RESUMEN

Introduction: Bruton's tyrosine kinase (BTK) and interleukin (IL)-2 Inducible T-cell Kinase (ITK) inhibitors have anti-inflammatory properties. We investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK/ITK inhibitor, in a mouse model of Graves' orbitopathy (GO). Methods: Genetic immunization was performed through intramuscular administration of the recombinant plasmid, pCMV6-hTSHR cDNA, to 8-week-old female BALB/c mice. Serum levels of T3, T4, and thyroid-stimulating hormone receptor (TSHR) antibodies (TRAbs) were quantified using enzyme-linked immunosorbent assay. Histopathological changes in orbital tissues were examined using immunohistochemistry (IHC) staining for TSHR and various inflammatory markers. Following successful genetic immunization, ibrutinib was orally administered daily for 2 weeks in the GO model mice. After treatment, the mRNA and protein expression levels of BTK, ITK, IL-1ß, and IL-6 in orbital tissues were evaluated using real-time PCR and Western blotting. Results: In total, 20 mice were sacrificed to confirm successful genetic immunization. The GO mouse group exhibited significantly increased serum T3, T4, and TRAb levels. IHC revealed increased expression of TSHR, IL-1ß, IL-6, transforming growth factor-ß1, interferon-γ, CD40, CD4, BTK, and ITK in the GO mouse model. The orbital inflammation was significantly attenuated in ibrutinib-treated mice. The mRNA and protein expression levels of BTK, ITK, IL-1ß, and IL-6 in orbital tissue were lower in ibrutinib-treated GO mouse group compared to the phosphate-buffered saline-treated GO mouse group. Conclusion: The GO mouse model demonstrated enhanced BTK and ITK expression. Ibrutinib, a BTK/ITK inhibitor, suppressed the inflammatory cytokine production. These findings highlight the potential involvement of BTK/ITK in the inflammatory pathogenesis of GO, suggesting its role as a novel therapeutic target.

Asunto(s)

Adenina , Agammaglobulinemia Tirosina Quinasa , Modelos Animales de Enfermedad , Oftalmopatía de Graves , Inflamación , Ratones Endogámicos BALB C , Piperidinas , Pirimidinas , Animales , Oftalmopatía de Graves/tratamiento farmacológico , Oftalmopatía de Graves/metabolismo , Oftalmopatía de Graves/patología , Adenina/análogos & derivados , Piperidinas/uso terapéutico , Ratones , Femenino , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/patología , Pirimidinas/uso terapéutico , Pirazoles/uso terapéutico , Pirazoles/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Receptores de Tirotropina/metabolismo , Receptores de Tirotropina/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico

RESUMEN

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.

Asunto(s)

Biomarcadores de Tumor , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , MicroARNs , Receptores de Tirotropina , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Humanos , MicroARNs/sangre , MicroARNs/genética , Femenino , Masculino , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/sangre , Cáncer Papilar Tiroideo/diagnóstico , Cáncer Papilar Tiroideo/patología , Persona de Mediana Edad , Neoplasias de la Tiroides/sangre , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/patología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Adulto , Receptores de Tirotropina/genética , Estudios de Casos y Controles , Curva ROC

RESUMEN

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.

Asunto(s)

Predisposición Genética a la Enfermedad , Enfermedad de Graves , Enfermedad de Hashimoto , Polimorfismo de Nucleótido Simple , Receptores de Tirotropina , Humanos , Receptores de Tirotropina/genética , Enfermedad de Graves/genética , Enfermedad de Hashimoto/genética , Tiroiditis Autoinmune/genética , Tiroiditis Autoinmune/epidemiología

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito , Oxidasas Duales , Secuenciación de Nucleótidos de Alto Rendimiento , Receptores de Tirotropina , Tiroxina , Humanos , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/tratamiento farmacológico , Hipotiroidismo Congénito/diagnóstico , Hipotiroidismo Congénito/sangre , Femenino , Masculino , Estudios Retrospectivos , Oxidasas Duales/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Tiroxina/uso terapéutico , Receptores de Tirotropina/genética , Preescolar , Lactante , Recién Nacido , Tiroglobulina/genética , Tiroglobulina/sangre , Proteínas de la Membrana

RESUMEN

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.

