An analysis of GNAS and THRA gene mutations in children with congenital hypothyroidism / 中国当代儿科杂志

OBJECTIVE@#To preliminarily investigate the relationship between stimulatory G protein α subunit (GNAS) and thyroid hormone receptor α (THRA) gene mutations and clinical phenotypes in children with congenital hypothyroidism (CH).@*METHODS@#A total of 70 children with CH diagnosed by neonatal screening were enrolled. Their peripheral blood samples were collected to extract genomic DNA. GNAS and THRA genes were screened for mutations using next-generation sequencing. Bioinformatics software was used to analyze the pathogenicity of gene mutations.@*RESULTS@#Of the 70 children with CH, nine missense mutations (three known mutations and six novel mutations) in the GNAS gene were detected in three patients (4%), and one gene polymorphism, c.508A>G(p.I170V), in the THRA gene was detected in four patients. The analysis results of bioinformatics software and ACMG/AMP guidelines showed that the two GNAS gene mutations [c.301C>T(p.R101C) and c.334G>A(p.E112K)] were more likely to be pathogenic. Three children with GNAS gene mutations showed different degrees of hypothyroidism.@*CONCLUSIONS@#GNAS gene mutations are related to the development of CH, and children with CH have different clinical manifestations. THRA gene mutations may not be associated with CH.

Short-term effects of triiodothyronine on thyroid hormone receptor alpha by PI3K pathway in adipocytes, 3T3-L1 / Efeitos rápidos da triiodotironina no receptor de hormônio tireoidiano alfa pela via PI3K em adipócitos, 3T3-L1

Objective The present study aimed to examine the effects of thyroid hormone (TH), more precisely triiodothyronine (T3), on the modulation of TH receptor alpha (TRα) mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway in adipocytes, 3T3-L1, cell culture. Materials and methods: It was examined the involvement of PI3K pathway in mediating T3 effects by treating 3T3-L1 adipocytes with physiological (P=10nM) or supraphysiological (SI =100 nM) T3 doses during one hour (short time), in the absence or the presence of PI3K inhibitor (LY294002). The absence of any treatment was considered the control group (C). RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukey’s test was used at 5% significance level. Results T3 increased TRα mRNA expression in P (1.91±0.13, p<0.001), SI (2.14±0.44, p<0.001) compared to C group (1±0.08). This increase was completely abrogated by LY294002 in P (0.53±0.03, p<0.001) and SI (0.31±0.03, p<0.001). To examine whether TRα is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). The presence of CHX completely abrogated levels TRα mRNA in P (1.15±0.05, p>0.001) and SI (0.99±0.15, p>0.001), induced by T3. Conclusion These results demonstrate that the activation of the PI3K signaling pathway has a role in T3-mediated indirect TRα gene expression in 3T3-L1 adipocytes. .

Objetivo O objetivo do presente estudo foi analisar os efeitos do hormônio tireoidiano (HT), triiodotironina (T3), na modulação da expressão de mRNA do receptor alfa (TRα) de HT e o envolvimento da via de sinalização da via fosfatidilinositol 3-quinase (PI3K) em adipócitos, 3T3-L1. Materiais e métodos: Foi examinado o envolvimento da via PI3K nos efeitos do T3 nos tratamentos de adipócitos, 3T3-L1, nas doses fisiológica (P=10nM) ou suprafisiológica (SI =100 nM) durante uma hora (tempo curto), na ausência ou na presença do inibidor da PI3K (LY294002). A ausência de qualquer tratamento foi considerada o grupo controle (C). RT-qPCR foi utilizado para analisar a expressão do mRNA. Para as análises dos dados, utilizou-se ANOVA complementada com o teste de Tukey a 5% de significância. Resultados O T3 aumentou a expressão de mRNA de TRα em P (1,91±0,13, p<0,001) e SI (2,14±0,44, p<0,001) em comparação com o grupo C (1±0,08). Esse aumento foi completamente abolido por LY294002 em P (0,53±0,03, p<0,001) e SI (0,31±0,03, p<0,001). Para examinar se a expressão de TRα foi diretamente induzida pelo T3, utilizou-se o inibidor de tradução, ciclohexamida (CHX). A presença de CHX reduziu os níveis de mRNA de TRα em P (1,15±0,05, p>0,001) e SI (0,99±0,15, p>0,001), induzidos pelo T3. Conclusão Esses resultados demonstram que a ativação da via de sinalização de PI3K tem um papel importante na expressão do gene TRα mediada indiretamente pelo T3, em adipócitos 3T3-L1. .

