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Objective:Exploring the role of thyrotropin receptor(TSHR) in lipotoxicity-induced thyroid function damage.Methods:Rat thyroid follicular epithelial cells(RTC) were stimulated with different doses of palmitic acid(PA), and the lipid content of the cells was observed through Oil Red O staining. The expression levels of TSH receptor(TSHR), Ttf1, and SSBP1 mRNA and protein in each group were detected using RT-PCR and Western blot. The TSHR protein level in the cell culture supernatant was measured using ELISA. Membrane TSHR was assessed through immunofluorescence and compared with the control group. We used PA to stimulate the TSHR over-expression(TSHR OE) and normal RTC, as PA+ TSHR OE group and PA group respectively, then testing Tg mRNA and protein, cAMP and Tg in cell supernatants levels, then comparing with the control.Results:RTC were stained into peau d′orange in PA groups. Compared with the control group, we found TTf1, SSBP1 and TSHR mRNA as well as protein levels in PA groups were decreased(all P<0.05), TSHR of the cell membrane and supernatants were reduced(all P<0.05), characterizing dose-dependent changes partly. Moreover, we found in PA group Tg mRNA level was downregulated( P<0.05), Tg protein levels were reduced in the supernatants and cells( P<0.05), cAMP level was decreased in cells( P<0.05); in TSHR OE group, Tg mRNA level was upregulated( P<0.05), Tg protein levels in cells and supernatants were increased(all P<0.05), cAMP level was similar. Compared with the PA group, we found in PA+ TSHR OE group Tg mRNA level was upregulated( P<0.05), Tg protein levels were increased in the supernatants and cells(all P<0.05), cAMP level was elevated in cells( P<0.05). Conclusion:PA induces lipid deposition in RTC, decreased synthesis and secretion of Tg. This effect is likely achieved through the downregulation of the TSHR/cAMP signaling pathway.
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Objective To construct a model of Graves'disease ( GD ) with ( or ) Graves'ophthalmopathy ( GO) in BALB/c mice by immunization with pcDNA3. 1/TSHR289. Methods pcDNA3. 1/TSHR289 was injected into the bilateral gastrocnemius muscle of 35 model mice and electroporation was immediately performed. 10 control mice were injected with sterile saline and electroporated, while 5 blank mice were injected with sterile saline only. Each group of mice was immunized at 1, 4, 7, and 10 weeks, respectively. Serum total T4 , TSH, TSAb, and TSBAb were measured before immunization, 2 weeks after each immunization, as well as 5 and 8 weeks after the last immunization. CT scan was used to evaluate the morphological changes of the eyes of the mice.99m TcO4- imaging was used to measure the thyroid uptake function, and the pathological changes of the thyroid and orbital tissues were evaluated by HE staining. Results After the 2nd time immunization, the serum concentrations of TT4 , TSAb and TSBAb in GD mice were significantly higher than those of control and blank groups( F=13.781, 31.435, 36.112, P<0.01, respectively).The TSH continued to be significantly lower than that of control and blank groups(F=13.966, P<0.01) . After the 4th time immunizations, the ability of uptaking99m TcO4- in GD mice thyroid was significantly enhanced compared with the control group. The thyroid goiter with a large amount of lymphocyte infiltration, and the thyroid follicle was thin. CT scan of GO mice showed thickening and swelling of the extraocular muscles, and no abnormalities in tendon and muscle attachment points. HE staining showed thickening of extraocular muscle fibers, lymphocyte infiltration of extraocular muscles and orbital tissue, increased hyaluronic acid, and infiltration of fat cells. Conclusion GD or GO model can be successfully induced by multiple intramuscular injection of pcDNA3.1/TSHR289 in BALB/c mice.
