RESUMO
Background: The sodium/iodide symporter (NIS) mediates active iodide accumulation in the thyroid follicular cell. Autosomal recessive iodide transport defect (ITD)-causing loss-of-function NIS variants lead to dyshormonogenic congenital hypothyroidism due to deficient iodide accumulation for thyroid hormonogenesis. Here, we aimed to identify, and if so to functionally characterize, novel ITD-causing NIS pathogenic variants in a patient diagnosed with severe dyshormonogenic congenital hypothyroidism due to a defect in iodide accumulation in the thyroid follicular cell, as suggested by nondetectable radioiodide accumulation in a normally located thyroid gland, as well as in salivary glands. Methods: The proposita NIS-coding SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of the novel NIS variants were performed. Results: Sanger sequencing revealed novel compound heterozygous SLC5A5 gene variants (c.970-3C>A and c.1106A>T, p.D369V). In silico analysis suggested that c.970-3C>A disrupts the canonical splice acceptor site located in intron 7. Splicing minigene reporter assay revealed that c.970-3C>A causes exon 8 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variant p.Y324Hfs*148. Moreover, in silico analysis indicated p.D369V as pathogenic. Functional in vitro studies demonstrated that p.D369V NIS does not mediate iodide accumulation, as p.D369V causes NIS to be retained in the endoplasmic reticulum. Mechanistically, we propose an intramolecular ionic interaction involving the ß carboxyl group of D369 and the guanidinium group of R130, located in transmembrane segment 4. Of note, an Asp residue at position 369-which is highly conserved in SLC5A family members-is required for functional NIS expression at the plasma membrane. Conclusions: We uncovered a critical intramolecular interaction between R130 and D369 required for NIS maturation and plasma membrane expression. Moreover, we identified the first intronic variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape in the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.
Assuntos
Membrana Celular/efeitos dos fármacos , Hipotireoidismo Congênito/tratamento farmacológico , Simportadores/efeitos dos fármacos , Membrana Celular/fisiologia , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Humanos , Glândula Tireoide/metabolismoRESUMO
Differentiated thyroid carcinoma (DTC) combined with congenital hypothyroidism (CH) is a rare situation, and there is no well-established causal relationship. CH is a common congenital endocrine, while DTC occurring in childhood represents 0.4-3% of all malignancies at this stage of life. The association of CH with DTC could be related to dyshormonogenetic goiter (DHG) or developmental abnormalities. This review will explore the clinical features and the molecular mechanisms potentially associated with the appearance of DTC in CH: sporadic somatic driver mutations, chronic increase of thyroid-stimulating hormone (TSH) levels, higher concentrations of hydrogen peroxide (H2O2), cell division cycle associated 8 (Borelain/CDC8) gene mutations, and in others genes associated with CH - either alone or associated with the mechanisms involved in dyshormonogenesis. There are some pitfalls in the diagnosis of thyroid cancer in patients with CH with nodular goiter, as the proper cytological diagnosis of nodules of patients with dyshormonogenesis might be demanding due to the specific architectural and cytological appearance, which may lead to an erroneous interpretation of malignancy. The purpose of this article is to suggest an analytical framework that embraces the fundamental relationships between the various aspects of CH and CDT. In face of this scenario, the entire genetic and epigenetic context, the complex functioning, and cross talk of cell signaling may determine cellular mechanisms promoting both the maintenance of the differentiated state of the thyroid follicular cell and the disruption of its homeostasis leading to cancer. Whereas, the exact mechanisms for thyroid cancer development in CH remain to be elucidated.
Assuntos
Hipotireoidismo Congênito , Neoplasias da Glândula Tireoide , Hipotireoidismo Congênito/diagnóstico , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Humanos , Peróxido de Hidrogênio , Mutação , Neoplasias da Glândula Tireoide/genéticaRESUMO
The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.
