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Endocrinology, doi:10.1210/en.2007-1517
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Endocrinology Vol. 149, No. 4 2001-2009
Copyright © 2008 by The Endocrine Society

Shared and Unique Susceptibility Genes in a Mouse Model of Graves’ Disease Determined in BXH and CXB Recombinant Inbred Mice

Sandra M. McLachlan, Holly A. Aliesky, Pavel N. Pichurin, Chun-Rong Chen, Robert W. Williams and Basil Rapoport

Autoimmune Disease Unit (S.M.M., H.A.A., P.N.P., C.-R.C., B.R.), Cedars-Sinai Research Institute and University of California, Los Angeles, School of Medicine, Los Angeles, California 90048; and Department of Anatomy and Neurobiology (R.W.W.), University of Tennessee Health Science Center, Memphis, Tennessee 38163

Address all correspondence and requests for reprints to: Sandra M. McLachlan, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: mclachlans{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Susceptibility genes for TSH receptor (TSHR) antibodies and hyperthyroidism can be probed in recombinant inbred (RI) mice immunized with adenovirus expressing the TSHR A-subunit. The RI set of CXB strains, derived from susceptible BALB/c and resistant C57BL/6 (B6) mice, were studied previously. High-resolution genetic maps are also available for RI BXH strains, derived from B6 and C3H/He parents. We found that C3H/He mice develop TSHR antibodies, and some animals become hyperthyroid after A-subunit immunization. In contrast, the responses of the F1 progeny of C3H/He x B6 mice, as well as most BXH RI strains, are dominated by the B6 resistance to hyperthyroidism. As in the CXB set, linkage analysis of BXH strains implicates different chromosomes (Chr) or loci in the susceptibility to induced TSHR antibodies vs. hyperthyroidism. Importantly, BXH and CXB mice share genetic loci controlling the generation of TSHR antibodies (Chr 17, major histocompatibility complex region, and Chr X) and development of hyperthyroidism (Chr 1 and 3). Moreover, some chromosomal linkages are unique to either BXH or CXB strains. An interesting candidate gene linked to thyroid-stimulating antibody generation in BXH mice is the Ig heavy chain locus, suggesting a role for particular germline region genes as precursors for these antibodies. In conclusion, our findings reinforce the importance of major histocompatibility complex region genes in controlling the generation of TSHR antibodies measured by TSH binding inhibition. Moreover, these data emphasize the value of RI strains to dissect the genetic basis for induced TSHR antibodies vs. their effects on thyroid function in Graves’ disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID-STIMULATING antibodies (TSAb) and hyperthyroidism characteristic of Graves’ disease can be induced in some, but not all, mouse strains using immunization approaches involving in vivo expression of the TSH receptor (TSHR). For example, mice of the BALB/c strain are susceptible to hyperthyroidism induced by injecting TSHR-expressing B cells (1) or dendritic cells (2) and by immunization with adenovirus encoding the TSHR or its A-subunit (3, 4). On the other hand, C57BL/6 (B6) strain mice immunized with TSHR- or A-subunit adenovirus develop TSHR antibodies but not hyperthyroidism (3, 5). CBA/J mice immunized with either TSHR-expressing fibroblasts (6) or TSHR-adenovirus (3) have poor TSHR antibody responses and do not become hyperthyroid. Non-major histocompatibility complex (non-MHC) genes contribute to this divergence of responses. For example, BALB/k and BALB/c strains, with different MHC but the same background genes, become hyperthyroid after TSHR-adenovirus immunization (4, 7). Studies on other mouse strains immunized with TSHR-fibroblasts or TSHR-adenovirus provide support for the involvement of non-MHC genes in the development of hyperthyroidism (6, 7, 8) (reviewed in Ref. 9).

More detailed information on the genetic susceptibility to A-subunit adenovirus immunization has recently been obtained using recombinant inbred (RI) strains of mice whose genomes have been characterized (10). The first whole genome scan in a murine model of Graves’ disease was performed using RI strains derived by repeated brother x sister matings of the progeny of BALB/c and B6 parents (hence termed CXB mice). The outcome of TSHR A-subunit adenovirus immunization of CXB strains pointed to several loci on different chromosomes (Chr) that separately controlled TSHR antibody generation and hyperthyroidism (11).

