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T F Davies

Publications and source records attributed to T F Davies.

At least 55 records · Page 3Linked to original sources
PubMed1999

Mapping the major susceptibility loci for familial Graves' and Hashimoto's diseases: evidence for genetic heterogeneity and gene interactions.

The autoimmune thyroid diseases (AITDs), comprising Graves' disease (GD) and Hashimoto's thyroiditis (HT), appear to develop as a result of a complex interaction between predisposing genes and environmental triggers. The goals of the present study were to identify the susceptibility loci for GD and HT and to study the relationships between them. We performed a whole genome linkage study on a dataset of 56 multiplex, multigenerational AITD families (354 individuals), using 387 microsatellite markers. We identified 6 loci that showed evidence for linkage to AITD. Only one locus, on chromosome 6 [AITD-1; 80 centimorgans (cM)], was linked with both GD and HT [maximum LOD score (MLS), 2.9]. This locus was close to, but distinct from, the human leukocyte antigen region. One locus on chromosome 13 (HT-1; 96 cM) was linked to HT (MLS, 2.1), and another locus on chromosome 12 (HT-2; 97 cM) was linked to HT in a subgroup of the families (MLS, 3.8). Three loci showed evidence for linkage with GD: GD-1 on chromosome 14 (99 cM; MLS, 2.5), GD-2 on chromosome 20 (56 cM; MLS, 3.5), and GD-3 on chromosome X (114 cM; MLS, 2.5). Since GD-2 showed the strongest evidence for linkage to GD we fine-mapped this locus to a 1-cM interval between markers at 55 and 56 cM on chromosome 20. These results demonstrated that 1) Graves' and Hashimoto's diseases are genetically heterogeneous, with only one locus in common to both diseases on chromosome 6; 2) only one HT locus was identified in all families, probably due to heterogeneity of the HT phenotype; and 3) three loci were shown to induce genetic susceptibility to GD by interacting with each other. One of them (GD-2) was fine-mapped to a 1-cM interval.

Chromosome Mapping↗
PubMed1999

Post-translational processing of the natural human thyrotropin receptor: demonstration of more than two cleavage sites.

Epitope-mapped monoclonal and polyclonal antibodies to the TSH receptor (TSHR) were used as immunoblot probes to detect and characterize the molecular species of the receptor present in normal human thyroid tissue. In reduced membrane fractions, both full-length (uncleaved) holoreceptor and cleavage-derived subunits of the holoreceptor were detected. Uncleaved holoreceptor species included a nonglycosylated form of apparent molecular mass 85 kDa and two glycosylated forms of approximately 110 and 120 kDa. The membranes also contained several forms of cleavage-derived TSHR-alpha and TSHR-beta subunits. TSHR-alpha subunits were detected by antibodies to epitopes localized within the amino terminal end of the TSHR ectodomain and migrated diffusely between 45-55 kDa, reflecting a differentially glycosylated status. TSHR-beta subunits were detected by antibodies to epitopes within the carboxyl end of the TSHR ectodomain. Several species of TSHR-beta subunit were present, the most abundant having apparent molecular masses of 50, 40, and 30 kDa. These data demonstrated that post-translational processing of the TSHR in human thyroid tissue involved multiple cleavage sites.

Animals↗
PubMed1999

T-cell receptors and autoimmune thyroid disease--signposts for T-cell-antigen driven diseases.