Modulation of thyroid hormone receptors, TRα and TRβ, by using different doses of triiodothyronine (T3) at different times / Modulação dos receptores de hormônio tireoidiano, TRα e TRβ, utilizando diferentes doses de triiodotironina (T3) em diferentes tempos

OBJECTIVE: To examine the effect of different doses of triiodothyronine (T3) on mRNA levels of thyroid hormone receptors, TRα and TRβ, at different times. MATERIALS AND METHODS: 3T3-L1 adipocytes were incubated with T3 (physiological dose: F; supraphysiological doses: SI or SII), or without T3 (control, C) for 0.5, 1, 6, or 24h. TRα and TRβ mRNA was detected using real-time polymerase chain reaction. RESULTS: F increased TRβ mRNA levels at 0.5h. After 1h, TRα levels increased with F and SI and TRβ levels decreased with SII compared with C, F, and SI. After 6h, both genes were suppressed at all concentrations. In 24h, TRα and TRβ levels were similar to those of C group. CONCLUSIONS: T3 action with F began at 1h for TRα and at 0.5h for TRβ. These results suggest the importance of knowing the times and doses that activate T3 receptors in adipocytes.

OBJETIVO: Examinar o efeito de diferentes doses de triiodotironina (T3) sobre a expressão gênica dos receptores TRα e TRβ em diferentes tempos. MATERIAIS E MÉTODOS: Adipócitos, 3T3-L1, foram incubados com T3 nas doses fisiológica (F, 10nM) e suprafisiológicas (SI, 100nM ou SII, 1000nM) ou veículo (controle, C) durante 0,5, 1, 6 ou 24h. mRNA dos TRs foram detectados utilizando PCR em tempo real. RESULTADOS: Níveis de TRβ aumentaram em F em 0,5h. Após 1h, níveis de TRα aumentaram em F e SI comparado ao C, enquanto TRβ diminuiu no SII comparado com C, F, e SI. Após 6h, ambos os genes foram suprimidos em todas concentrações. Em 24h, níveis de TRα e TRβ retornaram aos do C. CONCLUSÕES: Ação do T3 em F iniciou-se em 1h para TRα e 0,5h para TRβ. Esses resultados são importantes para determinar tempo inicial e dose de T3 em que os receptores de HT são ativados em adipócitos.

Insulin-like growth factor binding protein-3 interacts with the thyroid hormone receptor α1 and modulates transcription of thyroid hormone responsive gene / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the interaction between insulin-like growth factor binding protein-3 (IGFBP-3) and thyroid hormone receptor α1 (TRα1) and the modulatory effect of IGFBP-3 on transcription of the thyroid hormone responsive gene.</p><p><b>METHODS</b>The interaction between IGFBP-3 and TRα1 was detected with glutathione-S-transferase pull-down method, co-immunoprecipitation, fluorescence resonance energy transfer test. The cellular distribution of these two proteins was observed by confocal laser scanning microscopy. The effect of IGFBP-3 on the growth hormone promoter activity stimulated by triiodothyronine (T3) was determined by dual-luciferase reporter assay.</p><p><b>RESULTS</b>IGFBP-3 interacted with TRα1 both in vivo and in vitro. IGFBP-3 and TRα1 were shown to co-localize in the nucleus of HEK-293 cells. The overexpressed IGFBP-3 inhibited the growth hormone promoter activity stimulated by T3 (P<0.01).</p><p><b>CONCLUSIONS</b>IGFBP-3 interacts with TRα1 and inhibits T3 responsive gene transcription. This finding further confirms the insulin-like growth factor-independent role of IGFBP-3 in the nucleus.</p>

