TSH Receptor mRNA Research Articles

Investigation on peripheral blood TSHR mRNA for differentiation of benign and malignant thyroid nodules

Objective To assess the value of peripheral blood thyroid stimulating hormone receptor(TSHR) mRNA determination in differential diagnosis of benign and malignant thyroid nodules.Methods Fine needle aspiration cytology (FNAC) and (or) postoperative histopathology as the gold standard were carried out,the expression of circulating TSHR mRNA was determined by RT-PCR in 33 patients with benign thyroid nodules,39 patients with thyroid cancer,and 20 normal controls.Results TSHR mRNA signals were not detected in normal controls,the positive rate of TSHR mRNA was higher in the group with malignant nodules than the group of benign nodules (91.2％ vs 48.5％,P＜0.01).TSHR mRNA level in the preoperative malignant group was significantly higher than that in the normal,benign,and postoperative cancer groups (all P ＜ 0.01).Using peripheral blood TSHR mRNA for differentiating benign or malignant of thyroid nodule had a sensitivity,specificity,and accuracy of 91.2％,51.5％ and 71.6％,respectively.The sensitivities of TSHR mRNA,FNAC,and these two methods combined in detecting malignant nodules were 91.3％,86.9％,and 100.0％ respectively,while diagnostic accuracies were respectively 84.0％,80.0％,and 92.0％.TSHR mRNA expression showed no significant relationship with sex,age,size,and number of nodule in these patients (all P ＞ 0.05),but it did exhibit significant difference between benign and malignant nodules(P＜0.01).Conclusion The peripheral blood TSHR mRNA could be used as a molecular marker for thyroid cancer,and it would help enhance the preoperative differentiation of benign and malignant thyroid nodules. Key words: Thyroid cancer; Peripheral blood; Thyroid stimulating hormone receptor; Molecular markers

Information for Clinicians: Commercially Available Molecular Diagnosis Testing in the Evaluation of Thyroid Nodule Fine-Needle Aspiration Specimens

TSH receptor mRNA reverse transcription-polymerase chain reaction, the Veracyte and Asuragen commercial methods, and the noncommercial use of BRAF, RAS, RET/PTC, and PAX8/PPARγ testing have promising roles in the diagnosis and treatment of patients with nodular thyroid disease and thyroid cancer. However, at this time, experience with these molecular methods remains limited, and no test has perfect sensitivity and specificity. Peer-reviewed data evaluating the diagnostic performance of these tests are increasingly available. The American Thyroid Association (ATA) feels that until an expert consensus review of existing data (now underway by the ATA Guidelines Task Force) can be completed, no evidence-based recommendation for or against the use of these methods can be made. Clinicians are therefore advised to consider the use of these genetic diagnosis methods with appropriate caution, and to remain cognizant of the limitations of the data supporting their use. Patients who are interested in the use of these tests in their own care should discuss them thoroughly with their care providers. Until evidence-based recommendations are available, determining whether or not the limited data available support the use of these methods should be considered on a case-by-case basis.

