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Feline hyperthyroidism (FHT) is a debilitating disease affecting > 10% of elderly cats. It is generally characterised by chronic elevation of thyroid hormone in the absence of circulating TSH. Understanding of the molecular pathogenesis of FHT is currently limited. However, FHT shares clinical and histopathological similarities with human toxic multinodular goitre, which has been associated with activating mutations in TSH receptor (TSHR) and G s α encoding genes. Using RNA-seq transcriptomic analysis of thyroid tissue from hyperthyroid and euthyroid cats, we identified differentially expressed genes and dysregulated pathways in FHT, many of which are downstream of TSHR. In addition, we detected missense variants in thyroid RNA-seq reads that alter the structure of both TSHR and G s α. All FHT-associated mutations were absent in germline sequence from paired blood samples. Only a small number of hyperthyroid cats demonstrated TSHR variation, however all thyroids from advanced cases of FHT carried at least one missense variant affecting G s α. The activating nature of the acquired G s α mutations was demonstrated by increased cAMP production in vitro. These data indicate that constitutive activation of signalling downstream of TSHR is central to the TSH-independent production of thyroid hormone in FHT, offering a novel therapeutic target pathway in this common disease., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Dysfunction of CD8 + T cells in the tumor microenvironment (TME) contributes to tumor immune escape and immunotherapy tolerance. The effects of hormones such as leptin, steroid hormones, and glucocorticoids on T cell function have been reported previously. However, the mechanism underlying thyroid-stimulating hormone (TSH)/thyroid-stimulating hormone receptor (TSHR) signaling in CD8 + T cell exhaustion and tumor immune evasion remain poorly understood. This study was aimed at investigating the effects of TSH/TSHR signaling on the function of CD8 + T cells and immune evasion in colorectal cancer (CRC)., Methods: TSHR expression levels in CD8 + T cells were assessed with immunofluorescence and flow cytometry. Functional investigations involved manipulation of TSHR expression in cellular and mouse models to study its role in CD8 + T cells. Mechanistic insights were mainly gained through RNA-sequencing, Western blotting, chromatin immunoprecipitation and luciferase activity assay. Immunofluorescence, flow cytometry and Western blotting were used to investigate the source of TSH and TSHR in CRC tissues., Results: TSHR was highly expressed in cancer cells and CD8 + T cells in CRC tissues. TSH/TSHR signaling was identified as the intrinsic pathway promoting CD8 + T cell exhaustion. Conditional deletion of TSHR in CD8 + tumor-infiltrating lymphocytes (TILs) improved effector differentiation and suppressed the expression of immune checkpoint receptors such as programmed cell death 1 (PD-1) and hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3) through the protein kinase A (PKA)/cAMP-response element binding protein (CREB) signaling pathway. CRC cells secreted TSHR via exosomes to increase the TSHR level in CD8 + T cells, resulting in immunosuppression in the TME. Myeloid-derived suppressor cells (MDSCs) was the main source of TSH within the TME. Low expression of TSHR in CRC was a predictor of immunotherapy response., Conclusions: The present findings highlighted the role of endogenous TSH/TSHR signaling in CD8 + T cell exhaustion and immune evasion in CRC. TSHR may be suitable as a predictive and therapeutic biomarker in CRC immunotherapy., (© 2024 The Author(s). Cancer Communications published by John Wiley & Sons Australia, Ltd on behalf of Sun Yat‐sen University Cancer Center.)

The expression of thyroid-stimulating hormone receptor (TSHR) has been documented on various immune cells, including B lymphocytes, T lymphocytes, Natural Killer (NK) cells, monocytes, and dendritic cells (DCs). Natural Killer T (NKT) cells serve as a crucial link between innate and adaptive immunity, playing significant roles in immunological interactions and autoimmune diseases. The aim of the present study was to evaluate the presence of TSHR on NKT cells. Our research involved patients with thyroid disease, as well as healthy controls. Peripheral blood mononuclear cells (PBMCs) and, thereafter, NKT cells were isolated from 86 patients with benign nodular thyroid disease with and without autoimmune thyroid disease (AITD) (28 and 56 cases, respectively), and TSHR expression was analyzed using fluorescence-activated cell sorting (FACS). In order to confirm the results, the reverse-transcription polymerase chain reaction (RT-PCR) method was used in cells obtained from healthy individuals. Our findings obtained with application of the FACS method revealed that TSHR is not expressed on NKT cells in either AITD or non-AITD patients, though TSHR was detected in the total PBMC population (TSHR+ cells 2.77%). The absence of TSHR on NKT cells was further confirmed with RT-PCR in healthy individuals ( p < 0.0001). These results questioned the previously suggested direct influence of NKT cells on AITD development.

Extra-thyroidal effects of TSH have been reported in various tissues expressing the TSH receptor (TSHR) including several areas of the brain. However, the influence of TSH on neuronal phenotypes has not been examined. Using a well-characterized human neuroblastoma cell line (SH-SY5Y), we have examined TSH signaling effects on the phenotype of these cells after their neuronal differentiation. Following an 18-day differentiation protocol, we successfully redifferentiated the SH-SY5Y cells into ~100% neuronal cells as indicated by the development of extensive neurofilaments with SMI-31 expression. Furthermore, using absolute digital PCR, we quantified TSHR mRNA, and also TSHR protein expression, in the redifferentiated cells and found that the neuronal cells expressed high quantities of both TSHR message and protein at baseline. Exposure to TSH induced primary, secondary, and tertiary neurite outgrowths, which are essential for cell-cell communication. Quantitative analysis of neurites using ImageJ showed a dose-dependent increase in neurites. The addition of TSH up to 1 mU/ml resulted in a ~2.5-fold increase in primary, and ~1.5-fold in secondary and tertiary neurites. The lengths of the neurites remained unaffected with the dosage of TSH treatment. Furthermore, TSHR signaling in the differentiated cells resulted in enhanced generation of cAMP, pPI3K, pAKT, and pNFkB pathways and suppression of pMAPK suggesting an influence of these signals in driving neurite outgrowth. These data showed that the TSH/TSHR axis in neurons may contribute to enhanced neurite outgrowth. The potential pathophysiological effects of TSH on the induction of neurite outgrowth and its relationship to neurodegenerative diseases remain to be explored., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Mansoori, Latif, Morshed, Zaidi and Davies.)

Purpose: The study aimed to establish a mouse model of Graves' disease (GD) with Graves' orbitopathy (GO; GD + GO) that can represent the clinical disease characteristics., Methods: A eukaryotic expression plasmid of insulin-like growth factor 1 receptor (IGF-1R) α subunit (pcDNA3.1/IGF-1Rα) and a thyrotropin receptor (TSHR) A subunit plasmid (pcDNA3.1/TSHR-289) were injected in female BALB/c mice followed by immediate electroporation to induce a GD + GO model. Grouping was performed according to the frequency of injection (2- to 4-week intervals) and type of injected plasmids: T: pcDNA3.1/TSHR-289( +), I: pcDNA3.1/IGF-1Rα( +), or co-injection T + I: pcDNA3.1/TSHR-289( +) and pcDNA3.1/IGF-1Rα( +). Serum TSH, T4, TSAb, TSBAb, body weight, and blood glucose levels were evaluated. Thyroid 99m TcO 4- imaging and retrobulbar magnetic resonance imaging (MRI) were performed, and bilateral eye muscle volumes were measured. Immunohistochemistry and hematoxylin-eosin staining were performed on the relevant tissues, and semi-quantitative analysis was performed., Results: A total of 60% of mice (3/5, one mouse died) in the T group developed GD + GO. In the T + I group, 83.3% of mice (5/6) developed GD + GO. Mice in the I group did not develop GD. Compared with the control group, serum T4, TSAb, and TSBAb of the mice in the GD + GO model groups were increased to varying degrees (P < 0.05), and serum TSH and body weight were significantly lower compared to the control group (P < 0.05). The thyroid uptake capacity of 99m TcO 4- and the volume of eye muscle of mice in the GD + GO group were significantly higher compared to the control group (P < 0.05). The thyroid and retrobulbar muscles of these mice showed varying inflammatory infiltration and interstitial muscle edema. The severity of GD + GO in the co-injection group was not related to injection frequency; however, GD and ocular signs in co-injection mice were more severe compared to the T group., Conclusions: We successfully induced a GD + GO mouse model by a repeated co-injection of pcDNA3.1/IGF-1Rα and pcDNA3.1/TSHR-289 plasmids. Injection of pcDNA3.1/IGF-1Rα alone failed to induce GD. Co-injection of two plasmids induced more severe GD + GO than pcDNA3.1/TSHR-289( +) alone., (© 2024. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