High-resolution genetic maps have been generated for four other sets of RI strains that, like CXB, share B6 as one of the parental strains (10, 12). One of these RI strains is designated BXH because it is derived from crosses of B6 and C3H/He parents. The C3H/He parental strain is known to develop Graves’ hyperthyroidism after TSHR-fibroblast injection (6), but its response to TSHR-adenovirus immunization has not been determined.

In the present study, we tested C3H/He mice and the F1 offspring of this strain crossed to B6 mice for their susceptibility to hyperthyroidism induced by immunization with TSHR A-subunit adenovirus. In addition, we investigated the responses of 13 BXH strains immunized in the same way. Our data confirm the findings in CXB mice (11) that genes on multiple Chr contribute separately to the variations in TSHR antibody generation and the induction of hyperthyroidism. Moreover, the observations from the previous (CXB) and present (BXH) studies provide evidence for shared as well as unique genetic loci that influence the induction of TSHR antibodies and hyperthyroidism in the TSHR-adenovirus mouse model of Graves’ disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunization of mice with TSHR A-subunit adenovirus
Adenovirus expressing the human TSHR A-subunit (A-subunit-Ad, amino acid residues 1–289) and null adenovirus [control adenovirus (Con-Ad)] have been described (4, 13). Adenoviruses were propagated in HEK293 cells (American Type Culture Collection, Manassas, VA), purified on CsCl density gradients and viral particle concentration determined from the absorbance at 260 nm (14). Female mice of the following strains (5–8 wk of age; Jackson Laboratory, Bar Harbor, ME) were studied:

C3H/HeJ, C57BL/6J, and the F1 offspring of these two parental strains, B6C3F1. These mice are subsequently referred to as C3H, B6, and F1. Immunization was with A-subunit-Ad (108 particles per injection; n = 20 mice per strain) or Con-Ad (108 particles per injection; n = 10 mice per strain).

BXH strains BXH2/TyJ, BXH4/TyJ, BXH6/TyJ, BXH7/TyJ, BXH8/TyJ, BXH9/TyJ, BXH10/TyJ, BXH11/TyJ, BXH14/TyJ, BXH19/TyJ, BXH20/KccJ, and BXH22/KccJ and B6C3-1/KccJ. For simplification, these strains are referred to as BXH2, BXH4, etc. A maximum of six mice of each BXH strain can be provided by the Jackson Laboratory over a period of at least 6 months, a number sufficient for most genetic studies. Preimmunization, baseline serum TSHR antibody and T4 levels were determined (usually five to six mice per strain but, due to limited amounts of serum, two to four mice for some strains). Subsequently, mice were immunized with A-subunit-Ad (108 particles per injection).

For both groups of mice above, immunization was performed on three occasions at three-weekly intervals. Blood was drawn 1 wk after the second immunization, and mice were euthanized 4 wk after the third injection.

C3H/HeJ, C3H/OuJ, C57BL/10J, and C57BL/10ScNJ mice (Jackson Laboratory). The latter two are abbreviated to B10 and B10.ScN. These mice received two injections 3 wk apart of A-subunit-Ad (108 particles per injection; n = 6 mice per strain) or Con-Ad (108 particles per injection; n = 4 mice per strain). Mice were euthanized 2 wk after the second injection.

All studies were approved by the Institutional Animal Care and Use Committee and performed with the highest standards of care in a pathogen-free facility.

TSHR antibody assays
Three assays were used: TSH binding inhibition (TBI), TSAb (generation of cAMP), and ELISA using A-subunit protein. TBI was measured using a commercial kit (Kronus, Boise, ID). Serum aliquots (25 µl) were incubated with detergent-solubilized TSHR; [125I]TSH was added, and the TSHR-antibody complexes were precipitated with polyethylene glycol. TBI values were calculated from the following formula: [1 – (TSH binding in test serum – nonspecific binding)/(TSH binding in normal serum – nonspecific binding)] x 100.

TSAb activity was assayed as reported previously (5) with the following modifications: TSHR-CHO cells in 96-well plates were incubated (60 min, 37 C) with test sera diluted 1:20 in Ham’s F12 supplemented with 10 mM HEPES (pH 7.4) and 1 mM isobutylmethylxanthine. After aspirating the medium, intracellular cAMP was extracted with ethanol, evaporated to dryness, and resuspended in Dulbecco’s PBS. Samples (20 µl) were assayed using the LANCE cAMP kit (PerkinElmer, Boston, MA). TSAb activity was expressed as a percentage of cAMP values attained with sera from control, unimmunized mice.