Human fibrocytes coexpress thyroglobulin and thyrotropin receptor

Thyroglobulin (Tg) is the macromolecular precursor of thyroid hormones and is thought to be uniquely expressed by thyroid epithelial cells. Tg and the thyroid-stimulating hormone receptor (TSHR) are targets for autoantibody generation in the autoimmune disorder Graves disease (GD). Fully expressed GD is characterized by thyroid overactivity and orbital tissue inflammation and remodeling. This process is known as thyroid-associated ophthalmopathy (TAO). Early reports suggested that in TAO, both Tg and TSHR become overexpressed in orbital tissues. Previously, we found that CD34(+) progenitor cells, known as fibrocytes, express functional TSHR, infiltrate the orbit, and comprise a large subset of orbital fibroblasts in TAO. We now report that fibrocytes also express Tg, which resolves as a 305-kDa protein on Western blots. It can be immunoprecipitated with anti-Tg Abs. Further, (125)iodine and [(35)S]methionine are incorporated into Tg expressed by fibrocytes. De novo Tg synthesis is attenuated with a specific small interfering RNA targeting the protein. A fragment of the Tg gene promoter fused to a luciferase reporter exhibits substantial activity when transfected into fibrocytes. Unlike fibrocytes, GD orbital fibroblasts, which comprise a mixture of CD34(+) and CD34(-) cells, express much lower levels of Tg and TSHR. When sorted into pure CD34(+) and CD34(-) subsets, Tg and TSHR mRNA levels become substantially higher in CD34(+) cells. These findings indicate that human fibrocytes express multiple "thyroid-specific" proteins, the levels of which are reduced after they infiltrate tissue. Our observations establish the basis for Tg accumulation in orbital GD.

Thyroid stimulating hormone receptor expression in human placenta and decidua

ObjectivesElevated Thyroid Stimulating Hormone (TSH) concentrations have been associated with adverse pregnancy outcomes. We hypothesise that TSH could have a direct impact on uteroplacental development. We, therefore, sought to describe...

Review of Molecular Markers for Thyroid Cancer

The incidence of thyroid cancer has been rapidly increased in Korea. Although fine needle aspiration cytology is recommended for diagnosis of cancer, there are some limitations. Patients with indeterminate or suspicious cytology category in which malignancy cannot be ruled out usually undergone a thyroidectomy, however, only 10∼25% of them finally diagnosed as cancer. According to the progress in understanding molecular mechanism, some mutations or other molecular alterations have been studied for the diagnostic and prognostic markers for thyroid cancer. The majority of papillary thyroid cancers have BRAF and RAS mutations or RET/PTC rearrangement, and approximately 80% of follicular thyroid cancers harbor a RAS mutation or PAX8/PPARγ rearrangement. These genetic alterations are mostly studied and current clinical guidelines suggested that these molecular markers may help management for patients with indeterminate cytology. In addition, recent studies demonstrated the high sensitivity and specificity of thyroid-stimulating hormone receptor mRNA in diagnosing cancer in patients with indeterminate cytology. For the detection of recurrent or residual thyroid cancer, serum thyroglobulin is the only circulating marker in clinical practice. However, it lacks sensitivity and is unreliable specifically in the presence of antibodies to thyroglobulin. Recent studies demonstrated a significant role of measuring the mRNA of thyroglobulin, thyroid peroxidase, thyroid-stimulating hormone receptor, and sodium/iodine symporter in peripheral blood for monitoring of the recurrence of thyroid cancer. (Ewha Med J 2012;35(1):3-10)

Expression and correlation of sodium/iodide symporter and thyroid stimulating hormone receptor in human thyroid carcinoma

To investigate the expression of sodium/iodide symporter (NIS) and thyroid stimulating hormone receptor (TSHR) in human thyroid cancer. NIS and TSHR mRNA levels quantified by real-time PCR as well as NIS and TSHR proteins evaluated by immunohistochemistry were examined in surgical specimens including 38 benign nodules, 32 thyroid carcinomas and 36 normal thyroid samples. NIS and TSHR mRNA levels in thyroid carcinomas were significantly lower than in benign nodules and normal thyroid samples (P <0.001). Interestingly, we found that NIS and TSHR mRNA expression in benign nodules had similar levels to those in normal thyroid tissues. However, NIS and TSHR protein expression in benign nodules and thyroid carcinomas was stronger than in normal thyroid samples (P <0.05) but mainly located in cytoplasm. In addition, there was a significant positive correlation between NIS and TSHR in benign nodules and normal thyroid samples (r = 0.551 and 0.667, respectively, P = 0.001 and 0.000, respectively) but there was no such correlation in thyroid carcinomas (r = 0.222, P = 0.376). In thyroid carcinomas, NIS and TSHR mRNA levels were lower but the proteins were overexpressed. The NIS protein mainly locates in the cytoplasm, which therefore lacks the ability of transporting and absorbing iodine in patients with thyroid carcinoma. In addition, there was no correlation between NIS and TSHR in thyroid cancer, which may explain why, even after TSH stimulation, 10-20% of these malignant tumors are unable to concentrate enough radioiodine for effective therapy.

