Chromosome 14 Contains Determinants That Regulate Susceptibility to Theiler's Virus–Induced Demyelination in the Mouse

Corresponding author: M. Rodriguez, Department of Immunology, Mayo Clinic/Foundation, 200 First St., SW, Rochester, MN, 55901, rodriguez.moses{at}mayo.edu (E-mail).

Communicating editor: N. A. JENKINS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Theiler's murine encephalomyelitis virus causes a chronic demyelinating disease in susceptible strains of mice that is similar to human multiple sclerosis. Several nonmajor histocompatibility complex–linked genes have been implicated as determinants of susceptibility or resistance to either demyelination or virus persistence. In this study, we used linkage analysis of major histocompatibility complex identical H-2^d (DBA/2J x B10.D2) F₂ intercross mice to identify loci associated with susceptibility to virus-induced demyelinating disease. In a 20-cM region on chromosome 14, we identified four markers, D14Mit54, D14Mit60, D14Mit61, and D14Mit90 that are significantly associated with demyelination. Because two peaks were identified, one near D14Mit54 and one near D14Mit90, it is possible that two loci in this region are involved in controlling demyelination.

THE genetic factors involved in the development of the most common human demyelinating disease, multiple sclerosis (MS), have been the subject of great study. Epidemiological studies indicate that both environmental and genetic factors are implicated in pathogenesis (reviewed by EBERS and SADOVNICK 1994 ). More recently, three independent groups used microsatellite analysis to screen the entire genome of affected individuals and their family members (EBERS et al. 1996 ; MULTIPLE SCLEROSIS STUDY GROUP 1996; SAWCER et al. 1996 ). Regardless of the method chosen to identify genes influencing MS, no agreement has been reached regarding the required genetic factors. The most consistent associations made to date are between particular major histocompatibility complex (MHC) alleles and the development of demyelination (reviewed by EBERS and SADOVNICK 1994 ). The lack of complete penetrance of the MHC genes, as well as the potential existence of multiple undefined genes that influence pathology, provide a formidable challenge for investigators in the field.

There are two commonly accepted animal models of MS. Experimental autoimmune encephalomyelitis provides investigators with a T-cell-mediated autoimmune model of MS, while Theiler's murine encephalomyelitis virus (TMEV) provides a means to study both the genetic and environmental factors that impact demyelination. Intracerebral infection of TMEV, a naturally occurring enteric pathogen of mice, causes a biphasic disease in susceptible strains of mice that resembles human multiple sclerosis. The acute disease is characterized by encephalitis and, subsequently, demyelination, and viral persistence develops in susceptible strains of mice (LIPTON 1975 ). Resistant mice have the capacity to clear the virus after the first phase of the disease, and demyelination does not occur. Susceptibility to TMEV-induced disease is multifactorial, involving virus persistence, spinal cord inflammation, subsequent demyelination, and the development of neurological deficits. Some of the endpoints used in defining susceptibility have been clinical deficits, viral RNA persistence, and morphological evidence of demyelination.

The range of susceptibility or resistance to demyelination varies greatly between strains. SJL/J, DBA/2J, and PL/J mice are highly susceptible; C57BL/6J, C57BL/10J, and BALB/cJ are resistant; and C3H/J and AKR/J are moderately susceptible. Previous studies have identified several loci associated with susceptibility or resistance to TMEV-induced demyelination, both MHC and non-MHC linked. These include the D region of the H-2 locus (CLATCH et al. 1985 ; RODRIGUEZ and DAVID 1985 , RODRIGUEZ and DAVID 1986b; MELVOLD et al. 1987 ), a region on chromosome 3 near the Car-2 gene (MELVOLD et al. 1990 ), and another area on chromosome 6 near the Tcrb locus (MELVOLD et al. 1986 ; RODRIGUEZ et al. 1992 , RODRIGUEZ et al. 1994 ). In the H-2 D region, the D gene itself is implicated in resistance/susceptibility, as shown with transgenic mice (AZOULAY et al. 1994 ; LIPTON et al. 1995 ; RODRIGUEZ and DAVID 1995 ). More recently, a locus near the gene for interferon- on chromosome 10 has been implicated in viral persistence, as well as a potential gene near Mbp on chromosome 18 (BUREAU et al. 1993 ). One caveat to these genetic studies is that investigators have used different endpoints in defining resistance and susceptibility to TMEV.

