A Novel TSHR Gene Mutation in a Family with Non-autoimmune Hyperthyroidism.

Background: Familial non-autoimmune hyperthyroidism is a rare disorder characterized by the absence of thyroid autoimmunity, particularly TSH receptor antibody [TRAb]. Objective: The aim of this study was to describe a novel TSHR mutation identified in a family of two siblings and their father. Methods: Two siblings presented for endocrine assessment at ages 7 and 14 years with mild T3 toxicosis, and the father presented at 30 years of age with non-autoimmune thyrotoxicosis. Both siblings were treated with oral antithyroid therapy to achieve reasonable symptom control and thyroid function normalization. The father was treated with oral antithyroid therapy, radioactive iodine, thyroidectomy, and thyroid replacement therapy. Peripheral blood DNA was extracted from both affected siblings and father. Mutation analysis of TSHR was carried out by PCR and Sanger sequencing of both strands of the extracted DNA. Results: Both siblings and their father were heterozygous for the missense TSHR variant c.1855G>C, p.[Asp619His], in exon 10. Conclusions: This novel TSHR variant is associated with T3 toxicosis during childhood. Therefore, early identification and treatment may improve patient outcomes.

TSHR Variant Screening and Phenotype Analysis in 367 Chinese Patients With Congenital Hypothyroidism.

Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype-phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions: We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.

Thyroid hormone deprival and TSH/TSHR signaling deficiency lead to central hypothyroidism-associated intestinal dysplasia.

BACKGROUND: Central hypothyroidism (CH) is characterized by low T4 levels and reduced levels or bioactivity of circulating TSH. However, there is a lack of studies on CH-related intestinal maldevelopment. In particular, the roles of TH and TSH/TSHR signaling in CH-related intestinal maldevelopment are poorly understood. Herein, we utilized Tshr-/- mice as a congenital hypothyroidism model with TH deprival and absence of TSHR signaling. METHODS: The morphological characteristics of intestines were determined by HE staining, periodic acid-shiff staining, and immunohistochemical staining. T4 was administrated into the offspring of homozygous mice from the fourth postnatal day through weaning or administrated after weaning. RT-PCR was used to evaluate the expression of markers of goblet cells and intestinal digestive enzymes. Single-cell RNA-sequencing analysis was used to explore the cell types and gene profiles of metabolic alternations in early-T4-injected Tshr-/- mice. KEY FINDINGS: Tshr deletion caused significant growth retardation and intestinal maldevelopment, manifested as smaller and more slender small intestines due to reduced numbers of stem cells and differentiated epithelial cells. Thyroxin supplementation from the fourth postnatal day, but not from weaning, significantly rescued the abnormal intestinal structure and restored the decreased number of proliferating intestinal cells in crypts of Tshr-/- mice. Tshr-/- mice with early-life T4 injections had more early goblet cells and impaired metabolism compared to Tshr+/+ mice. SIGNIFICANCE: TH deprival leads to major defects of CH-associated intestinal dysplasia while TSH/TSHR signaling deficiency promotes the differentiation of goblet cells and impairs nutrition metabolism.

TSHR signaling promotes hippocampal dependent memory formation through modulating Wnt5a/ß-catenin mediated neurogenesis.

Subclinical hyperthyroidism is defined biochemically as a low or undetectable thyroid-stimulating hormone (TSH) with normal thyroid hormone levels. Low TSHR signaling is considered to associate with cognitive impairment. However, the underlying molecular mechanism by which TSHR signaling modulates memory is poorly understood. In this study, we found that Tshr-deficient in the hippocampal neurons impairs the learning and memory abilities of mice, accompanying by a decline in the number of newborn neurons. Notably, Tshr ablation in the hippocampus decreases the expression of Wnt5a, thereby inactivating the ß-catenin signaling pathway to reduce the neurogenesis. Conversely, activating of the Wnt/ß-catenin pathway by the agonist SKL2001 results in an increase in hippocampal neurogenesis, resulting in the amelioration in the deficits of memory caused by Tshr deletion. Understanding how TSHR signaling in the hippocampus regulates memory provides insights into subclinical hyperthyroidism affecting cognitive function and will suggest ways to rationally design interventions for neurocognitive disorders.

