RESUMO
Thyrotropin receptor (TSH-R), thyroglobulin (Tg), thyroperoxidase (TPO), thyroid specific transcription factor-1 (TTF-1), paired box 8 transcription factor (PAX-8), insulin like growth factor-1 (IGF-1) and estrogen receptor alpha (ERα) transcripts were determined by real-time PCR in follicular carcinoma and contralateral (CL) lobes, and healthy thyroid canine glands. Concentrations of TSH-R, PAX-8, and ERα mRNA were not different among groups; the carcinoma group had lower Tg and TPO mRNA than healthy and CL groups, while no differences were found between the two latter groups, suggesting that the carcinoma tissue presents an altered capacity to synthesize thyroid hormones. The transcription factor that promotes thyrocytes proliferation, TTF-1 as well as IGF-1, presented a greater mRNA expression in the CL group, suggesting that the CL lobe may function in a compensatory state.
Assuntos
Doenças do Cão/metabolismo , Receptor alfa de Estrogênio/biossíntese , Fator de Crescimento Insulin-Like I/biossíntese , Iodeto Peroxidase/biossíntese , Proteínas Nucleares/biossíntese , Fatores de Transcrição Box Pareados/biossíntese , Receptores da Tireotropina/biossíntese , Tireoglobulina/biossíntese , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cães , Feminino , Masculino , Fator de Transcrição PAX8 , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/biossínteseRESUMO
CONTEXT: Serum thyroglobulin is a sensitive tumor marker in the follow-up of patients with differentiated thyroid carcinoma (DTC), but the presence of endogenous anti-thyroglobulin antibodies (TgAb) can interfere on its measurement. To prevent interference by TgAb, several investigators have tried to quantify blood mRNA Tg by real-time RT-PCR, but the results have been variable, not reporting a correlation between mRNA Tg and the presence of metastases. OBJECTIVE: The aim of the study was to evaluate the development of a sensitive and specific quantitative RT-PCR assay for blood mRNA Tg in the follow-up of patients with DTC. DESIGN AND PATIENTS: An assay employing primers located in a region not affected by alternative splicing or single nucleotide polymorphisms was developed to study 104 DTC patients (13 of 104 with positive TgAb). RESULTS: The assay is specific for thyroid tissue because we found mRNA Tg expression in normal thyroid tissue, but we did not find any mRNA Tg expression in any extrathyroidal tissues. Quantitative mRNA Tg levels were significantly different between patients "free of disease" (82 of 104) and those with metastases (22 of 104) (2.61 +/- 0.26 vs. 27.58 +/- 1.62 pg mRNA Tg/microg RNA) (P < 0.0001). A cutoff point of 5.51 was able to discriminate between the two groups. In addition, the measurement of mRNA Tg was not affected by the presence of TgAb. CONCLUSION: This new mRNA Tg quantification is a reliable method that allowed us to differentiate patients free of disease from those with metastases, and it could represent an appropriate molecular marker for the follow-up of patients with DTC, especially those with positive TgAb.
Assuntos
RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tireoglobulina/biossíntese , Tireoglobulina/genética , Neoplasias da Glândula Tireoide/diagnóstico , Adolescente , Adulto , Idoso , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , Feminino , Humanos , Assistência de Longa Duração , Masculino , Pessoa de Meia-Idade , Pescoço/diagnóstico por imagem , Metástase Neoplásica/diagnóstico , Recidiva Local de Neoplasia/diagnóstico , RNA Mensageiro/genética , Curva ROC , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Neoplasias da Glândula Tireoide/patologia , Ultrassonografia , Adulto JovemRESUMO
BACKGROUND: Thyroglobulin (TG) deficiency is an autosomal-recessive disorder that results in thyroid dyshormonogenesis. A number of distinct mutations have been identified as causing human hypothyroid goitre. OBJECTIVES: The purpose of this study was to identify and characterize new mutations in the TG gene in an attempt to increase the understanding of the genetic mechanism responsible for this disorder. A total of six patients from four nonconsanguineous families with marked impairment of TG synthesis were studied. METHODS: Single-strand conformation polymorphism (SSCP) analysis, sequencing of DNA, genotyping, expression of chimeric minigenes and bioinformatic analysis were performed. RESULTS: Four different inactivating TG mutations were identified: one novel mutation (c.7006C>T [p.R2317X]) and three previously reported (c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.6725G>A [p.R2223H]). Consequently, one patient carried a compound heterozygous for p.R2223H/p.R2317X mutations; two brothers showed a homozygous p.A2215D substitution and the remaining three patients, from two families with typical phenotype, had a single p.R277X mutated allele. We also showed functional evidences that premature stop codons inserted at different positions in exon 7, which disrupt exonic splicing enhancer (ESE) sequences, do not interfere with exon definition and processing. CONCLUSIONS: In this study, we have identified a novel nonsense mutation p.R2317X in the acetylcholinesterase homology domain of TG. We have also observed that nonsense mutations do not interfere with the pre-mRNA splicing of exon 7. The results are in accordance with previous observations confirming the genetic heterogeneity of TG defects.