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Objective To explore the characterization of thyroid stimulating hormone receptor(TSHR) gene mutational spectrum in children with hyperthyroidism from Guangzhou. Methods Ninety children were diagnosed with hyperthyroidism from July 2009 to July 2014 in our institute. Their median age at diagnosis was(7.5± 3.4) years, and there were 28 males and 62 females. Mutational analysis were performed by performing polymerase chain reaction (PCR) and DNA direct sequencing of exon 10 of TSHR gene. TSHR gene mutations from 50 unrelated healthy children were served as controls. The correlation between TSHR gene and hyperthyroidism in children was explored. Results A total of 3 mutations were identified in ninety children who were diagnosed with hyperthyroidism, one synonymous mutations(p.V614V), and two missense mutations( p. R707W and p. D727E). Mutation of p. V614V do not change amino acid and do not influence the structure and function of TSHR, no pathogenicity. p.R707W is a SNP associated with human cancers. The frequency of C allele of the D727E in children with hyperthyroidism was 86.7%, while 55.0% in the controls, significant different between the children with hyperthyroidism and the controls( P＜0. 01). In this study, a very high association between the D727E SNP and hyperthyroidism ( OR=18. 86, P＜0. 01) was found. Conclusion Three different mutations of TSHR gene exon 10 were identified in 90 children with hyperthyroidism, (c.1842A＞G,p.V614V、c.2119C＞T,p.R707W、c.2181G＞C,p.D727E), there were association between p.D727E and hyperthyroidism, nor p. V614V and p. R707W. Finally, p. D727E may be correlated with hyperthyroidism in children.
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Objective To identify the association of thyroid stimulating hormone receptor ( TSHR ) gene intron 1 susceptible loci and 4p14 susceptible locus rs6832151 polymorphisms with Graves’ disease ( GD) in Han Chinese population in Bengbu, Anhui, China. The gene-gene interaction among TSHR intron 1 susceptible loci and 4p14 susceptible locus rs6832151 was also investigated. Methods The genotypes of the single-nucleotide polymorphisms ( SNPs) were analyzed by Taqman probe technique on Fluidigm EP1 platform in 611 patients with GD and 555 control subjects, and linkage analysis, correlation analysis, haplotype analysis, and epistasis analysis with them were performed. Results Six SNPs in two candidate genes(rs12101261, rs4903964,rs179247, rs2284722 and rs17111394 in TSHR, rs6832151 in 4p14) were associated with GD (all P<0. 05). The frequency distributions of haplotypes of SNPs in TSHR intron 1 ( AGTA, GGCG, AATA, and CC) were significantly different between GD and control groups(all P<0. 01). There existed the interactions between rs179247 and rs12101261 in TSHR(P=0. 001) and among rs179247(TSHR),rs4903964(TSHR) and rs6832151(4p14) (P=0. 001). Conclusions rs683215 in 14p14 and rs12101261, rs4903964, rs179247, rs2284722 and rs17111394 in TSHR intron 1 were susceptible loci of GD in the Chinese Han population from Bengbu. The haplotypes in TSHR intron 1 were associated with GD. There exists the interaction between the SNPs in TSHR and 4p14,which may change the risk of GD.
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Objective:To study the pathological mechanism of the inducible co-stimulator molecular and ligand ( ICOS/ICOSL) in Graves disease animal.Methods:45 out-bred BALB/c mice were randomly divided into three groups with 15 rats in each group;using gene gun to deliver different plasmid injection.Group A was delivered with pCDNA3.0-mICOSL and pCDNA3.0-hTSHR, Group B with pCDNA3.0-hTSHR and null pCDNA3.0 with Group C for immunization as the control group.The concentration of serum free thyroxine immunization was deter mined with immunoassay and serum thyrotropin receptor antibody ( TRAb ) with ELISA, supernatant of IFN-γconcentration in mouse spleen cells was measured with radioimmunoassay,and hTSHR transected CHO cells were incubated to detect the concentration of cAMP to deter mine autoantibody TRAb activity.Results: After plasmid injection serum FT4 level in Group A (0.49±0.25) pg/ml ( q=6.571,P=0.023) was higher than that in Group C,the standard rate was higher than Group B and C (χ2=14.47,P=0.005).IFN-γconcentration of mice spleen cultured supernatant in Group A (1.88±0.41) pmol/L was significantly higher than the other two groups.The activity of autoantibody TRAb in Group A 188.3 (179.7-260.2) %was higher than that in the other two groups ( P=0.027 ) .Conclusion: Exogenous delivery of pCDNA3.0-mICOSL plasmid in GD mice could stimulate the spleen lymphocytes to secrete more IFN-γ,increase the activity of TRAb autoantibodies and might lead to upregulation of immune response in Graves animal model in vivo.