Can inactivation mutation in the thyroid stimulating hormone receptor gene and hyperthyroidism coexist?: A case report.

INTRODUCTION: We found the G132R heterozygous mutation of thyroid stimulating hormone receptor (TSHR) gene in a patient with recurrent hypokalemia. Because the patient had a medical history of hyperthyroidism, the mutation was suspected to be related to hyperthyroidism at first. Subsequently, the expression and function studies in vitro were conducted. METHODS: Wide-type TSHR and mutant TSHR (mutTSHR) were constructed in the phage vector and pEGFP-C1 vector. After transfection, the samples were collected for detection of mRNA level, protein expression, cell activity and cAMP content. RESULTS: Compared with the wild-type TSHR, the mRNA level of the mutTSHR was not significantly different. But the protein expression, cell activity and cAMP content of the mutTSHR were significantly lower. So this indicated that the G132R mutation is a loss-of-function mutation. CONCLUSION: We identified the G132R monoallelic heterozygous mutation of TSHR gene in a patient with hyperthyroidism. Based on disease history of the patient, we speculated that the heterozygous mutation did not cause thyroid dysplasia or hypothyroidism for her. Our study enriched experiment content in vitro studies and clinical phenotype about the G132R mutation in TSHR gene.

Mechanisms of thyrotropin receptor-mediated phenotype variability deciphered by gene mutations and M453T-knockin model.

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.

A Novel TSH Receptor Gene Variant Associated with Non-Autoimmune Hyperthyrotropinemia: A Case Report.

BACKGROUND: Resistance to TSH is defined as reduced sensitivity to normal, biologicallyactive TSH, and abnormally high levels of TSH are needed to achieve normal levels of thyroid hormones. CASE PRESENTATION: A 15-year-old female patient, having been treated since childhood with levothyroxine for hyperthyrotropinemia was referred to our institution complaining of tachycardia after the levothyroxine therapy had been increased. Thyroid ultrasound features were normal, and thyroid antibodies were negative. The therapy was gradually tapered in light of the symptoms, although subclinical hypothyroidism was evident at thyroid function tests. First-degree relatives were tested for thyroid function, and the father was also found to have a previously-unknown subclinical hypothyroidism. The patient underwent genetic testing for TSH receptor (TSHR) gene mutations, which revealed a gene variant hitherto not described: p.C598R (c.1792T>C). The father was also tested and was found to carry the same mutation, while other first-degree relatives were wild-type for the TSHR gene. An in-silico analysis was performed, which revealed a loss-of-function phenotype corresponding to the described variant, suggesting a novel loss-of-function TSH receptor gene mutation. CONCLUSION: In this case report, we present a novel loss-of-function gene mutation in the TSH receptor gene associated with a TSH resistance phenotype.

Regulation of Thyroid Hormone Gatekeepers by Thyrotropin in Tanycytes.

Background: Tanycytes are specialized glial cells within the mediobasal hypothalamus that have multiple functions, including hormone sensing and regulation of hypophysiotropic hormone secretion. There are ongoing discussions about the role of tanycytes in regulating the supply of hypothalamic thyroid hormones (THs) through the expression of TH transporters (Slc16a2, Slco1c1) and deiodinases (Dio2, Dio3). In this study, we investigated the potential feedback effect of thyrotropin (TSH) on the transcription of these gatekeeper genes on tanycytes. Methods: We analyzed the changes in the expression of TH-gatekeeper genes, in TSH-stimulated primary tanycytes, using quantitative polymerase chain reaction (qPCR). We also used RNAScope® in brain slices to further reveal the local distribution of the transcripts. In addition, we blocked intracellular pathways and used small-interfering RNA (siRNA) to elucidate differences in the regulation of the gatekeeper genes. Results: TSH elevated messenger RNA (mRNA) levels of Slco1c1, Dio2, and Dio3 in tanycytes, while Slc16a2 was mostly unaffected. Blockade and knockdown of the TSH receptor (TSHR) and antagonization of cAMP response element-binding protein (CREB) clearly abolished the increased expression induced by TSH, indicating PKA-dependent regulation through the TSHR. The TSH-dependent expression of Dio3 and Slco1c1 was also regulated by protein kinase C (PKC), and in case of Dio3, also by extracellular signal-regulated kinase (ERK) activity. Importantly, these gene regulations were specifically found in different subpopulations of tanycytes. Conclusions: This study demonstrates that TSH induces transcriptional regulation of TH-gatekeeper genes in tanycytes through the Tshr/Gαq/PKC pathway, in parallel to the Tshr/Gαs/PKA/CREB pathway. These differential actions of TSH on tanycytic subpopulations appear to be important for coordinating the supply of TH to the hypothalamus and aid its functions.