TSHR antibodies of IgG class were measured by ELISA as described previously (4). Recombinant A-subunit protein secreted by Chinese hamster ovary cells with an amplified transgenome (15) was purified from culture supernatants by affinity chromatography (16). ELISA wells were coated with A-subunit protein (1 µg/ml) and incubated with test sera (duplicate aliquots, diluted 1:100). Antibody binding was detected with horseradish peroxidase-conjugated mouse anti-IgG (Sigma Chemical Co., St. Louis, MO), and the signal was developed with o-phenylenediamine and H2O2. Data are reported as the OD at 490 nm.

Serum T4
Total T4 in mouse sera was measured in undiluted serum (25 µl) by RIA using a kit (Diagnostic Products Corp., Los Angeles, CA). T4 concentrations were computed from standards provided in the kit.

Statistical analyses
Significant differences between responses in different groups were determined by Mann Whitney U rank sum test or, when normally distributed, by Student’s t test. Multiple comparisons were performed using ANOVA. Tests were performed using SigmaStat (Jandel Scientific Software, San Rafael, CA).

Genetic linkage analysis
Putative quantitative trait loci (QTL) involved in BXH strain traits before and after A-subunit-Ad immunization were mapped using the genotype files for BXH RI strains generated by Williams et al. (10) available at http://www.nervenet.org and embedded in GeneNetwork. This genotype file consists of 1384 markers with unique strain distribution patterns (www.genenetwork.org/dbdoc/CXBGeno.html). The probability of linkage between our traits and previously mapped genotypes was estimated at about 1-cM intervals (~2 Mb) along the entire genome, except for the Y Chr . To establish criteria for suggestive and significant linkage, a permutation test was performed (1000 permutations at 1-cM intervals) (17). This test compares the peak likelihood ratio statistics (LRS = LOD x 4.6) obtained for a given data set with the peak LRS score obtained for 1000 random permutations of the same data set.

The primary phenotype data (10 traits) have been entered into the mouse BXH Phenotype database on GeneNetwork (www.genenetwork.org) under the trait accession identifiers 10145 through 10155 and can be found as a group by searching for the name McLachlan. In Results, we refer to the identification (GN) numbers of specific traits so that readers can verify and extend the data analysis. Because GN interval maps connect directly to the UCSC (University of California Santa Cruz) Genome Browser, it is possible to explore the gene complement of chromosomal intervals together with the QTL profile.

The statistical power of RI sets with 13 members is small (18). Therefore, we combined the data for BXH strains and our previously reported findings for CXB strains (11) to provide a data set from 26 RI strains sharing one parental strain (B6). As previously described for the variation in neuron number in two sets of RI mice (BXH and BXD) (19), we calculated the probability associated with a {chi}2 value equal to 2(lnPBXD + lnPCXD) with 4 degrees of freedom, where lnPBXD and lnPCXD are the natural logarithms of the probabilities derived independently for the two RI strains in the same chromosomal interval.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TSHR antibodies and thyroid function in C3H/He and B6 mice and their F1 offspring
TBI activity was induced in all strains after two and three A-subunit-Ad immunizations and, despite variability in some animals, at significantly lower levels after the third injection in C3H/He vs. B6 and F1 mice (Fig. 1AGo, P < 0.05). Similarly, TSAb was extremely low in all but two C3H mice and higher in B6 and F1 mice (Fig. 1BGo). TSHR antibodies measured by ELISA in sera after the third A-subunit-Ad immunization were low with no significant differences between the strains: OD at 490 nm (mean ± SEM) in C3H/He, F1, and B6 mice of 0.38 ± 0.17, 0.87 ± 0.24, and 0.27 ± 0.05, respectively.


Figure 1
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  		FIG. 1. TSHR antibodies and thyroid function in C3H/He (C3H) and C57BL/6 (B6) mice and the B6C3 F1 offspring (F1) in response to immunization with A-subunit-Ad (A-sub-Ad). Data for individual mice are presented for sera tested 1 wk after two immunizations (2x) and 4 wk after three immunizations (3x). Values for mice immunized with Con-Ad (3x) are included. A, TSHR antibodies measured by inhibition of TSH binding to its receptor (TBI). TBI values (percent) are provided for Con-Ad immunized animals (n = 10 per strain) and for A-subunit-Ad immunized animals (n = 20 per strain). Values significantly different between C3H vs. F1 and B6 after 3 immunizations: *, P < 0.05 (ANOVA). B, TSAb. Stimulation values (percent control) are for Con-Ad (n = 5 per strain) and A-subunit-Ad immunized animals (n = 10 per strain). Values significantly different: *, P < 0.05, C3H vs. F1 and B6 after two immunizations (ANOVA); #, P < 0.05, C3 vs. F1 and B6 after three immunizations (ANOVA). C, Serum T4 (µg/dl) in Con-Ad (n = 10 per strain) and A-subunit-Ad immunized animals (n = 20 per strain). Values significantly different: *, P < 0.05, C3H vs. B6 after two immunizations (ANOVA).