A stimulatory TSH receptor antibody enhances adipogenesis via phosphoinositide 3-kinase activation in orbital preadipocytes from patients with Graves' ophthalmopathy

Graves' ophthalmopathy (GO) is characterized by expanded volume of the orbital tissues associated with elevated serum levels of TSH receptor (TSHR) autoantibodies. Because previous studies have demonstrated evidence of adipogenesis within the GO orbit, we sought to determine whether M22, a human monoclonal antibody directed against TSHR, enhances adipogenesis in orbital fibroblasts from patients with GO and, if so, to identify signaling mechanisms involved. GO orbital fibroblast cultures (n=10) were treated for 10 days with bovine TSH (1 or 10·0 U/l) or M22 (1 or 10 ng/ml) in serum-free adipocyte differentiation medium. Some cultures also received a phosphoinositide 3-kinase (PI3K) inhibitor or an inhibitor of cAMP production. In other experiments, confluent cultures (n=8) were treated for between 1 and 30 min with TSH (0·1–10·0 U/l) or M22 (0·1–100 ng/ml) with measurement of cAMP production or levels of phosphorylated AKT (pAKT). We found levels of adiponectin, leptin, and TSHR mRNA to be increased in GO cultures treated for 10 days with either M22 (2·6 mean fold ±0·7; P=0·03) or TSH (13·2±5·8-fold, P=0·048). In other studies, M22 and TSH stimulated cAMP production and pAKT levels in GO cells. Inhibition of PI3K activity during 10 days in culture decreased the levels of M22-stimulated mRNA encoding adiponectin (67±12%; P=0·021), as well as adiponectin and CCAAT/enhancer-binding protein α protein levels. In conclusion, M22 is a pro-adipogenic factor in GO orbital preadipocytes. This antibody appears to act via the PI3K signaling cascade, suggesting that inhibition of PI3K signaling may represent a potential novel therapeutic approach in GO.

Circulating Thyrotropin Receptor mRNA as a Novel Marker of Thyroid Cancer

Since thyroglobulin, no new blood tests for differentiated thyroid cancer (DTC) have been introduced into routine clinical practice. In initial studies, the detection of circulating DTC cells by thyrotropin receptor (TSHR) mRNA measurement distinguished benign from malignant thyroid diseases. This prospective validation study tests the ability of TSHR mRNA to diagnose DTC preoperatively and to detect cancer recurrence. TSHR mRNA was measured by quantitative RT-PCR from blood drawn perioperatively in patients undergoing thyroid surgery (n = 526), postoperatively in patients undergoing DTC follow-up (n = 418) and in patients monitored for known benign disease (n = 151). The reference range and applications for TSHR mRNA were previously defined from 663 samples from patients with normal, benign, and malignant thyroid disease. In patients with follicular neoplasms or suspicious cytology, preoperative TSHR mRNA >1 ng/μg had 96% predictive value for DTC, whereas 95% of patients with undetectable mRNA and benign thyroid sonography had benign disease. In patients with DTC, elevated TSHR mRNA levels became undetectable in all patients (n = 64) on the first postoperative day, except in 5 who manifested persistent or recurrent cervical disease within the year. In long-term follow-up of DTC patients with thyroglobulin antibodies, 96% with undetectable TSHR mRNA also had no evidence of cancer recurrence. TSHR mRNA provides an additional clinical tool for the evaluation of patients with thyroid nodules. It is particularly useful in guiding appropriate initial surgery for follicular neoplasms. TSHR mRNA also represents a new blood test to aid assessment of disease status in thyroid cancer follow-up.