Genetic defects in the TSH receptor ( TSHR ) can cause poor thyroid differentiation (thyroid dysgenesis) and/or thyroid malfunction (thyroid dyshormonogenesis). The phenotype spectrum is wide: from severe congenital hypothyroidism to mild hyperthyrotropinemia. Over 250 TSHR variants have been published, many uncharacterized in vitro. We aimed to genetically characterize patients with thyroid dyshormonogenesis with TSHR defects and to study in vitro the effect of the genetic variants to establish the genotype-phenotype relationship. Pediatric patients with thyroid dyshormonogenesis (160 patients, Catalan CH neonatal screening program, confirmation TSH range: 18.4-100 mIU/L), were analyzed by a high-throughput gene panel. In vitro studies measuring the TSH-dependent cAMP-response-element activation were performed. Five patients with mild or severe thyroid dyshormonogenesis presented six TSHR variants, two unpublished. Each variant showed a different in vitro functional profile that was totally or partially deleterious. Depending on the genotype, some of the variants showed partial deficiency in both genotypes, whereas others presented a different effect. In conclusion, the percentage of patients with thyroid dyshormonogenesis and candidate variants in TSHR is 3.13%. Our in vitro studies contributed to the confirmation of the pathogenicity of the variants and highlighted the importance of studying the effect of the patient's genotype for a correct diagnostic confirmation.

Thyroid stimulating hormone (TSH), a glycoprotein synthesized and secreted from thyrotrophs of the pituitary gland, is composed of a glycoprotein hormone common alpha subunit (CGA) and a specific beta subunit (TSHB). The major biological function of TSH is to stimulate thyroidal follicles to synthesize and secrete thyroid hormones through activating its cognate receptor, the thyroid stimulating hormone receptor (TSHR). In the present study, polyclonal antisera against ricefield eel Tshb and Tshr were generated respectively, and the expression of Tshb and Tshr was examined at mRNA and protein levels. RT-PCR analysis showed that tshb mRNA was expressed mainly in the pituitary as well as in some extrapituitary tissues including the ovary and testis. Tshr mRNA was also expressed in a tissue-specific manner, with transcripts detected in tissues including the kidney, ovary, and testis. The immunoreactive Tshb signals in the pituitary were shown to be localized to the inner areas of adenohypophysis which are close to the neurohypophysis of adult ricefield eels. Tshb-immunoreatvie cells in the pituitary of ricefield eel larvae were firstly observed at hatching. The expression of immunoreactive Tshb and Cga was also detected in ricefield eel ovary and testis together with Tshr. In the ovary, immunoreactive Tshb, Cga, and Tshr were observed in oocytes and granulosa cells. In the testis, immunoreactive Tshb was mainly observed in Sertoli cells while immunoreactive Cga and Tshr were detected in germ cells as well as somatic cells. Results of the present study suggest that Tsh may be synthesized both in the ovary and testis locally, which may play paracrine and/or autocrine roles in gonadal development in ricefield eels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

One of the sensitive markers for autoimmune thyroid disease (AITD) clinical identification is thyroid-stimulating hormone receptor antibodies (TRAbs). To quickly distinguish TRAb with distinct antigenic epitopes, a straightforward and uncomplicated technique has not yet been created. The objective of this study is to search for molecular diagnostic targets for different types of AITD {Graves' disease (GD), Graves' orbitopathy (GO), GD with third-degree goiter [GD(3)], hypothyroidism combined with positive TRAb [HT(TRAb+)]} as molecular diagnostic targets. Following action on thyroid cells, differential genes (DEGs) generated by TRAb with distinct antigenic epitopes were detected and identified by RNA sequencing (RNA-Seq), bioinformatics analysis, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) in the serum of patients with AITD. Using the 5-ethynyl-2'-deoxyuridine (EdU) assay, the effect of coculturing thyroid cells with different antigenic TRAb epitopes on the cells' capacity to proliferate was investigated. Bioinformatics analysis and RT-qPCR validation identified one GD key gene alpha 2-HS glycoprotein ( AHSG ), two GO key genes [adrenoceptor alpha 1D ( ADRA1D ) and H2B clustered histone 18 ( H2BC18 )], two GD(3) key genes [suppressor of cytokine signaling 1 ( SOCS1) and cytochrome b-245 beta ( CYBB )], and one HT(TRAb+) key gene (MASP2). Correlation analysis and ROC curves showed that the abovementioned genes could be used as molecular diagnostic targets for different types of AITD. Finally, EdU results showed that TRAb inhibited thyroid cell proliferation in the HT(TRAb+) group compared with the normal control group, whereas the remaining three groups promoted thyroid cell proliferation, with a statistically significant difference ( P < 0.05). We identified six key genes for different types of AITD, which have diagnostic value for different types of AITD. Meanwhile, we found that TRAbs with different antigenic epitopes in AITD have different biological functions. NEW & NOTEWORTHY We identified six molecular targets of different types of AITD [GD, GO, GD(3), and HT(TRAb+)], which have diagnostic value for different types of AITD. Meanwhile, we found that TRAb with different antigenic epitopes extracted from the sera of patients with AITD had different biological functions, which also provided a new idea for further research on the mechanism of action of TRAb with different antigenic epitopes in AITD.

Background: Early diagnosis of thyroid cancer is hampered by the inability of fine-needle aspiration biopsy (FNAB) to accurately classify ∼30% of cases while preoperative cancer staging detects lymph nodal involvement in only half of cases. Liquid biopsy may present an accurate, non-invasive alternative for preoperative thyroid nodule assessment. Thyrotropin receptor (TSHR) mRNA, a surrogate marker for circulating cancer cells (CTC), may be an option for early detection of malignancy from peripheral blood, but requires methodological improvements. We aimed to investigate if TSHR mRNA can be detected in low sample volumes by employing an ultrasensitive method - droplet digital PCR (ddPCR)., Methods: Less than 5 mL of blood was collected from 47 patients with thyroid nodules (25 benign and 22 malignant). RNA was isolated from the fraction of mononuclear cells where CTCs segregate. Samples were analysed for the presence of TSHR mRNA by ddPCR., Results: Thyrotropin receptor mRNA was detectable in 4 mL sample volumes, with the test having good specificity (80%) but modest diagnostic accuracy (68.1%). Combining TSHR mRNA with ultrasound features and FNAB diagnosis, the test reaches high rule-out performances (sensitivity = 90% and NPV = 88.2%). Strikingly, TSHR mRNA correctly classified all samples with thyroid capsule invasion, lymph node metastasis and extrathyroidal extension. If aggressiveness is defined using these parameters, TSHR mRNA test reaches 100% sensitivity and 100% NPV for detecting high-risk cases., Conclusions: Employing ddPCR for TSHR mRNA improves its measurement by enabling detection in sample volumes common for laboratory testing. The test displays high prognostic performance, showing potential in preoperative risk assessment., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Thyroid cancer is the most common endocrine cancer, with differentiated thyroid cancers (DTCs) accounting for 95% of diagnoses. While most DTC patients are diagnosed and treated with radioiodine (RAI), up to 20% of DTC patients become RAI refractory (RAI-R). RAI-R patients have significantly reduced survival rates compared to patients who remain RAI-avid. This study explores [ 89 Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [ 89 Zr]Zr-TR1402 was synthesized with a molar activity of 25.9 MBq/nmol by conjugating recombinant human TSH (rhTSH) analogue TR1402 to chelator p-SCN-Bn-deferoxamine (DFO) in a molar ratio of 3:1 (DFO/TR1402) and radiolabeling with 89 Zr ( t 1/2 = 78.4 h, β + = 22.7%). As TSHR is absent in commonly available DTC-derived cell lines, TSHR was reintroduced via stable transduction by delivering a lentivirus containing the full-length coding region of the human TSHR gene. Receptor-mediated uptake of [ 89 Zr]Zr-TR1402 was evaluated in vitro in stably transduced TSHR+ and wild-type TSHR- DTC cell lines. In vivo PET imaging was performed on Days 1-3 postinjection in male and female athymic nude mice bearing TSHR+ and TSHR- xenografts, along with ex vivo biodistribution on Day 3 postinjection. In vitro uptake of 1 nM [ 89 Zr]Zr-TR1402 was significantly higher in TSHR+ THJ529T ( P < 0.0001) and FTC133 ( P < 0.01) cells than in TSHR- THJ529T and FTC133 cells. This uptake was shown to be specific in both TSHR+ THJ529T ( P < 0.0001) and TSHR+ FTC133 ( P < 0.0001) cells by blocking uptake with 250 nm DFO-TR1402. In vivo PET imaging showed accumulation of [ 89 Zr]Zr-TR1402 in TSHR+ tumors, which was the highest on Day 1. In the male FTC133 xenograft model, ex vivo biodistribution confirmed a significant difference ( P < 0.001) in uptake between FTC133+ (1.3 ± 0.1%ID/g) and FTC133- (0.8 ± 0.1%ID/g) tumors. A significant difference ( P < 0.05) in uptake was also seen in the male THJ529T xenograft model between THJ529T+ (1.8 ± 0.6%ID/g) and THJ529T- (0.8 ± 0.4%ID/g) tumors. The in vitro and in vivo accumulation of [ 89 Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.