 
Turning to thyroid function, after two A-subunit -Ad injections, serum T4 was significantly elevated (P < 0.05) in the C3H/He group compared with B6 mice (Fig. 1CGo). After the third immunization, a few C3H/He mice and one F1 mouse retained high T4 levels, but at this time interval, there were no significant differences between the three groups. Overall, despite variability and lower TSHR antibody levels (TBI and TSAb), C3H/He mice had a greater susceptibility to induced hyperthyroidism than either B6 or F1 mice.

Susceptibility to hyperthyroidism on the C3H/He background
C3H/He mice have mutations in the Toll-like receptor 4 gene (Tlr4) that prevent recognition of lipopolysaccharide (LPS) (20). Because of this defect, we compared C3H/He mice with their wild-type counterparts, C3H/Ou, that express the wild-type Tlr4. In addition, we studied B10 mice (wild-type Tlr4) and B10.ScN mice with a mutated Tlr4 that prevents LPS recognition (20). All four strains developed comparable TBI levels after two A-subunit-Ad injections (Fig. S1, A and B, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). In contrast, hyperthyroidism developed in C3H/He mice but not in C3H/Ou, B10. ScN, and B10 mice, which remained euthyroid (supplemental Fig. S1, C and D). These findings indicated that neither the TSHR antibody nor thyroid responses to A-subunit-Ad immunization were related to the Tlr4 mutation. Likewise, induction of TSHR antibodies and induction of hyperthyroidism did not differ between mice lacking Tlr2 and wild-type mice on the C57BL/6 background (supplemental Table S1).

Responses of BXH RI mouse strains to A-subunit-Ad immunization
TSHR antibodies measured in the TBI assay were detectable in all 13 BXH strains after two and three A-subunit-Ad immunizations, with lower levels in some strains, notably BXH6 (Fig. 2AGo). TSAb levels were variable, being extremely high in some strains (such as C3-1, BXH11, and BXH14 after three immunizations) and low or undetectable in others (including BXH2, -19, -6, and -4) (Fig. 2BGo).


Figure 2
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  		FIG. 2. Thyroid autoantibodies in 13 strains of RI BXH mice immunized with A-subunit-Ad. Sera were tested 1 wk after two immunizations (2x) and 4 wk after three immunizations (3x). A, TBI. Data are presented as percent inhibition (mean + SEM, five to six mice per strain). Dashed line represents mean + 2 SD of preimmunization TBI values for the 13 strains. B, TSAb. Data are presented as percentage of control cAMP values obtained with sera from unimmunized mice (mean + SEM, five to six mice per strain). Dashed line represents mean + 2 SD of sera from Con-Ad immunized parental mice (n = 8).

 
Preimmunization T4 levels varied markedly between strains, being lowest in BXH14 and highest in BXH4 and C3-1 strains (Fig. 3AGo). Relative to these baseline levels, hyperthyroidism developed in some individual mice in the BXH6, BXH7, and BXH11 strains (Fig. 3AGo). To provide a single parameter incorporating thyroid function at baseline, {Delta}T4 values were calculated by subtracting the mean preimmunization strain value from T4 levels in individual mice after two or three injections of A-subunit-Ad (Fig. 3Go, B and C, respectively). After the second immunization, the BXH6 strain had the highest {Delta}T4 values, followed by BXH4, BXH7, BXH9, and BXH11 strains.


Figure 3
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  		FIG. 3. Thyroid function in BXH mice before and after immunization with A-subunit-Ad. A, Serum T4 before immunization and after two (2x) and three (3x) immunizations with A-subunit-Ad. Data are presented as µg/dl T4 (mean + SEM, five to six mice per strain) and ranked according to preimmunization values from the lowest (left, BXH14) to the highest (right, BXH4). B and C, {Delta}T4 levels (difference between T4 for individual animals and mean preimmunization value) after two immunizations (B) and three immunizations (C) with A-subunit-Ad.