Thyroid-stimulating hormone receptor and thyroid hormone receptors are involved in human endometrial physiology

To study the expression, distribution, and function of thyroid-stimulating hormone receptor (TSHR) and thyroid hormone receptors (TR) α1, α2, and β1 in human endometrium. Experimental clinical study. University hospital. 31 fertile women. Endometrial biopsy samples obtained throughout the menstrual cycle. Real-time reverse transcriptase polymerase chain reaction, immunohistochemistry and Western blot to study the expression of TSHR, TRα1, TRα2, and TRβ1 messenger RNA (mRNA) and proteins in human endometrium. We found TSHR, TRα1, TRα2 and TRβ1 mRNA and proteins expressed in human endometrium. Immunostaining for TSHR in the luminal epithelium and TRα1 and β1 in the glandular and luminal epithelium increased statistically significantly on luteinizing hormone (LH) days 6 to 9, coinciding with appearance of pinopodes. Endometrial stromal and Ishikawa cells expressed mRNA for TSHR, TR, and iodothyronine deiodinases 1-3. After 48 hours, TSH significantly increased leukemia inhibitory factor (LIF) and LIF receptor (LIFR) messenger RNA (mRNA) in endometrial stromal cells, but decreased their expression in Ishikawa cells. Glucose transporter 1 mRNA was up-regulated by TSH in Ishikawa cells. We found that TSH statistically significantly increased secretion of free triiodothyronine (T3) and total thyroxin (T4) by Ishikawa cells compared with nonstimulated cells. Thyroid hormones are directly involved in endometrial physiology.

Blood thyroglobulin and TSH receptor mRNA detection by RT-PCR in the follow-up of differentiated thyroid cancer patients

Objective During the last years several groups have used the technique of RT-PCR for the detection of circulating thyroid cells, through the amplification of thyroglobulin (Tg) and TSH receptor(TSH-R) mRNA; however the published results are controversial. In this study we investigated the utility for the detection of Tg and TSH-R mRNA by RT-PCR in patients with differentiated thyroid cancer (DTC) during treatment with levothyroxine. Subjects and methods We investigated the expression of Tg and TSH-R mRNA by single and nested RT-PCR in the blood of 3 groups of subjects: (A) 34 patients with DTC and no evidence of disease, (B) 8 patients with DTC and evidence of local or distant metastasis and (C) 13 normal subjects. Expression levels of Tg mRNA were also analysed by comparative semi-quantitative RT-PCR. Results Tg and TSH-R mRNA signals were detected in all subjects (patients with DTC with and without evidence of disease and in normal subjects) by single or nested RT-PCR. By semi-quantitative RT-PCR and densitometric analysis of PCR products, mean levels of circulating Tg mRNA of the 3 groups were: Group A 0.182±0.107, Group B 0.329±0.298 and Group C 0.305±0.217. Conclusions Single or nested RT-PCR for Tg and TSH-R mRNA is not a suitable tool in the follow-up of patients with DTC. Lower levels of Tg mRNA in patients with DTC without evidence of disease, although not significant, may indicate that small numbers of thyroid cells may be normally present in the circulation or may represent an ectopic transcriptionof messengers from blood cells

Impact of Moderate Calorie Restriction on the Reproductive Neuroendocrine Axis of Male Rhesus Macaques.

