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Endocrinology Vol. 147, No. 6 2789-2800
Copyright © 2006 by The Endocrine Society

Probing the Genetic Basis for Thyrotropin Receptor Antibodies and Hyperthyroidism in Immunized CXB Recombinant Inbred Mice

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

Autoimmune Disease Unit (H.A.A., P.N.P., C.-R.C., B.R., S.M.M.), Cedars-Sinai Research Institute and UCLA 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
 
Immunization with adenovirus encoding the TSH receptor (TSHR) or its A-subunit induces Graves’ hyperthyroidism in BALB/c and BALB/c x C57BL/6 offspring but not C57BL/6 mice. High-resolution genetic maps are available for 13 recombinant inbred CXB strains generated from BALB/c x C57BL/6 progeny by repeated brother x sister matings to establish fully inbred lines. CXB strains were studied before and after A-subunit adenovirus immunization for TSHR antibodies (TBI, inhibition of TSH binding), serum T4, and thyroid histology. All strains developed TBI activity (at variable levels), six strains became hyperthyroid, and one was overtly thyrotoxic. No low TBI responders became hyperthyroid, but high TBI did not predict hyperthyroidism. Preimmunization T4 levels varied in different CXB strains and was unrelated to subsequent T4 elevation. Linkage analysis indicated that different chromosomes were involved in generating TSHR antibodies and serum T4 before and after immunization. TBI activity was linked in part with major histocompatibility (MHC) genes on chromosome 17 (Chr 17) but induced Graves’ disease involved non-MHC genes (Chr 19 and 10). The Chr 10 locus is close to the Trhde gene that encodes TSH-releasing hormone degrading enzyme. Expression of Trhde is controlled by thyroid hormones and linkage with a thyroid function-related gene is intriguing. Our data, the first genome scan in murine Graves’ disease, provides insight into the role of MHC and non-MHC genes in human and murine Graves’ disease. Finally, our study demonstrates the potential of recombinant inbred mice for discriminating between immune-response genes and thyroid function susceptibility genes in Graves’ disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPERTHYROIDISM IN GRAVES’ disease is caused by autoantibodies to the TSH receptor (TSHR) that mimic the actions of TSH. TSHR antibodies and Graves’-like hyperthyroidism are induced in some strains of mice by im injections of adenovirus encoding the TSHR or its A-subunit (reviewed in Ref.1). CBA and DBA/1 mice are poor responders to TSHR adenovirus immunization because they develop undetectable or low levels of TSHR antibody, and all mice remain euthyroid (2). BALB/c and C57BL/6 (abbreviated to B6) strains are good responders to TSHR adenovirus immunization with respect to developing high levels of TSHR antibodies. However, BALB/c mice are susceptible, whereas B6 mice are resistant, to induction of elevated levels of T4 (2). Adenovirus expressing the TSHR A-subunit is more effective than adenovirus encoding the full-length or noncleaving TSHR for inducing hyperthyroidism in BALB/c mice (3). Moreover, in this strain, low-dose A-subunit adenovirus immunization induces hyperthyroidism as effectively as the high dose and the properties of the induced TSHR antibodies resemble those in Graves’ patients (4). In contrast, B6 mice remained resistant to becoming hyperthyroid even when immunized with low-dose A-subunit adenovirus (5).

Filial (F) 1 hybrids of BALB/c and B6 mice respond similarly to BALB/c mice in becoming hyperthyroid after low dose A-subunit adenovirus immunization by developing elevated serum T4 levels as in BALB/c mice (5). Thus, the BALB/c strain carries one or more dominant genes for susceptibility to induced hyperthyroidism. Studies of strains bearing different major histocompatibility (MHC) genes on the same genetic background vs. strains with the same MHC on different background genes supported a role for non-MHC genes in TSHR-adenovirus induced hyperthyroidism induced by TSHR-adenovirus immunization (2, 6). Similar conclusions were drawn from the Shimojo model in which hyperthyroidism is induced by injecting H2-K strain mice with fibroblasts coexpressing the TSHR and syngeneic MHC class II (7, 8). However, there is no information on the non-MHC genes responsible for susceptibility to hyperthyroidism in immunized mice.

Recombinant inbred (RI) strains of mice are generated from the F1 progeny of two inbred strains, followed by repeated brother x sister matings for 20 generations or more to establish multiple homozygous lines (9). Because they are inbred, individual RI lines are renewable, which is an advantage compared with F2 progeny or backcross populations (10). Most important, high-resolution genetic maps have been generated for five sets of RI strains that share B6 as one of the parental strains (11). Diverse Mendelian and quantitative traits have been mapped using such RI strains, including the size of the mouse cerebellum (12), resistance to cytomegalovirus infection (13), and spontaneous erosive arthritis vs. generalized autoimmunity (14).

One panel of genetically characterized RI mice, the CXB strain, was derived by crossing the hyperthyroid susceptible BALB/c strain (abbreviation "C") with the resistant B6 strain (abbreviation "B"). In the present study, we used thirteen CXB RI strains to explore the genetic factors that control the development of TSHR antibodies and hyperthyroidism after low-dose TSHR A-subunit adenovirus immunization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunization of mice with TSHR A-subunit adenovirus
Adenovirus expressing the TSHR A-subunit (amino acid residues 1–289, A-Subunit-Ad) and control ß-galactosidase adenovirus (Con-Ad) have been described (2, 3). Both adenoviruses were propagated in human embryonic kidney 293 cells (American Type Culture Collection, Manassas, VA), purified on CsCl density gradients and viral particle concentration determined by measuring the absorbance at 260 nm (15).