Asunto(s)

Anguilas , Receptores de Tirotropina , Animales , Receptores de Tirotropina/metabolismo , Receptores de Tirotropina/genética , Femenino , Masculino , Anguilas/metabolismo , Anguilas/genética , Testículo/metabolismo , Gónadas/metabolismo , Comunicación Paracrina/fisiología , Ovario/metabolismo , Hipófisis/metabolismo , Tirotropina de Subunidad beta/metabolismo , Tirotropina de Subunidad beta/genética , Comunicación Autocrina/fisiología

RESUMEN

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.

Asunto(s)

Epítopos , Enfermedad de Graves , Receptores de Tirotropina , Humanos , Receptores de Tirotropina/inmunología , Receptores de Tirotropina/genética , Epítopos/inmunología , Enfermedad de Graves/inmunología , Enfermedad de Graves/sangre , Enfermedad de Graves/diagnóstico , Oftalmopatía de Graves/inmunología , Oftalmopatía de Graves/genética , Oftalmopatía de Graves/sangre , Autoanticuerpos/inmunología , Autoanticuerpos/sangre , Femenino , Masculino , Inmunoglobulinas Estimulantes de la Tiroides/sangre , Inmunoglobulinas Estimulantes de la Tiroides/inmunología , Glándula Tiroides/inmunología , Adulto , Persona de Mediana Edad , Proliferación Celular , Tiroiditis Autoinmune/inmunología , Tiroiditis Autoinmune/genética , Hipotiroidismo/inmunología

RESUMEN

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 [89Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [89Zr]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 89Zr (t1/2 = 78.4 h, ß+ = 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 [89Zr]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 [89Zr]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 [89Zr]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 [89Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.

Asunto(s)

Ratones Desnudos , Tomografía de Emisión de Positrones , Receptores de Tirotropina , Neoplasias de la Tiroides , Circonio , Animales , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Humanos , Ratones , Circonio/química , Tomografía de Emisión de Positrones/métodos , Línea Celular Tumoral , Femenino , Receptores de Tirotropina/metabolismo , Receptores de Tirotropina/genética , Radiofármacos/farmacocinética , Radiofármacos/química , Distribución Tisular , Masculino , Radioisótopos/química

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito , Secuenciación del Exoma , Yoduro Peroxidasa , Receptores de Tirotropina , Humanos , Taiwán , Femenino , Masculino , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/diagnóstico , Recién Nacido , Yoduro Peroxidasa/genética , Receptores de Tirotropina/genética , Oxidasas Duales/genética , Tiroglobulina/genética , Proteínas de Unión a Hierro/genética , Preescolar , Variación Genética , Mutación , Disgenesias Tiroideas/genética , Disgenesias Tiroideas/diagnóstico , Lactante , Autoantígenos

RESUMEN

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 99mTcO4- 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 99mTcO4- 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.

Asunto(s)

Modelos Animales de Enfermedad , Oftalmopatía de Graves , Ratones Endogámicos BALB C , Receptor IGF Tipo 1 , Receptores de Tirotropina , Animales , Oftalmopatía de Graves/genética , Oftalmopatía de Graves/patología , Ratones , Receptor IGF Tipo 1/genética , Femenino , Receptores de Tirotropina/genética , Receptores de Tirotropina/inmunología , Inmunización/métodos , Enfermedad de Graves/genética , Enfermedad de Graves/patología

RESUMEN

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.

Asunto(s)

Hipertiroidismo , Neoplasias de la Tiroides , Humanos , ADN , Hipertiroidismo/genética , Radioisótopos de Yodo , Mutación , Receptores de Tirotropina/genética

RESUMEN

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.

Asunto(s)

Hipertiroidismo , beta Catenina , Ratones , Animales , beta Catenina/metabolismo , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Vía de Señalización Wnt/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Hipocampo/metabolismo , Neurogénesis/fisiología , Hipertiroidismo/metabolismo

RESUMEN

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.