The human autoimmune thyroid diseases (AITDs) are characterized by profuse infiltrates of both CD4+ and CD8+ T cells. The intrathyroidal T-cell-receptor repertoire in Graves' disease, more than in Hashimoto's disease, has been shown to be biased as evidenced by phenotypic analysis and by the use of a restricted T-cell-receptor variable (V) gene repertoire seen in both TCR alpha and beta chains. Evidence for a bias in the T-cell repertoire has also been observed in animal models of induced and spontaneous autoimmune thyroiditis. We found a similar phenomenon of autoimmune thyroid-related T-cell bias in thyroid-humanized scid mice. In these studies we transplanted lymphocyte-depleted thyrocytes and autologous peripheral lymphocytes from AITD patients with a basement membrane preparation which allowed the formation of an artificial thyroid which we have called an "organoid". T-cell clonal expansion was present in these artificial mixed-cell organoids which appeared to mimic the in vivo process. Such clonal expansion was suggestive of an antigen-driven immune response and could also be identified in thyroid tissue from patients with Graves' disease. Our data on scid mice grafted with human mixed-cell thyroid organoids, therefore, suggested that the major antigens driving T-cell selection in patients with AITD were most likely to be thyroid specific. These antigens include thyroglobulin, thyroid peroxidase, and the receptor for thyroid stimulating hormone (TSHR) on the surface of thyroid epithelial cells and we found significant T-cell proliferation to synthetic TSHR peptides in patients with AITD as compared with normals. Our search for a TCR recognition motif for the autoantigen TPO did not reveal any specific sequence motifs. Instead, analysis of the physico-chemical characteristics i.e. hydrophobicity of the amino acids in the CDR3 (N) region of the TCR alpha chain, revealed a strong negative linear correlation between strength of stimulation and the average hydrophobicity of N-region amino acids. This led us to hypothesize that lower affinity T-cell clones were commonly more hydrophobic in their CDR3 alpha region amino acids in keeping with potential crossreactivity of such T cells as a consequence of promiscuous, hydrophobic CDR3 regions. This phenomenon would be analogous to polyreactive, natural autoantibodies which tend to be crossreactive and 'sticky'. Thus, the physico-chemical characteristics of the TCR alpha CDR3 region supported the interaction with antigen/MHC by potentially cross-reactive T cells of low affinity. It would seem likely that such low-affinity autoreactive T-cell populations serve as a pool of potentially pathogenetic cells. These cells would be able to respond to an insult which, via a number of possible mechanisms such as molecular mimicry, would initiate a thyroid lymphocytic infiltration in an antigen-driven fashion with intrathyroidal T-cell expansion and a marked bias in the utilization of T-cell-receptor V genes.

Animals↗
PubMed1998

Characterization of the murine immune response to the murine TSH receptor ectodomain: induction of hypothyroidism and TSH receptor antibodies.

The thyrotropin receptor (TSHR) is the major autoantigen of human Graves' disease. In order to define the antigenicity of the TSHR in a defined model, we examined the immune response of BALB/c mice to immunization with a new bioactive, recombinant preparation of the ectodomain of the murine TSHR (mTSHR-ecd). Mice (n = 10) were immunized with 25-50 microg of insect cell expressed, purified and refolded, mTSHR-ecd in alum adjuvant containing pertussis toxin, on days 0, 21, 36, 50 and 70. Control mice received wild-type baculovirus-infected insect cell protein lysate, in a similar way. After 28 days, murine serum contained high titres of antibodies specific to mTSH-ecd and their titres continued to increase over 90 days. Antibody epitope mapping, using 26 peptides spanning the human TSHR-ecd, showed that a variety of regions of the ectodomain were antigenic. The earliest epitope included aa 22-41, but later two regions of reactivity were noted clustered towards the mid portion and carboxyl terminus of the ectodomain. The murine TSHR autoantibodies (TSHR-Abs) inhibited up to 78% of the binding of labelled TSH to native TSHR, demonstrating the presence of antibodies capable of blocking the native TSHR. We showed that these TSHR antibodies acted, in vitro, as TSH blocking antibodies, inhibiting TSH-induced generation of cyclic AMP in chinese hamster ovary (CHO) cells transfected with the hTSHR. Hence, the antibody response to mTSHR-ecd was potentially antagonistic in its influence on the TSHR. Assessment of thyroid function in the immunized mice showed a fall in serum total T3 by 90 days and markedly elevated murine TSH levels (from 64.0 to 239.6 ng/ml), confirming the onset of thyroid failure. However, thyroid histology remained grossly normal. These data demonstrate that mTSHR-ecd is a potent antigen with three major immunogenic regions. The induced mTSHR-Abs blocked TSH action in vivo and reduced murine thyroid function.