Backgruound: Both Graves' disease (GD) and Hashimoto's thyroiditis (HT) are classified as autoimmune thyroid diseases (AITDs). It has been hypothesized that changes in the thyroid-stimulating hormone receptor (TSHR) gene may contribute to the development of these conditions. This study aimed to analyze the correlation between the TSHR rs179247 gene polymorphism and susceptibility to AITD., Methods: We conducted a thorough search of the Google Scholar, Scopus, Medline, and Cochrane Library databases up until March 2, 2024, utilizing a combination of relevant keywords. This review examines data on the association between TSHR rs179247 and susceptibility to AITD. Random-effect models were employed to assess the odds ratio (OR), and the findings are presented along with their respective 95% confidence intervals (CIs)., Results: The meta-analysis included 12 studies. All genetic models of the TSHR rs179247 gene polymorphism were associated with an increased risk of developing GD. Specifically, the associations were observed in the dominant model (OR, 1.65; P<0.00001), recessive model (OR, 1.65; P<0.00001), as well as for the AA genotype (OR, 2.09; P<0.00001), AG genotype (OR, 1.39; P<0.00001), and A allele (OR, 1.44; P<0.00001). Further regression analysis revealed that these associations were consistent regardless of the country of origin, sample size, age, and sex distribution. However, no association was found between TSHR rs179247 and the risk of HT across all genetic models., Conclusion: This study suggests that the TSHR rs179247 gene polymorphism is associated with an increased risk of GD, but not with HT, and may therefore serve as a potential biomarker.

This study aimed to investigate the expression of microRNAs (miRNAs) -146b-3p, -221-5p, -222-3p, and -21a-3p and the methylation pattern of the thyroid-stimulating hormone receptor ( TSHR ) gene in blood plasma samples from papillary thyroid cancer (PTC) patients before and after thyroidectomy compared to healthy controls (HCs). This study included 103 participants, 46 PTC patients and 57 HCs, matched for gender and age. Significantly higher preoperative expression levels of miRNAs and TSHR methylation were determined in the PTC patients compared to HCs. Post-surgery, there was a notable decrease in these biomarkers. Elevated TSHR methylation was linked to larger tumor sizes and lymphovascular invasion, while increased miRNA-222-3p levels correlated with multifocality. Receiver operating characteristic (ROC) analysis showed AUCs below 0.8 for all candidate biomarkers. However, significant changes in the expression of all analyzed miRNAs and TSHR methylation levels indicate their potential to differentiate PTC patients from healthy individuals. These findings suggest that miRNAs and TSHR methylation levels may serve as candidate biomarkers for early diagnosis and monitoring of PTC, with the potential to distinguish PTC patients from healthy individuals. Further research is needed to validate these biomarkers for clinical application.

Thyroid dyshormonogenesis (TDH) is responsible for 15%-25% of congenital hypothyroidism (CH) cases. Pathogenetic variants of this common inherited endocrine disorders vary geographically. Unraveling the genetic underpinnings of TDH is essential for genetic counseling and precise therapeutic strategies. This study aims to identify genetic variants associated with TDH in Southern Taiwan using whole exome sequencing (WES). We included CH patients diagnosed through newborn screening at a tertiary medical center from 2011 to 2022. Permanent TDH was determined based on imaging evidence of bilateral thyroid structure and the requirement for continuous medication beyond 3 years of age. Genomic DNA extracted from blood was used for exome library construction, and pathogenic variants were detected using an in-house algorithm. Of the 876 CH patients reviewed, 121 were classified as permanent, with 47 (40%) confirmed as TDH. WES was conducted for 45 patients, and causative variants were identified in 32 patients (71.1%), including DUOX2 (15 cases), TG (8 cases), TSHR (7 cases), TPO (5 cases), and DUOXA2 (1 case). Recurrent variants included DUOX2 c.3329G>A, TSHR c.1349G>A, TG c.1348delT, and TPO c.2268dupT. We identified four novel variants based on genotype, including TSHR c.1135C>T, TSHR c.1349G>C, TG c.2461delA, and TG c.2459T>A. This study underscores the efficacy of WES in providing definitive molecular diagnoses for TDH. Molecular diagnoses are instrumental in genetic counseling, formulating treatment, and developing management strategies. Future research integrating larger population cohorts is vital to further elucidate the genetic landscape of TDH., (© 2024 The Author(s). The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Congenital hypothyroidism (CHT) is a diverse condition with various genetic etiologies. This study aimed to investigate the utility of next-generation sequencing (NGS) analysis in guiding treatment decisions and predicting prognosis for CHT patients with gland in situ (GIS). A retrospective analysis was conducted on 33 CHT patients with GIS who underwent NGS analysis at a single institution between 2018 and 2023. Patients were classified as having permanent (PCH), transient congenital hypothyroidism, or ambiguous congenital hypothyroidism (ACH) CHT based on their response to levothyroxine discontinuation at 3 years of age. Among the 33 patients, genetic variants were identified in 26, with the most prevalent variants found in DUOX2 (26.92%), TSHR (30.77%), TG (19.35%), and DUOXA2 (19.23%). Patients with high initial thyroid-stimulating hormone levels (>50 mIU/L) and low free thyroxine levels (<0.89 ng/dL) at diagnosis tended to have compound heterozygous or homozygous variants in DUOX2, DUOXA2, and TG, and were more likely to develop PCH. In contrast, patients with heterozygous variants in these genes often exhibited ACH. TSHR variants were associated with diverse clinical manifestations, ranging from PCH to ACH, and were more common in patients with initial thyroid-stimulating hormone levels <50 mIU/L. The study highlights the potential utility of NGS analysis in predicting the clinical course and guiding treatment decisions for CHT patients with GIS. Genetic analysis may aid in determining the appropriate duration of levothyroxine therapy and monitoring strategies, particularly in cases where traditional clinical indicators are inconclusive., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

The clinical spectrum of thyrotropin receptor-mediated (TSHR-mediated) diseases varies from loss-of-function mutations causing congenital hypothyroidism to constitutively active mutations (CAMs) leading to nonautoimmune hyperthyroidism (NAH). Variation at the TSHR locus has also been associated with altered lipid and bone metabolism and autoimmune thyroid diseases. However, the extrathyroidal roles of TSHR and the mechanisms underlying phenotypic variability among TSHR-mediated diseases remain unclear. Here we identified and characterized TSHR variants and factors involved in phenotypic variability in different patient cohorts, the FinnGen database, and a mouse model. TSHR CAMs were found in all 16 patients with NAH, with 1 CAM in an unexpected location in the extracellular leucine-rich repeat domain (p.S237N) and another in the transmembrane domain (p.I640V) in 2 families with distinct hyperthyroid phenotypes. In addition, screening of the FinnGen database revealed rare functional variants as well as distinct common noncoding TSHR SNPs significantly associated with thyroid phenotypes, but there was no other significant association between TSHR variants and more than 2,000 nonthyroid disease endpoints. Finally, our TSHR M453T-knockin model revealed that the phenotype was dependent on the mutation's signaling properties and was ameliorated by increased iodine intake. In summary, our data show that TSHR-mediated disease risk can be modified by variants at the TSHR locus both inside and outside the coding region as well as by altered TSHR-signaling and dietary iodine, supporting the need for personalized treatment strategies.

The possibility that thyrotropin receptor (TSHR) expression in non-thyroid tissue is well-documented. However, there is insufficient data on the expression of TSHR in medulla oblongata regions, particularly when focusing on the background of encephalopathy associated with hyperthyroidism. In this study, we explored the expression of the functional TSHR in Graves' disease (GD) mouse cerebral vascular endothelial cells and the effects of thyrotropin receptor autoantibody (TRAb) on its expression. A mouse model of GD was constructed with an adenovirus overexpressing TSHR289. The location and expression of the TSHR gene and protein in vivo were determined via RT-qPCR, Western blot, and immunofluorescence techniques. The effect of TRAb on the expression of functional TSHR in vitro was investigated using bEnd.3 cells. Our results show that medulla oblongata vascular endothelial cells from GD mice expressed higher levels of TSHR compared to control mice. In an in vitro experiment, novel results demonstrated that after treatment with a monoclonal TSHR-specific agonistic antibody (M22), the expression of TSHR on the bEnd.3 cells increased at both the protein and mRNA levels. Furthermore, compared with bEnd.3 cells were treated with IBMX only, those treated with M22 showed increased cAMP production. This study suggested that TSHR is expressed and functionally active in the mouse medulla oblongata and in vitro-cultured bEnd.3 cells and TRAb (M22) increased the expression of TSHR on bEnd.3 cells.