Female CXB1 through CXB13 mice and the parental strains BALB/cByJ, C57/BL/6ByJ (5–8 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME). For most of the 13 strains, a maximum of only six mice can be provided by Jackson over a period of numerous months, and genetic studies have indicated that this number is sufficient for most purposes. The parental strains were immunized with A-subunit-Ad (108 particles/injection, n = 7 mice/strain) or Con-Ad (108 particles/injection, n = 3 mice/strain) on three occasions at three weekly intervals. CXB1-CXB13 mice were immunized with A-subunit-Ad (108 particles per injection; n = 6 mice/strain except CXB5, for which only two animals were available). Before immunization, blood was drawn to provide base-line values for TSHR antibodies by ELISA and TBI and serum T4 (5–6 mice/strain for ELISA and, due to limited amounts of serum, four mice/strain for TBI and T4). For all strains, blood was drawn 1 wk after the second injection, and mice were euthanized 4 wk after the third injection to obtain blood and thyroid glands. 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.

Assays for TSHR antibodies
TSHR antibodies were studied using two assays: TBI and A-subunit protein ELISA. 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 formula:

Formula
TSHR antibodies of IgG class were measured by ELISA as previously described (3). Recombinant A-subunit protein secreted by Chinese hamster ovary cells with an amplified transgenome (16) was purified from culture supernatants by affinity chromatography (17). 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.

Assay standardization
Preimmunization samples and sera obtained after two and three injections were analyzed simultaneously for TBI, ELISA, and T4 for each CXB strain. Subsequently, sera from all CXB mice after the third immunization, as well as preimmunization sera from two mice per CXB strain, were retested in a single assay for each parameter (TBI, ELISA, or T4). Values obtained in the first assay correlated closely with those in the second assay: r = 0.89 for T4 (P = 0.016); r = 0.96 for TBI (P < 0.001); r = 0.96 for ELISA (P < 0.001).

Thyroid histology
At euthanasia, thyroids were removed from three individual mice/strain and goitrous glands were noted. The tissues were fixed in buffered formaldehyde (pH 7.4) and embedded in paraffin wax; 16–18 serial sections for each gland were stained with hematoxylin and eosin.

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

Genetic linkage analysis
Putative quantitative trait loci (QTL) involved in traits of CXB strains before and after immunization with A-subunit adenovirus were mapped using the genotype files for CXB RI strains generated by Williams et al. (11) and available at http://www.nervenet.org and embedded in GeneNetwork. This genotype file consists of 1384 markers with unique strain distribution patterns (see www.genenetwork.org/dbdoc/CXBGeno.html). The quantitative phenotypic data for each CXB strain were used without discriminating between C and B phenotypes, resulting in statistics that are essentially two tailed and conservative. The probability of linkage between our traits and previously mapped genotypes was estimated at approximately 1-cM intervals (~2 megabase; Mb) along the entire genome, except for the Y chromosome (not relevant to our study because all mice were female). To establish criteria for suggestive and significant linkage, a permutation test was performed (1000 permutations at 1-cM intervals) (18). This test compares the peak likelihood ratio statistics (LRS; LRS = LOD x 4.6) obtained for a given data set with the peak LRS score obtained for 1000 random mutations of the same data set.

All of the primary phenotype data (11 traits total) have been entered into the mouse CXB Phenotype database on GeneNetwork (www.genenetwork.org) under the trait accession identifiers 10511 through 10521 and can be found as a group by searching for the term "Graves." In Results we refer to the identification GeneNetwork (GN) numbers of specific traits so that readers can verify and extend the data analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Parental Bailey (By) strain responses to A-subunit-Ad immunization
Previously, we used Jackson (J) substrains of BALB/c, C57BL/6, and their F1 hybrids to compare responses to A-subunit-Ad (5). The recombinant inbred CXB strains were derived from the By substrains, which were separated in 1935 for BALB/c and in 1951 for C57BL/6 mice (10). Differences are likely to arise from genetic drift in separately maintained lines (for example Ref.18). Indeed, five single nucleotide polymorphism differences distinguish the Jackson and By strains of B6 mice (10). Therefore, we tested the By parental strains for their response to immunization with A-subunit adenovirus (A-subunit-Ad).

All BALB/cBy and B6By mice immunized with A-subunit-Ad, unlike control adenovirus immunized animals of the same strains, developed TSHR antibodies measured by TSH binding inhibition, TBI (Fig. 1AGo). On TSHR A-subunit ELISA, IgG-class antibodies were detected in some but not all A-subunit-Ad immunized mice (Fig. 1BGo). After two A-subunit-Ad injections, three of six BALB/cBy mice had markedly elevated T4 levels compared with mice injected with control adenovirus. At the same time point, serum T4 was slightly elevated in only one of seven B6By mice (Fig 1CGo). After the third immunization, one BALB/cBy mouse remained hyperthyroid, whereas all other animals of both strains were euthyroid or possibly hypothyroid. Overall, after A-subunit-Ad immunization, By substrains BALB/c and B6 displayed similar characteristics to those we observed in their Jackson substrain counterparts (5).


Figure 1
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  		FIG. 1. Response of parental strains BALB/cByJ and B6ByJ to immunization with A-subunit adenovirus. Mice were immunized three times with A-subunit-Ad (A-sub) or Con-Adenovirus (Con-Ad) and sera were studied 1 wk after two (2x) or a month after three (3x) injections. Data are shown for individual Con-Ad immunized mice (white; three mice/strain, pooled data for two and three injections) and for individual A-subunit-Ad immunized mice (black; data shown separately for two and three injections, six to seven mice/strain). A, TSHR antibody activity measured as TSH binding inhibition (%). Values significantly greater than for Con-Ad immunized mice: *, P < 0.05 (ANOVA). B, TSHR IgG class antibodies measured by A-subunit ELISA. Data are shown for OD values at 490 nm. Values significantly greater than for Con-Ad immunized mice: *, P < 0.05 (ANOVA). C, Serum T4 (µg/dl). The shaded area represents the mean ± 2 SD for the two strains of mice immunized with Con-Ad.