In this study, susceptible DBA/2J were crossed to resistant B10.D2 mice, and microsatellite analysis was performed to systematically search the genomes of 108 F2 mice for additional loci influencing the susceptibility or resistance to TMEV-induced demyelination. Morphological criteria were used to define susceptibility and resistance. The use of these two strains of mice allows for the identification of non-MHC-linked genes because the parental strains are both H-2 ^d. This approach identified four markers within a 20-cM region on chromosome 14 that are significantly associated with inflammation and demyelination. After the initial genome scan, an additional experiment was performed to directly test the hypothesis that this region of chromosome 14 was involved in conferring susceptibility to TMEV-induced demyelination. This second experiment confirmed the results of the genome scan and solidified the finding that this 20-cM region is involved in determining susceptibility to demyelination.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Virus:
Daniel's strain of TMEV was used in these experiments. The passage history has been published previously (RODRIGUEZ et al. 1983 ).

Animals:
DBA/2J and B10.D2 mice were purchased from The Jackson Laboratories (Bar Harbor, ME). (DBA/2J x B10.D2) F₂ mice were bred at the Mayo Clinic (Rochester, MN). Four- to six-week-old mice were intracerebrally infected with 2 x 10⁵ pfu of TMEV in a 10-µl volume. At day 45 after infection, animals were perfused with Trump's fixative, and the spinal cords were processed for morphometric analysis, as described previously (RODRIGUEZ et al. 1986A ). Before perfusion, pieces of liver and tail were removed and stored in liquid nitrogen until DNA was extracted using standard procedures (DAVIS et al. 1986 ). Handling of all animals conformed to the guidelines of both the National Institutes of Health and the Mayo Clinic/Foundation Animal Care and Use Committee.

Quantitative morphology:
Every third spinal cord block (1 mm) was embedded in glycol-methacrylate (JB4) and stained with a modified erichrome stain for myelin, as described previously (PIERCE and RODRIGUEZ 1989 ). Gray matter inflammation, meningeal inflammation, and demyelination were analyzed on 12–15 coronal sections per animal. This study involved the analysis of 11,880 spinal cord quadrants in Experiment 1 and 468 spinal cord quadrants in Experiment 2. Data are expressed as the percentage of quadrants in the spinal cord sections from an individual mouse with a morphologic abnormality. The maximum score for each criteria was 100, which indicates that every spinal cord quadrant of every block from each mouse examined had gray matter disease, meningeal inflammation, or demyelination. The pathological score for each mouse was determined without knowledge of genotype.

In situ hybridization:
In situ hybridization was performed using an ³⁵S-labeled 253-bp (nucleotides 3053–3305) cDNA probe specific for the VP1 region of TMEV (Daniel's strain) per our published methodology (PATICK et al. 1990A ). The central nervous system area was measured with an IBAS Image Analysis System (Kontron, Munich, West Germany) attached to an Axiphot microscope (Carl Zeiss, Inc., Thornwood, NY).

Experimental design:
Two independent experiments were performed to identify and confirm the existence of loci involved in susceptibility to virus-induced demyelinating disease.

Genome scan (Experiment 1): In the initial experiment, morphological and genetic data were analyzed for 108 (DBA/2J x B10.D2) F₂ mice. Ninety-two microsatellites were used to screen the entire genome of these mice. The statistics used to test for an association between a locus and susceptibility to demyelination are described below.

Test of hypothesis (Experiment 2): A second independent experiment was performed to confirm the results obtained in Experiment 1. In this study, 148 mice were screened at D14Mit54 and D14Mit90 using microsatellite analysis, and DBA/2 (n = 23) or B10.D2 (n = 13) homozygous (DBA/2J x B10.D2) F₂ mice were identified. Morphological studies were then performed on mice that were homozygous at both loci. In this experiment, Student's t-test was used to determine if there were statistical differences between the groups.