The thyroid stimulating hormone receptor ( TSHR ), a glycoprotein hormone receptor, plays an important role in metabolic regulation and photoperiod control in the time of reproduction in birds and mammals. Previous genome-wide association studies revealed that the TSHR gene was related to reproduction and its function was identified in female reproduction, but rare studies reported the polymorphism of TSHR gene. However, the molecular mutations of the TSHR gene in sheep have not been reported so far. Herein, we explored potential polymorphisms of the sheep TSHR gene, and a 29 bp nucleotide sequence variant (rs1089565492) was identified in the AUW sheep. There were three genotypes of the 29 bp variant locus detected which named 'II' 'DD' and 'ID' been identified. Association analysis results showed the 29 bp variant was significantly associated with the litter size of the AUW sheep ( p  < 0.05). This finding suggests that the 29 bp nucleotide sequence variant within TSHR gene could be a candidate marker of reproduction traits for sheep breeding improving through the marker-assisted selection (MAS).

Objectives: To evaluate the frequency and risk of malignancy of TSHRpI568T mutations discovered in indeterminate thyroid nodules (ITN) within the Veracyte CLIA laboratory undergoing Afirma ® Genomic Sequencing Classifier (GSC) testing, and to evaluate a broader cohort of TSHR variants and their categorization as Afirma GSC benign (GSC-B) or suspicious (GSC-S). Finally, we seek to assess the risk of malignancy (ROM) of this group of TSHR mutated ITN in the GSC-S category., Methods: ITN submitted to Veracyte for Afirma GSC testing between October 2017 and February 2022 were analyzed for TSHR variants and rates of GSC-B and GSC-S were calculated based upon BIII or IV cytology, by TSHR variant codon amino acid (AA) substitution, age, and gender. For GSC-S samples, surgical pathology reports were requested, and the rate of malignancy was calculated., Results: Five percent of the ITN samples harbored an isolated TSHR variant and 5% of those were classified as GSC-S. Among TSHRpI568T samples, 96% were GSC-B and of the GSC-S samples, 21% were malignant. Among an unselected group of TSHR, absent TSHRpI568T mutations, 16.3% of GSC-S samples were malignant, all but one with codon mutations in the transmembrane subdomains of the TSHR. This prompted a dedicated evaluation of transmembrane codons which revealed a malignancy rate of 10.7% among GSC-S nodules. In total, 13/85 (15.3%) TSHR mutated ITN with Afirma GSC-S results were found to be malignant., Conclusions: TSHR variants are rare in ITN, and most are categorized as benign under Afirma GSC testing which carries a < 4% risk of malignancy. For GSC-S ITN with TSHR mutations, the risk of malignancy is ≥= 15%, which is clinically meaningful and may alter treatment or monitoring recommendations for patients., Competing Interests: SW, YH, JH, JPK, RTK, and GCK are employees and equity owners of Veracyte, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Whitmer, Phay, Holt, O’Donnell, Nguyen, Joseph, Chi, Wu, Hao, Huang, Klopper, Kloos, Kennedy and Shin.)

Proximity ligation assay (PLA) is a methodology that permits detection of protein-protein closeness, that is, proteins that are within 40 nanometers of each other, in cells or tissues at endogenous protein levels or after exogenous overexpression. It detects the protein(s) with high sensitivity and specificity because it employs a DNA hybridization step followed by DNA amplification. PLA has been used successfully with many types of proteins. In this methods paper, we will describe the workings of PLA and provide examples of its use to study TSH/IGF-1 receptor crosstalk in Graves' orbital fibroblasts (GOFs) and TSH receptor homodimerization in primary cultures of human thyrocytes., Competing Interests: MCG is a consultant with Crosstalk Therapeutics LTD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 Krieger, Boutin, Neumann and Gershengorn.)

The development of low-molecular-weight antagonists of thyroid-stimulating hormone (TSH) receptor is a promising trend in the treatment of autoimmune hyperthyroidism. We studied the effect of thieno[2,3-d]-pyrimidine derivative TPY1 on TSH-stimulated synthesis of thyroid hormones in the culture of FRTL-5 thyrocytes and on thyroliberin-stimulated production of thyroid hormones in rat blood. Preincubation of FRTL-5 cells with TPY1 suppressed the stimulatory effect of TSH on the synthesis of thyroxine and triiodothyronine. Intraperitoneal injection of TPY1 in a dose of 25 mg/kg reduced thyroliberin-stimulated levels of thyroid hormones in the blood and inhibited the expression of genes encoding thyroid peroxidase, thyroglobulin, and Na + /I- cotransporter responsible for thyroxine synthesis. In the absence of thyroliberin stimulation, TPY1 did not affect the levels of thyroid hormones and expression of thyroidogenesis genes. Thus, a new TPY1 antagonist of TSH receptor can be a prototype of a drug for the treatment of autoimmune hyperthyroidism., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

We aimed to investigate the relationship between the effects excessive of fluoride on thyroid health in children and the moderating role of thyroid stimulating hormone receptor (TSHR) or protein tyrosine phosphatase nonreceptor-22 (PTPN22) gene polymorphisms. Four hundred thirteen children (141 with dental fluorosis and 198 boys) were enrolled from both historical endemic and non-endemic areas of fluorosis in Tianjin, China. The fluoride exposure levels, thyroid health indicators, and TSHR (rs2268458) and PTPN22 (rs3765598) polymorphisms were examined. Multiple logistic models were applied to evaluate the relationship between dental fluorosis and thyroid abnormalities. Children over 9 year old with dental fluorosis have lower FT 4 and TGAb levels and thyroid volume and higher TPOAb levels (all P < 0.05). In overall participants, children with dental fluorosis were more likely to have thyroid antibody single positive issues (adjusted P = 0.039) and less likely to have a goiter according to age or body surface area (age or BSA) (adjusted P = 0.003); In the TSHR (rs2268458) SNP = CC/CT or PTPN22 (rs3765598) SNP = CC subgroup, dental fluorosis may cause thyroid antibody single positive (adjusted P = 0.036; adjusted P = 0.002); in the TSHR (rs2268458) SNP = TT or PTPN22 (rs3765598) SNP = CC subgroup, dental fluorosis may protect children from goiter (age or BSA) (adjusted P = 0.018; adjusted P = 0.013). Excessive fluoride may induce thyroid antibody single positive and reduce goiter in children. Heterogeneity exists in the relationship between excessive fluoride and thyroid antibody single positive or goiter issues across children carrying different TSHR (rs2268458) or PTPN22 (rs3765598) genotypes., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Thyroid-stimulating hormone receptor (TSHR) is widely expressed in human tissues and cells. TSHR is not only involved in thyroid disease but also in the neuroendocrine-immune regulatory network. However, no study has exclusively focused on the expression and function of TSHR in natural killer (NK) cells., Methods: We studied TSHR expression using reverse transcription PCR to verify TSHR mRNA transcripts in human and mouse NK cells. Human and mouse thyroid and liver tissues as well as peripheral blood mononuclear cells (PBMCs) or spleen lymphoid cells (SLCs) were used as controls. The TSHR protein levels in NK-92 cells were determined by immunofluorescence staining. The function of TSHR in NK cells was investigated by measuring the TSH-stimulated cAMP levels., Results: TSHR mRNA was detected in human and mouse NK cells as well as in NK-92 cells and had the same sequence as that of thyroid-derived, PBMC-derived, and liver-derived mRNA. The TSHR protein was also expressed in the cell membrane of NK-92 cells. Furthermore, the cAMP levels in NK-92 cells were significantly higher after adding 102 mIU/mL of bovine TSH at p < 0.05, which stimulated cAMP production in NK-92 cells., Conclusions: Our findings confirm that TSHR is present and functional in NK cells and provide key clues for the potential regulatory effects of TSH on TSHR in NK cells in the immune system., (© 2021 S. Karger AG, Basel.)