Prokaryotic expression and polyclonal antibody preparation of TRalphaA in Japanese flounder Paralichthys olivaceus / 生物工程学报

To study the role of the thyroid hormone receptor TRalphaA involved in the process of the metamorphic development of Japanese flounder, we firstly cloned the TRalphaA gene, then ligated into the fusion expression vector pET30a and expressed in Escherichia coli DE3 (BL21) host cells. After induced for 4 h with 1 mmol/L Isopropyl beta-D-Thiogalactoside, the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The recombinant protein was denatured and purified by His-Bind resin, then renatured through gradient washing on His-bind resin column. After that, polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein. Dot blotting analysis showed the antibody with the titer of 1:200 000 reacted specifically to the expressed recombinant protein. Furthermore, a chromatin immunoprecipitation assay was performed to identify the specific binding between the antibody and TRalphaA in living cells of Japanese flounder. The result showed that thyroid hormone was involved in the alkaline phosphatase (ALP) gene transcriptional regulation through TRalphaA in vivo.

Depresión refractaria y resistencia periférica a hormona tiroidea: el efecto de altas dosis de triyodotironina / Refractory depression and peripheral resistance to thyroid hormone: the effect of high doses of triodothyronine

We describe a 39-year-old woman with a severe chronic mood disorder, refractory to antidepressive treatment, who showed a marked improvement after a self prescription of very high doses of liothyronine (T3). A modified Refetoff protocol was carried out to study the rol of high doses of thyroid hormones on her clinical and biochemical responses. Depression severity and change was assessed by the HAM-D and MADRS rating scales. Sequencing of thyroid receptors TRalpha1 and TRbeta1 was done and no abnormal findings were obtained. At the final stage of the study plasma T3 and free T3 were >800 ng/dl (80-180) and 1.409 pg/dl (230-420),respectively. No changes in the cardiovascular parameters, alcaline phosphatase isoenzymes or ferritine were observed. An improvement in mood was confirmed by a marhed drop in the rating scales scores (HAM-D 24 to 8; MADRS 40 to 11). These results support the existence in this patient of a peripheral resistance to thyroid hormone (RTH) and the response of depressive symptoms to high dosis of T3 Screening for RTH in refractory chronic depression may pro vide access to alternative and more efficacious treatments for this psychiatric condition.

Describimos el caso de una mujer de 39 años portadora de un trastorno del ánimo crónico y refractario a tratamiento, que experimentó una marcada mejoría de sus síntomas depresivos luego de auto-prescribirse altas dosis de liotiranina (T3). Se le sometió a un protocolo de Refetoff modificado a fin de estudiar los efectos de altas dosis de hormona tiroidea en sus síntomas clínicos y en variables bioquímicas. La severidad y cambio en la intensidad de la depresión se evaluó mediante la aplicación de las escalas HAM-D y MADRS. Se secuenciaron los receptores de hormona tiroidea TRalfa1 y TRbeta1, sin hallazgos anormales. Al final del estudio los niveles plasmáticos de T3 y T31ibre fueron >800 ng/dl (80-180) y 1.409 pg/dl (230-420), respectivamente. No hubo cambios en los parámetros cardiovasculares, fosfatasas alcalinas ni ferritina. Una marcada mejoría del ánimo fue confirmada por la disminución de los puntajes de las escalas aplicadas (HAM-D24 a 8; MADRS 40 a 11). Estos resultados apoyan la existencia de una resistencia periférica a hormona tiroidea (RHT) en esta paciente y la respuesta de los síntomas anímicos a altas dosis de liotironina. La búsqueda de RHT en pacientes con depresión crónica refractaria podría dar acceso a tratamientos alternativos más eficaces para esta condición psiquiátrica.