Assuntos
Proteínas de Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Códon sem Sentido , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Sequência Conservada , Cães , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios PDZ/genética , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/química , Simportadores/genética , Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
ABSTRACT Objective Determine the milk quality effect during lactation on the metabolic and thyroid programming of hypothyroid offspring. Materials and methods Ten-week-old female Wistar rats were divided into two groups: euthyroid and thyroidectomy-caused hypothyroidism. The rats were matted and, one day after birth, the pups were divided into three groups: euthyroid offspring (EO), hypothyroid offspring (HO) and hypothyroid with a euthyroid replacement wet nurse (HRO). During lactation, the milk quality and offspring body length were evaluated. The body weight and energy intake were determined on a weekly basis, as well as the metabolic profile at the prepubertal (P35-36) and postpubertal (P55-56) ages. At P56, the animals were sacrificed, the adipose tissues were weighed and the thyroid glands were dissected for histological processing. Results The milk of the hypothyroid wet nurse decreases proteins (16-26%), lipids (22-29%) and lactate (22-37%) with respect to euthyroid. The HO has a lower body weight gain (23-33%), length (11-13%) and energy intake (15-21%). In addition, HO presents impaired fasting glucose and dyslipidemia, as well as a reduction in seric thyroid hormone (18-34%), adipose reserves (26-68%) and thyroid gland weight (25-34%). The HO present thyroid gland cytoarchitecture alteration. The HRO develop the same metabolic alterations as the HO. However, the thyroid gland dysfunction was partially prevented because the HRO improved under about 10% of the serum thyroid hormone concentration, the thyroid gland weight although histological glandular changes presented. Conclusions The replacement of hypothyroid offspring with a euthyroid wet nurse during lactation can improve the thyroid programming without modifying metabolic programming.
Assuntos
Animais , Feminino , Ratos , Hormônios Tireóideos/metabolismo , Lactação/metabolismo , Hipotireoidismo Congênito/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Glândula Tireoide/patologia , Ratos Wistar , Modelos Animais de DoençasRESUMO
OBJECTIVE: Determine the milk quality effect during lactation on the metabolic and thyroid programming of hypothyroid offspring. MATERIALS AND METHODS: Ten-week-old female Wistar rats were divided into two groups: euthyroid and thyroidectomy-caused hypothyroidism. The rats were matted and, one day after birth, the pups were divided into three groups: euthyroid offspring (EO), hypothyroid offspring (HO) and hypothyroid with a euthyroid replacement wet nurse (HRO). During lactation, the milk quality and offspring body length were evaluated. The body weight and energy intake were determined on a weekly basis, as well as the metabolic profile at the prepubertal (P35-36) and postpubertal (P55-56) ages. At P56, the animals were sacrificed, the adipose tissues were weighed and the thyroid glands were dissected for histological processing. RESULTS: The milk of the hypothyroid wet nurse decreases proteins (16-26%), lipids (22-29%) and lactate (22-37%) with respect to euthyroid. The HO has a lower body weight gain (23-33%), length (11-13%) and energy intake (15-21%). In addition, HO presents impaired fasting glucose and dyslipidemia, as well as a reduction in seric thyroid hormone (18-34%), adipose reserves (26-68%) and thyroid gland weight (25-34%). The HO present thyroid gland cytoarchitecture alteration. The HRO develop the same metabolic alterations as the HO. However, the thyroid gland dysfunction was partially prevented because the HRO improved under about 10% of the serum thyroid hormone concentration, the thyroid gland weight although histological glandular changes presented. CONCLUSIONS: The replacement of hypothyroid offspring with a euthyroid wet nurse during lactation can improve the thyroid programming without modifying metabolic programming.