The impact of moderate calorie restriction on reproductive neuroendocrine function was investigated in young adult male rhesus macaques (Macaca mulatta). The animals were subjected to either 30% calorie restriction (CR; n=5), or were fed a standard control diet (CON; n=5), starting during their peripubertal period. Plasma LH and testosterone concentrations were examined after 7 years of differential dietary treatment, and were found to be similar in both groups, both during the day and during the night. Microarray profiling of pituitary gland and testicular gene expression was performed after 8 years of treatment, using GeneChip® Rhesus Macaque Genome Arrays (Affymetrix), and showed very little effect of caloric restriction. Using a 1.5-fold difference threshold, our microarray analysis revealed differential expression of only 145 probesets in the pituitary gland and 260 in the testes, out of a total of >54,000. Semi-quantitative RT-PCR performed on pituitary gland mRNA corroborated the microarray findings for selected modulated genes, including TSH receptor (TSHR) and sperm-specific antigen 2 (SSFA2). Most notably, significantly lower expression of TSH receptor mRNA was observed in the pituitary of CR compared to CON animals. Also, significantly lower expression of the glycoprotein hormone alpha subunit (CGA) was observed in CR animals, and this finding was further corroborated using quantitative real-time RT-PCR. No significant diet-induced changes were detected in the testis for genes associated with reproduction, circadian clocks, or oxidative stress. There is mounting evidence that CR may promote health and longevity in a wide range of organisms, including nonhuman primates. Importantly, our data suggest that moderate CR has no obvious lasting detrimental effect on the reproductive neuroendocrine axis of long-lived primates, and has only a modest influence on pituitary and testicular gene expression.

Detection of circulating thyroid cancer cells in patients with thyroid microcarcinomas

Circulating thyroid cancer cells detected by peripheral blood thyroid-stimulating hormone receptor (TSHR) mRNA have demonstrated usefulness for thyroid cancer diagnosis and long-term surveillance. The aim of this study was to determine detectability and clinical importance of TSHR mRNA in patients with microcarcinomas. We compared clinical characteristics of 37 patients with papillary thyroid microcarcinomas (PTMC; tumor size </=1 cm) having undetectable (-) versus detectable (+)TSHR mRNA. 59 Of the PTMC patients, 59% had (+)TSHR mRNA levels, similar to those with tumors >1 cm (72%; P = NS) and distinctly higher than false (+) rates in benign goiters (15%; P < .001). All patients with (-)TSHR mRNA had classical PTMC, whereas variants (32%) occurred with (+)mRNA (P = .001). Mean tumor size (5 mm) and multifocality rates (45%) were similar in both mRNA groups. Of the PTMC patients, 35% had concurrent cervical nodal metastases, which occurred more frequently with tumors >/=5 mm (P = .04) and with (+)TSHR mRNA in pre-operatively known PTMC (P < .05). No patients with incidentally detected PTMC and (-)TSHR mRNA had metastases. This study is the first to demonstrate that TSHR mRNA, reflecting circulating thyroid cancer cells, is detectable even with thyroid microcarcinomas. PTMC with (+)TSHR mRNA may characterize patients with potentially more aggressive histology at initial operation.

Underexpression of PPARγ is associated with aneuploidy and lower differentiation of thyroid tumours of follicular origin

Peroxisome proliferator-activated receptor gamma (PPARgamma) gene is a nuclear receptor that is involved in thyroid tumourigenesis. Recently, our group has shown that follicular carcinomas underexpressing PPARgamma protein are more prone to develop distant metastases, to invade locally, to present poorly differentiated areas and to persist after surgery. Aneuploidy is also observed in some thyroid tumours, particularly in the more advanced cases. The aim of the present study was to investigate the association of PPARgamma expression with the degree of differentiation and ploidy status of benign and malignant thyroid neoplasias. DNA cytometric studies, ploidy and S-phase fraction (SPF) determination, and quantitative RT-PCR analysis of molecular markers specific for thyroid follicular cells, namely Tg (thyroglobulin), TSHR (TSH receptor) and NIS (Na+/I- symporter) were compared between thyroid lesions with positive or negative PPARgamma protein expression. We observed that PPARgamma-negative tissues expressed lower levels of Tg mRNA [4.66 x 10(6) a.u. (arbitrary units) +/- 1.49 x 10(6)], and were more frequently aneuploid (36%), and presented higher SPF (3.1%+/-0.4) than PPARgamma-positive samples (Tg mRNA = 2.54 x 10(7) a.u. +/- 9.72 x 10(6), P=0.0006; aneuploidy=8%, P=0.0031; SPF=2.2%+/-0.2, P=0.0430). A similar trend was also observed for TSHR and NIS mRNA, although not reaching statistical significance. This study showed that underexpression of PPARgamma is associated with poor tumour differentiation, aneuploidy and higher cell proliferative activity. Therapies designed to modulate expression of PPARgamma may have an impact on the growth of thyroid neoplasias.

