unaffected progeny. Therefore, we determined the genotypes of a total of 120 unaffected progeny at these two markers. For D4Arb7 (Npy) the allele frequency in 120 unaffected progeny was F344 108:LEW 122 (P = 0.4). These data demonstrated that the observed deviation from the expected allele frequency in the affected animals was likely due to linkage to a disease controlling locus. Conversely, for D2Mgh15, the allele frequency in 120 unaffected progeny was F344 91:LEW 131 (P = 0.007). These data implied that the observed deviation from the expected allele frequency at D2Mgh15 resulted from segregation distortion, i.e. a lack of F344 alleles in the F2 generation, and it was therefore not possible to evaluate the presence of a disease susceptibility locus in this region.


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Table 2. Twelve markers on chromosome 2, 4, 10 and 12 analyzed in 117 F2 rats with significant EAU and in 40 F2 unaffected rats
 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Significant variation in EAU susceptibility among inbred rodent strains indicates that EAU is genetically controlled. We have identified two candidate non-MHC genomic regions on rat chromosomes 4 and 12 that are likely to contain genes that control EAU susceptibility in the progeny of LEW (EAU-susceptible) and F344 (EAU-resistant) rats. The MHC region on rat chromosome 20 did not co-segregate with disease susceptibility in progeny of these two strains, presumably because they have very similar MHC, RT1l and RT1lv1. The class II genes of these MHC haplotypes are identical. In a previous study, MHC control of EAU was tentatively mapped to class II, supporting the interpretation that MHC affects susceptibility largely through presentation of (class II-restricted) pathogenic epitopes (4). Our present genetic analysis, as well as previous functional studies demonstrating that both the LEW and the F344 strains can effectively recognize and present the R16 peptide antigen (3,6), confirm that mechanisms different from MHC class II recognition of the immunizing antigen are responsible for the observed differences in EAU susceptibility between LEW and F344 rats. In this study we detected two non-MHC genomic regions that are likely to contain genes that regulate susceptibility to EAU.

The non-MHC region associated with EAU susceptibility on rat chromosome 4 (D4Arb8 (Prss1), D4Arb7 (Npy), D4Mit17) is particularly interesting, because this genomic interval is associated with susceptibility to a number of other autoimmune disorders in rats. These include collagen-induced arthritis (Cia3) and experimental autoimmune encephalomyelitis in DA rats (12,13), adjuvant-induced arthritis in DA rats (Aia3), and spontaneous diabetes (Idd1) and thyroiditis in BB rats (15,16). The homologous chromosomal region on mouse chromosome 6 also controls diabetes (18), lupus-like disease, experimental autoimmune orchitis (19) and experimental allergic encephalomyelitis (20). Furthermore, a number of human autoimmune diseases analyzed by genome scans (multiple sclerosis, ulcerative colitis, Crohn's disease, psoriasis, rheumatoid arthritis, and insulin-dependent diabetes mellitus) demonstrated at least suggestive evidence for disease-associated genes within the homologous regions of the human genome (21,22). The possibility that a single locus may control development of a variety of autoimmune diseases (14) is intriguing and is consistent with familial clustering of autoimmune diseases (2327). A large number of immunologically relevant candidate genes are expected to reside within the EAU chromosome 4 susceptibility region (based on homology with mouse) (14). These include the TCR Vß loci, the {kappa} light chain of Ig, lymphocyte antigen-36, CD8 antigen {alpha} and ß chains, transforming growth factor-{alpha}, CD27 antigen, CD4 antigen, CD69 antigen, and tumor necrosis factor receptor 1.

The second putative EAU-susceptibility region we identified in this genome-wide scan was located at the end of the rat chromosome 12 linkage group [D12Arb2 (Gpr12), D12Arb8]. The homologous human chromosomal region is 13q12 and in mouse is the telomeric end of chromosome 5. Lbw3, a locus that controls lupus susceptibility in New Zealand mice, is located in this portion of mouse chromosome 5 (18).

We also identified a region on chromosome 2 near the marker D2Mgh15 which demonstrated significant segregation distortion in 237 rats including 117 affected and 120 unaffected (P = 0.00005). We interpret these data to mean that this region harbors a gene for which the LEW allele provides a selective survival advantage in F344xLEW hybrids and cannot evaluate this region for EAU susceptibility loci. Interestingly, this marker (D2Mgh15) is located only 15 cM away from the peak marker for Cia7, a locus that controls collagen-induced arthritis in progeny of DAxACI rats (28).

We cannot exclude the existence of additional susceptibility loci that control EAU in this cross. Loci with small effects or effects that are dependent upon interactions with other loci (epistasis) may not have been detected in this study. Two genomic regions on chromosomes 7 and 10 exhibited a trend toward co-segregation of genotypes with EAU phenotypes. Interestingly, they were located near Cia8 (unpublished) and Cia5 (12) respectively, two loci we have previously shown to regulate collagen-induced arthritis in F2 progeny of DAxF344 rats. Further evaluation of all these genomic regions will require generation of congenic strains with the resistant strain alleles on the susceptible strain background and/or vice versa.

In conclusion, identification of the genes within the chromosomal regions that control EAU susceptibility may provide insights into uveitis as well as into other human autoimmune diseases. These genes may also represent novel targets for therapy of autoimmune disease.


    Abbreviations
 
EAUexperimental autoimmune uveoretinitis
IRBPinterphotoreceptor retinoid-binding protein

    Notes
 
Transmitting editor: R. L. Coffman

Received 4 June 1998, accepted 12 October 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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