 
Antibody responses in CXB strains immunized with A-subunit-Ad
The 13 CXB RI strains were immunized with A-subunit-Ad (six mice per strain except for CXB5; only two mice available). Preimmunization blood samples were drawn to establish base-line values. As for the parental BALB/cBy and B6By strains, CXB mice received three injections of A-subunit-Ad; sera were obtained 1 wk after the second injection and at euthanasia, 4 wk after the third injection. Sera from all CXB strains were TBI positive after two and three A-subunit-Ad immunizations, whereas preimmunization TBI levels were very low or negative (Fig. 2AGo). However, compared with the other 10 strains, CXB1, CXB4, and CXB9 mice were poor TBI responders. In general, within a particular CXB strain, individual animals had similar TBI activities, as reflected by the small SE bars. In CXB12 and CXB5 strains (the latter with only two mice), TBI values were variable and the SE bars were larger.


Figure 2
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  		FIG. 2. TSHR antibodies induced by immunizing CXB RI mice with A-subunit adenovirus. Sera were studied before immunization, 1 wk after two injections and a month after three injections of A-subunit-Ad. Values for TBI or IgG class antibody in A-subunit ELISA are shown as the mean + SEM; five to six sera per strain after immunization (except CXB5, where n = 2); four preimmunization sera per strain. A, High levels of TBI activity develop in all CXB RI strains except CXB1, CXB4, and CXB9. CXB8 mice had the highest mean TBI (~87% inhibition of TSH binding); strains with significantly lower TBI after two and three immunizations: *, P < 0.05 (ANOVA). B, Antibody binding in ELISA presented as OD 490 nm. CXB10 mice had the highest ELISA OD (>3.5 after two and three immunizations); strains with significantly lower ELISA levels: *, after two injections; #, after three injections, P < 0.05 (ANOVA).

 
On A-subunit binding in ELISA, no TSHR antibodies were present in preimmunization sera but were detectable after two or three injections of A-subunit-Ad (Fig. 2BGo). In this assay, the poor responder strains were CXB3, CXB4, CXB7, CXB8, CXB11, and CXB13, of which only CXB4 mice were low TBI responders. Whereas TBI activities were similar in individual mice of the same CXB strain (Fig. 2AGo), TSHR antibodies detected by ELISA were variable within strains, as reflected by the large SE bars (Fig. 2BGo). For CXB mice, the contrast between the consistency of TBI responses vs. the variability of TSHR antibodies by ELISA (Fig. 2Go, A vs. B) resembles our observations for the parental strains (Fig. 1Go, A vs. B).

Hyperthyroidism induced in CXB mice
A minority of CXB strains became hyperthyroid in response to A-subunit-Ad immunization (Fig. 3AGo), contrasting with the induction of TSHR antibodies in all CXB strains (Fig. 2Go). CXB11 mice were strikingly different from all other strains because of their elevated T4 levels after the second and particularly after the third immunization. Unexpectedly, however, we found that preimmunization T4 levels varied markedly between strains, being high in CXB1 and CXB5 and lower in CXB7 and CXB13. Consequently, in one analysis, hyperthyroidism was assessed for each mouse strain by comparing T4 levels at base-line vs. the values after immunization. After two A-subunit-Ad injections, T4 levels were significantly elevated compared with base-line in CXB6, CXB10, and CXB11 and, after the third immunization, in CXB2, CXB8, CXB11, and CXB13 (the latter with very low maximum T4 levels).


Figure 3
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  		FIG. 3. Hyperthyroidism develops in a few CXB strains after immunization with A-subunit-Ad. A, Serum T4 levels (µg/dl) were studied before immunization, 1 wk after two injections, and a month after three injections of A-subunit-Ad. Data are the mean + SEM for five to six sera after immunization per strain for CXB1 through CXB13 (except CXB5, where n = 2) and four preimmunization sera per strain. T4 levels significantly higher than in preimmunization sera for the same strain: *, P < 0.05 (t test) or 0.01 (rank sum test); **, P < 0.008 (t test); + P = 0.07 (t test). B and C, {Delta}T4 values were calculated by subtracting the average preimmunization level for each strain from T4 values in individual mice after two (panel B) or three (panel C) injections of A-subunit-Ad. Data are the mean + SEM for five to six CXB1 through CXB13 mice (except CXB5, where n = 2). The dashed line is an arbitrary cut-off value of 2.00 to emphasize the difference between strains defined as hyperthyroid from the data in panel A, namely CXB6, CXB10, and CXB11 after two immunizations and CXB2, CXB8, CXB11, and CXB13 after three immunizations.

 
In another approach, {Delta}T4 was calculated by subtracting the mean preimmunization value for a particular CXB strain from the T4 level in individual mice of that strain after A-subunit-Ad immunization (Fig. 3Go, B and C). These values incorporate thyroid function at baseline and provide a single parameter for subsequent use in the linkage analysis. Moreover, this approach emphasizes differences between strains, with elevated serum T4 after two injections reverting to normal after the third injection in CXB6 and CXB10 vs. high T4 values being maintained or increased after the third immunization (CXB8 and particularly CXB11).