Microsatellite mapping:
The sequences of the PCR primers have been described by DIETRICH et al. 1994 . Gnrh is a microsatellite from the Nuffield Department of Surgery, Oxford, England (HEARNE et al. 1991 ). Amplifications were performed with an annealing temperature of 55°, as described previously (MONTAGUTELLI et al. 1991 ). Map distances were calculated from recombination frequencies using the Map-Manager program (MANLY 1993 ). Depending on the pair of adjacent markers, in Experiment 1, 77–108 mice were used to calculate genetic distances. To account for the deviation of demyelination scores from the Laplace-Gauss distribution, an empirical distribution was obtained by a Monte Carlo method (data not shown). The F distribution was evaluated in two simulations of 20,000 random replicates, each under the assumption of no linkage between the genotype and the phenotype. For these simulations, the demyelination scores were those observed in the experiment, and the genotypes were randomly assigned to the members of the F₂ cross. The simulated distribution was very close to the F distribution of the table used during the remainder of this study. For each locus, the mean and standard error of the mean (SEM) of the demyelination score were calculated for heterozygotes and each of the two homozygotes. Analysis of variance was performed using the StatView program. A robust nonparametric test, the Kruskal-Wallis test, gave results similar to those of this analysis. An analysis with Mapmaker/QTL, a program that uses a maximum likelihood algorithm with "interval mapping" and "simultaneous search," permitted better localization of the loci and exclusion mapping (LANDER et al. 1987 ). The distance chosen was defined by the function of Kosambi (DAVIES et al. 1995 ).

To test the hypothesis of the existence of a second locus on chromosome 14, an empirical significance level of the F distribution for this locus was obtained by a Monte Carlo method. The F distribution was evaluated in two simulations of 20,000 random replicates under the assumption of a linkage between the genotype at the D14Mit54 locus and the phenotype. For these simulations, the demyelination scores were those observed in the experiment, and the genotypes were randomly assigned to the members of the F₂ cross under the assumption that the second locus was 20 cM away from the D14Mit54 marker. The empirical significance level was obtained from the simulated distribution by adding the F values greater than those calculated for the D14Mit90 marker and dividing the sum by the number of replicates.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DBA/2J mice are susceptible to TMEV-induced demyelination, whereas B10.D2 mice are resistant (Table 1). Both strains have the same H-2 ^d haplotype because the B10.D2 strain is a C57BL/10 strain congenic for the H-2 region of the DBA/2J strain. Therefore, comparing the DBA/2J and B10.D2 strains should allow the identification of non-H-2 loci involved in the susceptibility/resistance to demyelination. F₁ (DBA/2J x B10.D2) mice had demyelination scores similar to that of the DBA/2J strain (Table 1). This was in contrast to previous results from our laboratories that demonstrated MHC-conferred resistance to be conferred as a dominant trait in B10 congenic mice (PATICK et al. 1990B ). We conclude from this finding that genes mapping outside the MHC can override the host's ability to clear infecting virus and prevent demyelination. It would be of great interest to identify the nature of these modifying determinants. In Experiment 1 (genome scan), 108 F₂ (DBA/2J x B10.D2) animals were bred and studied both histologically and genetically. There was a wide range of susceptibility to demyelination in the F₂ progeny, ranging from 0 to 55% of the spinal cord quadrants with demyelination (Figure 1A and Figure B, and Figure 2). Ninety-two microsatellites spread over most of the genetic map were used to screen the entire genome of each mouse by PCR. Between two and 12 loci were analyzed for each chromosome, depending on its size and the possible existence of loci controlling demyelination. Particular attention was taken to choose the most telomeric and centromeric polymorphic markers among the microsatellites from DIETRICH et al. 1992 . The distance between adjacent loci varied between 1 and 42 cM. The mean of demyelination scores from mice heterozygous or homozygous at each locus were compared by analysis of variance (Table 2). The differences were considered statistically significant when the probability of observing these differences by chance was <0.0016, which is equivalent to an LOD score >2.8. This level of significance was chosen according to the criteria proposed by LANDER and KRUGLYAK 1995 and corresponds to a "suggestive linkage" because, in an F₂ cross, such an event is expected to occur once by chance in a genome scan.