Chicken folliculo-stellate cells express thyrotropin receptor mRNA

We investigated the presence of thyrotropin receptor (TSHR) mRNA in chicken pituitary and brain, and quantified the changes in its expression during the last week of embryonic development. We found that in the pituitary gland, TSHR mRNA co-localizes with folliculo-stellate cells but not with thyrotropic cells, suggesting the existence of a paracrine ultra-short thyrotropin feedback loop. TSHR mRNA was also present throughout the diencephalon and various other brain regions, which implies a more general function for thyrotropin in the avian brain. During late embryogenesis, when the activity of the hypothalamo-pituitary-thyroidal axis increases markedly, a significant rise in TSHR mRNA expression was observed in pituitary, which may signify an important change in pituitary ultra-short thyrotropin feedback regulation around the period of hatching.

Gene expression of differentiation- and dedifferentiation markers in normal and malignant human thyroid tissues.

Steady state mRNA transcript levels of thyroid differentiation markers such as TSH receptor (TSHR), thyroglobulin (Tg) and thyroid peroxidase (TPO) as well as a potential marker of dedifferentiation, c-myc, marker were investigated in patients with thyroid tumors and in normal controls using Northern blot analysis. Blots were normalized by acridine orange staining whereas analysis of beta-actin mRNA levels revealed highly variable levels already in normal tissue suggesting regulation of this "constitutively" expressed gene. Determination of c-myc mRNA revealed increased steady state mRNA levels in anaplastic carcinomas (ATC) as compared to normal tissues. However, in some patients c-myc transcript levels were lower in the tumor than in the adjacent normal tissue reducing the significance of c-myc as a marker of dedifferentiation. High levels of TSH mRNA were found in control thyroids, whereas in ATC no normal TSHR mRNA was detected. In PTC and follicular thyroid carcinomas (FTC) the transcripts varied from increased to markedly reduced levels. In one patient with FTC 2 independent preparations of the tumor revealed different results, undetectable and clearly detectable TSHR mRNA levels. Xenotransplantation of this tissue on nude rats showed a variable expression pattern in the individual xenotransplantations suggesting heterogeneity of the tumor tissue. Tg and TPO mRNA were strongly expressed in normal tissues and completely lost in all ATC. In differentiated thyroid tumors the transcript levels of Tg and TPO varied from normal to complete loss of expression of either Tg or TPO, or both.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of perfluorooctane sulfonate on rat thyroid hormone biosynthesis and metabolism