Assuntos
Hipotireoidismo Congênito/metabolismo , Lactação/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Hormônios Tireóideos/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Ratos , Ratos Wistar , Glândula Tireoide/patologiaRESUMO
The goal of this study is to investigate whether congenital hypothyroidism induced by MMI during gestation (G) or gestation plus lactation (GL) would affect the leptin action upon body weight control on hypothalamus. Six to eight pups per group were killed at 90 days of age. For statistical analysis one-way ANOVA followed by the Holm-Sìdak post hoc test was used. Hypothyroidism resulted in a significant increase in leptin serum levels in G 20% and GL 25% (p<0.04). There was a significant expression decrease of OBR in G 45% and GL 63%; pSTAT3 in G 56% and GL 51%; pERK in G 50% and GL 48%; POMC in G 41% and GL 46% (p<0.04), while a significant increase was assigned to SOCS3 in G 52% and GL 170% (p<0.04) protein expression. We can conclude that hypothyroxinemia condition in rats on adulthood results in impairment of the leptin signaling pathway via ObRb-STAT3 in the hypothalamus, which is likely to be involved in the leptin resistance.
Assuntos
Envelhecimento/metabolismo , Hipotireoidismo Congênito/metabolismo , Hipotálamo/metabolismo , Leptina/metabolismo , Transdução de Sinais , Animais , Peso Corporal , Hipotireoidismo Congênito/sangue , Comportamento Alimentar , Feminino , Hormônios/sangue , Leptina/sangue , Masculino , Ratos WistarRESUMO
BACKGROUND: Hypothyroidism has been related to low-weight births, abortion and prematurity, which have been associated with changes in the content of glycogen and vascularization of the placenta. Since hypothyroidism can cause dyslipidemia, it may affect the lipid content in the uterus affecting the development of fetuses. OBJECTIVE: To investigate the effect of hypothyroidism on the lipid levels in serum and uterus during pregnancy and their possible association with the size of fetuses. METHOD: Adult female rabbits were grouped in control (n = 6) and hypothyroid (n = 6; treated with methimazole for 29 days before and 19 days after copulation). Food intake and body weight were daily registered. At gestational day 19 (GD19), dams were sacrificed under an overdose of anesthesia. Morphometric measures of fetuses were taken. Total cholesterol (TC), triglyceride (TAG), and glucose concentrations were quantified in blood, uterus and ovaries of dams. The expression of uterine 3ß- hydroxysteroid dehydrogenase (3ß-HSD) was quantified by Western blot. RESULTS: Hypothyroidism reduced food intake and body weight of dams, as well as promoted low abdominal diameters of fetuses. It did not induce dyslipidemia and hyperglycemia at GD19 and did not modify the content of lipids in the ovary. However, it reduced the content of TAG and TC in the uterus, which was associated with uterine hyperplasia and an increased expression of 3ß-HSD in the uterus. CONCLUSION: Hypothyroidism alters the lipid content in the uterus that might subsequently affect the energy production and lipid signaling important to fetal development.
Assuntos
Desenvolvimento Fetal/fisiologia , Hipotireoidismo/metabolismo , Hipotireoidismo/patologia , Metabolismo dos Lipídeos , Útero/metabolismo , Animais , Hipotireoidismo Congênito/metabolismo , Hipotireoidismo Congênito/patologia , Modelos Animais de Doenças , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Peso Fetal/efeitos dos fármacos , Peso Fetal/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/análise , Placenta/química , Placenta/efeitos dos fármacos , Placenta/metabolismo , Placenta/patologia , Gravidez , Coelhos , Hormônios Tireóideos/farmacologia , Útero/efeitos dos fármacos , Útero/patologiaRESUMO
The goal of this study was to evaluate the influence of hypothyroidism induced by MMI, during gestation (G) or gestation plus lactation (GL) on testis and its relation with leptin in rats. Six to eight pups were killed at 90 days of age. For statistical analysis One-way ANOVA followed by the Holm-Sìdak post hoc test was used. Hypothyroidism resulted in a significant reduction in LH, FSH and testosterone and an increase in leptin serum levels (p < 0.04). There was a significant decrease in StAR, AR, FSHR, LHR, pSTAT3 and SOCS3 (p < 0.04) protein expression and in the fertility parameters (p < 0.04). We can conclude that hypothyroidism is associated with reduction of steroidogenesis and spermatogenesis leading to a low fertility potential in these animals. This outcome could be a consequence of low pituitary stimulus and testicular response and probably are not related with leptin hormone since its signaling pathway is down-regulated in the testis.
