Identification of susceptibility genes for experimental autoimmune encephalomyelitis that overcome the effect of protective alleles at the eae2 locus

Johan Jirholt, Anna-Karin Lindqvist, Jenny Karlsson, Åsa Andersson and Rikard Holmdahl

Section of Medical Inflammation Research, CMB, Lund University, I11 BMC, 221 84 Lund, Sweden

Correspondence to: J. Jirholt, AstraZeneca R & D Mölndal, 431 83 Mölndal, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have previously identified a locus on mouse chromosome 15 (eae2) that regulates susceptibility to experimental autoimmune encephalomyelitis in a cross between the susceptible strain B10.RIII and the resistant strain RIIIS/J. In an effort to verify the protective effect from having two RIIIS/J alleles at eae2, the resistant locus was bred into the susceptible strain in homozygous form. However, the expected effect was not as clear as in the original study. This might be due to an epistatic effect conferred by several unidentified genes in the genome of the resistant strain or due to the environment by genotype interactions, possibly overcoming the effect of protective alleles at eae2. To further the genetic understanding in this disease, a genome-wide linkage screening approach was employed on an F2 intercross that carried the protective allele at eae2in homozygous form while the rest of the genome segregated between the B10.RIII and RIIIS/J strains as in the original investigation. In the present study we find one region on chromosome 7, not previously identified in this strain combination, that affects the disease at significant logarithm of the odds score and six regions showing suggestive evidence for linkage to disease phenotypes.

Keywords: congenic strain, disease susceptibility, epistasis, genetic crosses, linkage analysis, mouse, multiple sclerosis, quantitative trait


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Multiple sclerosis (MS) is a disease affecting ~0.1% of the Caucasian population (1,2). It is a disease with disabling properties, where myelin sheets surrounding nerve fibers in the CNS are attacked by immune cells that likely have autoaggressive properties (3). The reasons behind autoreactivity are largely unknown today, but there is a significant genetic influence on disease development in addition to environmental factors (4,5). MS is thought to be a complex disease with polygenic inheritance, but to date the only identified genetic factor comes from the established association with the MHC region (6,7). Several genome-wide linkage analyses have been performed and they have confirmed the HLA association, but no other loci have reached the level of significant linkage by meta-analysis (8–14). This might be a stochastic event resulting from the detection power in the different experiments, but more likely depends upon differences in the underlying genetics of disease (5,15,16) or differences in diagnosis. Here animal models are useful in investigating the mechanisms and causes of this disease in a more homogenous genetic milieu and under a controlled environment.

Experimental autoimmune encephalomyelitis (EAE) is a commonly used animal model for MS (17). The disease is induced by immunizing mice or rats with CNS components in combination with adjuvants. Myelin basic protein (MBP), myelin oligodendrocyte glycoprotein, proteolipid protein as well as spinal cord homogenate are commonly used to induce the disease in genetically susceptible recipients. Following immunization, EAE develops with characteristic symptoms such as tail weakness, varying degrees of hind leg paralysis and balance disturbances. The disease progresses along three major paths: acute, chronic progressive and chronic relapsing, similarly as in MS (18).