Goiter was clearly evident in some mice and occurred only in animals with elevated serum T4 levels. Because of the relatively short duration of thyroid stimulation and resolution of thyrotoxicosis in some animals, a diagnosis of goiter was not readily made. However, histologically, thyrocyte hypertrophy was more easily discerned (Fig. 4Go). Although T4 levels in CXB8 mice after three immunizations did not achieve statistical significance compared with preimmunization values (P = 0.07, Fig. 3AGo), glands from all three mice examined exhibited thyroid epithelial hypertrophy. Similarly, thyrocyte hypertrophy in a single CXB12 mouse was consistent with its elevated T4 level after three immunizations (responsible for the large SE bars in Fig. 3Go, A and C). However, thyroid histology did not support a diagnosis of hyperthyroidism in CXB13 mice, despite the statistically significant difference between serum T4 before and after three injections of A-subunit-Ad (Fig. 3AGo). This strain had the lowest T4 levels before and after immunization. Consequently, the maximum T4 level (Fig. 3BGo) appears to be an important consideration in the diagnosis of hyperthyroidism.


Figure 4
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  		FIG. 4. Thyroid histology in CXB mice immunized with A-subunit adenovirus and thyroid epithelial thickness. Representative histology of normal tissue in euthyroid mice (A) and hypertrophied thyroid tissue (B).

 
Segregation of responses in CXB mice
Strains that were low TBI responders were not low responders as measured by A-subunit antibody ELISA, except for CXB4 mice, which were poor responders for both traits. None of the low TBI responders became hyperthyroid but the magnitude of TBI did not directly predict hyperthyroidism: high TBI levels (>80%) were characteristic of mice that became hyperthyroid (CXB8) or remained euthyroid (CXB3). Moreover, the strain with the highest, persistently elevated T4 levels (CXB11) had moderately high TBI activity (~68%). There was no relationship between base-line serum T4 and development of elevated T4 levels after A-subunit-Ad immunization. However, as would be expected, sustained elevation of T4 was linked with goiter) and/or thyroid epithelial hypertrophy.

Genetic linkage analysis
QTL for TBI, ELISA, T4, and {Delta}T4 (data from Figs. 2Go, and 3Go, respectively) were mapped using the CXB RI strain database files (11). The results summarized below can be remapped using the GN data sets to generate precise LRS scores, permutation P values, and estimates of additive effects. 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.

Given the small size of the CXB RI panel, LRS values (see Materials and Methods) are usually quite modest (maximum LRS is about 60 and an LRS >25 is usually required to reach P > 0.05 genome wide). The whole genome interval mapping revealed linkage data for most parameters that are interesting but provisional (LRS values ~10–12 and LOD scores of 2.0–2.5); however, higher values (LRS >15 and LOD >3.0) were observed for TBI. The analysis highlighted the following chromosomes (Fig. 5GoGo; identifiers for the traits are provided in the legend):


Figure 5
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  		FIG. 5A. Whole genome interval mapping (Chr 1–19 and X) for traits in CXB mice before or after immunization with A-subunit-Ad. In each panel, the dashed line indicates the LRS value above which a trait is linked with a particular chromosome (asterisks). Interval mapping is shown for traits after two immunizations (2x) and/or after three immunizations (3x) for the following traits (their GeneNetwork identifiers in parentheses). A, TBI (2x) (trait 10512); a similar profile was obtained for TBI (3x) (trait 10511, not shown); A-subunit ELISA (2x) (trait 10518); T4 (2x) (trait 10514) and (3x) (trait 10520). No association were observed for ELISA (3x) (trait 10515, not shown). B, Preimmunization T4 (trait 10516); {Delta}T4 (2x) (trait 10519) and {Delta}T4 (3x) (trait 10521).

 

Figure 5
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  		FIG. 5B. Continued

 
1) TBI after two immunizations (2x) with Chr 17 and Chr X; similar data were observed for TBI after three immunizations (3x, not shown) (Fig. 5AGo).

2) ELISA for TSHR antibody (2x) with Chr X (Fig. 5AGo).

3) Serum T4 (2x) and T4 (3x) with Chr 19 (Fig. 5AGo).

4) Preimmunization T4 levels were linked with Chr 13 (Fig. 5BGo).

5) {Delta}T4 (3x) with Chr 10 (and a secondary locus on Chr 7) (Fig. 5BGo).

6) Borderline linkage was observed for {Delta}T4 (2x) and Chr 3 (Fig. 5BGo) as well as for T4 (3x) and Chr 10 (Fig. 5AGo). No linkage was observed for ELISA (3x) (data not shown).

At the level of individual chromosomes, interval mapping for TBI after two immunizations (2x) showed two discrete LRS peaks on Chr 17 (~35 Mb and ~45 Mb) and a single Chr X peak (~75 Mb) for the same trait (Fig. 6AGo; upper two panels). The same loci on Chr 17 and X were linked with TBI after three immunizations with similar LRS values (data not shown). Different Chr X markers (~35 Mb) were linked with TSHR antibody ELISA (2x) (Fig. 6AGo, middle panel). For serum T4, the LRS peaked on Chr 19 at approximately 44 Mb after two immunizations and approximately 23 Mb after three immunizations; however, both interval maps showed similar profiles, with a shift in the location of the dominant peak after two or three immunizations (Fig. 6AGo, lower panels).


Figure 6
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  		FIG. 6A. Analysis of individual chromosomes linked with particular traits in CXB mice immunized with A-subunit-Ad. In each panel, the solid trace is the LRS value for the trait indicated to the right. The faint trace indicates the extent of enhancement (magnitude on the right) by the parental gene labeled [c] or [b] for BALB/c or B6. Lower horizontal dashed line, LRS value suggestive of association. Upper horizontal dashed line, LRS value required for highly significant linkage. Numbers indicate the location (Mb) of chromosomal markers; the approximate size of each chromosome is provided in parentheses. A, Chr 17 and TBI (2x) (trait 10512); Chr X and TBI (2x) (trait 10512); Chr X and ELISA (2x) (trait 10518); Chr 19 and T4 (2x) (trait 10514); Chr 19 and T4 (3x) (trait 10520). B, Chr 13 and pre-T4 (trait 10516); Chr 10 and {Delta}T4 (3x) (trait 10521); Chr 10 and T4 (3x) (trait 10520); Chr 7 and {Delta}T4 (3x) (trait 10521).