Assuntos
Hipotireoidismo Congênito/metabolismo , Hipotireoidismo Congênito/patologia , Hormônios/metabolismo , Testículo/patologia , Animais , Peso Corporal , Hipotireoidismo Congênito/sangue , Comportamento Alimentar , Feminino , Fertilidade/genética , Regulação da Expressão Gênica , Hormônios/sangue , Lactação , Masculino , Modelos Biológicos , Gravidez , Ratos Wistar , Espermatozoides/metabolismo , Testículo/metabolismoRESUMO
Abstract Background: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH) in rats has not been elucidated thus far. Objective: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC) and β-MHC isoforms. Methods: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and β-MHC were measured by qPCR. Results: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05) and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05) were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, β-MHC expression was higher (201%) and α- MHC expression was lower (47%) than control. Conclusion: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of β-MHC to α- MHC ratio in the heart.
Resumo Fundamento: Deficiência de hormônio da tireoide durante vida fetal pode afetar a função cardíaca no futuro. O mecanismo subjacente dessa ação em hipotireoidismo fetal (HF) em ratos ainda não tem explicação. Objetivo: O objetivo desse estudo é avaliar o efeito de HF na função cardíaca em ratos macho e determinar a contribuição da α-miosina de cadeia pesada (α-MCP) e de isoformas β-MCP. Métodos: Seis ratos fêmea gestantes foram aleatoriamente divididas em dois grupos. O grupo do hipotireoidismo recebeu água contendo 6-propil-2-tiouracil durante a gestação, e os ratos no grupo de controle receberam água de torneira. Os filhotes dos ratos foram testados quando atingiram idade adulta. O coração dos ratos HF e controle foram isolados e submetidos a perfusão pelo método de Langendorff para medição de parâmetros hemodinâmicos. Também foram medidas as expressões de mRNA do coração de α-MCP e β-MCP por qPCR. Resultados: PVED de base (74,0 ± 3,1 vs. 92,5 ± 3,2 mmHg, p < 0,05) e pressão arterial (217 ± 11 vs. 273 ± 6 batidas/min, p < 0,05) mostraram-se mais baixas em ratos HF do que em ratos controle. Além disso, esses resultados mostraram a mesma significância em ±dp/dt. Em ratos HF, a expressão de β-MCP foi mais alta (201%) e a de α-MCP foi mais baixa (47%) do que em ratos controle. Conclusão: Deficiência de hormônio da tireoide durante a vida fetal pode enfraquecer funções cardíacas normais em ratos adultos, efeito devido em parte à expressão aumentada de β-MCP em relação a α-MCP no coração.
Assuntos
Animais , Masculino , Feminino , Gravidez , Peso Corporal/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Hipotireoidismo Congênito/metabolismo , Miocárdio/metabolismo , Propiltiouracila , Antitireóideos , Tiroxina/sangue , Tri-Iodotironina/sangue , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos Wistar , Pressão Ventricular , DNA Complementar/metabolismo , Hipotireoidismo Congênito/induzido quimicamente , Hipotireoidismo Congênito/sangue , Modelos Animais de Doenças , Frequência CardíacaRESUMO
BACKGROUND: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH) in rats has not been elucidated thus far. OBJECTIVE: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC) and ß-MHC isoforms. METHODS: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and ß-MHC were measured by qPCR. RESULTS: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05) and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05) were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, ß-MHC expression was higher (201%) and α- MHC expression was lower (47%) than control. CONCLUSION: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of ß-MHC to α- MHC ratio in the heart.