Assuntos
Hipotireoidismo Congênito/genética , Bócio/genética , Polimorfismo de Nucleotídeo Único , Tireoglobulina/deficiência , Tireoglobulina/genética , Pré-Escolar , Códon sem Sentido/genética , Códon sem Sentido/fisiologia , Hipotireoidismo Congênito/complicações , Hipotireoidismo Congênito/etiologia , Análise Mutacional de DNA/métodos , Éxons , Feminino , Expressão Gênica , Testes Genéticos , Bócio/complicações , Bócio/congênito , Bócio/etiologia , Células HeLa , Humanos , Lactente , Recém-Nascido , Masculino , Polimorfismo de Nucleotídeo Único/fisiologia , Polimorfismo Conformacional de Fita Simples , Estrutura Terciária de Proteína/genética , Tireoglobulina/biossíntese , Tireoglobulina/química , Transfecção , Transgenes/genéticaRESUMO
CONTEXT: Thyroglobulin (TG) is a large glycoprotein and functions as a matrix for thyroid hormone synthesis. TG gene mutations give rise to goitrous congenital hypothyroidism (CH) with considerable phenotype variation. OBJECTIVES: The aim of the study was to report the genetic screening of 15 patients with CH due to TG gene mutations and to perform functional analysis of the p.A2215D mutation. DESIGN: Clinical evaluation and DNA sequencing of the TG gene were performed in all patients. TG expression was analyzed in the goitrous tissue of one patient. Human cells were transfected with expression vectors containing mutated and wild-type human TG cDNA. RESULTS: All patients had an absent rise of serum TG after stimulation with recombinant human TSH. Sequence analysis revealed three previously described mutations (p.A2215D, p.R277X, and g.IVS30+1G>T), and two novel mutations (p.Q2142X and g.IVS46-1G>A). Two known (g.IVS30+1G/p.A2215D and p.A2215D/p.R277X) and one novel (p.R277X/g.IVS46-1G>A) compound heterozygous constellations were also identified. Functional analysis indicated deficiency in TG synthesis, reduction of TG secretion, and retention of the mutant TG within the cell, leading to an endoplasmic reticulum storage disease, whereas small amounts of mutant TG were still secreted within the cell system. CONCLUSION: All studied patients were either homozygous or heterozygous for TG gene mutations. Two novel mutations have been detected, and we show that TG mutation p.A2215D promotes the retention of TG within the endoplasmic reticulum and reduces TG synthesis and secretion, causing mild hypothyroidism. In the presence of sufficient iodine supply, some patients with TG mutations are able to compensate the impaired hormonogenesis and generate thyroid hormone.