Although the experimental model of MS has been studied extensively, the mechanisms of disease have yet to be revealed. To investigate this disease, we initiated a segregation experiment aimed at isolating regions harboring susceptibility genes by linkage analysis. In our previous study (19) we identified two loci linked to disease in an F2 intercross between the EAE-susceptible mouse strain B10.RIII (20) and the resistant strain RIIIS/J (21). One region was identified on chromosome 15 (eae2), where the B10.RIII allele promotes disease development in a dominant fashion. In fact, in a cohort of 193 animals we did not observe one single affected animal that carried two copies of the resistant RIIIS/J genotype at the marker showing the peak logarithm of odds (LOD) score (D15Nds2). The other locus that we identified was located to a region on chromosome 3 (eae3). At this locus, alleles from the B10.RIII strain added to both severity and incidence of the disease in an additive fashion. In both the present and the previous study the MHC region has been eliminated as a segregating factor since both strains share the same MHC region (H-2r).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
B10.RIII animals were originally provided by Jan Klein (Tübingen, Germany) and kept in our breeding colony. RIIIS/J animals were purchased from Jackson Laboratories (Bar Harbor, ME). The B10RIII.chr15 animals were initiated from a (B10.RIIIxRIII S/J)F2 intercross founder and then backcrossed to B10.RIII for four generations while selecting for the RIIIS/J genotype in the fragment on chromosome 15. Finally, the animals were intercrossed to produce the B10RIII.chr15 4N1i strain where chromosome 15 segregates. All of the B10RIII.chr15 4N1i animals were homozygous for the B10.RIII allele at eae3 as expected. To produce animals for the segregation analysis, one founder that was homozygous for RIIIS/J in the fragment on chromosome 15 and developed EAE (B10 RIII.eae2RIIIS/J) was selected from the B10RIII.chr15 4N1i generation and bred with RIIIS/J females. The resulting B10 RIII.eae2RIIIS/J F1 offspring were intercrossed to produce in total 259 B10 RIII.eae2RIIIS/J F2 animals. Animals were kept under clean conditions in our animal facilities at Lund University.

Disease induction
The MBP89–101 peptide was synthesized on an Applied Biosystems (Foster City, CA) peptide synthesizer model ABI430A using the FastMoc program. Mass spectrometric analysis of the peptide showed the expected m/z as a major signal in the spectrum. Animals were immunized at the root of the tail with 0.1 ml of an emulsion containing: 250 µg MBP89–101 in PBS mixed with an equal amount of Freund's complete adjuvant (Difco, Detroit, MI) containing 100 µg Mycobacterium tuberculosis H37ra (Difco). The animals were also injected i.p. with 400ng Bordetella pertussis toxin (Sigma, St Louis, MO) in 100 µl PBS the day of immunization and 2 days later. Experiments were initiated with animals 8–16 weeks of age.

Disease assessment
The disease was assessed by scoring the symptoms of the animals every second or third day as indicated below: 0, no abnormality; 1, tail weakness; 2, tail paralysis; 3, tail paralysis and mild waddle; 4, tail paralysis and severe waddle; 5, tail paralysis and paralysis of one limb; 6, tail paralysis and paralysis of a pair of limbs; 7, tetraparesis; 8, moribund or deceased. In addition to the classical paralytic symptoms above, additional symptoms indicating the involvement of the brain were assessed using a similar scale as shown below: 1, mildly affected; 2, affected gait, cramps; 3, balance disturbance; 4, severe balance disturbance; 5, immobilized due to lack of coordination; 6, moribund; 7, diseased.

Animals were assigned to different phenotypic groups depending on a set of criteria described in Table 1Go.


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Table 1. Phenotypes and criteria used for assessment of disease in the B10 RIII.eae2RIIIS/J F2 linkage analysis
 
Genotyping and data analysis
DNA was prepared from tail biopsies as previously described (22). A set consisting of 111 fluorescently labeled microsatellite markers (INTERACTIVA Biotechnologie, Ulm, Germany), evenly spread over the autosomal chromosomes and the X chromosome, was used for genotyping. PCR was performed with 15 ng of DNA in a 9-µl reaction volume containing: 10 mM Tris–HCl, 50 mM KCl, pH 8.3, 1.5 mM MgCl2, 1.5 pmol of each primer, 0.83 mM dNTP and 0.18 U AmpliTaq Gold (Applied Biosystems). Amplification was performed in ABI 887 (Applied Biosystems) thermal cyclers using a protocol consisting of: 95°C 10 min, 13 cycles of denaturation 95°C 30 s, annealing 50°C 75 s and elongation 72°C 75 s; 20 cycles of denaturation 89°C 30 s, annealing 50°C 75 s and elongation 72°C 75 s; and 72°C 7 min. The PCR products were diluted 1:20 for FAM- and TET-labeled products or 1:10 for HEX labeled products. Then 1 µl PCR product was mixed with 1.5 µl standard loading buffer containing a TAMRA-labeled size marker (Applied Biosystems) GS350 or GS500.