Assuntos
Peso Corporal/efeitos dos fármacos , Hipotireoidismo Congênito/metabolismo , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Animais , Antitireóideos , Hipotireoidismo Congênito/sangue , Hipotireoidismo Congênito/induzido quimicamente , DNA Complementar/metabolismo , Modelos Animais de Doenças , Feminino , Frequência Cardíaca , Masculino , Gravidez , Propiltiouracila , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos Wistar , Tiroxina/sangue , Tri-Iodotironina/sangue , Pressão VentricularRESUMO
Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264_3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5' ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position -14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91(phox)/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of IOD. Cryptic splicing sites have been characterized in DUOX2 gene for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects.
Assuntos
Hipotireoidismo Congênito/genética , Mutação , NADPH Oxidases/genética , Sítios de Splice de RNA , Criança , Hipotireoidismo Congênito/metabolismo , Oxidases Duais , Células HeLa , Heterozigoto , Humanos , Masculino , NADPH Oxidases/metabolismo , Linhagem , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNA , Glândula Tireoide/metabolismoRESUMO
Astrocytes clearly play a role in neuronal development. An indirect mechanism of thyroid hormone (T3) in the regulation of neuronal development mediated by astrocytes has been proposed. T3 alters the production and organization of the extracellular matrix (ECM) proteins and proteoglycans, producing a high-quality substrate for neuronal differentiation. The present study investigated the effect of hypothyroidism on the astrocyte production of fibronectin (FN) and laminin (LN) as well as their involvement in neuronal growth and neuritogenesis. Our results demonstrated that the amount of both FN and LN were significantly reduced in cultures of hypothyroid astrocytes from rat cerebellum compared with normal cells. This effect was accompanied by reduced numbers of neurons and neuritogenesis. Similarly, the proportions of neurons and neurons with neurites were reduced in cultures on ECM prepared from hypothyroid astrocytes in comparison with normal cells. The proportion of both normal and hypothyroid neurons is strongly reduced in astrocyte ECM compared with cocultures on astrocyte monolayers, suggesting that extracellular factors other than ECM proteins are involved in this process. Moreover, treatment of hypothyroid astrocytic cultures with T3 restored the area of both FN and LN immunostaining to normal levels and partially reestablished neuronal survival and neuritogenesis. Taken together, our results demonstrated that hypothyroidism involves impairment of the astrocytic microenvironment and affects the production of ECM proteins. Thus, hypothyroidism is implicated in impaired neuronal development.
Assuntos
Astrócitos/metabolismo , Hipotireoidismo Congênito/patologia , Matriz Extracelular/metabolismo , Neurogênese/fisiologia , Neurônios/patologia , Animais , Western Blotting , Células Cultivadas , Cerebelo/metabolismo , Cerebelo/patologia , Hipotireoidismo Congênito/complicações , Hipotireoidismo Congênito/metabolismo , Matriz Extracelular/química , Fibronectinas/biossíntese , Imunofluorescência , Técnicas In Vitro , Laminina/biossíntese , Ratos , Ratos WistarAssuntos
Anormalidades Congênitas/embriologia , Hipotireoidismo Congênito/embriologia , Hormônios Tireóideos/metabolismo , Criança , Pré-Escolar , Anormalidades Congênitas/genética , Anormalidades Congênitas/metabolismo , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Humanos , Rim/anormalidades , Rim/embriologia , Glândula Tireoide/anormalidades , Glândula Tireoide/embriologiaRESUMO
En 1995 se implementó en la provincia de Buenos Aires el programa de pesquisa de hipotiroidismo congénito para su diagnóstico y tratamiento precoz. El objetivo de este trabajo fue analizar los registros durante el periodo 2000-2004 y compararlos con los del periodo previo (1995-1999).