Animals↗
PubMed1998

A new Graves disease-susceptibility locus maps to chromosome 20q11.2. International Consortium for the Genetics of Autoimmune Thyroid Disease.

The autoimmune thyroid diseases (AITDs) include two related disorders, Graves disease (GD) and Hashimoto thyroiditis, in which perturbations of immune regulation result in an immune attack on the thyroid gland. The AITDs are multifactorial and develop in genetically susceptible individuals. However, the genes responsible for this susceptibility remain unknown. Recently, we initiated a whole-genome linkage study of patients with AITD, in order to identify their susceptibility genes. We studied a data set of 53 multiplex, multigenerational AITD families (323 individuals), using highly polymorphic and densely spaced microsatellite markers (intermarker distance <10 cM). Linkage analysis was performed by use of two-point and multipoint parametric methods (classic LOD-score analysis). While studying chromosome 20, we found a locus on chromosome 20q11.2 that was strongly linked to GD. A maximum two-point LOD score of 3.2 was obtained at marker D20S195, assuming a recessive mode of inheritance and a penetrance of.3. The maximum nonparametric LOD score was 2.4 (P=.00043); this score also was obtained at marker D20S195. Multipoint linkage analysis yielded a maximum LOD score of 3.5 for a 6-cM interval between markers D20S195 and D20S107. There was no evidence for heterogeneity in our sample. In our view, these results indicate strong evidence for linkage and suggest the presence of a major GD-susceptibility gene on chromosome 20q11.2.

Autoantibodies↗
PubMed1998

Development and characterization of monoclonal antibodies specific for the murine thyrotropin receptor.

We have characterized 10 monoclonal antibodies (Mabs) to recombinant murine thyrotropin receptor extracellular domain (mTSHR-ecd). Affinity purified mTSHR-ecd (amino acids 22-415), expressed in a baculovirus-insect cell system, was refolded in vitro and used to hyperimmunize female Balb/c mice. Spleens were removed 10 days after a final boost of 25 microg mTSHR-ecd intraperitoneally and intravenously, and the cells were fused to SP-2 cells and cloned. Hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) with folded mTSHR-ecd antigen. Ten of 18 higher affinity hybridomas were selected at random and ascites fluids prepared. Nine of the monoclonals were of IgG 1 isotype, and one was IgM. Five Mabs (M3, M4, M5, M6, and M9) inhibited the binding of 125I-TSH to functional hTSHR expressed on Chinese hamster ovary (CHO) cells, and four (M1, M3, M5, and M9) blocked the TSH-stimulated generation of cyclic adenosine monophosphate (cAMP), using the same cells. The remaining Mabs appeared to be neutral in their interaction with native TSHR. The Mabs were also compared for their reactivity to mTSHR-ecd under folding (ELISA) and unfolding (reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) conditions. Most Mabs demonstrated reactivity to both conformational (folded) and linear (unfolded) forms of mTSHR-ecd, suggesting that they were generated primarily against linear epitopes although one Mab (M4) showed affinity for only folded antigen indicating a preference for a conformational epitope. Mapping the Mab epitopes using 26 overlapping peptides spanning the human (h)TSHR-ecd showed that 6 bound peptide 397-415, 1 bound peptide 352-371, and 1 peptide 22-41. These epitope mapped Mabs to the mTSHR-ecd, both receptor blocking and receptor neutral, will provide further insight into the structure-function of the TSHR ectodomain.

Animals↗
PubMed1998

Linkage analysis of candidate genes in autoimmune thyroid disease. III. Detailed analysis of chromosome 14 localizes Graves' disease-1 (GD-1) close to multinodular goiter-1 (MNG-1). International Consortium for the Genetics of Autoimmune Thyroid Disease.