 
Preimmunization T4 was linked with loci on Chr 13 (~99 Mb) and {Delta}T4 after three immunizations (3x) with loci on Chr 10 (~114 Mb). The markers for both of these traits are located at the distal end of their respective chromosomes (Fig. 6BGo, upper two panels). Intriguingly, a secondary locus for T4 (3x) was also on the distal portion of Chr 10 in almost the same location (~114 Mb) (Fig. 6BGo, third panel). {Delta}T4 after three immunizations (3x), a complex trait, was also linked with Chr 7 (~136 Mb) (Fig. 6BGo, fourth panel).


Figure 6
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  		FIG. 6B. Continued

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used recombinant inbred CXB mice to investigate the genetic basis for TSHR antibodies and hyperthyroidism induced by immunization with TSHR A-subunit adenovirus (A-subunit-Ad). High-resolution genetic maps are available for CXB mice (11) that were derived from the F1 of (BALB/c x B6) by repeated brother x sister matings for at least 20 generations (9). The rationale for using CXB strains was based on observations that BALB/c and BALB/c x B6 F1 offspring are susceptible, but B6 mice are resistant, to hyperthyroidism induced by TSHR- or A-subunit-adenovirus (2, 5). In our present study, linkage analysis suggests that induction of TSHR antibodies and hyperthyroidism are regulated by different genes, each trait involving several chromosomes.

In previous studies of BALB/c, B6 and their F1 offspring, we used Jackson parental substrains (5), whereas CXB recombinant inbred mice were derived from the By substrains, bred separately from their Jackson counterparts for over 50 yr (10). We, therefore, tested (and confirmed) the different susceptibilities of the two substrains to A-subunit-Ad-induced hyperthyroidism. This difference was initially described for Charles Rivers Laboratories (N) substrains (2). Consequently, the susceptibility-resistance pattern is common to all three substrains. However, unlike uniformly high values induced in Jackson substrains (5), TBI and A-subunit ELISA (particularly the latter) were more variable in By substrains.

In assessing the responses of CXB mice to A-subunit-Ad immunization, defining a precise, quantitative phenotype was essential for our genetic studies. We, therefore, focused on assays with the greatest precision, reproducibility, and sensitivity, namely TSHR receptor antibodies measured by TBI and ELISA, as well as serum T4. As explained in Materials and Methods, we also repeated the TBI, T4, and ELISA to clarify these traits, and insufficient serum was available to study thyroid-stimulating antibodies (TSAb) or thyroid-blocking antibodies (TBAb), assays with less precision and reproducibility. Moreover, although elevated T4 is often associated with TSAb levels (for example 2), nevertheless striking discordances between these parameters have been reported (high TSAb, no TBAb and euthyroidism) (19). In our view, the most sensitive functional antibody read-out of hyperthyroidism is the serum T4 that measures the net effect of TSAb vs. TBAb on the mouse’s own thyroid.

The responses to A-subunit-Ad immunization in CXB mice were complex. Low TBI responder strains were, in most cases, not low antibody responders on A-subunit ELISA. The TBI assay (which measures antibodies to the native TSHR) is specific for TSHR antibodies in human Graves’ sera, whereas the latter assay does not distinguish between Graves’ and control sera. TBI values were high in all strains that became hyperthyroid. However not all high TBI strains developed hyperthyroidism. Preimmunization serum T4 levels were variable in different CXB strains and required consideration in diagnosing hyperthyroidism in a particular strain. Thus, in addition to postimmunization serum T4 levels, we used {Delta}T4 values to provide a single parameter incorporating thyroid function at baseline and after immunization. Linkage analysis indicated that different chromosomes, or loci on the same chromosome, are involved in TSHR antibody induction, as well as in pre- and postimmunization serum T4 levels. We discuss these CXB loci in relation to the candidate genes or loci revealed by linkage analysis in human disease (reviewed in Ref.20).

In CXB mice, TBI was linked with two marker sets on Chr 17 (Table 1Go), one of which is in the vicinity of MHC genes. Experimentally induced T cell-mediated murine thyroiditis is controlled by MHC (reviewed in Ref.21). Moreover, MHC (HLA in humans, Chr 6) has long been associated with Graves’ disease (for example Refs.22 and 23). However, the relative risks are low, and the lack of linkage (24) suggests that HLA is not a major gene in Graves’ disease. We previously hypothesized (25) that HLA associations in hyperthyroid Graves’ disease (26) and hypothyroidism due to TSHR-blocking antibodies (27) reflected the underlying autoimmune process (whether humoral or cell-mediated) rather than overt clinical disease. Our findings in CXB mice support a role for MHC in TBI generation. However, a second Chr 17 locus is also involved. Of interest, a susceptibility gene for both Graves’ and Hashimoto’s diseases mapped close to, but distinct from, the HLA region (28).


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  		TABLE 1. Chromosomes revealed by linkage analysis for CXB strains immunized with A-subunit adenovirus

 
Both TBI activity and TSHR antibodies measured by ELISA were linked with the X chromosome but involved different loci (Table 1Go). For ELISA, the markers are in the vicinity of the gene for lysosome-associated membrane protein 2 (Lamp2) (29) (human Chr X) and programmed cell death domain 8 protein (Pdcd8) (30) (human Chr Xq25-q26), which could be involved in antigen processing or antibody regulation, respectively. Some associations with the X chromosome (albeit at different loci) have been reported for human Graves’ disease (28, 31, 32) but not confirmed in other data sets (33, 34). The X chromosome markers in CXB mice linked with ELISA are in the vicinity of Xq21-q22 reported for human Graves’ disease in one study (31). However, as mentioned above, the relevance of antibodies detected by A-subunit ELISA in human disease and in CXB mice is unclear.