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AlM:To construct recombination eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes. METHODS:We amplified the target gene of shuttle vector PHMCMVTSHR289, conjugated the target gene and eukaryotic expression plasmid pcDNA3. 1 +, and accredited whether pcDNA3. 1+/TSHR289 was connected or not by enzymatic digestion and sequencing. Cationic liposomes encapsulated the recombination plasmid pcDNA3. 1+/TSHR289. RESULTS: Recombination plasmid pcDNA3. 1+/TSHR289 digested with enzyme Hindlll and the fragment through 0. 8% gel electrophoresis showed 512bp strip. Recombination plasmid pcDNA3. 1+/TSHR289 were found synonymous mutation through forward ( AAC to AAT ) and reverse sequencing ( GCG to GCT) . The volume ratio of cationic liposomes and recombinant plasmid was 3:1. CONCLUSlON: lt is successful to construct the recombination plasmid pcDNA3. 1+/TSHR289 by accredit it through enzymatic digestion and sequencing.
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Objective: To investigate the aberrant methylation status of hMLH1 (human mutL homologue 1) gene in PTC (papillary thyroid carcinoma) tissues, and its correlations with the aberrant methylation of NIS (sodium iodide symporter) and TSHR (thyroid-stimulating hormone receptor) genes. Methods: qMSP (quantitative methylation-specific PCR) was carried out to detect the promoter methylation status of hMLH1, NIS and TSHR genes in PTC tissues and adjacent normal thyroid tissues from 152 patinets with PTC. The correlation between the clinicopathologic characteristics and the promoter methylation status of hMLH1 gene was analyzed. The relationships among the methylation status of hMLH1, NIS and TSHR genes and their independent or synergistic effects on the progression of PTC were investigated. Results: The promoter methylation rate of hMLH1 gene in PTC tissues (37.5%) was significantly higher than that in the adjacent normal thyroid tissues (5.3%) (P 0.05). There was a weak correlation among aberran promoter methylation of hMLH1, NIS and TSHR genes (r 0.05). Conclusion: The aberrant methylation of hMLH1 gene promoter in PTC tissues might be associated with the progression of the tumor. There exists no correlation among the aberrant methylation of hMLH1, NIS and TSHR genes, and their independent or synergistic effect also may not be associated with the progression of PTC. Copyright © 2013 by TUMOR.
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OBJECTIVES: To precisely classify the various forms of TD, and then to screen for mutations in transcription factor genes active in thyroid development. SUBJECTS AND METHODS: Patients underwent ultrasound, thyroid scan, and serum thyroglobulin measurement to accurately diagnose the form of TD. DNA was extracted from peripheral leukocytes. The PAX8, and NKX2.5 genes were evaluated in all patients, and TSH receptor (TSHR) gene in those with hypoplasia. RESULTS: In 27 nonconsanguineous patients with TD, 13 were diagnosed with ectopia, 11 with hypoplasia, and 3 with athyreosis. No mutations were detected in any of the genes studied. CONCLUSION: Sporadic cases of TD are likely to be caused by epigenetic factors, rather than mutations in thyroid transcription factors or genes involved in thyroid development.
OBJETIVOS: Classificar corretamente as várias formas de DT e depois rastrear por mutações em genes que participam no desenvolvimento da tireoide. SUJEITOS E MÉTODOS: Os pacientes realizaram ultrassonografia, cintilografia e tireoglobulina sérica para o diagnóstico preciso de DT. DNA foi extraído de leucócitos periféricos. Os genes PAX8 e NKX2.5 foram estudados em todos os pacientes e o gene do receptor do TSH (TSHR) foi estudado na hipoplasia. RESULTADOS: Avaliaram-se 27 pacientes sem consanguinidade com DT, dos quais 13 foram diagnosticados com ectopia, 11 com hipoplasia e 3 com atireose. Nenhuma mutação foi detectada nos genes estudados. CONCLUSÃO: Casos esporádicos de DT são provavelmente causados mais por fatores epigenéticos do que por mutações em fatores de transcrição ou genes envolvidos no desenvolvimento tireoidiano.