The autoimmune thyroid diseases [Graves' and Hashimoto's diseases (GD and HT)] develop in genetically susceptible individuals, but the genes responsible for this susceptibility remain unknown. To identify such genes, we have been testing candidate genes and chromosomal regions using highly polymorphic microsatellite markers. We recently reported evidence for the first locus linked to GD (GD-1) on chromosome 14q31 in a small group of families. We have now extended these studies and analyzed 53 multiplex families with GD and/or HT (323 individuals). Chromosome 14 was screened using 16 microsatellite markers spanning the entire chromosome. Three additional markers located inside candidate genes on chromosome 14 were also studied. Microsatellite markers were amplified using fluorescent-labeled primers and separated on an ABI-310 genetic analyzer. The data were analyzed using LIPED software for two-point logarithm of odds (LOD) score analysis and GeneHunter software for multipoint linkage analysis. No linkage of any marker was found to HT or autoimmune thyroid diseases (GD+HT). The previously identified GD-1 locus on 14q31 continued to show evidence of linkage to GD in this much larger set of families. The maximum LOD score was 2.1 obtained for marker D14S81 (theta=0.01), assuming a recessive mode of inheritance and a penetrance of 0.3. Multipoint analysis yielded a maximum LOD score of 2.5 between markers D14S81 and D14S1054. There was no evidence for heterogeneity in our sample. These data again suggest the presence of a major Graves' disease susceptibilitygene (GD-1) on chromosome 14q31. This locus is close to the recently identified multinodular goiter-1 locus.

Autoimmune Diseases↗
PubMed1998

Linkage analysis of candidate genes in autoimmune thyroid disease: 1. Selected immunoregulatory genes. International Consortium for the Genetics of Autoimmune Thyroid Disease.

Graves' and Hashimoto's diseases are autoimmune thyroid diseases in which the genetic contribution is complex. For this reason, identification of necessary susceptibility genes has been difficult. However, a number of immunoregulatory genes have been implicated by association studies, including: CTLA-4, a recently described protein involved in antigen presentation, located on chromosome 2q33; the T-cell receptor V alpha and V beta gene complexes, located on 14q11 and 7q35, respectively; and the Ig gene complex (IgH), located on 15q11. We used polymorphic microsatellite markers located within these genes, or gene complexes, to test for linkage (rather than association), to each of these candidates. Using markers within the loci allowed us to assume a fixed recombination fraction of 0.01 in the tested model. Three hundred eight subjects from 48 multiplex families were studied, with 142 affected subjects. Using this set of families, we have previously shown evidence of linkage with a major susceptibility locus for Graves' disease (GD-1) on 14q24.3-31, with a maximum lod (logarithm + odds) score of 2.1, at a penetrance of 80% and with a dominant mode of inheritance. In the present study, we obtained consistently negative lod scores for each of the candidate genes, assuming either dominant or recessive modes of inheritance. These data, therefore, showed evidence against linkage with all the candidate genes. Unlike association studies, linkage analyses detect major genetic influences on disease susceptibility exerted by the linked loci. The lack of linkage for the immunoregulatory genes that were studied indicated, therefore, that they were not major contributors to disease etiology.

Abatacept↗
PubMed1998

Linkage analysis of candidate genes in autoimmune thyroid disease. II. Selected gender-related genes and the X-chromosome. International Consortium for the Genetics of Autoimmune Thyroid Disease.