View larger version (160K): In this window In a new window Download PPT slide   Figure 1. —Demyelination and virus RNA in (DBA/2J x B10.D2) F2 intercross mice. A wide range of demyelination and inflammation is observed in the spinal cords (w, white matter; g, gray matter) of F2 mice (A and B). The large arrows indicate demyelinated axons, and the smaller arrows indicate normal white matter with intact myelin sheaths. In situ hybridization for virus RNA on spinal cords from the mice in A and B demonstrate that virus RNA (arrows) is localized to the white matter of the spinal cord (C and D). The mouse in A and C is homozygous for DBA/2J at D14Mit54 and D14Mit90. The mouse in B and D is homozygous for B10.D2 at D14Mit54 and D14Mit90. A total of 19 mice were homozygous for DBA/2J at D14Mit54 and D14Mit90 and were processed for morphology in Experiment 1. Twelve of these mice had demyelination scores of >10%. A total of 15 mice were homozygous for B10.D2 at D14Mit54 and D14Mit90 and were processed for morphology in Experiment 1. Thirteen of these mice had demyelination scores of <7%.

View larger version (17K): In this window In a new window Download PPT slide   Figure 2. —Individual demyelination scores of mice in Experiment 1 (genome scan). Each point on the graph represents the demyelination score of an individual mouse with the indicated genotype at D14Mit54 and D14Mit90. Data for mice heterozygous at both D14Mit54 and D14Mit90 are not shown.

Four markers located on a 20-cM region of chromosome 14 were significantly associated with demyelination: D14Mit54 (P = 0.0008), D14Mit60 (P = 0.0011), D14Mit61 (P = 0.0015), and D14Mit90 (P = 0.0013) (Table 2). The effect of the D14Mit54 locus accounted for 12.5% of the total variance. The data were analyzed more precisely with the Mapmaker/QTL program, which showed linkage in this region as a large plateau limited by small peaks (Figure 3). The most probable position of a locus controlling demyelination is close to the D14Mit54 locus (LOD score = 3.26). The confidence interval for this localization is very large, however, extending >55 cM (Figure 3, hatched region). The allele conferring susceptibility at this locus is probably dominant (LOD score = 3.01) but could also be additive (LOD score = 2.85) (Table 2 and Figure 3). A second, lower peak is detected between Gnrh and D14Mit90 (LOD score = 3.14). At this position, inheritance is most consistent with codominant inheritance (LOD score = 3.14) and not dominant (LOD score = 1.83 for dominance of the susceptible allele) (LOD score = 2.30 for dominance of the resistant allele; Table 2 and Figure 3). Further evidence for the possible existence of more than one locus was provided by a permutation test (CHURCHILL and DOERGE 1994 ) using QTL Cartographer (BASTEN et al. 1994 ). These two peaks satisfied the suggestive linkage criteria and were separated by a depression with a depth of 0.5 LOD score units.

View larger version (17K): In this window In a new window Download PPT slide   Figure 3. —Region on chromosome 14 significantly associated with demyelination, generated using the Mapmaker/QTL program. The linkage in this region is a large plateau limited by small peaks (D14Mit54 and an area between Gnrh and D14Mit90) that encompass a 20-cM region. The confidence interval is the 54-cM region indicated by the hatched bar. Four likelihood curves were generated considering four modes of inheritance for linkage on chromosome 14: unconstrained, additive, dominant, and recessive.

To directly test the influence of gene(s) linked to D14Mit54 and D14Mit90 on susceptibility to demyelination, a second independent group of mice was studied. In Experiment 2 (test of hypothesis), mice were screened at D14Mit54 and D14Mit90, and animals bearing homozygous microsatellites from the B10.D2 or DBA/2J homozygous mice were chosen for morphological study. Mice with the DBA/2J genotype at these two loci had significantly higher levels of demyelination compared to mice of the B10.D2 genotype at the same markers (P = 0.05; Table 3 and Figure 4). Taken together with the initial genome scan, these results strongly support the role of the region between D14Mit54 and D14Mit90 as containing at least one determinant(s) of susceptibility to TMEV-induced demyelination. Furthermore, at least one additional unidentified factor that controls demyelination must be involved in the susceptibility of the DBA/2J strain because the genetic status of the F₂ mice in the region defined above does not fully explain the demyelination score of their inbred parents and of the F₁ mice (Table 1). A simultaneous search for the rest of the genome with chromosome 14 fixed was performed. No other loci were suggestive. The sex of the animal did not influence the development of demyelination with respect to these loci.

