Arginine at position 74 of the HLA-DR beta1 chain is associated with Graves' disease.

Graves' disease (GD) is associated with HLA-DR3 (DRB1*03) in Caucasians, but the exact amino-acid sequence in the DR beta1 chain conferring susceptibility to GD is unknown. Therefore, the aim of our study was to identify the critical sequence among the HLA-DRB1 amino-acid residues occupying the peptide-binding pocket, which conferred susceptibility to GD. We sequenced the HLA-DRB1 locus in 208 Caucasian GD patients and 149 Caucasian controls. Sequence analysis showed an increased frequency of DR beta-Arg-74 in GD patients compared to controls (41.8 and 13.4%, respectively; P=2.3 x 10(-8), OR=4.6). Moreover, subset analyses showed that DR beta-Arg-74 was also significantly more frequent in the HLA-DR3 negative GD patients than in controls (7.6 vs 0.8%, P=0.02, OR=10.5), suggesting that the association with DR beta-Arg-74 is independent of the association with HLA-DR3. Structural modeling studies demonstrated that the change at position 74 from the neutral amino acids Ala or Gln to the positively charged amino-acid Arg significantly modifies the three-dimensional structure of the DR peptide-binding pocket. Our results suggested that structural heterogeneity of the DR beta-chain peptide-binding pocket P4 at residue 74 predispose some at risk individuals to GD.

Analysis of the CTLA-4, CD28, and inducible costimulator (ICOS) genes in autoimmune thyroid disease.

The cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) gene on 2q33 is associated with autoimmune thyroid diseases (AITDs). Our earlier study in 56 families showed linkage of 2q33 to the presence of thyroid antibodies (TAbs). The goals of this study were to confirm the linkage of the 2q33 region to TAbs, to fine map this region, and study the ICOS gene. We performed a linkage study in an expanded data set of 99 multiplex AITD-TAb families (529 individuals). The highest two-point LOD score of 2.9 was obtained for marker D2S325 on 2q33. To fine map this locus, we genotyped 238 Caucasian AITD patients and 137 controls for five additional markers in the linked locus, which contained the CTLA-4, CD28, and ICOS genes. The A/G single-nucleotide polymorphism at position 49 of CTLA-4 was associated with AITD (P=0.01, OR=1.5), while markers inside CD28 and ICOS were not. Functional studies have shown that the G allele was associated with reduced inhibition of T-cell proliferation by CTLA-4. We concluded that: (1) the AITD gene in the 2q33 locus is the CTLA-4 gene and not the CD28 or ICOS genes; and (2) the G allele is associated with decreased function of CTLA-4.

Autoimmune thyroid disease susceptibility loci in a large Chinese family.

OBJECTIVES: The autoimmune thyroid diseases (AITDs) comprising Graves' disease (GD) and Hashimoto's thyroiditis (HT) are complex genetic diseases, which result from an interaction between predisposing genes and environmental triggers. The aim of our study was to dissect the genetic predisposition to GD and HT in one large Chinese family with multiple members affected with AITD. PATIENTS: We completed a whole genome screen of a large multiplex Chinese-American family. We enrolled 27 family members from three generations. Eight members were affected with AITD, six had GD and two had HT. DESIGN: We determined the information limits of the family. Power calculations indicated that the maximum attainable LOD scores were 5.1 assuming dominant inheritance, and 3.4 assuming recessive inheritance. These estimates both assumed 100% penetrance and one gene. Whole genome screening was performed using 400 highly polymorphic and densely spaced microsatellite markers spanning the entire human genome (intermarker distance < 10 cM). Linkage analysis was performed using two-point and multipoint parametric and nonparametric methods. RESULTS: Initial whole genome screening performed with 400 microsatellite markers identified two markers that showed evidence for linkage to AITD in this family, D11S4191 and D9S175, with two-point LOD scores of 2.31 and 2.05, respectively. Multipoint linkage analysis focusing on the regions containing these markers revealed a maximum multipoint LOD score (MLS) of 2.13 and a nonparametric linkage score (NPL) of 6.1 for D11S4191 and an MLS of 2.01 and NPL of 7.5 for D9S175. CONCLUSIONS: These results showed that this Chinese family harboured susceptibility loci for AITD which were distinct from those previously found in the Caucasian population. This suggests that different susceptibility loci exist between different ethnic groups. Furthermore, even within a single family from a genetically homogenous population, more than one gene was involved in the genetic susceptibility to AITD, supporting the notion that AITDs are caused by multiple genes of varying influences.