Hashimoto's thyroiditis (HT) and Graves' disease (GD) are autoimmune thyroid diseases (AITD) in which multiple genetic factors are suspected to play an important role. Until now, only a few minor risk factors for these diseases have been identified. Susceptibility seems to be stronger in women, pointing toward a possible role for genes related to sex steroid action or mechanisms related to genes on the X-chromosome. We have studied a total of 45 multiplex families, each containing at least 2 members affected with either GD (55 patients) or HT (72 patients), and used linkage analysis to target as candidate susceptibility loci genes involved in estrogen activity, such as the estrogen receptor alpha and beta and the aromatase genes. We then screened the entire X-chromosome using a set of polymorphic microsatellite markers spanning the whole chromosome. We found a region of the X-chromosome (Xq21.33-22) giving positive logarithm of odds (LOD) scores and then reanalyzed this area with dense markers in a multipoint analysis. Our results excluded linkage to the estrogen receptor alpha and aromatase genes when either the patients with GD only, those with HT only, or those with any AITD were considered as affected. Linkage to the estrogen receptor beta could not be totally ruled out, partly due to incomplete mapping information for the gene itself at this time. The X-chromosome data revealed consistently positive LOD scores (maximum of 1.88 for marker DXS8020 and GD patients) when either definition of affectedness was considered. Analysis of the family data using a multipoint analysis with eight closely linked markers generated LOD scores suggestive of linkage to GD in a chromosomal area (Xq21.33-22) extending for about 6 cM and encompassing four markers. The maximum LOD score (2.5) occurred at DXS8020. In conclusion, we ruled out a major role for estrogen receptor alpha and the aromatase genes in the genetic predisposition to AITD. Estrogen receptor beta remains a candidate locus. We found a locus on Xq21.33-22 linked to GD that may help to explain the female predisposition to GD. Confirmation of these data in HT may require study of an extended number of families because of possible heterogeneity.

Aromatase↗
PubMed1997

Comparison of administration of recombinant human thyrotropin with withdrawal of thyroid hormone for radioactive iodine scanning in patients with thyroid carcinoma.

BACKGROUND: To detect recurrent disease in patients who have had differentiated thyroid cancer, periodic withdrawal of thyroid hormone therapy may be required to raise serum thyrotropin concentrations to stimulate thyroid tissue so that radioiodine (iodine-131) scanning can be performed. However, withdrawal of thyroid hormone therapy causes hypothyroidism. Administration of recombinant human thyrotropin stimulates thyroid tissue without requiring the discontinuation of thyroid hormone therapy. METHODS: One hundred twenty-seven patients with thyroid cancer underwent whole-body radioiodine scanning by two techniques: first after receiving two doses of thyrotropin while thyroid hormone therapy was continued, and second after the withdrawal of thyroid hormone therapy. The scans were evaluated by reviewers unaware of the conditions of scanning. The serum thyroglobulin concentrations and the prevalence of symptoms of hypothyroidism and mood disorders were also determined. RESULTS: Sixty-two of the 127 patients had positive whole-body radioiodine scans by one or both techniques. The scans obtained after stimulation with thyrotropin were equivalent to the scans obtained after withdrawal of thyroid hormone in 41 of these patients (66 percent), superior in 3 (5 percent), and inferior in 18 (29 percent). When the 65 patients with concordant negative scans were included, the two scans were equivalent in 106 patients (83 percent). Eight patients (13 percent of those with at least one positive scan) were treated with radioiodine on the basis of superior scans done after withdrawal of thyroid hormone. Serum thyroglobulin concentrations increased in 15 of 35 tested patients: 14 after withdrawal of thyroid hormone and 13 after administration of thyrotropin. Patients had more symptoms of hypothyroidism (P<0.001) and dysphoric mood states (P<0.001) after withdrawal of thyroid hormone than after administration of thyrotropin. CONCLUSIONS: Thyrotropin stimulates radioiodine uptake for scanning in patients with thyroid cancer, but the sensitivity of scanning after the administration of thyrotropin is less than that after the withdrawal of thyroid hormone. Thyrotropin scanning is associated with fewer symptoms and dysphoric mood states.

Adult↗
PubMed1997

The genetic susceptibility to Graves' disease.

Graves' disease (GD) develops as a result of a complex interaction between genetic susceptibility genes and likely environmental factors. Most epidemiological data support an important genetic contribution to the development of GD. The concordance rate of GD in monozygotic twins is 30-60% and in dizygotic twins 3-9%, and thyroid autoantibodies have been reported in up to 50% of the siblings of patients with GD. For many years now, HLA studies have consistently shown an increased frequency of HLA-DR3 in Caucasian patients with GD; but with only a risk ratio of 3-5. However, recent advances in human genome mapping techniques have enabled the study of many other candidate genes. Of these additional, non-HLA genes, only CTLA-4 has been consistently found to be associated with GD. Using a linkage based approach which only detects highly significant susceptibility genes we have recently reported preliminary results which demonstrated that a marker located approximately 25 cM from the TSH receptor gene on chromosome 14q31 is linked to GD and in the same vicinity as the IDDM-11 locus. Such results, if confirmed, may signal the presence of a gene family related to endocrine autoimmunity on chromosome 14q31.