 
Genetic linkage analysis
QTL were mapped using the BXH strain database files (10, 12) for the following parameters: TBI and TSAb (Fig. 2Go, A and B), T4 and {Delta}T4 (Fig. 3Go, A–C), and ELISA (not shown). LRS values were generally modest, with values ranging from 12.0–20.0 (LRS > 25 is usually required to reach P < 0.05 genome-wide). The following Chr were highlighted by the analysis: 1) Chr 17 with TBI after two and three immunizations (abbreviated 2x and 3x, respectively): Chr X with TBI (3x) (Fig. 4Go, A and B), 2) Chr 9 and Chr 12 with TSAb (3x) (Fig. 4CGo) and Chr 12 for TSAB (2x) (not shown), 3) Chr 1 and 13 with preimmunization T4 levels (Fig. 4DGo), 4) Chr 2 and 16 with serum T4 (2x) (Fig. 4EGo), 5) Chr 3, 8 and 9 with serum T4 (3x) (Fig. 4FGo), 6) Chr 9 with {Delta}T4 (2x) (Fig. 4GGo), and 7) Chr 13 and Chr 3 with {Delta}T4 (3x) (Fig. 4HGo).


Figure 4
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  		FIG. 4. Whole genome interval mapping for traits in BXH mice before or after A-subunit-Ad immunization. Chr 1–19 and X are indicated at the top of each panel, and LRS values on the vertical axis. The horizontal line indicates the LRS values above which a trait is associated with a particular Chr as indicated by asterisks. Interval mapping is shown for values obtained after two immunizations (2x) and/or after three immunizations (3x), with GeneNetwork (GN) BXH identifiers are in parentheses for the following panels: A, TBI 2x (GN 10145); B, TBI 3x (GN 10146); C, TSAb 3x (GN 10148); D, pre-T4 (GN 10150); E, T4 2x (GN 10151); F, T4 3x (GN 10152); G, Del T4 2x (difference between serum T4 after two immunizations and preimmunization) (GN 10153); and H, Del T4 3x (difference between serum T4 after three immunizations and preimmunization) (GN 10154). No associations were observed for ELISA (3x) (GN 10149).

 
Focusing on TSHR antibodies (Table 1Go), the highest LRS value (19.9, LOD score 4.3) was observed for TSAb (3x) and Chr 9, but there are no obvious candidate genes in this region. However, TSAb (3x) and TSAb (2x) were linked to a Chr 12 locus for which a potential candidate gene is the Ig{gamma}2b constant region. TBI (2x and 3x) were linked to loci on Chr 17 including MHC region genes (Chr 6 in humans). The Chr X region linked to TBI (3x) corresponds to human Chr X; a potential candidate gene is IL-13 receptor-{alpha}.


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  		TABLE 1. Chr associations revealed by linkage analysis for TSHR antibodies induced in BXH strains immunized with A-subunit-Ad

 
Turning to thyroid function, the traits {Delta}T4 (3x), T4, and pre-T4 were linked to a similar region on Chr 13 (LRS values 14.0, 12.3, and 12.0, respectively) (Table 2Go and Fig. 5Go, lower right panel). {Delta}T4 was linked to a second region on Chr 13 (Fig. 5Go) close to the Cd38 gene. Surprisingly, different chromosomal linkages were found for T4 (2x) (Chr 16 and Chr 2) and for {Delta}T4 (2x) (Chr 9). Although linkage was observed between Chr 9 and both TSHR antibodies (TSAb 3x) and thyroid function ({Delta}T4 2x), the responsible loci are different, being located at opposite ends of the Chr (~90 vs. ~25 Mb, respectively). In addition to Chr 13, pre-T4 levels were linked to a region on Chr 1. Potential candidate genes for the Chr 13 locus include IL-6 signal transducer (IL6st) and IL-31 receptor A (Il31ra) (Chr 5q11 in humans). However, whether any of these (and other genes listed in Table 2Go) contribute to thyroid function remains to be determined.