Activation of mitogen-activated protein kinase signaling and development of papillary thyroid carcinoma in thyroid-stimulating hormone receptor D633H knockin mice.

Objective: Nonautoimmune hyperthyroidism (NAH) is rare and occurs due to a constitutively activating thyroid stimulating hormone receptor (TSHR) mutation. In contrast to other thyroid nodules, no further evaluation for malignancy is recommended for hot thyroid nodules. In the first model for NAH in mice nearly all homozygous mice had developed papillary thyroid cancer by 12 months of age. Methods: To further evaluate these mice, whole exome sequencing and phosphoproteome analysis were employed in a further generation of mice to identify any other mutations potentially responsible and to identify the pathways involved in thyroid carcinoma development. Results: Only three genes (Nrg1, Rrs1, Rasal2) were mutated in all mice examined, none of which were known primary drivers of papillary thyroid cancer development. Wild-type and homozygous TSHR D633H knockin mice showed distinct phosphoproteome profiles with an enrichment of altered phosphosites found in ERK/mitogen-activated protein kinase (MAPK) signaling. Most importantly, phosphosites with known downstream effects included BRAF p.S766, which forms an inhibitory site: a decrease of phosphorylation at this site suggests an increase in MEK/ERK pathway activation. The decreased phosphorylation at BRAF p.S766 would suggest decreased AMP-activated protein kinase (AMPK) signaling, which is supported by the decreased phosphorylation of STIM1 p.S257, a downstream AMPK target. Conclusion: The modified phosphoproteome profile of the homozygous mice in combination with human literature suggests a potential signaling pathway from constitutive TSHR signaling and cAMP activation to the activation of ERK/MAPK signaling. This is the first time that a specific mechanism has been identified for a possible involvement of TSH signaling in thyroid carcinoma development.

The Potential Role of Pyrroloquinoline Quinone to Regulate Thyroid Function and Gut Microbiota Composition of Graves' Disease in Mice.

Graves' disease (GD) is an autoimmune disorder disease, and its prevalence continues to increase worldwide. Pyrroloquinoline quinone (PQQ) is a naturally antioxidant compound in milk, vegetables, and meat. We aim to identify the treatment efficacy of PQQ on GD and its regulatory effect on intestinal microbiota. The GD mice model was built by an adenovirus expressing autoantigen thyroid-stimulating hormone receptor (Ad-TSHR289). Fecal samples were collected for 16S rDNA sequencing after PQQ pretreatments (20, 40, or 60 mg/kg BW/day) for 4 weeks. Thyroid and intestine functions were measured. The levels of serum TSHR and T4 were significantly raised, and the thyroid gland size was typically enlarged in the GD group than in controls, reversed by PQQ therapy. After PQQ replenishment, IL6 and TNFα levels in small intestine tissues were lower than those in the GD group, with Nrf2 and HO1 levels improved. Also, the PQQ supplement could maintain the mucosal epithelial barrier impaired by GD. In microbial analyses, PQQ treatment could prompt the diversity recovery of gut microbiota and reconstruct the microbiota composition injured by GD. Lactobacillus served as the most abundant genus in all groups, and the abundance of Lactobacillus was increased in the GD group than in control and PQQ groups. Besides, Lactobacillus was highly correlative with all samples and the top 50 genera. PQQ supplementation regulates thyroid function and relieves intestine injury. PQQ changes the primary composition and abundance of GD's intestine microbiota by moderating Lactobacillus, which may exert in the pathogenesis and progression of GD.