Unlike TSHR antibodies, hyperthyroidism in CXB mice did not involve MHC genes, consistent with data for other mouse strains (2, 6). Non-MHC candidate genes in human Graves’ disease include CTLA4 (35), CD40 (36), PTN22/LYP (37), the TSHR (38, 39), thyroglobulin (20, 40), FCRL3 (a B cell molecule), and MAP3K7IP2 (involved in nuclear factor-{kappa}B binding) (41). We obtained no evidence that any of these candidate genes are involved in the response of CXB mice to A-subunit-Ad immunization. The TSHR gene is unlikely to play a role in this model because the immunogen is the human TSHR A-subunit. Moreover, thyroglobulin could not be involved because the parental B6 and BALB/c mice have the same exon 10 thyroglobulin haplotype associated with resistance to thyroiditis induced with thyroglobulin and adjuvant (40).

Preimmunization serum T4 levels varied widely in CXB strains, a trait linked with Chr 3. Thyroid function has previously been investigated in several mouse strains. In particular, neonatal T4 levels were almost twice as high in DBA/2J as in BL6 mice (42). The genetic basis for this trait was studied in six recombinant inbred BXD strains and their parents (GeneNetwork identifiers BXD 10602 and 10603): serum T4 levels were linked with murine Chr 5 and estimated free T4 with Chr 13, 15, and 16. Although Chr 13 was linked with free T4 in BXD mice and total T4 in CXB mice (present study), the loci were not the same.

Postimmunization serum T4 was linked with Chr 19, with a shift in the marker profile from approximately 45 Mb after two A-subunit-Ad injections to approximately 23 Mb after three injections (Fig. 6AGo and Table 1Go). No obvious candidate genes lie in the vicinity of these markers. The increase ({Delta}) in T4 levels after three immunizations was linked with Chr 10 and a similar locus was a secondary marker for serum T4 at the same time interval (Table 1Go). These loci are close to the Trdhe gene (Chr 19; 114.056 Mb; human Chr 12q15-q21), which encodes an enzyme that degrades TSH-releasing hormone (43). Expression of TSH-releasing hormone-degrading enzyme is stringently controlled by thyroid hormones (44). Whether linkage with the Trdhe gene is a consequence, rather than a cause, of thyroid hormone changes in CXB mice, is unknown, but the suggested linkage with a thyroid function gene is intriguing.

In conclusion, we observed that induction in mice of TSHR antibodies with TBI activity is linked with MHC genes, at least in part, whereas hyperthyroidism involves non-MHC genes. Our data, the first genome scan in a murine model of Graves’ disease, provide insight into the role of MHC and non-MHC genes in this model, and perhaps also in human Graves’ disease. Human populations are heterogeneous and disease outcome is influenced by the environment. In contrast, recombinant inbred mice are more homogeneous and can be studied under pathogen-free conditions. Consequently, our study demonstrates the potential of recombinant inbred mice for discriminating between immune-response genes and thyroid function susceptibility genes in Graves’ disease.


   Footnotes
 
This work was supported by the National Institutes of Health Grants DK54684 (to S.M.M.), DK19289 (to B.R.), U01AA13499 and P20-DA-21131 (to R.W.W.) and a Winnick Family Clinical Research Scholar Award (to S.M.M.). R.W.W. and GeneNetwork are supported by Integrative Neuroscience Initiative on Alcoholism-National Institute on Alcohol Abuse and Alcoholism (INIA-NIAAA); Biomedical Informatics Research Network-National Center for Research Resources (BIRN-NCRR); and Mouse Models of Human Cancers Consortium-National Cancer Institute (MMHCC-NCI) and a Human Brain Project. We are also grateful for contributions by Dr. Boris Catz, Los Angeles.

Disclosure of Potential Conflicts of Interest: H.A.A., P.N.P., C.-R.C., R.W.W., B.R. and S.M.M. have nothing to declare.

First Published Online March 16, 2006

Abbreviations: By, Bailey; Chr, chromosome; F1 or F2, filial 1 or 2; GN, GeneNetwork; LRS, peak likelihood ratio statistics; MHC, major histocompatibility; QTL, quantitative trait loci; RI, recombinant inbred; TBAb, thyroid-blocking antibodies; TBI, inhibition of TSH binding; TSAb, thyroid-stimulating antibodies; TSHR, TSH receptor.

Received February 8, 2006.