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  		TABLE 2. Chr associations for thyroid function traits in BXH strains before and after immunization with A-subunit-Ad

 

Figure 5
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  		FIG. 5. Individual Chr associated with particular traits in BXH (present study) and CXB (11 ) mice before and after A-subunit-Ad immunization. Chromosomal distance (megabases) is on the horizontal axes, and LRS values are on the vertical axes. GeneNetwork (GN) BXH identifiers are provided in parentheses for the following comparisons: left panels, Chr 17, TBI 2x for BXH mice (GN 10145) and CXB mice (GN 10512); Chr 3, Del T4 3x (difference between serum T4 after three immunizations and preimmunization) for BXH mice (GN 10154) and Del T4 2x (difference between serum T4 after two immunizations and preimmunization) for CXB mice (GN 10519); Chr 13, Pre-T4 (preimmunization serum T4) for BXH mice (GN 10150) and CXB mice (GN 10516); and right panels, Chr X, TBI 3x for BXH mice (GN10146) and ELISA 2x (TSHR antibodies measured by ELISA after two immunizations) for CXB mice (GN 10518); Chr 1, pre-T4 (preimmunization serum T4) for BXH mice (GN 10150) and CXB mice (GN 10516); Chr 13, pre-T4 (preimmunization serum T4), T4 3x, and Del T4 3x (difference between serum T4 after three immunizations and preimmunization) for BXH mice (GN 10150, 10152, and 10154).

 
Shared vs. unique genes in BXH vs. CXB mice
Chromosomal linkages were compared in BXH mice (present study) and CXB mice (11) (Table 3Go). TBI values were linked to the same locus on Chr 17 and adjacent regions on Chr X were associated with TBI (3x) in BXH strains and ELISA (2x) in CXB strains (Fig. 5Go, upper left and right panels, respectively). In terms of thyroid function, similar Chr 3 loci were associated with {Delta}T4 in CXB (2x) and BXH (3x) strains (Fig. 5Go, left middle panel). Also, similar loci were associated with Chr 1 and preimmunization T4 values in CXB and BXH strains (Fig. 5Go, right middle panel). The Chr 13 loci linked to thyroid function in BXH strains is adjacent to, but does not overlap, that for CXB mice (Fig. 5Go, left lower panel).


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  		TABLE 3. Shared and unique Chr loci (and syntenic human Chr) for traits studied in BXH and CXB mice before and after A-subunit-Ad immunization

 
Unlike the shared genetic loci, each set of RI mice had unique chromosomal linkages for several traits (Table 3Go). Some of these loci may be sampling errors (or false-positive peaks) associated with the size of these RI sets. Nevertheless, the following observations are of potential interest: 1) thyroid function in the BXH set was linked to Chr 16 and 2 (T4 2x), Chr 8 (T4 3x), Chr 9 ({Delta}T4 2x), and a nonshared Chr 13 locus ({Delta}T4 3x); 2) in the CXB RI set, linkage was observed between TBI (2x) and a second locus on Chr X as well as for {Delta}T4 (2x and 3x) (Chr 13 in BXH and Chr.10 in CXB mice).

Finally, the linkage data obtained separately for BXH and CXB sets (both derived from B6 parents) were combined to increase the statistical power provided by 26 strains. This analysis confirmed that the variability in four traits could be attributed to shared loci. In particular, the probability of the observed linkage occurring by chance for TBI activity and the Chr 17 MHC region is less than 0.000017, yielding a combined LOD score of 5.78. The {chi}2 values for the combined linkages for the appropriate chromosomal intervals were also statistically significant for Chr X and TBI/ELISA, Chr 3 and {Delta}T4, and Chr 3 and preimmunization T4 (Table 4Go, P = 0.00262 and P = 0.0058, respectively).


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  		TABLE 4. Combined analysis of the linkage data for BXH and CXB RI mice (26 strains in total)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
RI mice provide a valuable resource for mapping the genetic basis for Mendelian and quantitative traits in diverse fields including anatomical, functional, behavioral, and disease-related traits (21, 22, 23). Recently, we used CXB RI strains to begin a search for susceptibility loci for TSHR antibodies and hyperthyroidism induced by immunization with adenovirus expressing the A-subunit of the TSHR, the autoantigen in Graves’ disease (11). However, the relatively small number of strains in the CXB set did not permit identification of specific candidate genes.