DNA size fractioning and detection were performed on the Applied Biosystems ABI 377 sequencer using 4% polyacrylamide gels at the Genotyping Center (Uppsala, Sweden). Data collection and size determination was performed using the ABI Prism 377 Collection, Gene Scan 3.1 and Genotyper 2.5 software (Applied Biosystems).

Recombination fractions, possible genotyping errors and LOD score statistics were calculated using the Mapmaker software: Mapmaker version 3.0b (23,24) and Mapmaker QTL version 1.9 (25,26). Permutation analysis was performed using QTL Cartographer (27,28) where model 3 (interval mapping) was used to calculate the test statistics. Maps of the mouse chromosomes were calculated and compared to the Mouse Genome Informatics map (29). No significant discrepancy in map order was observed. All suspected genotyping errors were reanalyzed or retyped if necessary. The marker map covered >91% of the genome (not regarding the Y chromosome) at a 20 cM inter-marker distance. Arithmetic mean and maximum distance to the next marker or chromosome end was 11.7 and 33 cM respectively. The B10 RIII.eae2RIIIS/J founder was densely typed (146 microsatellites) and the F2 population analyzed for skewing in the segregation pattern of all markers to establish potentially segregating regions since this animal was not completely backcrossed. The regions identified were excluded from the linkage analysis. The congenic fragment on chromosome 15 showed the expected homozygous RIIIS/J genotype from the top marker D15Mit80 (9.4 cM) through D15Mit182 (19.8 cM), while the region defined by the markers D15Mit100 (21 cM) to D15Mit234 (34.2) segregated and the markers downstream, beginning with D15Mit239 (48.2 cM), were homozygous for B10.RIII. In addition, most of chromosome 19 segregated and was excluded from the linkage analysis. Markers from the centromeric D19Mit31 (7 cM) through D19Mit36 (52 cM) segregated, but from D19Mit1 (52 cM) and downstream the markers were homozygous for B10.RIII. Furthermore, one marker on chromosome10 (D10Mit70) and one marker on chromosome 14 (D14Mit185) segregated. In total this corresponds to between 3.1 and 5.6% segregation in the genome (depending on the location of cut-offs), strongly reducing the risk of having undetected residual regions segregating, as the expected average segregation in this cross is 3%.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effect of protective alleles at eae2
To reproduce our previously identified locus eae2, a large fragment of chromosome 15 was bred from the RIIIS/J strain onto the B10.RIII strain (Fig. 1Go). This strain was then backcrossed 4 times to produce a congenic animal and then intercrossed to obtain a 1:2:1 distribution of the chromosome 15 fragment. Of these 52 B10RIII.Chr15 4N1i animals (28 males and 24 females), 25% were RIIIS/J homozygous, 58% heterozygous and 17% B10.RIII homozygous at the chromosome 15 locus defined by the microsatellite marker D15Nds2. The observed distribution was in agreement with the expected Mendelian distribution for this number of animals. The animals were immunized to develop EAE and examined for a period of 60 days. Surprisingly, we found no significant effect of the congenic fragment on incidence (Table 2Go), in contrast to what was expected from the original study. However, there was a weak trend that heterozygous animals were more severely affected than either of the homozygous animals (Table 2Go), which is in agreement with the original finding. One of the animals that was homozygous for RIIIS/J at D15Nds2 and developed EAE, was crossed with RIIIS/J females to produce F1, and subsequently F2 animals, to investigate the genome without the effect conferred by the eae2 gene. This founder animal was named B10 RIII.eae2RIIIS/J (Fig. 1Go).