Accepted for publication March 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McLachlan SM, Nagayama Y, Rapoport B 2005 Insight into Graves’ hyperthyroidism from animal models. Endocr Rev 26:800–832[Abstract/Free Full Text]
  2. Nagayama Y, Kita-Furuyama M, Ando T, Nakao K, Mizuguchi H, Hayakawa T, Eguchi K, Niwa M 2002 A novel murine model of Graves’ hyperthyroidism with intramuscular injection of adenovirus expressing the thyrotropin receptor. J Immunol 168:2789–2794[Abstract/Free Full Text]
  3. Chen C-R, Pichurin P, Nagayama Y, Latrofa F, Rapoport B, McLachlan SM 2003 The thyrotropin receptor autoantigen in Graves’ disease is the culprit as well as the victim. JClin Invest 111:1897–1904[CrossRef][Medline]
  4. Chen C-R, Pichurin P, Chazenbalk GD, Aliesky H, Nagayama Y, McLachlan SM, Rapoport B 2004 Low-dose immunization with adenovirus expressing the thyroid-stimulating hormone receptor A-subunit deviates the antibody response toward that of autoantibodies in human Graves’ disease. Endocrinology 145:228–233[Abstract/Free Full Text]
  5. Chen CR, Aliesky H, Pichurin PN, Nagayama Y, McLachlan SM, Rapoport B 2004 Susceptibility rather than resistance to hyperthyroidism is dominant in a thyrotropin receptor adenovirus-induced animal model of Graves’ disease as revealed by BALB/c-C57BL/6 hybrid mice. Endocrinology 145:4927–4933[Abstract/Free Full Text]
  6. Nagayama Y, McLachlan SM, Rapoport B, Niwa M 2003 A major role for non-MHC genes, but not for micro-organisms, in a novel model of Graves’ hyperthyroidism. Thyroid 13:233–238[CrossRef][Medline]
  7. Shimojo N, Kohno Y, Yamaguchi K-I, Kikuoka S-I, Hoshioka A, Niimi H, Hirai A, Tamura Y, Saito Y, Kohn LD, Tahara K 1996 Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin repector and a class II molecule. Proc Natl Acad Sci USA 93:11074–11079[Abstract/Free Full Text]
  8. Yamaguchi K-I, Shimojo N, Kikuoka S, Hoshioka A, Hirai A, Tahara K, Kohn LD, Kohno Y, Niimi H 1997 Genetic control of anti-thyrotropin receptor antibody generation in H-2k mice immunized with thyrotropin receptor-transfected fibroblasts. J Clin Endocrinol Metab 82:4266–4269[Abstract/Free Full Text]
  9. Bailey DW 1971 Recombinant-inbred strains. An aid to finding identity, linkage, and function of histocompatibility and other genes. Transplantation 11:325–327[Medline]
  10. 1997 Handbook on genetically standardized Jax mice. 5th ed. Bar Harbor, ME: The Jackson Laboratory
  11. Williams RW, Gu L, Qi S, Lu L 2001 The genetic structure of recombinant inbred mice: high-resolution consensus maps for complex trait analysis. Genome Biol 2:Research0046.1–0046.18
  12. Airey DC, Lu L, Williams RW 2001 Genetic control of the mouse cerebellum: identification of quantitative trait loci modulating size and architecture. J Neurosci 21:5099–5109[Abstract/Free Full Text]
  13. Scalzo AA, Fitzgerald NA, Wallace CR, Gibbons AE, Smart YC, Burton RC, Shellam GR 1992 The effect of the Cmv-1 resistance gene, which is linked to the natural killer cell gene complex, is mediated by natural killer cells. J Immunol 149:581–589[Abstract]
  14. Mountz JD, Yang P, Wu Q, Zhou J, Tousson A, Fitzgerald A, Allen J, Wang X, Cartner S, Grizzle WE, Yi N, Lu L, Williams RW, Hsu HC 2005 Genetic segregation of spontaneous erosive arthritis and generalized autoimmune disease in the BXD2 recombinant inbred strain of mice. Scand J Immunol 61:128–138[CrossRef][Medline]
  15. Mittereder N, March KL, Trapnell BC 1996 Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol 70:7498–7509[Abstract]
  16. Chazenbalk GD, Jaume JC, McLachlan SM, Rapoport B 1997 Engineering the human thyrotropin receptor ectodomain from a non-secreted form to a secreted, highly immunoreactive glycoprotein that neutralizes autoantibodies in Graves’ patients’ sera. J Biol Chem 272:18959–18965[Abstract/Free Full Text]
  17. Chazenbalk GD, Wang Y, Guo J, Hutchison JS, Segal D, Jaume JC, McLachlan SM, Rapoport B 1999 A mouse monoclonal antibody to a thyrotropin receptor ectodomain variant provides insight into the exquisite antigenic conformational requirement, epitopes and in vivo concentration of human autoantibodies. J Clin Endocrinol Metab 84:702–710[Abstract/Free Full Text]
  18. Bailey DW 1982 How pure are inbred strains of mice? Immunol Today 3:210–214[CrossRef]
  19. Muehlberg T, Gilbert JA, Rao PV, McGregor AM, Banga JP 2004 Dynamics of thyroid-stimulating and -blocking antibodies to the thyrotropin receptor in a murine model of Graves’ disease. Endocrinology 145:1539–1545[Abstract/Free Full Text]
  20. Ban Y, Tomer Y 2005 Susceptibility genes in thyroid autoimmunity. Clin Dev Immunol 12:47–58[CrossRef][Medline]
  21. Kong YM 2004 Experimental models for autoimmune thyroid disease: recent developments. In: Volpe R, ed. Contemporary endocrinology: autoimmune endocrinopathies. Totawa, NJ: Humana Press; 91–111
  22. Farid NR, Stone E, Johnson G 1980 Graves’ disease and HLA: clinical and epidemiologic associations. Clin Endocrinol 15:535–544
  23. Simmonds MJ, Howson JM, Heward JM, Cordell HJ, Foxall H, Carr-Smith J, Gibson SM, Walker N, Tomer Y, Franklyn JA, Todd JA, Gough SC 2005 Regression mapping of association between the human leukocyte antigen region and Graves disease. Am J Hum Genet 76:157–163[CrossRef][Medline]