Subject(s)
Adolescent , Child , Child, Preschool , Female , Humans , Young Adult , Congenital Hypothyroidism/genetics , Homeodomain Proteins/genetics , Mutation , Paired Box Transcription Factors/genetics , Thyroid Dysgenesis/genetics , Thyrotropin/genetics , Transcription Factors/genetics , Thyroid Dysgenesis/diagnosisABSTRACT
Objective To investigate the association between single nucleotide polymorphisms in the intron 1 of thyroid stimnulating hormone receptor gene (TSHR) and Graves' disease (GD) in the Chinese Han population from Linyi city,Shandong Province.Methods A total of 1759 GD patients and 1740 control subjects were recruited for genotyping in TSHR intron 1 with genome-wide association study (GWAS) and Taqman probe technique.At the same time,serum thyroid hormone and TSH receptor antibody (TRAb) levels of patients were determined.Results Five SNPs were selected for further replication.The rs12101261 _T was significantly associated with GD risk ( OR=1.257,95％CI 1.137-1.390,P =8.23 × 10-6 ). Logistic regression identified that rs12101261 was an independent susceptibility locus of GD ( P=1.61 × 10-3 ).Furthermore,rs12101261 _T was strongly associated with GD ( OR =1.317,95％ CI 1.171-1.481,P=4.14× 10-4 ) in TRAb positive patients,but no association in TRAb negative patients ( OR=1.056,95％ CI 0.892-1.251,P=0.524 ).Serum TRAb concentration showed remarkable difference among three genotype groups of rs12101261.Conclusions Five SNPs in TSHR intron 1 are associated with GD.rs12101261 contributes to increased GD risk independently and is associated with serum TRAb level.
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Objective To investigate the association between the six single nucleotide polymorphisms ( SNP),named as rs179247,rsl2101261,rs2284722,rs4903964,rs2300525,rsl7111394 in the intron 1 of thyroid stimulating hormone receptor gene (TSHR) and Graves' disease (GD).MethodsThe genotypes of the six SNPs were genotyped by Taqman probe technique on Fluidigm EP1 platform in 618 GD patients and 646 control subjects.Meanwhile,TSH receptor antibodies (TRAb) of the patients were determined.ResultsAmong the six SNPs,five S NPs were strongly associated with GD,with the most signals at rs179247_G,rs12101261_C,rs4903964 _G (P=2.85×10-10,OR=1.73,95％CI1.46-2.05;P=1.74×10-10,OR=1.73,95％CI 1.46-2.05;P=2.24×10-10,OR=1.69,95％ CI 1.44-1.99 ).The results of logistic regression analysis indicated that rs12101261 and rs4903964 were main susceptibility loci of GD in the intron 1 of TSHR.rs179247_G,rs1210126 1_C,and rs4903964_G were associated with subset of the GD patients with positive TRAb (P=4.24× 10-13,p=5.48× 10-13,P =3.89×10-12 ).Conclusionrs179247,rs12101261,and rs4903964 in TSHR intron 1 were significantly associated with GD in the Chinese Han population from Bengbu city.rs12101261 and rs4903964 were the major susceptibility SNPs associated with GD.TSHR gene may play a main role of susceptibility gene in the subset of GD patients with persistent positive TRAb.
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Objective To investigate the association between polymorphisms of thyroid-stimulating hormone receptor(TSHR)gene intron 1(rs179247, rs12101261)and Graves′ disease(GD)in the China Han population from Xuzhou city, Jiangsu Province. Methods Total 1 066 GD patients and 1 107 control subjects were recruited for genotyping by Taqman probe technique on Fluidigm EP1 platform. Meanwhile, serum concentrations of thyroid hormone and TSH receptor antibodies(TRAb)were determined. Results The rs179247_A, rs12101261_T were significantly associated with GD risk(OR=1.35, 95%CI 1.19-1.54, P=5.92×10-6; OR=1.32, 95%CI 1.16-1.50, P=2.22×10-5). Logistic regression identified that rs179247 was an independent susceptibility locus of GD. Serum TRAb concentration showed a significant difference(P=0.015)among rs179247_AA, AG, and GG genotypes. Conclusion rs179247 and rs12101261 in TSHR intron 1 are both associated with GD, and rs179247 may contribute risk to GD independently. The polymorphism is associated with TRAb, but not with serum concentration of thyroid hormones, age of onset, diffused thyroid goiter, ophthalmic signs, and relapse.