The thyroid-stimulating hormone receptor (TSHR) is a Class A G protein-coupled receptor (GPCR) that mediates signalling through the hypothalamic-pituitary-thyroid axis. Inappropriate activation of TSHR by autoantibodies or mutations, results in human disease such as Grave's disease and Hashimito's thyroiditis. Therefore, there is a need to develop novel therapeutics targeting the TSHR. Understanding the structure and mechanism of activation of this receptor would help elucidate the pathogenesis of disease and aid drug development. Here, we describe a method for the expression of the human TSHR in a mammalian cell line generated through a lentiviral expression system. The receptor is then purified by affinity chromatography in the ligand-free state and is suitable for structure determination by single-particle electron cryo-microscopy (cryo-EM)., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Hormones are identified as key biological variables in tumor immunity. However, previous researches mainly focused on the immune effect of steroid hormones, while the roles that thyroid-stimulating hormone (TSH) played in the antitumor response were far from clear., Methods: The source of TSH was determined using single-cell transcriptomic, histologic, quantitative PCR, and ELISA analysis. The influence of TSH on tumor proliferation, invasion, and immune evasion was evaluated in multiple cell lines of thyroid cancer, glioma, and breast cancer. Then transcriptomic sequencing and cellular experiments were used to identify signaling pathways. TSH receptor (TSHR) inhibitor was injected into homograft mouse tumor models with or without anti-programmed cell death protein-1 antibody., Results: Monocyte-derived dendritic cells (moDCs) highly expressed TSHα and TSHβ2 and were the primary source of TSH in the tumor microenvironment. TSH released by moDCs promoted proliferation and invasion of tumors with high TSHR expressions, such as thyroid cancers and glioma. TSH also induced tumor programmed death-ligand 1 (PD-L1) expression through the TSHR-AC-PKA-JNK-c-JUN pathway. TSHR inhibitors reversed tumor immune evasion by inhibiting PD-L1 expression in tumor and myeloid cells and enhancing Teff activation., Conclusions: TSH-TSHR axis promotes tumor evasion in thyroid cancers and glioma. TSH suppression therapy is an effective therapeutic strategy for combination in immune checkpoint blockades., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: This study was designed to determine the value of miR-205-5p and thyroid-stimulating hormone receptor (TSHR) mRNA in differentiating benign thyroid nodules from malignant thyroid nodules., Methods: A total of 67 patients with malignant thyroid nodules admitted to our hospital from October 2016 to March 2018 were enrolled in the malignant group, and 71 patients with benign thyroid nodules were enrolled in the benign group. Another 56 healthy individuals, as determined by physical examination, were enrolled in the control group. Quantitative real-time polymerase chain reaction (qRT-PCR) was adopted to determine the relative expression of miR-205-5p and TSHR mRNA in serum, and receiver operating characteristic (ROC) curves were used to analyze the diagnostic value of miR-205-5p and TSHR mRNA in thyroid nodules and their benignancy or malignancy. The correlation of miR-205-5p and TSHR mRNA with clinical data of the patients with thyroid carcinoma was analyzed, and multivariate logistic regression was used to analyze risk factors for lymph node metastasis of thyroid carcinoma. In addition, Pearson's test was used to analyze the relationship between miR-205-5p and TSHR mRNA in serum of patients with thyroid carcinoma., Results: The malignant group showed significantly higher expression of miR-205-5p and TSHR mRNA than the benign group and control group (both p <0.001), and the benign group showed significantly higher expression of these than the control group (p <0.01). miR-205-5p expression was related to tumor size, TNM staging, lymph node metastasis, capsular infiltration, and BRAF mutation (p <0.05), and TSHR mRNA expression was related to lymph node metastasis, capsular infiltration, BRAF mutation, and RAS mutation (p <0.05). Gender, number of lesions, capsular infiltration, miR-205-5p, and TSHR mRNA were independent risk factors for lymph node metastasis of patients with thyroid carcinoma. Moreover, there was a positive correlation between miR-205-5p and TSHR mRNA in patients with thyroid carcinoma (r=0.735, p<0.001)., Conclusion: MiR-205-5p and TSHR mRNA may be potential diagnosis indexes of thyroid nodules, their benignancy, or malignancy, while the independent risk factors for patients with thyroid carcinoma include a large number of lesions, occurrence of capsular infiltration, and relatively high expression of miR-205-5p and TSHR mRNA., (© 2021. Hellenic Endocrine Society.)

Background: Loss-of-function mutations of thyrotropin receptor (TSHR) are one of the main causes of congenital hypothyroidism. As for many disease-associated G-protein coupled receptors (GPCRs), these mutations often affect the correct trafficking and maturation of the receptor, thus impairing the expression on the cell surface. Several retained GPCR mutants are able to effectively bind their ligands and to transduce signals when they are forced to the cell surface by degradation inhibition or by treatment with chaperones. Despite the large number of well-characterized retained TSHR mutants, no attempts have been made for rescue. Further, little is known about TSHR degradation pathways. We hypothesize that, similar to other GPCRs, TSHR retained mutants may be at least partially functional if their maturation and membrane expression is facilitated by chaperones or degradation inhibitors. Methods: We performed in silico predictions of the functionality of known TSHR variants and compared the results with available in vitro data. Western blot, confocal microscopy, enzyme-linked immunosorbent assays, and dual luciferase assays were used to investigate the effects of degradation pathways inhibition and of chemical chaperone treatments on TSHR variants' maturation and functionality. Results: We found a high discordance rate between in silico predictions and in vitro data for retained TSHR variants, a fact indicative of a conserved potential to initiate signal transduction if these mutants were expressed on the cell surface. We show experimentally that some maturation defective TSHR mutants are able to effectively transduce Gs/cAMP signaling if their maturation and expression are enhanced by using chemical chaperones. Further, through the characterization of the intracellular retained p.N432D variant, we provide new insights on the TSHR degradation mechanism, as our results suggest that aggregation-prone mutant can be directed toward the autophagosomal pathway instead of the canonical proteasome system. Conclusions: Our study reveals alternative pathways for TSHR degradation. Retained TSHR variants can be functional when expressed on the cell surface membrane, thus opening the possibility of further studies on the pharmacological modulation of TSHR expression and functionality in patients in whom TSHR signaling is disrupted.

The underlying molecular mechanisms that determine long day versus short day breeders remain unknown in any organism. Atlantic herring provides a unique opportunity to examine the molecular mechanisms involved in reproduction timing, because both spring and autumn spawners exist within the same species. Although our previous whole genome comparisons revealed a strong association of TSHR alleles with spawning seasons, the functional consequences of these variants remain unknown. Here we examined the functional significance of six candidate TSHR mutations strongly associated with herring reproductive seasonality. We show that the L471M missense mutation in the spring-allele causes enhanced cAMP signaling. The best candidate non-coding mutation is a 5.2 kb retrotransposon insertion upstream of the TSHR transcription start site, near an open chromatin region, which is likely to affect TSHR expression. The insertion occurred prior to the split between Pacific and Atlantic herring and was lost in the autumn-allele. Our study shows that strongly associated coding and non-coding variants at the TSHR locus may both contribute to the regulation of seasonal reproduction in herring.

Background: Thyrotropin (TSH) is well known as the hormone of the anterior pituitary thyrotrophs responsible for acting in the thyroid gland, where it stimulates synthesis and release of thyroid hormones through G s and G q/11 protein coupled TSH receptors (TSHRs). Methods: In this study, we examined whether the functional TSHRs are also expressed in cultured rat pituitary cells, using double immunocytochemistry, quantitative reverse transcription-polymerase chain reaction analysis, cAMP and hormone measurements, and single-cell calcium imaging. Results: Double immunocytochemistry revealed the expression of TSHRs in cultured corticotrophs and melanotrophs, in addition to previously identified receptors in folliculostellate cells. The functional coupling of these receptors to the G q/11 signaling pathway was not observed, as demonstrated by the lack of TSH activation of IP 3 -dependent calcium mobilization in these cells when bathed in calcium-deficient medium. However, TSH increased cAMP production in a time- and concentration-dependent manner and facilitated calcium influx in single corticotrophs and melanotrophs, indicating their coupling to the G s signaling pathway. Consistent with these findings, TSH stimulated adrenocorticotropin and β-endorphin release in male and female pituitary cells in a time- and concentration-dependent manner without affecting the expression of proopiomelanocortin gene. Conclusions: These results indicate that TSH is a potential paracrine modulator of anterior pituitary corticotrophs and melanotrophs, controlling the exocytotic but not the transcriptional pathway in a cAMP/calcium influx-dependent manner.