Evidence for association of polycystic ovary syndrome in caucasian women with a marker at the insulin receptor gene locus.

The polycystic ovary syndrome (PCOS) is one of the commonest female endocrinopathies affecting 5-10% of women of reproductive age. The disorder, characterized by chronic anovulation and signs of hyperandrogenism, results from a complex interaction between genetic predisposing factors and environmental triggers. We have studied 85 Caucasian PCOS patients and 87 age-matched Caucasian control women for associations with four candidate genes: follistatin, CYP19 (aromatase), CYP17a, and the insulin receptor (INSR). These genes were analyzed using microsatellite markers located near or inside the genes. We found that only the insulin receptor gene marker D19S884 was significantly associated with PCOS (p=0.006 and even after a conservative correction p=0.042). The INSR gene region was then fine mapped with an additional panel of 9 markers but only marker D19S884, located 1 cM telomeric to the INSR gene, was again associated with PCOS. In conclusion, our results suggested that a susceptibility gene for PCOS was located on chromosome 19p13.3 in the insulin receptor gene region. It remains to be determined if this susceptibility gene is the insulin receptor gene itself or a closely located gene. Since insulin stimulates androgen secretion by the ovarian stroma it is likely that INSR function in the ovary is involved in the genetic susceptibility ot PCOS.

Limited genetic susceptibility to severe Graves' ophthalmopathy: no role for CTLA-4 but evidence for an environmental etiology.

Graves' disease (GD) is an autoimmune thyroid disease (AITD) characterized by hyperthyroidism and by the occurrence of a distinctive ophthalmopathy (orbitopathy), which presents with varying degrees of severity. Graves' disease clusters in families but the importance of heredity in the pathogenesis of the associated ophthalmopathy is unclear. We have studied the family history of 114 consecutive, ethnically mixed patients with severe Graves' ophthalmopathy (GO). Patients were selected by unambiguous single ascertainment. Seventy-seven percent of patients were female and 59% smoked. The mean age at onset of their GD was 43 years (range 17-78 years). Forty-one patients (36%) had a family history of AITD, defined as a first- and/or a second-degree relative affected with either Graves' disease (GD) or Hashimoto's thyroiditis (HT). The segregation ratio for AITD in nuclear families in our ascertained Graves' ophthalmopathy families was 0.07 (0.12 in Caucasians only). Hence, the higher prevalence of AITD among relatives of Graves' ophthalmopathy patients agreed with the known genetic predisposition to AITD and this predisposition was stronger in women than in men. However, only 3 of the 114 patients had a family history of severe Graves' ophthalmopathy (all second-degree relatives) and the segregation ratio for GO was 0. These data did not support a major role for familial factors in the development of severe Graves' ophthalmopathy distinct from Graves' disease itself. In addition, we tested 4 candidate genes, human leukocyte antigen (HLA), tumor necrosis factor-beta (TNF-beta), CTLA-4 and the thyrotropin receptor (TSHR), for association with Graves' ophthalmopathy. These were negative except for the HLA and CTLA-4 genes, which were found to be weakly associated with GO giving similar relative risk (RR) as in GD patients without ophthalmopathy. These data suggested that environmental factors, rather than major genes, were likely to predispose certain individuals with AITD to severe Graves' ophthalmopathy. Smoking remains one example of such potential external insults.

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.

Endogenous antigen presentation by autoantigen-transfected Epstein-Barr virus-lymphoblastoid cells: T cell receptor N-region hydrophobicity relates to thyroid antigen recognition.