View larger version (10K): In this window In a new window Download PPT slide   Figure 4. —Individual demyelination scores of mice in experiment 2 (test of the hypothesis). Each point on the graph represents the demyelination score of an individual mouse. Mice are either B10.D2 or DBA/2 at both D14Mit54 and D14Mit90.

In situ hybridization was performed on a limited number of animals to determine if either the D14Mit54 or D14Mit90 locus affected TMEV RNA localization. Because both groups of mice experienced demyelinating disease, it would be expected that virus would be found in the white matter of the spinal cord. As expected, mice homozygous for either B10.D2 or DBA/2 at both D14Mit54 and D14Mit90 had TMEV RNA in the spinal cord white matter (Figure 1C and Figure D). TMEV-specific serum immunoglobulin levels were also assayed by ELISA using purified virus as the antigen in these same animals. All animals showed high-titer antibody responses (OD = 1.72 ± 0.12) compared to uninfected controls (OD = 0.45 ± 0.02), and no differences were found between the groups.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Human populations vary greatly in their potential to develop MS. A genetic component is only one risk factor associated with this disease. Population studies have implicated a virus(es) as a potential etiological agent, although no particular virus has been identified as the cause of the disease. The disease caused by TMEV serves as an excellent model for MS for the following reasons: (1) the pathology and neurological deficits seen in TMEV-induced demyelinating disease and MS are remarkably similar; (2) there is wide variability in the susceptibility of different inbred strains to TMEV-induced demyelination, thereby mimicking various human populations; (3) a great deal is known about the genetics of the mouse, allowing for facilitated identification of genes involved in demyelination; and (4) most strains of inbred mice are obtained and bred easily, allowing researchers access to large populations.

In the study presented here, we identified a 54-cM region of chromosome 14 that contains four markers significantly associated with the demyelination score. These markers are centromeric with respect to the TCR locus, but there is no association with the locus. These markers fulfill the criteria for "suggestive linkage," according to LANDER and KRUGLYAK 1995 . Our data therefore suggest the presence in this region of one or several gene(s) that affect susceptibility. To convincingly confirm that this finding is not an artifact of statistical analysis, a second independent experiment was performed on an additional set of animals. The results from this second experiment (Table 3) confirm the existence of at least one previously undefined determinant of demyelination located on chromosome 14. Other factors must be involved in the susceptibility of the DBA/2J strain to TMEV-induced demyelinating disease because the genetic status of the F₂ mice in the region defined above does not fully explain the demyelination score of their inbred parent and of the F₁ mice. The demyelination score of the F₂s that are B10.D2 homozygotes at the D14Mit54 locus (3.94 ± 0.94, n = 26) is significantly higher than that of the B10.D2 parent (0.89 ± 0.41, n = 17; P < 0.01; Table 2). However, no other genetic markers significantly associated with the demyelination were detected in the rest of the genome in spite of the number of markers used. It is possible that there are many of them, each with a small contribution to the overall effect.

Because the region we identified is large (54 cM) and contains two peaks of approximately the same height separated by 20 cM, the region may contain two loci that control demyelination, one located close to D14Mit54 and one near D14Mit90. Three observations support the hypothesis that more than one locus in this 54-cM region is involved in the development of demyelination. First, the pattern of genetic inheritance seems to be different for these two loci. The patterns are consistent with the hypothesis that the first one is dominant and the second one is codominant (Table 2). The statistical analysis, however, did not formally exclude a codominant inheritance pattern for the first locus. Second, the probability obtained by simulation, that a locus affecting demyelination is located by chance close to D14Mit90 and 20 cM away from a first locus cosegregating with D14Mit54, is between 0.03 and 0.045. Third, the analysis of a different trait, meningeal inflammation, in the same F₂ mice gave exactly the same LOD score plot as the one shown for demyelination (data not shown). Definite proof of the presence of more than one locus controlling susceptibility to TMEV-induced demyelination on chromosome 14 may be obtained only after analyzing mice congenic for this region.