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Objective To study the expressions of the tumor suppressor gene TSH receptor( TSHR),P16, and RAS in papillary thyroid carcinoma ( PTC ) , and the correlation between the occurrence of tumor and the aberrant promoter hypermethylation of three tumor suppressor genes. Methods RT-PCR was used to detect the mRNA expression of three tumor suppressor genes in tissues of 50 cases of PTC ,20 cases of nodular goiter,and 12 cases of thyroid adenoma. The promoter methylation status of three tumor suppressor genes was examined by methylation-specific PCR technique( MSP). Gene sequencing was used to test if the hypermethylation existed in the promoter of three tumor suppressor genes. Results In 68.0% (34/50) TSHR gene, 54.0% (27/50) P16 gene, and 60.0% ( 30/50 ) RAS gene in PTCs, hypermethylation in promoter region was detected, the respective results 21.9% (7/32) , 15. 6% (5/32) ,and 31. 3% (10/32) were found in control tissues. The rates of the three genes with promoter hypermethylation in PTC were significantly higher than those in control tissues ( all P<0. 05). The mRNA expressions of TSHR,P16,and RAS were significantly lower in PTC than those in control tissues (0. 41 ± 0.11 vs 0.63±0. 08,0. 51±0. 17 vs 0. 72±0. 22,0. 56±0. 10 vs 0. 67±0. 16, all P<0. 05). The sequencing confirmed that there was CC to TC transmission in the promoters of three tumor suppressor genes. Conclusions The methylation of three tumor suppressor genes in promoter region is a common molecule event and may be involved in the genesis and development of human PTC.
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Congenital hypothyroidism (CH) is detected at a rate of 1 in 3,000 to 4,000 live births, making it the most common congenital endocrine disorder worldwide. CH is most commonly caused by defects in thyroid development leading to thyroid dysgenesis or dyshormonogenesis. Congenital hypothyroidism is usually sporadic, but up to 2% of cases of thyroid dysgenesis are familial, and CH caused by organification defects is often inherited in a recessive manner. The candidate genes associated with this genetically heterogeneous disorder fall into two main groups: those causing thyroid gland dysgenesis and those causing dyshormonogenesis. Genes associated with thyroid gland dysgenesis include the TSHR gene in non-syndromic CH, and Gsa and the thyroid transcription factor (TTF-1, TTF-2, and Pax-8) genes, which are associated with different complex syndromes that include CH. Among genes associated with dyshormonogenesis, the TPO and TG genes were initially described, and more recently PDS, NIS, and THOX2 gene defects. There is some evidence for a third group of CH conditions associated with iodothyronine transporter defects that are, in turn, associated with severe neurological sequelae.
Subject(s)
Congenital Hypothyroidism , Genetic Variation , Live Birth , Thyroid Dysgenesis , Thyroid Gland , Transcription FactorsABSTRACT
Objective Gene linkage would be processed in order to make sure if an autosomal dominant congenital hyperthyroidism family has genetic linking relationship with the known hyperthyroidism disease genes,TSHR or THRB.Methods Microsatellite marked gene linkage was done with the use of three microsatellite markers,D14S74,D3S2338 and D3S1266,whose chromosomal locations were very close to TSHR or THRB gene,and the results were analyzed by Genemapper 3.5 Software.Results LOD scores of the three markers were all less than 1,revealing that there were no linking relationships between TSHR or THRB gene and this hyperthyroidism family,further reflecting this family might have a new disease gene other than TSHR and THRB.Conclusion There might be new disease genes responsible for autosomal dominant congenital hyperthyroidism.
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BACKGROUND: Recent studies reported that BRAF V600E mutation, the most prevalent genetic event found in papillary thyroid carcinoma, is an independent poor prognostic marker. Additionally, it correlates with a less differentiated tumor stage due to reduced expression of key genes involved in iodine metabolism. We previously described a patient with BRAF V600E mutation in primary tumor and a new mutation (V600E+K601del) in the matched-lymph node metastases. In the present study we report an unusual clinical behavior of PTC and correlate with BRAF mutational status and level of expression of TSHR and NIS. METHODS: Quantitative PCR (qPCR) was used to evaluate the NIS and TSHR level of expression in matched papillary thyroid carcinoma and adjacent normal tissue. RESULTS: In this study, we presented a seven-year follow up of a juvenile papillary thyroid carcinoma patient who had an aggressive tumor harboring BRAF mutation, and failed to conventional therapy. We found a markedly decrease of NIS and TSHR expression in primary PTC compared to adjacent normal thyroid tissue. CONCLUSION: Our findings suggest that BRAF mutational status and decreased NIS and TSHR expression in this patient may reduce radioiodine uptake and lead to a negative response to radioiodine therapy.