Seven human T cell lines from a patient with Graves' disease were raised against endogenously generated human thyroid peroxidase (hTPO) with stimulation indices ranging from 2.1 to 7.6. Clonal expansion within these T cell lines was demonstrated by sequencing multiple bacterial colonies containing RT-PCR-generated fragments derived from the expressed hTcRs. Some lines had more than one human T cell receptor (hTcR) alpha and beta chain mRNAs as judged by RT-PCR. Stopcodons present in several hTcR sequences indicated that only one V alpha and one V beta gene were translated. Both the V alpha/beta gene families and the J alpha/beta gene segments differed amongst the lines and no characteristic recognition sequences were discernable in the CDR3 regions. Using Kyte-Doolittle analysis we found hydrophobic peaks in most N alpha-regions (but not N beta regions) suggesting that hydrophobic interactions may be important in the recognition of hTPO. However, increasing affinity values, as measured by SI, were strongly correlated with decreasing hydrophobicity in the N alpha region (1st order regression, r = -0.93138, p < 0.01). Thus, lower affinity, self-reactive, T cells may be more hydrophobic ('sticky') in their N alpha regions while higher affinity cells may be characterized by TcRs with lower hydrophobicity. These findings demonstrate a substantial role for hydrophobic interactions in hTPO-reactive T cell receptors and further support a role for the TcR alpha chain in the recognition of thyroid autoantigen.

Graves' disease thyroid tissue transplants in scid mice: persistent selectivity in hTcR Va gene family use.

We have analyzed the human T-cell receptor (hTcR) V alpha gene repertoire in thyroid tissue transplants of a patient with hyperthyroid Graves' disease. Blocks of thyroid tissue were transplanted subcutaneously into 10 mice with severe immunodeficiency (scid) and 4 weeks later 5 of the mice were injected intraperitoneally with autologous peripheral blood mononuclear cells (PBMC) (10(7) cells per mouse). After a further 3 weeks, mice were sacrificed and total cellular RNA and cDNA prepared from each of the explants. We used specific olingonucleotides in polymerase chain reactions (PCR) to amplify 18 different human hTcR V alpha gene families and the identity of the PCR fragments was confirmed by Southern blot analysis. Different samples of the donor thyroid tissue consistently expressed 9-10 of the 18 hTcR V alpha gene families screened (V alpha 1-7, 11, 12 & 15). A more marked bias in hTcR V gene family use was seen in each of the explants with a mean of only 2.8 V alpha gene families detected. After 7 weeks of transplantation, the thyroid explants largely reflected some of the same genes seen in the hTcR V gene repertoire of the donor tissue with particularly pronounced expression of V alpha 2 and V alpha 3 gene families. The transplantation of PBMC into the scid mice showed evidence for their accumulation within the transplanted thyroid tissues as judged by the appearance of additional hTcR V gene families expressed in these samples although the specificity of such accumulation remains unclear.(ABSTRACT TRUNCATED AT 250 WORDS)

T-cell receptor V region beta-chain gene expression in the autoimmune thyroiditis of non-obese diabetic mice.

The non-obese diabetic (NOD) mouse develops both a spontaneous T-cell-mediated autoimmune insulitis and, in addition, a well characterized thyroiditis. We have examined the repertoire of murine T-cell receptor (TCR) variable (V) beta-chain genes used by intrathyroidal T cells with specific oligonucleotides that amplified 17 murine V beta gene families in cDNA samples prepared from intact NOD thyroid tissues. Normal NOD thyroid tissue contained only low levels of TCR V gene mRNA. In contrast, NOD mice with histologic thyroiditis showed the marked expression of up to 3 TCR V beta genes consistent with a restricted T-cell invasion. Sequencing of amplified TCR V beta cDNA showed that within each NOD thyroid sample at least one of the overexpressed V beta gene families was clonally expanded. However, the clonally expanded T-cell V gene family was not consistent in all animals. Even within the same TCR V beta gene families, various D and J segments had been rearranged with open reading frames and together with insertions and deletions gave no significant homology at the nucleotide or amino acid level. In summary, these data showed that the intrathyroidal T-cell infiltrate in NOD mice was markedly biased towards the use of a single, but variable, TCR V gene family within each animal. It also appeared that the choice of the TCR V beta chain determined the intrathyroidal infiltrative process rather than the choice of D and/or J regions. However, there was no consistent use of a single TCR V beta chain. As thyroiditis does not occur uniformly in apparently genetically homogeneous animals, reared under similar environmental conditions, it may not be surprising that different TCR V genes are involved in different animals.

Preservation of functioning human thyroid organoids in the scid mouse: II. Biased use of intrathyroidal T cell receptor V genes.