Epidemiologic Methods↗
PubMed1997

Preservation of functioning human thyroid "organoids" in the scid mouse. IV. In vivo selection of an intrathyroidal T cell receptor repertoire.

To study the in vivo influence of thyroid cells on the T cell receptor repertoire in human autoimmune thyroid disease, we mixed lymphocyte-free thyrocytes (approximately 1.2 x 10[6]) from patients with Graves' disease with autologous peripheral blood mononuclear cells (PBMC; approximately 1.5 x 10[6]) and transplanted this mixture sc into scid mice while suspended in a basement membrane gel (approximately 0.4 ml). Controls included mice that received either thyrocytes only or PBMC only. The resulting artificial mixed cell thyroid organoids were explanted after 5 weeks, and their T cell receptor repertoire was examined. Of a total of 63 organoids constructed, 60 were recovered (95.2%). Total RNA was extracted and then analyzed by reverse transcription-PCR primarily for human T cell receptor (hTcR) Vbeta gene expression using 21 hTcR Vbeta amplimers. A restricted pattern of hTcR Vbeta gene expression was found, with 6 Vbeta genes (Vbeta5, 6, 7, 8, 13.1, and 18) predominantly expressed [P < 0.05, by ANOVA on ranks and Student-Newman-Keul's (SNK) test]. PBMC and control organoids showed no preferential selection of particular hTcR V gene-expressing T cells. This reductionist, mixed cell, thyroid model reflected earlier observations in human and murine autoimmune thyroid diseases in which a bias in hTcR V gene family expression had been observed. The model permitted in vivo T cell selection and/or enrichment of potentially disease relevant human T cells.

Animals↗
PubMed1997

Characterization of soluble, disulfide bond-stabilized, prokaryotically expressed human thyrotropin receptor ectodomain.

To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein; it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21-415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21-35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain alpha F'), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

Antibodies↗
PubMed1997

Folding-dependent binding of thyrotropin (TSH) and TSH receptor autoantibodies to the murine TSH receptor ectodomain.

The mouse TSH receptor ectodomain (mTSHR-ecd) was amplified from murine thyroid complementary DNA and ligated into the pAcGP67B insect cell vector, and the nucleotide sequence was confirmed. Employing a baculovirus-insect cell system, the mTSHR-ecd (amino acids 22-415) was expressed as a fusion protein with the gp67 insect cell signal sequence at the NH2-terminus and a C-terminal six-histidine tag. Protein expression was assessed by Western blot using a murine monoclonal antibody (recognizing amino acids 22-35) and a rabbit antipeptide antibody (recognizing amino acids 397-415). These antibodies detected two principal species of mTSHR-ecd, one glycosylated (66 kDa) and one nonglycosylated (52 kDa), in cell lysates of infected insect cells. More than 10% of these species were present in a water-soluble (cytosolic) fraction. This fraction was then used to purify, under native conditions, 100-microg amounts of mTSHR-ecd using nickel-nitrilo-triacetic (Ni-NTA) resin chromatography. The purified cytosolic mTSHR-ecd migrated as a homogeneous 66-kDa band visible on Coomassie blue-stained gels and was confirmed by Western blotting. We also purified the mTSHR-ecd from total cell lysates under denaturing conditions, followed by "in vitro" refolding on the Ni-NTA column. Under these conditions, milligram amounts of soluble mTSHR-ecd were obtained. This material consisted primarily of the 66-kDa glycosylated form, but in addition contained four or five lower molecular mass, partially glycosylated intermediates and the 52-kDa nonglycosylated form. Deglycosylation with either endoglycosidase F or H, reduced all mTSHR-ecd glycosylated species to a 52-kDa nonglycosylated form. Both the cytosolic and refolded mTSHR-ecd preparations inhibited the binding of [125I]TSH to the full-length human TSHR expressed in Chinese hamster ovary cells in a dose-dependent manner, with similar affinities. The affinity of such interactions was 3 orders of magnitude less than observed with native porcine TSHR and was further reduced by unfolding the mTSHR-ecd preparations. The cytosolic and refolded mTSHR-ecd were also recognized by hTSHR autoantibodies in the serum of patients with hyperthyroid Graves' disease. Such autoantibody binding to mTSHR-ecd was also markedly reduced by unfolding the antigen. These results demonstrated the successful production of large quantities of well characterized, biologically active, mTSHR-ecd antigen. In addition, the data showed that although the ectodomain of the mTSHR bound TSH, intact holoreceptor may be required for high affinity ligand binding. Whether the transmembrane region is required for direct ligand binding, as seen for other G protein-linked receptors, or whether it is needed to stabilize the ligand binding to the ectodomain and maintain a correctly folded state, remains unclear.