Assuntos
Criança , Hipotireoidismo Congênito/complicações , Hipotireoidismo Congênito/diagnóstico , Hipotireoidismo Congênito/metabolismo , Hipotireoidismo Congênito/terapia , Hipotireoidismo/diagnóstico , Hipotireoidismo/terapiaRESUMO
Developmental thyroid hormone (TH) deficiency leads to mental retardation and neurological deficits in humans. In this study, congenital hypothyroidism was induced in rats by adding 0.05% 6-propyl-2-thiouracil in the drinking water during gestation and suckling period. This treatment induced hyperphosphorylation of neurofilaments, the neuronal intermediate filament (IF) proteins, of heavy, medium and low molecular weight (NF-H, NF-M and NF-L, respectively) without altering the phosphorylation level of astrocyte IF proteins, glial fibrillary acidic protein (GFAP) and vimentin in cerebral cortex of rats. NF-H was hyperphosphorylated on KSP repeats in the carboxy-terminal tail domain. Furthermore, the immunocontent of GFAP and NF subunits was down-regulated, while vimentin was unaltered both in tissue homogenate and in cytoskeletal fraction of hypothyroid animals. Moreover, we verified the immunocontent of astrocyte glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) as well as activation of mitogen-activated protein kinases (MAPKs) in hypothyroid rats. Results showed that hypothyroidism is associated with decreased GLAST and GLT-1 immunocontent. Additionally, we demonstrated increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation without altering Jun N-terminal kinase (JNK) and p38(MAPK) phosphorylation. However, total JNK levels were down-regulated. Taken together, these results suggest that the thyroid status could modulate the integrity of neuronal cytoskeleton acting on the endogenous NF-associated phosphorylating system and that such effect could be related to glutamate-induced excitotoxicity, as well as ERK1/2 and JNK modulation. These events could be somehow related to the neurological dysfunction described in hypothyroidism.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Hipotireoidismo Congênito/metabolismo , Regulação para Baixo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Filamentos Intermediários/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Córtex Cerebral/metabolismo , Hipotireoidismo Congênito/induzido quimicamente , Hipotireoidismo Congênito/patologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas In Vitro , Masculino , Fosfatos/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Propiltiouracila , Ligação Proteica/efeitos dos fármacos , Ensaio Radioligante , Ratos , Ratos WistarRESUMO
PURPOSE: The expression of S- and M-opsins in the murine retina is altered in different transgenic mouse models with mutations in the thyroid hormone receptor (TR)-beta gene, demonstrating an important role of thyroid hormone (TH) in retinal development. METHODS: The spatial expression of S- and M-opsin was compared in congenital hypothyroidism and in two different TR mutant mouse models. One mouse model contains a ligand-binding mutation that abolishes TH binding and results in constitutive binding to nuclear corepressors. The second model contains a mutation that blocks binding of coactivators to the AF-2 domain without affecting TH binding. RESULTS: Hypothyroid newborn mice showed an increase in S-opsin expression that was completely independent of the genotype. Concerning M-opsin expression, hypothyroidism caused a significant decrease (P < 0.01) only in wild-type animals. When TRbeta1 and -beta2 were T3-binding defective, the pattern of opsin expression was similar to TRbeta ablation, showing increased S-opsin expression in the dorsal retina and no expression of M-opsin in the entire retina. In an unexpected finding, immunostaining for both opsins was detected when both subtypes of TRbeta were mutated in the helix 12 AF-2 domain. CONCLUSIONS: The results show, for the first time, that the expression of S- and M-opsin is dependent on normal thyroid hormone levels during development.