Assuntos
Hipotireoidismo Congênito/genética , Mutação , Tireoglobulina/genética , Adulto , Células Cultivadas , Criança , Pré-Escolar , Hipotireoidismo Congênito/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Fenótipo , RNA Mensageiro/análise , Tireoglobulina/análise , Tireoglobulina/biossíntese , Tireotropina/farmacologia , TransfecçãoRESUMO
Thyroxine (T4) withdrawal or recombinant TSH is used for the stimulation of thyroglobulin (Tg), whole-body scanning (WBS) and iodine-131 treatment in patients with thyroid carcinoma. This study evaluated the T4 dose reduction protocol as an alternative for patients' preparation. Fifty-one patients were submitted to total T4 withdrawal for WBS and Tg measurement. T4 treatment was then resumed and maintained until TSH reached levels < 0.3 mIU/l. The T4 dose was then decreased to 0.8 microg/kg/day and TSH was measured weekly. Tg was assayed when TSH was > 30 mIU/l. Patients diagnosed with the disease upon initial evaluation were treated. We also evaluated the clinical and laboratory changes observed for both preparations. Using the reduced dose protocol, TSH levels > 30 mIU/l were reached within 6 and 8 weeks in 84.6 and 100% of the patients, respectively. T4 withdrawal was associated with more common symptoms of hypothyroidism and elevation of creatine kinase (CK) and LDL cholesterol. The T4 dose reduction protocol proved to be useful for Tg stimulation and ablative therapy, without the complication of severe hypothyroidism or the cost of recombinant TSH.
Assuntos
Carcinoma/radioterapia , Radioisótopos do Iodo/uso terapêutico , Tireoglobulina/sangue , Neoplasias da Glândula Tireoide/radioterapia , Tiroxina/administração & dosagem , Adulto , Carcinoma/sangue , Carcinoma/cirurgia , Estudos de Casos e Controles , LDL-Colesterol/sangue , Creatina Quinase/análise , Creatina Quinase/sangue , Feminino , Humanos , Ensaio Imunorradiométrico , Masculino , Pessoa de Meia-Idade , Tireoglobulina/biossíntese , Neoplasias da Glândula Tireoide/sangue , Neoplasias da Glândula Tireoide/cirurgia , Tireoidectomia , Tireotropina/sangue , Tiroxina/efeitos adversos , Resultado do Tratamento , Imagem Corporal TotalRESUMO
The bacterial lipopolysaccharide (LPS) is a biological activator that induces expression of multiple genes in several cell types. LPS has been proposed as an etiopathogenic agent in autoimmune diseases. However, whether LPS affects the expression of autoantigens has not been explored. Thyroglobulin (TG) is a key protein in thyroid hormonogenesis and one of the major thyroid autoantigens. This study aimed to analyze the action of LPS on TG gene expression in Fisher rat thyroid cell line FRTL-5 thyroid cells. We demonstrate that LPS increases the TSH-induced TG protein and mRNA level. Evidence that the effect of LPS is exerted at the transcriptional level was obtained by transfecting the minimal TG promoter. The C element of the TG promoter, which contains sequences for paired box domain transcription factor 8 (Pax8) and thyroid transcription factor (TTF)-1 binding, is essential for full TG promoter expression under TSH stimulation. The transcriptional activity of a construct containing five tandem repeats of the C site is increased by LPS, indicating a possible involvement of the C site in the LPS-induced TG gene transcription. We demonstrate that the TG promoter mutated at the Pax8 or TTF-1 binding element in the C site does not respond to LPS. In band shift assays, binding of Pax8 and TTF-1 to the C site is increased by LPS. The Pax8 and TTF-1 mRNA and protein levels are augmented by LPS. The half-lives of TG, Pax8, and TTF-1 are increased in endotoxin-treated cells. Our results reveal the ability of LPS to stimulate the expression of TG, a finding of potential pathophysiological implication.