The severe combined immunodeficiency (scid) mouse, which lacks functional B cells and T cells, has proven a useful model for exploring the survival of transplanted human lymphocytes and thyrocytes. In order to further characterize T cell infiltrates in reconstituted sc human thyroid organoids, we examined for the presence of 18 human T cell receptor (hTcR) V alpha and 21 hTcR V beta gene families using polymerase chain reaction (PCR) analysis. Human TcR V gene activity was confirmed by Southern blot analysis of the PCR fragments from all but one of the thyroid organoids, confirming the continued survival of human T cells within the thyroid organoids. However, only 3.5 out of 18 V alpha and 5.9 out of 21 V beta gene families were detected in these human thyroid organoids indicating a marked bias in T cell survival. Sequencing of the V-D-J regions of the amplified TcR fragments showed that approximately 60% of the sequences were representative of clonally expanded T cells. Hence, these passenger T cells exhibited highly biased use of particular TcR V gene families similar to that observed previously in thyroid tissue and intrathyroidal T cell cultures. Furthermore, variations in the V-D-J regions of sequences from similar V gene families indicated that the V gene region was important in T cell selection rather than the CD3 region.

T-cell receptor V gene use in autoimmune thyroid disease: direct assessment by thyroid aspiration.

We have examined the hTcR V gene family use of T-cells present in the aspiration thyroid biopsy specimens of patients with hyperthyroid Graves' disease (n = 8) and Hashimoto's autoimmune thyroiditis (n = 5). Nine of the 13 specimens had cytologically identified thyroid follicular cells, and 12 of the 13 contained human thyroglobulin-specific mRNA, confirming successful sampling. Of 18 hTcR V alpha and 19 hTCR V beta gene families tested for in the individual aspirates, a mean +/- SEM of 6.8 +/- 0.9 V alpha and 9.6 +/- 1.4 V beta gene families were present in the Graves' aspirates, while 12.2 +/- 1.7 and 16.8 +/- 0.4 V alpha and V beta gene families were present in the aspirates of patients with Hashimoto's thyroiditis. These samples, which offer a window onto the natural history of autoimmune thyroid disease, demonstrate significant hTcR V alpha and beta gene restriction in hyperthyroid Graves' disease, but much less restriction of both V alpha and V beta gene families in Hashimoto's disease. Such data extend our earlier information based only on examination of highly selected surgical specimens of patients with autoimmune thyroid disease to the much more typical patient. We conclude that hTcR V gene restriction of varying degrees is present in the majority of patients with autoimmune thyroid disease, but appears to be more easily detected in Graves', rather that Hashimoto's, disease.

Lack of disease associated HLA-DQ restriction fragment length polymorphisms in families with autoimmune thyroid disease.

Autoimmune thyroid disease (AITD) is often familial and serological HLA disease associations have been described in many different populations. However, such HLA disease associations are weak and the precise molecular contribution of HLA antigens to thyroid disease susceptibility remains unknown. Much of the data available are cross-sectional and few studies have explored familial inheritance of AITD at the molecular level. We have, therefore, examined the inheritance of AITD in multiplex and multi-generational families using restriction fragment length polymorphism (RFLP) analysis of DNA digested with the restriction enzyme BamH1 and probed with a full length human HLA-DQ beta cDNA probe. Thirty seven subjects in 7 informative families were available for study. Eleven subjects had Graves' disease and 4 were diagnosed as having Hashimoto's thyroiditis. Segregation of polymorphic fragments enabled genotyping of each individual to produce fully informative families. LOD scores were computed, using the LIPED program, for dominant and recessive models of inheritance, for recombination fractions of 0.01 to 0.5 for each sex, and for penetrances of 0.1 to 1.0. The results showed that maximum LOD scores were negative for all of the inheritance models tested. If the primary locus for AITD were in the HLA region, LOD scores would be highly positive. These data, therefore, provide strong evidence against a disease locus for AITD in linkage disequilibrium with the HLA-DQ beta locus.

Evidence for selective accumulation of intrathyroidal T lymphocytes in human autoimmune thyroid disease based on T cell receptor V gene usage.