Background: Graves' orbitopathy (GO) is the most common and serious manifestation of Graves' disease (GD). It is characterized by orbital inflammation and tissue remodeling. Although several GO models have been reported, most lack a full assessment or mechanistic evaluation. Here, we established a promising mouse model mimicking many aspects of human GO with a frequency of 70% and characterized the key role of T cells in the progression of GO. Methods: An adenovirus expressing the human thyrotropin (TSH) receptor A subunit (Ad-TSHRA) was injected in the muscles of female BALB/C mice nine times to induce GO. At predetermined time points, histological examinations of retrobulbar tissues and thyroid glands were performed to dynamically monitor changes; serum autoantibodies and total thyroxine levels were examined to evaluate thyroid function. Flow cytometry of CD4+ T cell subgroups and RNA sequencing (RNA-Seq) of splenocytes were also performed to explore the underlying mechanism. Results: After nine injections, 7 of 10 mice challenged with Ad-TSHRA developed the orbital changes associated with GO. Seven mice manifested retrobulbar fibrosis, and four mice showed adipogenesis. Exophthalmia, conjunctival redness, and orbital lymphocyte infiltration were also observed in a subset of mice. The orbitopathy was first detected after seven injections and followed the hyperplastic change observed in thyroids after four injections. Flow cytometry revealed increased proportions of Th1 cells and decreased proportions of Th2 cells and regulatory T (Treg) cells in the splenocytes of GO mice. This change in CD4+ T cell subgroups was confirmed by orbital immunohistochemical staining. Genes involved in T cell receptor signaling, proliferation, adhesion, inflammation, and cytotoxicity were upregulated in GO mice according to the RNA-Seq; a trend of upregulation of these GO-specific genes was observed in mice with hyperthyroidism without orbitopathy after four injections. Conclusions: A GO mouse model was successfully established by administering nine injections of Ad-TSHRA. The model was achieved with a frequency of 70% and revealed the importance of T cell immunity. A potential time window from Graves' hyperthyroidism to GO was presented for the first time. Therefore, this model could be used to study the pathogenesis and novel treatments for GO.

The aim of this study was to investigate the potential of the new TSH-receptor antibody (TRAb) assays to predict remission or relapse of hyperthyroidism in patients with Graves' disease (GD) and Graves' orbitopathy (GO). TRAbs were measured retrospectively in sera from a cohort of GD patients with GO (n=117; remission n=38 and relapse n=79-Essen GO biobank) with automated binding immunoassays: TRAb Elecsys (Cobas Roche) and TRAb bridge assay (IMMULITE, Siemens), and the TSAb (thyroid stimulating Ab) cell-based bioassay (Thyretain, Quidel Corp.). To identify relapse risk/remission of hyperthyroidism patients were followed up at least 10 months after the end of antithyroid drug therapy (ATD) therapy. ROC plot analysis was performed to calculate cut-off levels of TRAb and TSAb for prediction of relapse and remission of hyperthyroidism. Cut-off serum levels are provided for timepoints around 3, 6, 10, and 15 months after the beginning of ATD. Repeated measurements of TRAb increase the rate of relapses predictions to 60% (Elecsys), 70% (IMMULITE), and 55% (Thyretain). Patients with remission have consistently TRAb levels below the cut off for relapse in repeated measurements. The cell-based bioassay was the most sensitive - and continued to be positive during follow up [at 15 months: 90% vs. 70% (IMMULITE) and 65% (Elecsys)]. Identification of relapsing hyperthyroidism is possible with automated immunoassays and cell-based bioassay especially with serial TRAb measurements during the course of ATD therapy. Patient who need eye surgery may profit from an early decision towards definitive treatment., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Domestic chickens are less fearful, have a faster sexual development, grow bigger, and lay more eggs than their primary ancestor, the red junglefowl. Several candidate genetic variants selected during domestication have been identified, but only a few studies have directly linked them with distinct phenotypic traits. Notably, a variant of the thyroid stimulating hormone receptor (TSHR) gene has been under strong positive selection over the past millennium, but it's function and mechanisms of action are still largely unresolved. We therefore assessed the abundance of the domestic TSHR variant and possible genomic selection signatures in an extensive data set comprising multiple commercial and village chicken populations as well as wild-living extant members of the genus Gallus. Furthermore, by mean of extensive backcrossing we introgressed the wild-type TSHR variant from red junglefowl into domestic White Leghorn chickens and investigated gene expression, hormone levels, cold adaptation, and behavior in chickens possessing either the wild-type or domestic TSHR variant. While the domestic TSHR was the most common variant in all studied domestic populations and in one of two red junglefowl population, it was not detected in the other Gallus species. Functionally, the individuals with the domestic TSHR variant had a lower expression of the TSHR in the hypothalamus and marginally higher in the thyroid gland than wild-type TSHR individuals. Expression of TSHB and DIO2, two regulators of sexual maturity and reproduction in birds, was higher in the pituitary gland of the domestic-variant chickens. Furthermore, the domestic variant was associated with higher activity in the open field test. Our findings confirm that the spread of the domestic TSHR variant is limited to domesticated chickens, and to a lesser extent, their wild counterpart, the red junglefowl. Furthermore, we showed that effects of genetic variability in TSHR mirror key differences in gene expression and behavior previously described between the red junglefowl and domestic chicken., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The objective was to study targeted therapies using a biologically active monoclonal antibody against intracellular adhesion molecule-1 (ICAM-1 mAb) and an siRNA targeting thyroid-stimulating hormone (TSH) receptor (TSHR) in a BALB/c mouse model of Graves' disease (GD)., Material and Methods: An improved method for establishing a stable model of GD in BALB/c mice was developed by immunization with pcDNA 3.1/TSHR 289 and electroporation (EP). The mice in which GD was successfully established were divided into a nontreated control group, which was treated with continuous immunization, and treated groups, which were treated with the siRNA and ICAM-1 mAb. Normal mice were included as a blank group. These groups were used to compare the effects of treatment with the ICAM-1 mAb and siRNA., Results: The two novel treatments markedly improved weight loss, serum thyroxine (T4) levels, thyroid-stimulating hormone antibody (TSAb) levels, thyroid-stimulating blocking antibody (TSBAb) levels and thyroid uptake of 99mTcO4 in GD model mice. Compared with the siRNA treatment, treatment with the ICAM-1 mAb produced more obvious benefits. The differences in the posttreatment indexes between the two treatment groups were statistically significant (p < 0.05)., Conclusions: These preliminary data suggest that both the biologically active ICAM-1 mAb and the siRNA targeting TSHR were effective. The ICAM-1 mAb exerted a better therapeutic effect than the siRNA targeting TSHR. Both treatments showed potential efficacy as novel treatments for GD and may therefore represent therapeutic options in addition to the existing drugs or interventions.

Context: Inactivating variants of thyrotropin (thyroid-stimulating hormone; TSH) receptor (TSHR) cause congenital hypothyroidism. More than 60 such variants have been reported so far, most of which were located in the extracellular or transmembrane domain., Objective: We report the identification and characterization of a frameshift TSHR variant in the intracytoplasmic C-tail region., Methods: Sequencing of TSHR was performed in a patient with congenital hypothyroidism. The functionality of the identified variants was assessed by expressing TSHR in HEK293 cells and measuring TSH-dependent activation of the cAMP-response element-luciferase reporter. A series of systematic mutagenesis experiments were performed to characterize the frameshifted amino acid sequence., Results: The proband was heterozygous for a known TSHR variant (p.Arg519His) and a novel frameshift TSHR variant (p.Val711Phefs*18), which removed 54 C-terminal residues and added a 17-amino acid frameshifted sequence. The loss of function of Val711Phefs*18-TSHR was confirmed in vitro, but the function of Val711*-TSHR was found to be normal. Western blotting showed the low protein expression of Val711Phefs*18-TSHR. Fusion of the frameshift sequence to green fluorescent protein or luciferase induced inactivation of them, indicating that the sequence acted as a degron. A systematic mutagenesis study revealed that the density of hydrophobic residues in the frameshift sequence determined the stability. Eight additional frameshift TSHR variants that covered all possible shifted frames in C-tail were created, and another frameshift variant (Thr748Profs*27) with similar effect was found., Conclusions: We characterized a naturally occurring frameshift TSHR variant located in C-tail, and provided a unique evidence that hydrophobicity in the C-terminal region of the receptor affects protein stability., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Constitutively activating mutations in the thyrotropin receptor ( TSHR ) and the guanine nucleotide-binding protein G subunit alpha ( GNAS ) are the primary cause of hot thyroid nodules (HTNs). The reported prevalence of TSHR and GNAS mutations in HTNs varies. Previous studies show TSHR mutations in 8-82% of HTNs and GNAS mutations in 8-75% of HTNs. With sensitive and comprehensive targeted next-generation sequencing (tNGS), we re-evaluated the prevalence of TSHR and GNAS mutations in HTNs. Methods: Samples from three previous studies found to be TSHR and GNAS mutation negative were selected and re-evaluated using high-resolution melting (HRM) PCR. Remaining mutation negative samples were further reanalyzed by tNGS with a sequencing depth between 3000 × and 10,000 × . Our tNGS panel covered the entire TSHR coding sequence along with mutation hot spots in GNAS . Sequencing reads were aligned to reference and variants were called using Torrent Suite software v5.8. Results: In total, 154 of 182 previously mutation negative HTNs were positive for TSHR or GNAS mutations, resulting in an 85% prevalence of TSHR and GNAS mutations in HTNs, 79% and 6%, respectively. In a subset of 25 HTNs with multiple samples per nodule, and analyzed by tNGS at high sequencing depth, TSHR mutations were detected in 23 (92%) HTNs and 1 GNAS mutation was detected in 1 (4%) HTN, 96% mutation positive HTNs in this subset. Conclusions: Owing to the higher sensitivity of tNGS as compared with denaturing gradient gel electrophoresis and HRM-PCR, TSHR or GNAS mutations could be detected in 85% of HTNs. The detection of TSHR and GNAS mutations occurred in 96% of HTNs in a sample set with multiple samples per nodule analyzed by tNGS. Taken together with the fact that no other driver mutations could be identified by whole exome sequencing, our study strongly supports the hypothesis that TSHR and GNAS mutations are the main somatic mutations leading to HTNs.