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Fig. 1. Schematic presentation of the breeding set-up of the animals in the experiments performed. The congenic strain B10RIII.Chr15 was initiated from a (B10.RIIIxRIIIS/J)F2 (a) that was homozygous for RIIIS/J genotypes for a large fragment on chromosome 15. This founder was backcrossed for four generations to the B10.RIII parental strain, halving the RIIIS/J genetic contamination for each generation (residual contamination shown as percentages in the figure). The B10RIII.Chr15 4N strain was intercrossed to allow a segregation of the chromosome 15 genotypes and 52 animals were tested for disease development at the B10RIII.Chr15 4N1i generation (b). One animal (c), that developed EAE although carrying RIIIS/J alles homozygously in the fragment on chromosome 15, was selected to produce F2 animals (d) to investigate the contribution of other genetic factors on the disease. In total 259 B10 RIII.eae2RIIIS/J F2 animals (d) were immunized for development of EAE.

 

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Table 2. Genotype distribution of the congenic fragment on chromosome 15 in the B10 RIII.chr15 4N1i animals
 
Analysis of the B10 RIII.eae2RIIIS/JF2intercross
259 B10 RIII.eae2RIIIS/J F2 animals were bred and immunized for development of EAE. The disease of each animal was scored and assigned to one of three phenotypic groups depending on the disease course (Table 1Go). Some animals showed symptoms, such as balance disturbances, indicating the involvement of the brain (30); however, this was not specific for any of the groups above, but rather occurred in all groups to some extent. To investigate this phenotype, all animals that showed signs of brain involvement were included in an additional group. A summary of the B10 RIII.eae2RIIIS/J F2 EAE experiment is provided in Table 3Go.


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Table 3. Characteristics of the B10 RIII.eae2RIIIS/J F2 animal experiment performed
 
In total 241 animals were screened with 111 microsatellite markers spread over the different chromosomes and linkage analysis was performed for all phenotypes using standard computer packages to calculate LOD scores. Due to the semi-quantitative nature of our phenotypes we decided to establish an empirical genome-wide significance level for our dataset by performing a permutation analysis (31,32). The observed genotypes of our B10 RIII.eae2RIIIS/J F2 animals were kept constant while the phenotypes were randomly shuffled over the genotype data, thereby efficiently disrupting any genotype–phenotype correlation. Linkage analysis was performed and the highest LOD score in each experiment was recorded. This procedure was repeated 1000 times and through this method the threshold for 95% significance was calculated to LOD = 3.36. With this threshold for significance, one region on chromosome 7 was linked to acute disease with a genome-wide significant LOD score (Table 4Go) while several more regions were linked to different aspects of disease but not at genome-wide significance levels. While still remaining interesting, these putative regions need more investigation to establish their importance on the disease.


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Table 4. LOD score results from screening of 241 B10 RIII.eae2RIIIS/J F2 animals
 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have identified a new locus on chromosome 7 associated with EAE in a B10.RIIIxRIIIS/J cross in which the previous identified loci, eae1, eae2 and eae3 (19) were neutralized. In addition, we found suggestive loci on chromosomes 8, 10, 11 and 18. Interestingly, all of the new loci are located in the vicinity of loci associated with EAE in other mouse crosses (33–38).

The eae1 locus, i.e. the MHC region, had earlier been demonstrated to affect the disease using congenic strains (20), and was also kept neutral in our earlier published F2 cross between B10.RIII and RIIIS/J (19). The eae2 and eae3 identified in this cross were found to interact and to explain a large part of the variance since no mice developed EAE that were homozygous for the RIIIS/J allele at eae2. However, we show that the eae2 locus, on chromosome 15, could not be reproduced in an intercrossed 4N congenic strain, with a fragment from RIIIS/J on the B10.RIII background. The failure to reproduce the effect of the eae2 linkage in the B10RIII.Chr15 4N1i can be attributed to two possible causes. One explanation might be that the gene at the eae2 locus works in an epistatic manner together with undetected genes from the RIIIS/J strain, since the B10 RIII.chr15 4N1i cross is reduced 10-fold of its RIIIS/J-derived genetic content compared to the genetic environment in which eae2 was established. Another explanation is that slight differences in environmental conditions could shift the control of the disease. Such factors can potentially overcome the protective effect of the eae2 locus, allowing for other genetic influences on the outcome, as has been seen by others (33). However, it is very unlikely that the original linkage was a false positive since further gene segregation experiments in B10.RIII and RIIIS/J strains have reproduced the finding (J. Karlsson et al., to be published).