Assuntos
Hipotireoidismo Congênito/metabolismo , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/metabolismo , Hormônios Tireóideos/fisiologia , Animais , Animais Recém-Nascidos , Contagem de Células , Modelos Animais de Doenças , Eletrorretinografia , Feminino , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Retina/metabolismo , Receptores beta dos Hormônios Tireóideos/genéticaRESUMO
Congenital hypothyroidism was induced in rats by adding 0.05% 6-propyl-2-thiouracil in the drinking water from day 9 of gestation, and continually up to postnatal day 15. Structural alterations observed by light microscopy of seminiferous tubules and by transmission electron microscopy of Sertoli cells of treated animals were consistent with hypothyroid condition. Hypothyroidism was also associated with high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 levels. Furthermore, the phosphorylation and the immunoreactivity of cytoskeletal-associated vimentin were increased without altering vimentin expression, suggesting an accumulation of insoluble and phosphorylated vimentin. These alterations in intermediate filament dynamics could result in loss of Sertoli cell cytoskeletal integrity and be somewhat related to the deleterious effects of hypothyroidism in testis. In addition, the mitochondrial alterations observed could also be related to defective cytoskeletal dynamics implying in cell damage. Moreover, we observed decreased oxygen consumption and unaltered lipid peroxidation in hypothyroid testis. However, we demonstrated decreased enzymatic and non-enzymatic antioxidant defenses, supporting an increased mitochondrial reactive oxygen species (ROS) generation, contributing to biochemical changes in hypothyroid testis. In addition, the changes in the testis histoarchitecture could be ascribed to cytoskeletal alterations, decreased antioxidant defenses, and increased ROS generation, leading to oxidative stress in the organ.
Assuntos
Antioxidantes/metabolismo , Hipotireoidismo Congênito/metabolismo , Propiltiouracila , Testículo/metabolismo , Vimentina/metabolismo , Animais , Hipotireoidismo Congênito/induzido quimicamente , Hipotireoidismo Congênito/patologia , Citoesqueleto/metabolismo , Peroxidação de Lipídeos , Masculino , Mitocôndrias/metabolismo , Consumo de Oxigênio , Fosforilação , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Túbulos Seminíferos/metabolismo , Túbulos Seminíferos/patologia , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Testículo/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Iodide organification defects are frequently but not always associated with mutations in the thyroid peroxidase (TPO) gene and characterized by a positive perchlorate discharge test. These mutations phenotypically produce a congenital goitrous hypothyroidism, with an autosomal recessive mode of inheritance. OBJECTIVES: In the present study we extended our initial molecular studies in six unrelated patients heterozygous for the TPO mutations, in order to identify the second mutation in this autosomal recessive disease. METHODS: The promoter and the complete coding regions of the human TPO and DUOXA2 genes, along with the flanking regions of each intron were analysed by direct DNA sequencing. RESULTS: Four different inactivating TPO mutations were identified in two patients: two novel mutations (c.215delA [p.Q72fsX86] and c.1159G-->A [p.G387R]) and two previously reported (c.387delC [p.N129fsX208] and c.2422T-->C [p.C808R]), confirming the inheritance of two different compound heterozygous mutations, c.215delA/c.2422T-->C and c.387delC/c.1159G-->A. The remaining four patients did not show additional inactivating mutations in the TPO gene and all had only the wild type sequencing in the DUOXA2 gene. CONCLUSIONS: We have reported two patients with iodide organification defect caused by two compound heterozygous mutations, c.215delA/c.2422T-->C [p.Q72fsX86/p.C808R] and c.387delC/c.1159G-->A [p.N129fsX208/p.G387R], in the TPO gene and four patients with monoallelic TPO defect. Identification of the molecular basis of this disorder might be helpful for understanding the pathophysiology of congenital hypothyroidism.