  24. Roman SH, Greenberg D, Rubinstein P, Wallenstein S, Davies TF 1992 Genetics of autoimmune thyroid disease: lack of evidence for linkage to HLA within families. J Clin Endocrinol Metab 74:496–503[Abstract]
  25. McLachlan SM 1993 The genetic basis of autoimmune thyroid disease: time to focus on chromosomal loci other than the major histocompatibility complex (HLA in man). J Clin Endocrinol Metab 77:605A–605C[CrossRef]
  26. Inoue D, Sato K, Sugawa H, Akamizu T, Maeda M, Inoko H, Tsuji K, Mori T 1993 Apparent genetic difference between hypothyroid patients with blocking-type thyrotropin receptor antibody and those without, as shown by restriction fragment length polymorphism analyses of HLA-DP loci. J Clin Endocrinol Metab 77:606–610[Abstract]
  27. Cho BY, Chung JH, Shong YK, Chang YB, Han H, Lee J-B, Lee HK, Koh C-S 1993 A strong association between thyrotropin receptor-blocking antibody-positive atrophic autoimmune thyroiditis and HLA-DR8 and HLA-DQB1*0302 in Koreans. J Clin Endocrinol Metab 77:611–615[Abstract]
  28. Tomer Y, Barbesino G, Greenberg DA, Concepcion E, Davies TF 1999 Mapping the major susceptibility loci for familial Graves’ and Hashimoto’s diseases: evidence for genetic heterogeneity and gene interactions. J Clin Endocrinol Metab 84:4656–4664[Abstract/Free Full Text]
  29. Cha Y, Holland SM, August JT 1990 The cDNA sequence of mouse LAMP-2. Evidence for two classes of lysosomal membrane glycoproteins. J Biol Chem 265:5008–5013[Abstract/Free Full Text]
  30. Daugas E, Nochy D, Ravagnan L, Loeffler M, Susin SA, Zamzami N, Kroemer G 2000 Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett 476:118–123[CrossRef][Medline]
  31. Barbesino G, Tomer Y, Concepcion E, Davies TF, Greenberg D 1998 Linkage analysis of candidate genes in autoimmune thyroid disease: II. Selected gender-related genes and the X-chromosome. J Clin Endocrinol Metab 83:3290–3295[Abstract/Free Full Text]
  32. Imrie H, Vaidya B, Perros P, Kelly WF, Toft AD, Young ET, Kendall-Taylor P, Pearce SH 2001 Evidence for a Graves’ disease susceptibility locus at chromosome Xp11 in a United Kingdom population. J Clin Endocrinol Metab 86:626–630[Abstract/Free Full Text]
  33. Tomer Y, Ban Y, Concepcion E, Barbesino G, Villanueva R, Greenberg DA, Davies TF 2003 Common and unique susceptibility loci in Graves and Hashimoto diseases: results of whole-genome screening in a data set of 102 multiplex families. Am J Hum Genet 73:736–747[CrossRef][Medline]
  34. Taylor JC, Gough SC, Hunt PJ, Brix TH, Chatterjee K, Connell JM, Franklyn JA, Hegedus L, Robinson BG, Wiersinga WM, Wass JA, Zabaneh D, Mackay I, Weetman AP 2006 A genome-wide screen in 1119 relative pairs with autoimmune thyroid disease. J Clin Endocrinol Metab 91:646–653[Abstract/Free Full Text]
  35. Yanagawa T, Hidaka Y, Guimaraes V, Soliman M, DeGroot LJ 1995 CTLA-4 gene polymorphism associated with Graves’ disease in a Caucasian population. J Clin Endocrinol Metab 80:41–45[Abstract]
  36. Tomer Y, Greenberg DA, Concepcion E, Davies TF 2002 A single C/T nucleotide polymorphism in the promoter region of the CD40 gene confers susceptibility to Graves’ disease. Thyroid 12:1129–1135[CrossRef][Medline]
  37. Velaga MR, Wilson V, Jennings CE, Owen CJ, Herington S, Donaldson PT, Ball SG, James RA, Quinton R, Perros P, Pearce SH 2004 The codon 620 tryptophan allele of the lymphoid tyrosine phosphatase (LYP) gene is a major determinant of Graves’ disease. J Clin Endocrinol Metab 89:5862–5865[Abstract/Free Full Text]
  38. Hiratani H, Bowden DW, Ikegami S, Shirasawa S, Shimizu A, Iwatani Y, Akamizu T 2005 Multiple SNPs in intron 7 of thyrotropin receptor are associated with Graves’ disease. J Clin Endocrinol Metab 90:2898–2903[Abstract/Free Full Text]
  39. Dechairo BM, Zabaneh D, Collins J, Brand O, Dawson GJ, Green AP, Mackay I, Franklyn JA, Connell JM, Wass JA, Wiersinga WM, Hegedus L, Brix T, Robinson BG, Hunt PJ, Weetman AP, Carey AH, Gough SC 2005 Association of the TSHR gene with Graves’ disease: the first disease specific locus. Eur J Hum Genet 13:1223–1230[CrossRef][Medline]
  40. Ban Y, Greenberg DA, Concepcion E, Skrabanek L, Villanueva R, Tomer Y 2003 Amino acid substitutions in the thyroglobulin gene are associated with susceptibility to human and murine autoimmune thyroid disease. Proc Natl Acad Sci USA 100:15119–15124[Abstract/Free Full Text]
  41. Simmonds MJ, Heward JM, Carr-Smith J, Foxall H, Franklyn JA, Gough SC 2006 Contribution of single nucleotide polymorphisms within FCRL3 and MAP3K7IP2 to the pathogenesis of Graves’ disease. J Clin Endocrinol Metab 91:1056–1061[Abstract/Free Full Text]
  42. Seyfried TN, Glaser GH, Yu RK 1984 Genetic analysis of serum thyroxine content and audiogenic seizures in recombinant inbred and congenic strains of mice. Exp Neurol 83:423–428[Medline]
  43. Schauder B, Schomburg L, Kohrle J, Bauer K 1994 Cloning of a cDNA encoding an ectoenzyme that degrades thyrotropin-releasing hormone. Proc Natl Acad Sci USA 91:9534–9538[Abstract/Free Full Text]
  44. Schomburg L, Bauer K 1995 Thyroid hormones rapidly and stringently regulate the messenger RNA levels of the thyrotropin-releasing hormone (TRH) receptor and the TRH-degrading ectoenzyme. Endocrinology 136:3480–3485[Abstract]



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