The potential toxicity of perfluorooctane sulfonate (PFOS), an environmentally persistent organic pollutant, is of great concern. The present study examines the ability of PFOS to disturb thyroid function and the possible mechanisms involved in PFOS-induced thyroid hormone alteration. Male Sprague-Dawley rats were exposed to 1.7, 5.0, and 15.0 mg/L of PFOS in drinking water for 91 consecutive days. Serum was collected for analysis of total and free thyroxine (T4), total triiodothyronine (T3), and thyrotrophin (TSH). Thyroid and liver were removed for the measurement of endpoints closely related to thyroid hormone biosynthesis and metabolism following PFOS exposure. Determined endpoints were the messenger RNA (mRNA) levels for two isoforms of uridine diphosphoglucuronosyl transferases (UGT1A6 and UGT1A1) and type 1 deiodinase (DIO1) in liver, sodium iodide symporter (NIS), TSH receptor (TSHR), and DIO1 in thyroid as well as the activity of thyroid peroxidase (TPO). Serum total T4 level decreased significantly at all applied dosages, whereas total T3 level increased markedly only at 1.7 mg/L of PFOS. No statistically significant toxic effects of PFOS on serum TSH were observed. Hepatic UGTIA1, but not UGT1A6, mRNA was up-regulated at 5.0 and 15.0 mg/L of PFOS. Treatment with PFOS lowered hepatic DIO1 mRNA at 15.0 mg/L but increased thyroidal DIO1 mRNA dose dependently. The activity of TPO, NIS, and TSHR mRNA in thyroid were unaffected by PFOS treatment. These results indicate that increased hepatic T4 glucuronidation via UGT1A1 and increased thyroidal conversion of T4 to T3 via DIO1 were responsible in part for PFOS-induced hypothyroxinemia in rats.

Expression of iodine metabolism genes in human thyroid tissues: evidence for age and BRAFV600E mutation dependency

Children present a higher susceptibility to developing thyroid cancer after radioiodine exposure and also a higher frequency of functional metastases than adults. To assess the mRNA expression of the sodium/iodide (Na(+)/I(-)) symporter (NIS), the Pendred syndrome gene (PDS), thyroperoxidase (TPO), thyroglobulin (Tg) and TSH receptor (TSH-R) in normal thyroid tissues (NTTs) and papillary thyroid carcinomas (PTCs) among different age groups. Analysis included 59 samples: 21 NTTs and 38 PTCs, of which 21 were the classic type (CPTC) and 17 the follicular variant (FVPTC). Patients were divided into three age groups: I (n = 16) 5-21 years, II (n = 13) 22-59 years, and III (n = 10) 60-91 years. The relative mRNA expression of the five target genes was determinate by quantitative reverse transcription polymerase chain reaction (QRT-PCR). Expression of all genes was significantly higher in NTTs than in PTCs, and it was not age dependent in the NTT group. Among PTCs, the mean expression of PDS, TPO and TSH-R was significantly lower in group II than in group I. PDS, TPO and Tg expression was significantly lower in classic PTCs than in FVPTCs. The difference was related to a higher frequency of the BRAF(V600E) mutation in the former group. The finding of higher PDS, TPO and TSH-R mRNA expression in paediatric vs. adult primary tumour tissues supports the hypothesis that this might contribute to the increased functional activity of metastases in the paediatric group. The finding that mRNA expression of the target genes in NTT was not age dependent does not provide an explanation for the higher susceptibility in the paediatric group.

Association of the thyroid stimulating hormone receptor gene (TSHR) with Graves' disease

Graves' disease (GD) is a common autoimmune disease (AID) that shares many of its susceptibility loci with other AIDs. The thyroid stimulating hormone receptor (TSHR) represents the primary autoantigen in GD, in which autoantibodies bind to the receptor and mimic its ligand, thyroid stimulating hormone, causing the characteristic clinical phenotype. Although early studies investigating the TSHR and GD proved inconclusive, more recently we provided convincing evidence for association of the TSHR region with disease. In the current study, we investigated a combined panel of 98 SNPs, including 70 tag SNPs, across an extended 800 kb region of the TSHR to refine association in a cohort of 768 GD subjects and 768 matched controls. In total, 28 SNPs revealed association with GD (P < 0.05), with strongest SNP associations at rs179247 (chi(2) = 32.45, P = 8.90 x 10(-8), OR = 1.53, 95% CI = 1.32-1.78) and rs12101255 (chi(2) = 30.91, P = 1.95 x 10(-7), OR = 1.55, 95% CI = 1.33-1.81), both located in intron 1 of the TSHR. Association of the most associated SNP, rs179247, was replicated in 303 GD families (P = 7.8 x 10(-4)). In addition, we provide preliminary evidence that the disease-associated genotypes of rs179247 (AA) and rs12101255 (TT) show reduced mRNA expression ratios of flTSHR relative to two alternate TSHR mRNA splice variants.