Assuntos
Hipotireoidismo Congênito/genética , Iodeto Peroxidase/genética , Iodetos/metabolismo , Mutação , Sequência de Aminoácidos , Animais , Sequência de Bases , Hipotireoidismo Congênito/metabolismo , Feminino , Genes Recessivos , Heterozigoto , Humanos , Iodeto Peroxidase/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Linhagem , Percloratos/metabolismo , Polimorfismo Conformacional de Fita Simples , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
BACKGROUND: Iodide organification defects are associated with mutations in the dual oxidase 2 (DUOX2) gene and are characterized by a positive perchlorate discharge test. These mutations produce a congenital goitrous hypothyroidism, usually transmitted in an autosomal recessive mode. METHODS: We studied the complete coding sequence of the human DUOX2 gene by single-strand conformational polymorphism (SSCP) analysis of DNA from 17 unrelated patients with iodide organification defects. Samples showing an aberrant pattern were directly sequenced. All mutations were validated by SSCP analysis. Finally, the effect of a splicing mutation was studied by construction of minigenes. RESULTS: Genomic DNA sequencing revealed 3 novel mutations [c.108G>C (p.Q36H), c.1253delG (p.G418fsX482), and g.IVS19-2A>C] and 1 previously reported mutation [c.2895-2898delGTTC (p.S965fsX994)] in 2 families with 1 (family 1) and 2 (family 2) affected members. This implies the inheritance of 2 compound heterozygous mutations, p.Q36H and p.S965fsX994 in family 1 and p.G418fsX482 and g.IVS19-2A>C in family 2. The c.1253delG mutation was associated with a c.1254C>A transversion. In vitro transcription analysis showed that exon 20 is skipped entirely when the g.IVS19-2A>C mutation is present. The wild-type glutamine residue at position 36 is strictly conserved. CONCLUSIONS: Two previously unknown compound heterozygous mutations in the DUOX2 gene, p.Q36H/p.S965fsX994 and p.G418fsX482/g.IVS19-2A>C, are responsible for iodide organification defects in 2 unrelated families. Identification of the molecular basis of this disorder might be helpful for understanding the pathophysiology of this congenital hypothyroidism.
Assuntos
Hipotireoidismo Congênito/genética , Flavoproteínas/genética , Iodetos/metabolismo , Mutação , Glândula Tireoide/metabolismo , Animais , Sequência de Bases , Pré-Escolar , Hipotireoidismo Congênito/enzimologia , Hipotireoidismo Congênito/metabolismo , Oxidases Duais , Feminino , Heterozigoto , Humanos , Masculino , Dados de Sequência Molecular , NADPH Oxidases , Linhagem , Polimorfismo Conformacional de Fita Simples , Homologia de Sequência do Ácido Nucleico , Testes de Função TireóideaRESUMO
Thyroid diseases constitute a heterogeneous collection of abnormalities associated with mutations in genes responsible for the development of thyroid: thyroid transcription factor-1 (TTF-1), thyroid transcriptions factor-2 (TTF-2) and PAX8, or in one of the genes coding for the proteins involved in thyroid hormone biosynthesis such as thyroglobulin (TG), thyroperoxidase (TPO), hydrogen peroxide-generating system (DUOX2), sodium/iodide symporter (NIS), pendrin (PDS), TSH and TSH receptor (TSHr). Congenital hypothyroidism occurs with a prevalence of 1 in 4000 newborns. Patients with this syndrome can be divided into two groups: nongoitrous (dysem/bryogenesis) or goitrous (dyshormonogenesis) congenital hypothyroidism. The dysembryogenesis group, which accounts for 85% of the cases, results from ectopy, agenesis and hypoplasia. In a minority of these patients, the congenital hypothyroidism is associated with mutations in TTF-1, TTF-2, PAX-8, TSH or TSHr genes. The presence of congenital goiter (15% of the cases) has been linked to mutations in the NIS, TG, TPO, DUOX2 or PDS genes. The congenital hypothyroidism with dyshormonogenesis is transmitted as an autosomal recessive trait. Somatic mutations of the TSHr have been identified in hyperfunctioning thyroid adenomas. Another established thyroid disease is the resistance to thyroid hormone (RTH). It is a syndrome of reduced tissue responsiveness to hormonal action caused by mutations located in the thyroid hormone receptor beta (TRbeta) gene. Mutant TRbetas interfere with the function of the wild-type receptor by a dominant negative mechanism. In conclusion, the identification of mutations in the thyroid expression genes has provided important insights into structure-function relationships. The thyroid constitutes an excellent model for the molecular study of genetic diseases.