Asunto(s)

Hipotiroidismo , Hormonas Tiroideas , Tirotropina , Animales , Ratones , Hipotiroidismo/complicaciones , Hipotiroidismo/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Transducción de Señal , Hormonas Tiroideas/metabolismo , Intestinos/patología

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito , Humanos , China , Hipotiroidismo Congénito/diagnóstico , Hipotiroidismo Congénito/genética , AMP Cíclico , Oxidasas Duales/genética , Mutación , Fenotipo , Receptores de Tirotropina/genética , Tirotropina

RESUMEN

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.

Asunto(s)

Hipertiroidismo , Hipotiroidismo , Humanos , Femenino , Receptores de Tirotropina/genética , Hipertiroidismo/genética , Mutación , Hipotiroidismo/complicaciones , Receptores Acoplados a Proteínas G/genética , ARN Mensajero

RESUMEN

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.

Asunto(s)

Hipertiroidismo , Yodo , Receptores de Tirotropina , Animales , Humanos , Ratones , Hipertiroidismo/congénito , Mutación , Fenotipo , Receptores Acoplados a Proteínas G/genética , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo

RESUMEN

PURPOSE: To evaluate the impact of high thyroid stimulating hormone (TSH) levels on human granulosa-luteal (hGL) cells. METHODS: hGL cells were isolated from follicular aspirates derived from patients undergoing IVF treatment without any thyroid disorder (serum TSH 0.5-2 mU/L). Cells were cultured at 37 °C in DMEM, supplemented with 5% FBS. The cells were treated with 1 nM LH and increasing concentrations of TSH. At the end of culture, conditioned medium and cells were collected to analyze progesterone production, cell viability, and mRNA levels of genes involved in the steroidogenesis process. Human ovarian tissues were analyzed for TSH receptor (TSHR) expression by IHC. RESULTS: The expression of TSHR was detected in human corpus luteum by IHC and in hGL by RT-PCR. In hGL cells, TSH treatment did not modulate progesterone production nor the expression of steroidogenic genes, such as p450scc and HSD3b 1/2. However, TSH induced a dose-dependent increase in cell death. Finally, TSH did not affect LH-induced p450scc and HSD3b1/2 expression while LH partially reverted TSH negative effect on cell death in hGL. CONCLUSIONS: Elevated TSH levels in hypothyroid women may be associated with impaired CL functioning and maintenance. These findings open a new line of research for the importance of the treatment of women with thyroid dysfunction that could contribute to the onset of infertility.

Asunto(s)

Cuerpo Lúteo , Tirotropina , Humanos , Femenino , Tirotropina/metabolismo , Cuerpo Lúteo/metabolismo , Cuerpo Lúteo/efectos de los fármacos , Progesterona/metabolismo , Células Cultivadas , Receptores de Tirotropina/metabolismo , Receptores de Tirotropina/genética , Hormona Luteinizante/metabolismo , Adulto , Células Lúteas/metabolismo , Células Lúteas/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito , Receptores de Tirotropina , Femenino , Humanos , Niño , Adolescente , Receptores de Tirotropina/genética , Tiroxina/uso terapéutico , Pruebas de Función de la Tiroides , Mutación , Tirotropina

RESUMEN

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.

Asunto(s)

Células Ependimogliales , Tirotropina , Humanos , Tirotropina/farmacología , Tirotropina/metabolismo , Células Ependimogliales/metabolismo , Hormonas Tiroideas/metabolismo , Glándula Tiroides/metabolismo , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Hormonas Liberadoras de Hormona Hipofisaria/metabolismo , Proteína Quinasa C/metabolismo

RESUMEN

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.

Asunto(s)

Microbioma Gastrointestinal , Enfermedad de Graves , Ratones , Animales , Microbioma Gastrointestinal/fisiología , Cofactor PQQ , Enfermedad de Graves/tratamiento farmacológico , Enfermedad de Graves/genética , Receptores de Tirotropina/genética

RESUMEN

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.

Asunto(s)

Neoplasias de la Tiroides , Nódulo Tiroideo , Ratones , Humanos , Animales , Cáncer Papilar Tiroideo/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Tirotropina/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Transducción de Señal/genética , Neoplasias de la Tiroides/genética , Tirotropina/metabolismo