Activation of mitogen-activated protein kinase signaling and development of papillary thyroid carcinoma in thyroid-stimulating hormone receptor D633H knockin mice.

Objective: Nonautoimmune hyperthyroidism (NAH) is rare and occurs due to a constitutively activating thyroid stimulating hormone receptor (TSHR) mutation. In contrast to other thyroid nodules, no further evaluation for malignancy is recommended for hot thyroid nodules. In the first model for NAH in mice nearly all homozygous mice had developed papillary thyroid cancer by 12 months of age. Methods: To further evaluate these mice, whole exome sequencing and phosphoproteome analysis were employed in a further generation of mice to identify any other mutations potentially responsible and to identify the pathways involved in thyroid carcinoma development. Results: Only three genes (Nrg1, Rrs1, Rasal2) were mutated in all mice examined, none of which were known primary drivers of papillary thyroid cancer development. Wild-type and homozygous TSHR D633H knockin mice showed distinct phosphoproteome profiles with an enrichment of altered phosphosites found in ERK/mitogen-activated protein kinase (MAPK) signaling. Most importantly, phosphosites with known downstream effects included BRAF p.S766, which forms an inhibitory site: a decrease of phosphorylation at this site suggests an increase in MEK/ERK pathway activation. The decreased phosphorylation at BRAF p.S766 would suggest decreased AMP-activated protein kinase (AMPK) signaling, which is supported by the decreased phosphorylation of STIM1 p.S257, a downstream AMPK target. Conclusion: The modified phosphoproteome profile of the homozygous mice in combination with human literature suggests a potential signaling pathway from constitutive TSHR signaling and cAMP activation to the activation of ERK/MAPK signaling. This is the first time that a specific mechanism has been identified for a possible involvement of TSH signaling in thyroid carcinoma development.

Thyrotropin receptor autoantibody (TRAb) enhance the expression of thyrotropin receptor on mouse brain vascular endothelial cells: in vivo and in vitro.

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.

Molecular investigation of TSHR gene in Bangladeshi congenital hypothyroid patients.

The disorder of thyroid gland development or thyroid dysgenesis accounts for 80-85% of congenital hypothyroidism (CH) cases. Mutations in the TSHR gene are mostly associated with thyroid dysgenesis, and prevent or disrupt normal development of the gland. There is limited data available on the genetic spectrum of congenital hypothyroid children in Bangladesh. Thus, an understanding of the molecular aetiology of thyroid dysgenesis is a prerequisite. The aim of the study was to investigate the effect of mutations in the TSHR gene on the small molecule thyrogenic drug-binding site of the protein. We identified two nonsynonymous mutations (p.Ser508Leu, p.Glu727Asp) in the exon 10 of the TSHR gene in 21 patients with dysgenesis by sequencing-based analysis. Later, the TSHR368-764 protein was modeled by the I-TASSER server for wild-type and mutant structures. The model proteins were targeted by thyrogenic drugs, MS437 and MS438 to perceive the effect of mutations. The damaging effect in drug-protein complexes of mutants was explored by molecular docking and molecular dynamics simulations. The binding affinity of wild-type protein was much higher than the mutant cases for both of the drug ligands (MS437 and MS438). Molecular dynamics simulates the dynamic behavior of wild-type and mutant complexes. MS437-TSHR368-764MT2 and MS438-TSHR368-764MT1 showed stable conformations in biological environments. Finally, Principle Component Analysis revealed structural and energy profile discrepancies. TSHR368-764MT1 exhibited much more variations than TSHR368-764WT and TSHR368-764MT2, emphasizing a more damaging pattern in TSHR368-764MT1. This genetic study might be helpful to explore the mutational impact on drug binding sites of TSHR protein which is important for future drug design and selection for the treatment of congenital hypothyroid children with dysgenesis.

A Feedback Loop Involving Exosomal miR-146a and NG2 to Propel the Development and Progression of Hypothyroidism.