The mechanism of how these loci result in increased demyelination is unknown, and at this time, we cannot determine whether the gene(s) we localized exerts its effect directly on the demyelinating process or affects viral persistence. No difference was identified in the localization of virus RNA in mice homozygous for either B10.D2 or DBA/2J at both D14Mit54 or D14Mit90. Because morphological studies and quantitative virological studies (i.e., plaque assays) cannot be performed on the same animal, we are unable to determine whether virus burden directly correlates with the level of demyelination. Studies are currently underway with an additional group of F₂ mice to address this issue.

There are four known genes under the peak at D14Mit54 (ABBADI and NADEAU 1997 ; Mouse Genome Database at http:/www.informatics.jax.org/mgd.html): glutamate dehydrogenase, retinal binding protein 3, surfactant protein 1, and bone morphogenic protein 4. There are seven known genes under the peak at D14Mit90 (ABBADI and NADEAU 1997 ; Mouse Genome Database at http:/www.informatics.jax.org/mgd.html): gonadotropin-releasing hormone, leuteinizing hormone, esterol 10, serotonin receptor, retinoblastoma I, formamidase 5, and cyclin B1. None of these genes are obvious candidates for the control of demyelination.

A study by Melvold's laboratory (MELVOLD et al. 1986 ) using recombinant inbred stains between BALB/c and SJL/J identified a region on chromosome 6 (near the TCR Vß locus) that contributes to clinical deficits. The present study did not identify any regions on chromosome 6 that affect the demyelinating disease process. In addition, even though DBA/2J mice have clonal deletions of TCR Vß 8.1 and Vß 6, whereas B10.D2 do not, no association by FACS staining of Vß 8.1 was found with the extent of demyelination (data not shown), even though previous genetic studies have implicated TCR Vß in resistance to demyelination (RODRIGUEZ et al. 1992 , RODRIGUEZ et al. 1994 ). Another study by MELVOLD et al. 1990 using recombinant inbred strains between DBA/2J and C57Bl/6 implicated a region near the carbonic anhydrase-2 enzyme locus as being important in clinical deficits after virus infection. The P values assigned to this region on chromosome 3 in our study do not fulfill the criteria of "suggestive linkage." There are, however, several possible explanations for this discrepancy: (1) susceptibility to different virus strains (BeAn vs. Daniel's) may involve different loci, (2) the genes associated with resistance in different mouse strains (C57Bl/6 and B10.D2) may involve different loci, and (3) Melvold's study used clinical deficits as its primary endpoint, with histology performed on a limited number of mice. Our study used a quantitative demyelinating score as the primary endpoint. Studies by BUREAU et al. 1993 , which have implicated a gene at the IFN- locus, used different strains of mice (SJL/J and B10.S) and a different endpoint, that is, viral persistence, for analysis. In the present study, we did not find any association between this locus and demyelination.

In conclusion, we describe a linkage with one and possibly two loci on chromosome 14 for the control of susceptibility to virus-induced demyelination. Further studies of this nature may aid in the identification of more demyelination-associated genes in the mouse. To our knowledge, sequencing of the area between D14Mit54 and D14Mit90 is not currently underway. Currently, lines of congenic mice are being bred that may aid in identifying the gene or genes located in the region between D14Mit54 and D14Mit90 on chromosome 14 involved in susceptibility to TMEV-induced demyelination.

	FOOTNOTES

¹ These authors contributed equally to this study; authors are listed alphabetically.

	ACKNOWLEDGMENTS

This work was supported by National Institute of Health grants N01-AI-4-5197-02 (M.R.), R01-NS32129 (M.R.), R01-NS24180-10 (M.R.), and CA09127 (K.M.D.), and by grants from the Institut Pasteur Foundation, the Centre National de la Recherche Scientifique, the National Multiple Sclerosis Society, and the Association pour la Recherche sur la Sclerose en Plaques (M.B.). K.M.D. is a fellow of the National Multiple Sclerosis Society.

Manuscript received October 9, 1997; Accepted for publication December 22, 1997.

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