We have investigated the T cell receptor V alpha and V beta gene family usage by T lymphocytes infiltrating affected thyroids in patients with autoimmune thyroid disease. We show that the intrathyroidal T lymphocytes from patients (n = 6) with autoimmune thyroid disease display a widespread usage of V beta gene families with an average of 14.4/19 V beta gene families similar to the peripheral T lymphocytes of the same patients. Because we recently reported that the utilization of V alpha gene families is markedly reduced within these mitogen-stimulated intrathyroidal T cell populations, as well as within intact tissue from similar patients (n = 4) (overall mean of 4.0/18 families detected), these results indicate that in thyroids of patients with autoimmune thyroid disease the lymphocytes are selectively accumulating based on their V alpha rather than V beta elements. This preferential hTcR V alpha and widespread V beta gene usage was not mimicked in most 7-d autologous mixed lymphocyte reactions using non-T cell stimulators (n = 6) or EB-virus immortalized autologous B cell lines (n = 3). Hence, the selective V gene utilization by intrathyroidal T cells is likely to be secondary to multiepitopic thyroidal autoantigens activating thyroid infiltrating T cells or to the presence of a superantigenlike thyroidal self-antigen, capable of determining a selective infiltration or activation of a variety of T lymphocytes on the basis of their V alpha gene usage.

Evidence of limited variability of antigen receptors on intrathyroidal T cells in autoimmune thyroid disease.

BACKGROUND: Patients with autoimmune thyroid diseases, including Graves' disease and Hashimoto's disease, have marked lymphocytic infiltration in their thyroid glands. We examined the gene for the variable regions of the alpha-chain of the human T-cell receptor (the V alpha gene) in intrathyroidal T cells to determine whether the infiltration is a secondary heterogeneous immune response or a more restricted, and therefore primary and presumably pathogenetic, reaction to thyroid autoantigens. METHODS: We used the polymerase chain reaction to detect small numbers of T cells expressing the variable region of the V alpha gene. Different oligonucleotides were used to amplify complementary DNA for the 18 known families of the V alpha gene in intrathyroidal T cells from 9 patients with autoimmune thyroid disease. We compared the findings with the results in patients with nonautoimmune thyroid disease as well as those in normal subjects. RESULTS: We found marked restriction in the expression of T-cell-receptor V alpha genes by T cells from the thyroid tissue of patients with autoimmune thyroid disease. An average of only 5 of the 18 V alpha genes were expressed in such samples, as compared with 17 V alpha genes expressed in peripheral-blood T cells from the same patients. No such restriction was found in thyroid tissue from patients with nonautoimmune thyroid disease. The predominantly expressed V alpha genes differed from patient to patient, however, with no clear association with the type of disease. CONCLUSIONS: Intrathyroidal T-cell accumulation in autoimmune thyroid disease is highly restricted and points to the primacy of T cells in causing thyroid disorders. These results present the possibility of using antibodies to the T-cell receptor for the specific inhibition of abnormal T-cell function in autoimmune thyroid disease.

Positive regulation of human thyrotropin receptor mRNA by thyrotropin.

We have assessed the influence of natural and recombinant thyrotropin (TSH) on mRNA specific for the human TSH receptor (TSHR) in normal and abnormal adult human thyroid monolayer cells. Using physiological concentrations of TSH (less than 100 mU/L), a marked increase in the level of TSHR mRNA was observed within 12 hours and reached a greater than 1000% increase after 24 hours exposure. At high TSH concentrations (greater than 1000 mU/L), this increase in TSHR-specific mRNA was markedly reduced. However, at no time were basal TSHR mRNA levels suppressed even with 100 U/L of TSH for 48 hours. These observations demonstrate ligand-induced up-regulation of the human TSHR mRNA by physiologically relevant concentrations of TSH.

Recombinant human thyroid-stimulating hormone: initial bioactivity assessment using human fetal thyroid cells.

We have assessed the bioactivity of newly available recombinant human TSH (rec-hTSH) using human fetal thyroid cells, with the longer term aim of assessing its use for clinical applications. Rec-hTSH caused a consistent and dose-related increase in thyroid monolayer cell cAMP release and human thyroglobulin (hTg) secretion, confirming its bioactivity. Repetitive studies (n = 5) allowed us to derive an estimated biopotency for the rec-hTSH preparation examined of 5.6 IU/mg compared to 10 IU/mg for commercially available bovine TSH for human use. The rec-hTSH had a bioimmune ratio of 0.55, similar to that of purified pituitary hTSH standards, Furthermore, rec-hTSH induced thyroid epithelial cell growth, as evidenced by a decrease in thyroid cell doubling time from 54 +/- 2.1 to 31 +/- 1.7 h (P less than 0.005). Hence, rec-hTSH is a potent glycoprotein hormone preparation when measured in a homologous human thyroid cell culture system. Rec-hTSH could serve as a future definitive International Standard and has the potential for a useful diagnostic and therapeutic reagent.