Background: Thyrotropin receptor (TSHR) plays a central role in maintaining thyroid function and TSHR impairment causes hypothyroidism, which is often associated with metabolic disarrangement. The most common type of hypothyroidism is autoimmune disease-related and the mechanism, particularly with respect to the role of microRNAs (miRNAs), has not been delineated. Methods: Serum from 30 patients with subclinical hypothyroidism (SCH) and 30 healthy individuals were collected and exosomal miR-146a (exo-miR-146a) was examined, followed by extensive mechanistic investigation using various molecular and cellular experimental approaches and genetic-knockout mouse models. Results: Our clinical investigation showed that exo-miR-146a was systemically elevated in the serum of patients with SCH (p = 0.04) compared with healthy individuals, prompting us to investigate the biological effects of miR-146a in cells. We found that miR-146a could target and down-regulate neuron-glial antigen 2 (Ng2), with consequent down-regulation of TSHR. We next generated a thyroid-specific Ng2 knockout (Thy-Ng2-/-) mouse model and found a significant down-regulation of TSHR in Thy-Ng2-/- mice, accompanied by the development of hypothyroidism and metabolic disorders. We further found that a decrease in NG2 resulted in decreased receptor tyrosine kinase-linked downstream signaling and down-regulation of c-Myc, consequently resulting in up-regulation of miR-142 and miR-146a in thyroid cells. Up-regulated miR-142 targeted the 3'-untranslated region (UTR) of TSHR messenger RNA (mRNA) and post-transcriptionally down-regulated TSHR, explaining the development of hypothyroidism above. Local up-regulation of miR-146a in thyroid cells augments the earlier cited processes initiated by systemically elevated miR-146a, thereby forming a feedback loop to propel the development and progression of hypothyroidism. Conclusions: This study has uncovered a self-augmenting molecular loop initiated by elevated exo-miR-146a to suppress TSHR through targeting and down-regulating NG2, thereby initiating and propelling the development and progression of hypothyroidism.

Establishment and Comparison of Two Different Animal Models of Graves' Orbitopathy.

Purpose: The purpose of this study was to construct an animal model of Graves' ophthalmopathy (GO) by comparing recombinant adenovirus expressing human thyrotropin receptor A subunit (Ad-TSHR A) gene immunization and dendritic cell (DC) immunization. We evaluated the animal models that are closer to the pathology of human GO, and laid the foundation for the study of GO. Materials and Methods: Ad-TSHR A was injected intramuscularly into female BALB/c mice to induce the GO animal model. A GO animal model was constructed using TSHR combined with IFN-γ-modified primary DC immunized female BALB/c. The animal models constructed by the above two methods were evaluated in terms of ocular appearance, serology, pathology, and imaging to assess the modeling rate of the animal models, respectively. Results: Both modeled mice exhibited increased serological indexes of free thyroxine (FT4) and TSH receptor antibodies (TRAbs) levels and decreased TSH (P < 0.01). Thyroid pathology analysis revealed the number of thyroid follicles increases, the size varies, and the follicular epithelial cells proliferate to varying degrees in a cuboidal or tall columnar pattern, with a small amount of lymphocytic infiltration visible. Adipose tissue behind the eyeball was accumulated, the muscle outside the eyeball was broken and fibrotic, and hyaluronic acid (HA) behind the eyeball was increased. The animal model of GO constructed by immunization of TSHR with IFN-γ-modified DC had a modeling rate of 60%, whereas that of Ad-TSHR A gene immunization was 72%. Conclusions: Both gene immunization and cellular immunization can be used to construct GO models, and the modeling rate of gene immunization is higher than that of cellular immunization. Translational Relevance: In this study, two innovative methods, cellular immunity and gene immunity, were used to establish GO animal models, which improved the success rate to a certain extent. To our knowledge, this study presents the first cellular immunity modeling idea of TSHR combined with IFN-γ for the GO animal model, which provides an animal model basis for understanding the pathogenesis of GO and developing new treatment methods.

Unraveling the significance of TSHR mutations in indeterminate thyroid cytology specimens.