High prevalence of congenital hypothyroidism (CH) among Indian newborns prompted us to establish population-specific reference ranges of TSH and to explore the contribution of the common genetic variants in TSHR, TPO, TG and DUOX2 genes towards CH. A total of 1144 newborns (593 males and 551 females) were screened for CH. SNV profiling (n = 22) spanning three candidate genes, i.e. TSHR, TPO and TG was carried out in confirmed CH cases (n = 45). In screen negative cases (n = 700), ten TSHR variants were explored to establish association with CH. No mutation found in DUOX2. The 2.5th to 97.5th percentiles of TSH in these newborns were 0.5 to 12.2 mU/L. In newborns with optimal birth weight, the cut-off was 10 mU/L. Lower or higher birth weight resulted in slightly higher TSH. Two TSHR variants, i.e. rs7144481 and rs17630128 were associated with agenesis, hypoplasia and goiter. The rs2268477 was associated with agenesis and hypoplasia. The rs1991517, rs2075176 and rs2241119 were associated with agenesis only. The rs7144481, rs17630128, rs1991517 and rs2268477 were associated with 2.17, 4.62, 2.91 and 2.29-fold increased risk for CH, respectively. Among the TPO variants, rs867983 and rs2175977 were associated with agenesis and goiter, respectively. Among the TG variants, rs2076740 showed association with agenesis and goiter. Two rare variants i.e. TPO g.IVS14-19 G>C and TG c.1262 C>T were observed in CH cases. No genetic variant identified in the two exons of DUOX2. To conclude, the current study established Indian population-specific normative values for TSH and demonstrates specific genotype-phenotype correlations among three candidate genes.

Background: In obesity, the expression level of thyroid stimulating hormone receptor in adipose tissue is reduced and the levels of thyroid stimulating hormone (TSH) are often elevated within the normal range., Purpose/aim: To investigate the role of TSHR in brown and white adipose tissue (AT) using TSHR knockout (KO) mice and the physiological phenotypes affected by the TSHR knockout., Methods: AT-specific TSHR KO male mice and wild type (WT) controls were given a high-fat diet (HFD) or a control diet (CD). Body weights and food consumption were recorded for 20 weeks and body temperatures for the first 3 weeks. At termination, white and brown adipocytes were isolated. Gene expressios was investigated using real-time PCR. In a subgroup of female KO mice, glucose tolerance was investigated., Results: TSHR were partially knocked out in KO mice, which gained more weight than WT mice when fed both a CD (p = .03) and HFD (p = .003). Body temperatures were lower in KO mice on CD (p <.001) and on HFD (p <.001) than WT controls. This was in agreement with reduced gene expression of UCP1 in brown adipocytes in the KO mice. Glucose tolerance was significantly impaired in KO mice on CD mice before termination (p <.01). Expression of adipogenic and lipolytic genes were reduced in KO mice, which was exacerbated by HFD. The mRNA levels of adipokines including ADIPOQ and LEP were altered in white adipocytes of KO mice., Conclusions: TSHR KO led to dysfunction of both white and brown AT and predisposition to excess body weight gain in mice. Our data show that TSHR in AT regulates glucose tolerance, lipid metabolism, adipokine profile, and thermogenesis., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

1. The thyroid-stimulating hormone receptor (TSHR) is a glycoprotein hormone receptor which has a pivotal role in metabolic regulation and photoperiod control during reproduction in birds and mammals. However, the molecular characterisation of TSHR in goose is unknown. 2. The goose TSHR cDNA (TSHR-1) is 2334 bp in length and encodes a protein of 763 amino acids. This trial identified another three novel splice variants of goose TSHR, TSHR-2 (lacking the exon 3 in TSHR-1 transcript), TSHR-3 ( lacking the exon 6 in the TSHR-1 transcript) and TSHR-4 (lacking 12 bp of exon 8 and the entire exon 9 in the TSHR-1 transcript). 3. Bioinformatics analysis indicated that all the deduced TSHR amino acid sequences contained seven putative transmembrane domains, and the TSHR-3 protein lacked one potential N-linked glycosylation site (N-E-S) compared to the other three deduced proteins. 4. A phylogenetic tree based on amino acid sequences showed that the goose TSHR protein was closely related to those of other avian species, especially duck and chickens. 5. One microsatellite and three single nucleotide polymorphisms (SNPs) were identified. For the c1109A/G locus, AA and GA genotypes were found in the Zhedong-White goose population, GG and GA genotypes were detected in the Landes goose population, but the AA genotype was only detected in the other four goose populations. 6. All the information derived from this study can facilitate further studies on the functions of the goose TSHR gene.

Graves' disease is an autoimmune disorder, which is characterized by stimulatory antibodies targeting the human thyrotropin receptor (TSHR), resulting in hyperthyroidism and multiple organ damage. We systematically investigated monomeric and dimeric fusion proteins of the A subunit of TSHR for efficacy to bind to the monoclonal patient antibody M22, to interact with Graves' patient serum samples, and to impact on anti-TSHR antibody titers, hyperthyroidism, tachycardia and other in vivo read-outs in a long-term mouse model of Graves' disease induced by immunization with a recombinant adenovirus encoding TSHR A. Binding assays and functional measurements of TSHR-dependent cAMP formation showed binding of monomeric TSHR-His and dimeric TSHR-Fc to the anti-TSHR antibody M22 at low-effective concentrations (EC50 of 5.7 nmol/L and 8.6 nmol/L) and inhibition of the effects of this antibody at high efficiencies (IC50 values of 16-20 nmol/L). Both proteins also block the effects of polyclonal anti-TSHR antibodies occurring in Graves' patient sera with somewhat lower average efficiencies (mean IC50 values of 29 nmol/L and 68 nmol/L). However, in vivo characterization of epicutaneous patch administrations of TSHR-Fc at doses of 0.3 and 0.6 mg/kg body weight in a murine Graves' disease model did not result in any improvement of disease parameters. In conclusion, high affinity binding of TSHR-Fc to pathological anti-TSHR antibodies was not matched by efficacy to improve Graves' disease parameter in a long-term mouse model., (© 2020 Society for Endocrinology.)

The thyroid stimulating hormone (TSH) and its cognate receptor (TSHR) are of crucial importance for thyrocytes to proliferate and exert their functions. Although TSHR is predominantly expressed in thyrocytes, several studies have revealed that functional TSHR can also be detected in many extra-thyroid tissues, such as primary ovarian and hepatic tissues as well as their corresponding malignancies. Recent advances in cancer biology further raise the possibility of utilizing TSH and/or TSHR as a therapeutic target or as an informative index to predict treatment responses in cancer patients. The TSH/TSHR cascade has been considered a pivotal modulator for carcinogenesis and/or tumor progression in these cancers. TSHR belongs to a sub-group of family A G-protein-coupled receptors (GPCRs), which activate a bundle of well-defined signaling transduction pathways to enhance cell renewal in response to external stimuli. In this review, recent findings regarding the molecular basis of TSH/TSHR functions in either thyroid or extra-thyroid tissues and the potential of directly targeting TSHR as an anticancer strategy are summarized and discussed.