In humans and in mice, reproducibility in different samples or strains is critical to establish validity of putative genetic loci for a trait. Fortunately, another set of RI strains (BXH) was available for comparison with the previously studied CXB set. The BXH strains comprise recombinants of the F1 generation of the hyperthyroid-resistant B6 mice and the C3H/He strain. C3H/He mice were known to be susceptible to hyperthyroidism induced by the Shimojo approach, injecting fibroblasts coexpressing MHC class II and the TSHR (6). However, some strain differences have been observed for Graves’ disease induced using the Shimojo vs. the adenovirus approach (reviewed in Ref. 9). Therefore, we tested the response of C3H/He mice to A-subunit-Ad immunization. Although hyperthyroidism was not induced as readily in C3H/He mice as in BALB/c mice (5), the former strain was nevertheless more responsive than B6 mice. A marked and unexpected difference between these BXH parental strains mice was that TSHR antibody induction, as measured in TBI and TSAb assays, was lower in C3H/He than in B6 mice despite the greater propensity of the former to become hyperthyroid. These data suggest that the C3H/He thyroid is particularly sensitive to TSAb stimulation.

Another interesting difference between the CXB and BXH parental strains was that the F1 offspring of the BALB/c x B6 F1 mice responded similarly to the BALB/c (not to the B6) mice, indicating that BALB/c susceptibility to hyperthyroidism was dominant and overcame resistance in B6 mice (5). In contrast, in the F1 offspring of the parental BXH strains, responses were biased toward those of B6 rather than toward those of C3H/He mice. Turning to the RI BXH strains, the B6 genetic background also dominated the outcome of A-subunit-Ad immunization. Several BXH strains developed high TBI levels, but overt hyperthyroidism was rare, particularly after the third immunization. However, the genetic influence of both C3H/He and B6 parents was evident from the TSAb levels, which ranged from undetectable or very low levels in some BXH strains to very high levels in others.

Variability in preimmunization T4 values was a feature of individual BXH strains, as previously observed in several inbred mouse strains (24) and in the CXB set (11). C3H/He mice have an impairment of the selenoenzyme type I deiodinase, but the magnitude of this defect is extremely variable and its biological consequences have not been examined (25). Recently, in a study of consomic rats, the higher levels of serum TSH in BN vs. SS rats appeared to involve reduced TSHR expression (26). Studies in additional strains of RI mice may provide insight into the genetic factors controlling (and the mechanism involved in) the variability of serum T4 levels in different strains of mice.

C3H/He mice have the interesting property of having a mutated Tlr4 that does not recognize LPS (20). We addressed the question of whether an abnormal Tlr4 could, at least in part, contribute to the TSHR antibody and T4 responses to TSHR A-subunit immunization in C3H/He mice. For this purpose, we compared C3H/He mice with the closest related strain bearing a wild-type Tlr4 (C3H/Ou mice) as well as another pair of mouse strains with an abnormal (B10.ScN) or normal (B10) Tlr4. All four strains developed TSHR antibody responses, consistent with observations for C3H/He and B10.BR mice immunized with TSHR-fibroblasts (6). In contrast, some C3H/He mice, but not C3H/Ou or B10 strain mice, became hyperthyroid in response to A-subunit-Ad immunization. Therefore, Toll-like receptor 4 function was not associated with hyperthyroidism, consistent with the lack of effect of exogenously administered LPS on TSHR-Ad-induced hyperthyroidism (7). Resistance to hyperthyroidism has been maintained in the B6 and B10 substrains of C57BL mice, which were separated in 1937. In contrast, susceptibility to induced hyperthyroidism has developed in C3H/He mice after their separation from the C3H/Ou strain in 1952 (27).

Linkage analysis of the data for BXH mice, derived from C3H/He and B6 F1 offspring, demonstrated that the chromosomal loci predisposing to the induction of TSHR antibodies and changes in serum T4 were not the same, corroborating our findings in CXB mice (11). Moreover, in the BXH strains, we observed chromosomal linkages for some traits previously found in CXB mice: 1) Chr 17 (MHC region) and TBI, 2) Chr X and TSHR antibodies (TBI in BXH and ELISA in CXB strains), 3) Chr 3 loci and the difference ({Delta}) in pre-and postimmunization T4 levels; and 4) Chr 1 and preimmunization T4 values. Chr 13 harbors a locus linked to three thyroid function traits in BXH mice (preimmunization T4, T4 after three immunizations, and {Delta}T4). However, this region appears to be different from the region on the same Chr linked to baseline T4 levels in CXB mice.