Amino Acid Sequence↗
PubMed1997

Detection of major T cell epitopes on human thyroid stimulating hormone receptor by overriding immune heterogeneity in patients with Graves' disease.

To examine the major immunogenic regions of the human TSH receptor (hTSHR), we examined 14 patients with Graves' disease and 14 healthy control subjects for their peripheral blood T cell proliferative responses to 29 synthetic peptides representing the entire ectodomain of the hTSHR (TSHR-ecd). By combining an analytical approach encompassing the grading of peptide-induced responses and nonparametric testing, we obtained evidence for highly significant differences (P = < 0.000001) in the patient group compared with minor differences in the control group (P = 0.045). To account for this difference, we identified four major T cell epitopes (amino acid 247-266, 202-221, 142-161, and 52-71), by multiple comparison analysis, in the patient group. Furthermore, we demonstrated by radiolabeled PCR that the responding T cells were clonally expanding. These findings demonstrate that despite likely differences in human leukocyte antigen type among patients with Graves' disease, several distinct hTSHR epitopes elicited significant responses in the immune system of patients with Graves' disease, and that such patients are most often poorly tolerant to particular epitopes of the TSH receptor ectodomain, The data support the notion of TSHR peptide antigens overriding human immune heterogeneity in patients with Graves' disease, and raise the possibility of applying analog peptide blockade to suppress T cell responsivity.

Adult↗
PubMed1997

Mapping of a major susceptibility locus for Graves' disease (GD-1) to chromosome 14q31.

The autoimmune thyroid diseases (AITD), encompassing Graves' disease (GD) and Hashimoto's thyroiditis (HT), occur in genetically susceptible individuals. In order to identify the AITD susceptibility genes, we have studied DNA markers in the regions of 8 candidate genes: (1) the HLA region, (2) the TSH receptor, (3) thyroid peroxidase, (4) thyroglobulin, (5) IDDM-4, (6) IDDM-5, (7) Immunoglobulin heavy chain gene and (8) CTLA-4. One hundred and seven subjects from 19 informative families were studied, 14 subjects had GD and 32 subjects had HT. LOD scores were maximized assuming both dominant and recessive modes of inheritance. No linkage was found for any marker in patients with HT. In patients with GD, negative LOD scores were obtained for all the candidate genes, except for markers in the TSH receptor region on chromosome 14q31. Positive LOD scores were found for several markers on 14q31. Marker D14S81 gave the highest score (Z max = 2.05, theta = 0.01) assuming a dominant mode of inheritance and a penetrance of 0.8. These data confirm our previous observations of a lack of a necessary disease locus for AITD in the HLA gene region. Further, the data suggest the presence of an important susceptibility gene on 14q31 but at a considerable distance from the TSH receptor gene.

Chromosome Mapping↗