INTRODUÇÃO: Estudos recentes demonstraram que a mutação V600E no gene BRAF é o evento genético mais freqüentemente encontrado em carcinoma papilífero da tiróide e um marcador de prognóstico independente. Adicionalmente, esta alteração genética tem sido correlacionada com a redução de expressão de genes envolvidos no metabolismo do iodo. Previamente, nosso grupo descreveu uma paciente com a mutação V600E no gene BRAF no tumor primário e uma mutação nova (V600E+K601del) em metástases pareadas. Neste estudo, reportamos um carcinoma papilífero com um comportamento clínico incomum e correlacionamos com a presença de mutação no gene BRAF e os níveis de expressão de TSHR e NIS. MÉTODO: Análise de expressão dos genes NIS e receptor de TSH (TSHR) através da técnica de PCR em tempo real. RESULTADOS: Descrevemos sete anos de acompanhamento de uma paciente jovem que apresentava um tumor com comportamento agressivo e baixa resposta aos tratamentos convencionais. Uma acentuada diminuição da expressão do TSHR e a ausência de expressão de NIS foram observadas no tumor primário desta paciente quando comparada com o tecido tiroidiano normal adjacente. CONCLUSÃO: Nossos dados sugerem que as mutações encontradas nesta paciente no gene BRAF com conseqüente perda de expressão dos genes NIS e TSHR podem ter reduzido a captação de iodo radioativo e a resposta ao tratamento.
Subject(s)
Adolescent , Female , Humans , Carcinoma, Papillary/genetics , Mutation , Proto-Oncogene Proteins B-raf/genetics , Thyroid Neoplasms/genetics , Case-Control Studies , Gene Expression , Iodine/metabolism , Receptors, Thyrotropin/genetics , Symporters/geneticsABSTRACT
Objective To investigate the relationship between the characteristics of promoter methylation of thyroid stimulating hormone receptor(TSHR) gene in papillary thyroid Carcinomas(PTC) and the clinical manifestation of PTC. Methods The methylation status of TSHR gene was detected by methylation specific PCR technique(MSP).Results (1) The methylation rate of TSHR gene in PTC tissues was 64.7%(22/34),while the methylation rate of TSHR gene in adjacent thyroid tissues(ATT) was 26.5%(9/34),and the rate of methylation of TSHR promoter in PTC was significantly higher than of ATT(P
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Objective To obtain more information concerning polymorphism of the thyrotropin (TSHR) in Graves diseases(GD). Methods (1)A family of GD was studied (including 3 patients and 9 healthy family members)to examine SNPs of TSHR through direct sequencing of all 10 exons and part of introns. (2)In the current case-control study, 30 patients with familiar GD, 48 sporadic patients and 96 healthy control individuals were used to assess whether SNP of TSHR was associated with GD. Genomic DNA was extracted from peripheral leukocytes isolated from ACD-anticoagulated blood. Ten exons were amplified by PCR, using primers designed by ourselves. After purifying, the products were sequenced. Results Eight polymorphisms were found. There was a novel polymorphism in exon 8. There were no significant differences between patients and controls. Conclusions These findings suggested that the novel and other polymorphisms of the TSHR gene may not be responsible for GD. There are racial differences in the distribution of polymorphisms of TSHR gene.
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DQA1*0501.TSHR peptides of 183～198、195～210、248～263、301～320、343～362 and 357～376 could bind with high affinity to both HLA-DR3 and HLA-DQA1*0501,all of their IC50 values less than(1 ?mol/L),but they could bind to neither HLA-DR7 nor HLA-DQA1*0201 with high affinity.Conclusion Epitopes of TSHR 183～198,195～210,248～263,301～320,343～362 and 357～376 on the TSHR extracellular domain might be the auto-antigens to cause GD.