Effectiveness of Peripheral Thyrotropin Receptor mRNA in Follow-Up of Differentiated Thyroid Cancer

Thyroid cells in peripheral circulation have been linked to thyroid cancer (TC). These cells express thyrotropin receptor (TSHR) messenger RNA (mRNA), which has been studied as a marker of initial TC diagnosis. We examined the utility of TSHR mRNA in long-term follow-up of TC patients. From 2002 to 2007, TSHR mRNA was prospectively measured by quantitative reverse-transcription polymerase chain reaction (RT-PCR) from peripheral blood samples in 259 patients, and those followed > or =3 months since initial thyroidectomy were studied. TSHR mRNA levels were correlated to thyroglobulin (Tg), imaging studies, and disease status during follow-up. Thirty-four patients underwent 20 +/- 14 months median follow-up for papillary (n = 31, 91%), follicular (n = 2) or Hurthle cell (n = 1) TC. Advanced-stage disease occurred in 24% at presentation, and 11 (32%) developed cervical node metastases or recurrence requiring reoperation during follow-up. Of 52 simultaneous TSHR mRNA and serum Tg measurements, 52% were concordant. TSHR mRNA missed disease in 21% patients, but was better than Tg in 27%, including all those with Tg antibodies. TSHR mRNA concurred with whole-body scan detectable disease for 11/14 patients (79%) and accurately predicted overall TC disease status in 77% patients. In discordant cases, TC recurrence was apparent from other imaging modalities [positron emission tomography (PET) scan or ultrasound]. TSHR mRNA in conjunction with Tg diagnosed TC recurrence with 90% sensitivity and 94% specificity. We conclude that TSHR mRNA demonstrates high concordance rates with present methods of detecting TC recurrence, and appears to be more accurate in patients with Tg antibodies. As a novel adjunct, TSHR mRNA may enhance long-term management of TC patients.

Role of the transgenic human thyrotropin receptor A-subunit in thyroiditis induced by A-subunit immunization and regulatory T cell depletion

Transgenic BALB/c mice that express intrathyroidal human thyroid stimulating hormone receptor (TSHR) A-subunit, unlike wild-type (WT) littermates, develop thyroid lymphocytic infiltration and spreading to other thyroid autoantigens after T regulatory cell (T(reg)) depletion and immunization with human thyrotropin receptor (hTSHR) adenovirus. To determine if this process involves intramolecular epitope spreading, we studied antibody and T cell recognition of TSHR ectodomain peptides (A-Z). In transgenic and WT mice, regardless of T(reg) depletion, TSHR antibodies bound predominantly to N-terminal peptide A and much less to a few downstream peptides. After T(reg) depletion, splenocytes from WT mice responded to peptides C, D and J (all in the A-subunit), but transgenic splenocytes recognized only peptide D. Because CD4(+) T cells are critical for thyroid lymphocytic infiltration, amino acid sequences of these peptides were examined for in silico binding to BALB/c major histocompatibility complex class II (IA-d). High affinity subsequences (inhibitory concentration of 50% < 50 nm) are present in peptides C and D (not J) of the hTSHR and mouse TSHR equivalents. These data probably explain why transgenic splenocytes do not recognize peptide J. Mouse TSHR mRNA levels are comparable in transgenic and WT thyroids, but only transgenics have human A-subunit mRNA. Transgenic mice can present mouse TSHR and human A-subunit-derived peptides. However, WT mice can present only mouse TSHR, and two to four amino acid species differences may preclude recognition by CD4+ T cells activated by hTSHR-adenovirus. Overall, thyroid lymphocytic infiltration in the transgenic mice is unrelated to epitopic spreading but involves human A-subunit peptides for recognition by T cells activated using the hTSHR.