OBJECTIVES: We investigated the clinical significance of thyroid-stimulating hormone receptor (TSHR) mutations detected in thyroid fine needle aspiration (FNA) specimens. METHODS: The pathology archives at our institution were reviewed between 2018 and 2021 for indeterminate (Bethesda category III and IV) specimens with Thyroseq® analysis showing TSHR mutations. RESULTS: A total of 2184 cases diagnosed as atypia/follicular lesion of undetermined significance (AUS/FLUS), and 2625 diagnosed as follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) were identified. A total of 1735 AUS/FLUS and 2339 SFN/FN underwent Thyroseq® analysis; 69 showed TSHR mutations (1.6%, 59 female, 10 male, average age: 55 years). Ten cases showed oncocytic features. Twelve patients underwent radionuclide scans within 1 year of FNA:11 were hyperfunctioning. Nodule size and TSH levels were weakly correlated. Twenty-two different TSHR mutations were identified (most common: M453T). A second mutation was found in five cases (EZH1 n = 2, and EIF1AX n = 3). The expression of sodium-iodide transporter (NIS) mRNA was in the range of 0.01%-62.43% out of all sequencing reads, and was elevated in 49 (71%) cases. Surgical pathology follow-up was available in five cases (all benign except one). On follow-up available for 38 cases (mean: 24 months; range: 7-47 months), 34 (89.5%) nodules remained stable and 3 (8%) increased in size. CONCLUSIONS: TSHR mutations in thyroid FNA samples classified as indeterminate are rare, generally benign, and commonly associated with autonomy on scan if performed.

Thyroid Stem Cell Speciation-a Major Role for PKC.

Instructive signals that delineate the formation of thyroid follicles by thyrotropin (TSH) in stem cells are complex. Here, we have examined the role of protein kinase C (PKC) by using a unique Gαq/11 biased small molecule (MSq1) to develop thyroid progenitor cells. Mouse embryonic stem cells (mESCs) were differentiated into anterior endoderm cells and treated with either TSH or MSq1 in the presence or absence of PKC inhibitors. The transcriptional and translational response of key thyroid markers-sodium iodide symporter (NIS), thyroglobulin (TG), and thyrotropin receptor (TSHR) as well as potential signaling molecules-were then analyzed. The data confirmed that MSq1 is a potent Gαq/11 activator with a major increase in Gαq/11 signaling when compared to TSH. MSq1 activation resulted in an increase in thyroid-specific genes, demonstrating that enhanced PKC signaling was able to induce their expression. The specificity of the PKC signals over the protein kinase A (PKA) pathway in regulating thyroid gene expression was shown by using a specific PKC enzyme inhibitor. The data revealed that TG and NIS expression were suppressed in the presence of the PKC inhibition but, in contrast, were not influenced by PKA inhibition. This indicated that PKC activation was the dominant pathway in the inductive process for thyroid hormone production. Furthermore, by examining PKC isoforms we found that PKCξ was the predominant form in the ES cells that mediated the effects. Since PKCξ can lead to activation of transforming growth factor-ß-activated kinase (pTAK1), and its downstream effector nuclear factor κB (NFκB) complex, this demonstrated the involvement of the TAK1/NFκB pathway in thyroid speciation.

[Pathogenic TSHR variants in children with thyroid dysgenesis].