Neonatal screening in Macedonia detects congenital hypothyroidism (CH) with an incidence of 1 in 1,585, and more than 50% of cases exhibit a normally located gland- in-situ (GIS). Monogenic mutations causing dyshormonogenesis may underlie GIS CH; additionally, a small proportion of thyroid hypoplasia has a monogenic cause, such as TSHR and PAX8 defects. The genetic architecture of Macedonian CH cases has not previously been studied. We recruited screening-detected, non-syndromic GIS CH or thyroid hypoplasia cases ( n = 40) exhibiting a spectrum of biochemical thyroid dysfunction ranging from severe permanent to mild transient CH and including 11 familial cases. Cases were born at term, with birth weight >3,000 g , and thyroid morphologies included goiter ( n = 11), thyroid hypoplasia ( n = 6), and apparently normal-sized thyroid. A comprehensive, phenotype-driven, Sanger sequencing approach was used to identify genetic mutations underlying CH, by sequentially screening known dyshormonogenesis-associated genes and TSHR in GIS cases and TSHR and PAX8 in cases with thyroid hypoplasia. Potentially pathogenic variants were identified in 14 cases, of which four were definitively causative; we also detected digenic variants in three cases. Seventeen variants (nine novel) were identified in TPO ( n = 4), TG ( n = 3), TSHR ( n = 4), DUOX2 ( n = 4), and PAX8 ( n = 2). No mutations were detected in DUOXA2, NIS, IYD , and SLC26A7 . The relatively low mutation frequency suggests that factors other than recognized monogenic causes (oligogenic variants, environmental factors, or novel genes) may contribute to GIS CH in this region. Future non-hypothesis-driven, next-generation sequencing studies are required to confirm these findings., (Copyright © 2020 Zdraveska, Kocova, Nicholas, Anastasovska and Schoenmakers.)

Subclinical hyperthyroidism, a condition characterized by decreased thyroid-stimulating hormone (TSH) and normal concentration of thyroid hormone, is associated with an elevated risk for cognitive impairment. TSH is the major endogenous ligand of the TSH receptor (TSHR) and its role is dependent on signal transduction of TSHR. It has not, however, been established whether TSHR signaling is involved in the regulation of cognition. Here, we utilized Tshr knockout mice and found that Tshr deletion led to significantly compromised performance in learning and memory tests. Reduced dendritic spine density and excitatory synaptic density as well as altered synaptic structure in CA1 subfield of the hippocampus were also noted. Furthermore, the synapse-related gene expression was altered in the hippocampus of Tshr -/- mice. These findings suggest that TSHR signaling deficiency impairs spatial learning and memory, which discloses a novel role of TSHR signaling in brain function.

Objective: An association between hypothyroidism (HT) and oral lichen planus (OLP) has been reported. However, the mechanisms that could explain this association remain unresolved. This study aimed to evaluate the expression of thyroid-stimulating hormone (TSH) and thyroid-stimulating hormone receptor (TSHR) in healthy oral mucosa and in OLP lesions of individuals with and without HT. Material and methods: Immunohistochemical expression of TSH and TSHR was studied in oral mucosal biopsies obtained from 14 OLP patients with HT, 14 OLP patients without HT and 10 healthy controls without oral mucosal lesions. Gene expression of TSHR was investigated by using three different PCR techniques in oral mucosal samples from 7 OLP patients with HT, 3 OLP patients without HT, 9 healthy controls and in cultured human oral epithelial cells. Gene expression of TSH was examined by employing 2 PCR techniques in oral mucosal samples from 2 OLP patients with HT, 2 OLP patients without HT and 4 healthy controls. Results: TSH and TSHR stainings were negative in the studied oral mucosal specimens. Gene quantification assays demonstrated negative gene expression of TSH and TSHR in clinical and in vitro samples. Conclusions: These results suggest that TSH and TSHR may not be commonly involved in the pathogenetic mechanism that could explain the association between OLP and hypothyroidism.

The thyrotropin receptor ( TSHR ) mutation database, consisting of all known TSHR mutations and their clinical characterizations, was established in 1999. The database contents are updated here with the same website (tsh-receptor-mutation-database.org). The new database contains 638 cases of TSHR mutations: 448 cases of gain of function mutations (7 novel mutations and 41 new cases for previously described mutations since its last update in 2012) and 190 cases of loss of function mutations (28 novel mutations and 31 new cases for previously described mutations since its last update in 2012). This database is continuously updated and allows for rapid validation of patient TSHR mutations causing hyper- or hypothyroidism or insensitivity to TSH.

Since its cloning more than 30 years ago, the thyrotropin receptor (TSHR) has emerged as a pivotal player in thyroid physiology and pathophysiology. In particular, hyperthyroidism due to autoimmune disease or thyroid autonomy is linked with TSHR activation via autoantibodies or mutations respectively. This review summarises clinical aspects of constitutive TSH receptor activation by naturally occurring somatic or germline TSHR mutations resulting in TSH-independent thyroid function and cell proliferation.

Thyrotropin hormone (TSH) was reported to exhibit biphasic regulation of cAMP production in human thyroid slices; specifically, upregulation at low TSH doses transitioning to inhibition at high doses. We observed this phenomenon in HEK293 cells overexpressing TSH receptors (TSHRs) but in only 25% of human thyrocytes (hThyros) in vitro . Because TSHR expression in hThyros in vitro was low, we tested the hypothesis that high, in situ levels of TSHRs were needed for biphasic cAMP regulation. We increased expression of TSHRs by infecting hThyros with adenoviruses expressing human TSHR (AdhTSHR), measured TSH-stimulated cAMP production and TSHR homodimerization. TSHR mRNA levels in hThyros in vitro were 100-fold lower than in human thyroid tissue. AdhTSHR infection increased TSHR mRNA expression to levels found in thyroid tissue and flow cytometry showed that cell-surface TSHRs increased more than 15-fold. Most uninfected hThyro preparations exhibited monotonic cAMP production. In contrast, most hThyro preparations infected with AdhTSHR expressing TSHR at in vivo levels exhibited biphasic TSH dose responses. Treatment of AdhTSHR-infected hThyros with pertussis toxin resulted in monotonic dose response curves demonstrating that lower levels of cAMP production at high TSH doses were mediated by G i /G o proteins. Proximity ligation assays confirmed that AdhTSHR infection markedly increased the number of TSHR homodimers. We conclude that in situ levels of TSHRs as homodimers are needed for hThyros to exhibit biphasic TSH regulation of cAMP production., (Copyright © 2020 Boutin, Krieger, Marcus-Samuels, Klubo-Gwiezdzinska, Neumann and Gershengorn.)

The thyroid-stimulating hormone receptor (TSHR) has been indicated as a putative domestication gene in chicken. Comparison of WGS identified a variant in residue 558 of the transmembrane domain (TM) of TSHR, where the domestic chicken (GGD) presents an arginine, whereas the red jungle fowl (RJF) shares a conserved glycine with other vertebrates. This variant has been demonstrated to be associated with phenotypes that are important for domestication and related to thyroid regulation, such as less fearful behavior, reduced aggressive behavior and reduced dependence on seasonal reproduction in GGD as compared with RJF. By means of molecular dynamics simulations, we highlighted the structural and dynamic differences of variant Gly558Arg in the TSHR TM domain. Alterations in TM helix flexibility, structure and protein overall motion are described. The so-called 'arginine snorkeling' of residue 568 in GGD is observed and we hypothesize it as the originating force that produces the observed whole-protein perturbation in the helix bundle dynamics, capable of altering the TSHR signal transduction. The results are discussed in the context of their implications for a better understanding of biological mechanisms in chicken under control of the thyroid, such as body metabolism, as well as for their usefulness in biomedical research., (© 2019 Stichting International Foundation for Animal Genetics.)

Hashimoto's encephalopathy (HE) is a syndrome occurring in some patients with Hashimoto's thyroiditis or, less frequently, Graves' disease. Three known autoantigens are involved in HE: alpha-enolase, dimethylargininase-I (DDAHI) and aldehyde reductase-I (AKRIAI). We searched for amino acid sequence homologies between these proteins and the three classical thyroid autoantigens (thyroperoxidase (TPO), thyroglobulin (Tg), TSH-receptor (TSH-R)), which are also expressed in the central nervous system (CNS). TSH-R shows homologies with alpha-enolase (n=4), DDAHI (n=2) and AKRIAI (n=5); of these segments, two, two and four, respectively, overlap totally or partially with epitope-containing TSH-R segments. Tg has 10 homologies with alpha-enolase, five with DDAHI, and eight with AKRIAI; epitope-containing segments of Tg overlap four, three and four segments, respectively. TPO has six segments homologous to alpha-enolase, three to DDAHI and seven to AKRIAI; of these segments, five, one and four, respectively, are located in epitope-containing parts. These data suggest that cross-reactivity between CNS autoantigens and thyroid autoantigens might contribute to the HE pathogenesis, together with other proposed mechanisms, including autoimmunity involving autoantigens common to CNS and thyroid.