In contrast to the similarities, some chromosomal associations were unique to the RI sets. Some of these loci may reflect noise levels and require confirmation. Nevertheless, TSAb activity (a trait not studied in CXB strains) was linked to a potentially interesting Chr 12 locus, the Ig{gamma}2b constant region. B6 (but not BALB/c) mice have TSHR antibodies of this subclass (5), and linkage to this region is enhanced by B6 genes (not shown). The Igh locus on Chr 12 is very large (~3 Mb) and polymorphic. Recently it was observed that this region varies considerably in B6 and BALB/c mice, including differences in some germline variable region genes (28). Germline heavy and light chains are the precursors that undergo affinity maturation to form high-affinity antibodies, such as thyroid autoantibodies. This region in C3H mice (termed Ighj) differs from that in B6 mice (Ighb) and BALB/c mice (Igha) (29). It is intriguing to speculate that a difference in the complement of germline heavy chain genes influences the susceptibility to generating TSAb in BXH mice.

Combined analysis of the data for BXH and CXB strains confirmed that loci on Chr 17 and Chr X play a role in the variable production of TSHR antibodies (measured by TBI and ELISA) in these two sets of RI strains. Indeed, the strongest linkage data were observed for TBI and MHC region genes on Chr 17. In addition, loci on Chr 1 and Chr 3 are involved in preimmunization serum T4 and the increase in T4 levels after TSHR A-subunit adenovirus immunization, respectively. [Incidentally, unlike in humans (30), we found no evidence for involvement of the TSHR gene in our mouse model of Graves’ disease, presumably because we used the human TSHR A-subunit for immunization.]

MHC associations with Graves’ disease in humans are well known (reviewed in Ref. 31). Confirmation of the importance of genes in this region comes from recent genome-wide studies in autoimmune thyroid disease (32) and Graves’ disease (33). In the Graves’ study, the strongest associations were observed for a broad 3-Mb region near MHC for markers outside this region and for the cytotoxic T lymphocyte activator 4 (CTLA4) (33). In the study of thyroid autoimmune disease, data from an extended group of Graves’ patients (32) confirmed associations with the TSHR (30) and Fc receptor-like (FCRL) genes but not with CTLA4 (34). The explanation for the different outcomes (other than for MHC) between two powerful genome-wide associations may relate to the use of different single-nucleotide polymorphism markers in the two studies. However, it is also possible that inadvertent combination of different patient subgroups could mask the contribution of genes in each group.

In conclusion, the genetic basis for the varying susceptibilities of different mouse strains in an induced Graves’ disease model are being explored using RI strains for which refined single-nucleotide polymorphism genome maps are available. The present study on 13 BXH strains identifies loci on Chr 17 (MHC region) and Chr X as contributing to TSHR antibody generation, confirming a similar relationship observed previously in CXB mice (11). Other susceptibility loci appear to be unique to a particular RI set. Remarkably, in both BXH and CXB strains, differences in susceptibility to hyperthyroidism are linked to a locus on Chr 3 distinct from those linked to TSHR antibody generation. These studies in RI mice complement whole genome array studies in humans. Dissecting phenotypic differences (stratification) between immune responses and their consequences in humans is complex because of genetic heterogeneity, variable penetrance with age and environmental effects. Ultimately, as more RI mouse strains are investigated, identification of additional candidate genes will facilitate stratification of human disease phenotypes and increase the power of whole genome studies in humans.


   Acknowledgments
 
We are grateful for contributions by Dr. Boris Catz, Los Angeles.


   Footnotes
 
This work was supported by the National Institutes of Health Grants DK54684 (S.M.M.), DK19289 (B.R), and U01AA13499 and P20-DA-21131 (R.W.W.) and a Winnick Family Clinical Research Scholar Award (S.M.M). R.W.W. and GeneNetwork are also supported by Integrative Neuroscience Initiative on Alcoholism-National Institute on Alcohol Abuse and Alcoholism, Biomedical Informatics Research Network-National Center for Research Resources, and Mouse Models of Human Cancers Consortium-National Cancer Institute and a Human Brain Project.

Disclosure Summary: The authors have nothing to disclose.

First Published Online December 27, 2007

Abbreviations: A-subunit-Ad, Adenovirus expressing the human TSHR A-subunit; B6, C57BL/6; Chr, chromosome; LPS, lipopolysaccharide; LRS, likelihood ratio statistics; MHC, major histocompatibility complex; QTL, quantitative trait loci; RI, recombinant inbred; TBI, TSH binding inhibition; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor.

Received November 6, 2007.

Accepted for publication December 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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