BACKGROUND: Loss-of-function mutations in the TSH receptor gene (TSHR) (NP_000360.2) are the potential causes of thyroid dysgenesis in patients with congenital hypothyroidism. Heterozygous variants of the TSHR gene lead to partial resistance to TSH, homozygous and compound heterozygous variants have been shown to cause CH due to thyroid hypoplasia or TSH resistance. Recently more and more articles in this field have appeared in the international literature sources, while local publications are limited. The studies are necessary to understand the etiology, pathogenesis of the disease, to improve the management of these patients. AIM: To assess the frequency of incidence of pathogenic variants of the TSHR gene in children with CH due to thyroid dysgenesis. To study inheritance and phenotypic patterns of CH in families. MATERIALS AND METHODS: In this single-center interventional one-stage non-comparative study a group of CH patients was examined. The patients underwent neck ultrasound and radionuclide imaging. The examination was performed 14 days after hormone replacement therapy suspension or prior to its initiation. The structure of thyroid dysgenesis was estimated, genetic testing for mutations in the TSHR gene was performed using the NGS method. RESULTS: The study included 95 children with primary CH (75 girls; 20 boys). The patients' median age at the time of examination was 6.2 years [4.5; 8.9], the median level of neonatal TSH was 157.5 mU/l [60.9; 257.2]. Ectopic thyroid was found in 52% of children, aplasia in 36%, hypoplasia and hemiagenesis in 10% and 2%, respectively. In 5.4% of cases (in 5 out of 95 patients), different variants of the TSH gene were detected. Two children had heterozygous p.R450H and p.D487N variants in TSHR gene, two patients was homozygous for the p.S49Afs * 9 variant, one child had compound heterozygous variants (p.A485D and p.R450H). According to ultrasound imaging, all patients had thyroid hypoplasia of varying severity. Three children underwent thyroid scintigraphy, which revealed decreased 99mТc pertechnetate uptake (0.3-0.9%). CONCLUSION: In our study, the incidence of different variants in the TSHR gene in children with CH was 5.3%. Our analysis uncovered two previously undescribed variants. Genetic testing may be able to help with making the diagnosis, patient's management, and genetic counseling.

Screen of small fragment mutations within the sheep thyroid stimulating hormone receptor gene associated with litter size.

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).

The role and molecular mechanism of gut microbiota in Graves' orbitopathy.

PURPOSE: Graves' orbitopathy (GO) is an autoimmune orbital disorder. Gut microbiota dysfunction plays a vital role in autoimmune diseases, including Graves' disease (GD) and GO. In the present study, we aimed to investigate the change of gut microbiota in GD/GO using mouse model. METHODS: The murine model of GD/GO was established by the challenge of adenovirus expressing thyroid-stimulating hormone (TSH) receptor (TSHR) (Ad-TSHR). The histological changes of orbital and thyroid tissues were analyzed by hematoxylin and eosin (H&E), Masson staining, and immunohistochemistry (IHC) staining. The fecal samples were collected for 16S rRNA gene sequencing and bioinformatics analysis. RESULTS: The GD/GO model was established successfully, as manifested as the broadened eyelid, exophthalmia and conjunctive redness, severe inflammatory infiltration among thyroid glands and between extraocular muscle space, hypertrophic extraocular muscles, elevated thyroxine (T4) and decreased TSH, and positive CD34, CD40, collagen I, and α-SMA staining. A total of 222 operational taxonomic units (OUTs) were overlapped between mice in the Ad-NC and Ad-TSHR groups. The microbial composition of the samples in the two groups was mainly Bacteroidia and Clostridia, and the Ad-NC group had a significantly lower content of Bacteroidia and higher content of Clostridia. KEGG orthology analysis results revealed differences in dehydrogenase, aspartic acid, bile acid, chalcone synthase, acetyltransferase, glutamylcyclotransferase, glycogenin, and 1-phosphatidylinositol-4-phosphate 5-kinase between two groups; enzyme commission (EC) analysis results revealed differences in several dehydrogenase, oxidase, thioxy/reductase between two groups; MetaCyc pathways analysis results revealed differences in isoleucine degradation, oxidation of C1 compounds, tricarboxylic acid (TCA) cycle IV, taurine degradation, and biosynthesis of paromamine, heme, colonic acid building blocks, butanediol, lysine/threonine/methionine, and histidine/purine/pyrimidine between two groups. CONCLUSION: This study induced a mouse model of GD/GO by Ad-TSHR challenge, and gut microbiota characteristics were identified in the GD/GO mice. The Bacteroidia and Clostridia abundance was changed in the GD/GO mice. These findings may lay a solid experimental foundation for developing personalized treatment regimens for GD patients according to the individual gut microbiota. Given the potential impact of regional differences on intestinal microbiota, this study in China may provide a reference for the global overview of the gut-thyroid axis hypothesis.