Assuntos
Regulação da Expressão Gênica , Lipopolissacarídeos/metabolismo , Proteínas Nucleares/fisiologia , Fatores de Transcrição Box Pareados/fisiologia , Tireoglobulina/biossíntese , Tireoglobulina/genética , Tireotropina/metabolismo , Fatores de Transcrição/fisiologia , Animais , Autoantígenos/química , Sequência de Bases , Dados de Sequência Molecular , Fator de Transcrição PAX8 , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/citologia , Fator Nuclear 1 de Tireoide , Transcrição GênicaRESUMO
A suspensão da tiroxina (T4) ou o TSH recombinante são usados para a estimulação da tireoglobulina (Tg), para o mapeamento de corpo inteiro (MCI) e para o tratamento com 131Iodo em pacientes com carcinoma tireoideano. Esse estudo avaliou um protocolo de redução de dose do T4 como alternativa para o preparo desses pacientes. Cinquenta e um pacientes submeteram-se à suspensão total de T4 para o MCI e a medida de Tg. Tratamento com T4 foi então reinstituído e mantido até que o TSH atingisse níveis < 0.3 mUI/l. A dose de T4 foi então dominuída para 0,8 µg/kg/dia e o TSH medido semanalmente. A Tg foi analisada quando o TSH estava > 30 mUI/l. Pacientes diagnosticados com a doença na fase inicial da avaliação foram tratados. Nós também avaliamos as alterações clínicas e laboratoriais observadas para ambos os preparos. Usando o protocolo de redução de dose, níveis de TSH > 30 mUI/l foram atingidos em 6 e 8 semanas em 84,6 and 100% dos pacientes, respectivamente. A suspensão do T4 esteve associada com sintomas mais comuns de hipotireoidismo e com elevação da creatino- quinase (CK) e LDL-colesterol. O protocolo de redução da dose de T4 mostrou-se útil para a estimulação da Tg e terapia ablativa, sem apresentar as complicações do hipotireoidismo severo ou chegar ao custo do TSH recombinante.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Carcinoma/radioterapia , Radioisótopos do Iodo/uso terapêutico , Tireoglobulina/sangue , Neoplasias da Glândula Tireoide/radioterapia , Tiroxina/administração & dosagem , Estudos de Casos e Controles , Carcinoma/sangue , Carcinoma/cirurgia , LDL-Colesterol/sangue , Creatina Quinase/análise , Creatina Quinase/sangue , Ensaio Imunorradiométrico , Tireoidectomia , Resultado do Tratamento , Tireoglobulina/biossíntese , Neoplasias da Glândula Tireoide/sangue , Neoplasias da Glândula Tireoide/cirurgia , Tiroxina/efeitos adversos , Imagem Corporal TotalRESUMO
Many types of evidence support a role of the sympathetic nervous system in the regulation of thyroid function, although there is no general consensus on the type of influence that catecholamines exert. Depending on the experimental approach, epinephrine and norepinephrine (NE) can stimulate, inhibit, or fail to act on thyroid function. The aim of this study was to determine the effect of NE on thyroglobulin (Tg) synthesis and gene expression in FRTL-5 cells. Tg content, measured by immunoprecipitation with a specific antibody, showed that NE caused a 45% inhibition of thyrotropin (TSH) effect. The content of Tg mRNA was analyzed by Northern blot, the relative inhibition in total Tg mRNA levels from NE-treated cells, compared to TSH alone, ran parallel with inhibition in Tg content, while total RNA did not change after incubation with NE. There was no alteration in Tg mRNA stability by NE. When plasmids harboring different sequences of Tg promoter fused to the CAT reporter gene were transfected into FRTL-5 cells, TSH treatment stimulated promoter activity while NE diminished this effect by 43%-55%. Northern blots were performed to analyze mRNA for thyroid transcription factors (TTF1, TTF2, Pax8), and no significant changes were observed with the different treatments. In conclusion these results suggest that NE inhibits Tg synthesis at the transcriptional level.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Norepinefrina/farmacologia , Tireoglobulina/biossíntese , Tireoglobulina/genética , Glândula Tireoide/metabolismo , Animais , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Processamento de Imagem Assistida por Computador , Metionina/metabolismo , Regiões Promotoras Genéticas/genética , RNA/biossíntese , RNA Mensageiro/biossíntese , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Tireotropina/biossíntese , TransfecçãoRESUMO
This is a report of two cases of congenital goiter in which it was possible to indicate, through immunohistochemistry method, the disorder in the hormonal synthesis. One of these cases was of a sixteen-year-old woman, with goiter since childhood who used thyroid hormone replacement for ten years. Pathology showed coloid goiter with follicular adenoma. Immunohistochemistry showed strong mainly apical follicular cell thyroglobulin. Thyroglobulin was not found in the coloid. This finding is compatible with a malfunction of thyroglobulin exocitosis. The reaction for thyroid peroxidase was adequate for the age. The other case was a 13 year-old-boy with goiter and thyroid hormone replacement since the age of 1 year. He presented slight mental and growth retardation. Pathology showed coloid goiter with follicular adenoma. Immunohistochemistry study showed a very weak and diffuse reaction for thyroid peroxidase while the reaction for thyroglobulin was adequate. These findings indicate quantitative defect of thyroid peroxidase.