It was apparent that we needed to identify other gene regions associated with the disease that were of importance in the absence of eae2. For doing this, we set up an experiment in which not only eae1 and eae2 were neutralized by using congenic parentals, but also eae3. Eae3 was not expected to be identified since it was not observed in the original F2 experiment when only animals homozygous for RIIIS/J at eae2 were present.

In this new cross seven new loci linked to disease were identified. The strongest linkage was for a region on mouse chromosome 7. This region has previously been implicated in studies of EAE (34,35,39), collagen-induced arthritis (40) and systemic lupus erythematosus (41). Although the end target structures for the autoimmune response in these diseases are not related, the mechanism for the break of tolerance could be accomplished by the same gene products. According to current knowledge the QTL on chromosome 7 does not harbor any obvious candidate genes and further studies in congenic mice will be needed to elucidate the role for genes in this region in tolerance induction. We show in the present study that the gene contained in this region most probably is inherited in an additive fashion from B10.RIII. Interestingly, similar data has been found in a cross between the EAE-susceptible strain SJL/J and the EAE-resistant strain B10.S/DvTe (35). Here the alleles from the resistant strain at the locus eae4 conferred susceptibility in a recessive fashion. Thus, we independently confirm the presence of a gene that affects EAE in the region containing eae4. We also identified a locus that controlled brain-related symptoms in a region on chromosome 10. This is in the same region as a recently identified locus on chromosome 10 also controlling lesions in the brain (39). Moreover, two of the loci were found to originate from the resistant strain (RIIIS/J), something that has been noted in other linkage studies (35,42). Two loci (chromosome 1 and 6) showed their effect only in a heterozygous state (heterosis). For this mode of inheritance there is no calculated significance thresholds, hence we cannot define these loci as suggestive. Interestingly, some disease phenotypes map to the same marker (on chromosome 6 and 7), implying a genetic factor that affects the development of the disease in general (Table 4Go). Of notable interest is the fact that we do not detect the eae3 region on chromosome 3 that we previously described. This is in agreement with the original data as eae2 interacted in an epistatic fashion with eae3 by over-riding the effects of alleles at eae3 if two RIIIS/J alleles were present at eae2.

This study emphasize that complex diseases like EAE are controlled by genes clustered in certain regions that are shared between different strain combinations and also shared with other inflammatory diseases, such as arthritis. This does not necessarily mean that the genes are shared or that their interactions influence the disease similarly. It can be anticipated that complexity conferred by interacting genes also is present in MS and this is one reason for the difficulties in identifying significant loci outside the MHC region. However, in the mouse it will now be possible to isolate the newly identified loci in new congenic strains and directly test if and how these will interact to produce the disease phenotype. For further studies on eae2 and the chromosome 7 region, mice congenic for eae1, eae2 and the QTL on chromosome 7 will be established to investigate if RIIIS/J alleles in a homozygous fashion on eae2 and chromosome 7 will have an expected negative influence on disease development. With a strong effect on the disease phenotype it will be possible to considerably decrease the important congenic regions in the search for the responsible genes.


    Acknowledgments
 
We wish to thank the Genotyping Center in Uppsala, founded by SSF, for excellent genotyping. We also wish to thank Carlos Palestro and Lennart Lindström for help with animal experiments, and Caroline Treschow for assistance concerning linguistic matters. This work was supported by grants from the Kock and Österlund Foundations, the Swedish Medical Research Council, and EU project BMH4-CT97-2522.


    Abbreviations
 
EAE experimental autoimmune encephalomyelitis
LOD logarithm of odds
MS multiple sclerosis
MBP myelin basic protein

    Notes
 
Transmitting editor: E. Möller

Received 27 August 2001, accepted 9 October 2001.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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