Showing papers by "Massimo Tonacchera published in 2007"

Genetic analysis of the PAX8 gene in children with congenital hypothyroidism and dysgenetic or eutopic thyroid glands: identification of a novel sequence variant

Abstract: Summary Objective To analyse the coding region of PAX8 in individuals with congenital (CH) or post neonatal hypothyroidism due to dysgenetic (TD) or eutopic thyroid glands. Design and patients Forty-three children with CH and TD (13 agenesis, 23 ectopia, and seven hypoplasia), one subject with post neonatal onset of hypothyroidism and thyroid ectopia, 15 children with CH and eutopic thyroid glands and six euthyroid adults with thyroid hemiagenesis were enrolled as cases, along with 120 healthy individuals as controls. Measurements Exons 2–8 of the PAX8 were directly sequenced. HeLa and HEK293 cells were transfected with PAX8 wild-type (PAX8-WT), mutant PAX8, p300, thyroid transcription factor 1 (TTF-1) and thyroglobulin promoter pGL3 (TG prom-pGL3). Synergism of TTF-1 with PAX8-WT vs. mutant and activity of PAX8-WT vs. mutant in accompaniment with p300 on TG prom-pGL3 were also assessed. The luminescence produced by PAX8-WT and mutant PAX8 was measured. Results Among patients and controls only a 15-year-old girl with thyroid ectopia showed a heterozygous transition of cytosine to thymine at position 674 in exon 6, which changed a conserved threonine at position 225 to methionine (PAX8-T225M). Her father and sister harboured PAX8-T225M without abnormal thyroid phenotypes. PAX8-T225M and PAX8-WT similarly increased luciferase activity and had a similar synergistic effect with TTF-1. At 500 ng p300, however, PAX8-T225M could not significantly increase TG promoter activity when compared to PAX8-T225M alone, while PAX8-WT +500 ng p300 induction was significantly higher than PAX8-WT alone (P < 0·001). Cotransfection of TTF-1 together with PAX8-T225M resulted in rescuing of the lack of synergism with p300. Conclusions PAX8 mutations in congenital hypothyroidism due to dysgenetic or orthotopic thyroid glands are rare. PAX8-T225M is probably a rare variant.

Identification of TSH receptor mutations in three families with resistance to TSH.

Abstract: Summary Objective Genetic analysis of the TSH receptor gene in seven subjects with subclinical hypothyroidism (SH), in whom the diagnosis of autoimmune thyroid disease had been excluded by laboratory and instrumental techniques currently available. Patients Three families where different members (2 children and 5 adults) affected by SH were studied. Genetic analysis Genomic DNA was extracted from peripheral lymphocytes and the entire coding sequence of the TSHr gene was sequenced. pSVL-TSHr construct harbouring a Q8fsX62 insertion was obtained by site-directed mutagenesis. COS-7 cells transfected with wild-type and mutant receptor were used for binding studies, flow cytometry, and cyclic AMP (cAMP) determination. Results A four base pair (bp) duplication in position 41 (41TGCAins), leading to a premature stop of translation at codon 62 (Q8fsX62), was found to be heterozygous in the proband, the father and the sister in Family 1. In Family 2 the proband and the sister were heterozygous for the mutation D410N. In Family 3 the proband and the father were heterozygous for the mutation P162A. After transfection in COS-7 cells, the mutant receptor Q8fsX62 displayed a low expression at the cell surface, and a reduced response to bovine TSH (bTSH) in terms of cAMP production. Conclusions We identified TSH receptor mutations in seven members of three families with subclinical hypothyroidism.

The Thyroid Disruptor 1,1,1-Trichloro-2,2-Bis(p-Chlorophenyl)-Ethane Appears to Be an Uncompetitive Inverse Agonist for the Thyrotropin Receptor

Abstract: In this study, we aimed at establishing whether two previously identified thyroid disruptors, the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and Aroclor 1254 (a complex mixture of polychlorinated water), may inhibit thyrotropin (TSH) receptor (TSHr) activity. DDT and Aroclor 1254 were shown to inhibit both the basal and bovine TSH (bTSH)-stimulated accumulation of cAMP in Chinese hamster ovary (CHO)-K1 cells stably transfected with the TSHr. Furthermore, both DDT and Aroclor 1254 did indeed prevent cAMP accumulation, as induced by the constitutive activity of a point mutant TSHr(I486M) transiently transfected in African green monkey kidney fibroblast (COS)-7 cells. Neither trypsin digestion of the extracellular domain (ECD) nor deletion of the ECD in a mutant TSHr trunk transiently transfected in COS-7 cells counteracted the inhibitory activity of DDT and Aroclor 1254. DDT exerted a weak inhibitory activity against forskolin in both CHO-K1 and COS-7 cells, whereas it was nil against the agonists dopamine and 5'-(N-ethyl-carboxamido)-adenosine (NECA) in CHO cells stably transfected with the dopamine D1 receptor and in COS-7 cells transiently transfected with the adenosine type 2a receptor (A2a) receptor. Furthermore, DDT was inactive against the stimulation by isoproterenol of the endogenously expressed beta2 adrenergic receptor in COS-7 cells. Conversely, Aroclor 1254 inhibited completely forskolin activity in CHO-K1 cells but not in COS-7 cells. Furthermore, it did not prevent accumulation of cAMP as induced by NECA in A2a transfected cells. The analog of DDT, diphenylethylene, was inactive against bTSH-induced increase in cAMP in CHO-K1 cells stably transfected with the TSHr. We interpreted these results as indicating that DDT and possibly Aroclor 1254 may have an uncompetitive inverse agonist activity for the TSHr.

Ras homolog enriched in striatum inhibits the functional activity of wild type thyrotropin, follicle-stimulating hormone, luteinizing hormone receptors and activating thyrotropin receptor mutations by altering their expression in COS-7 cells.

Abstract: Ras homolog enriched in striatum (Rhes) is a member of the Ras family of small GT-Pases detected in the thyroid. Rhes inhibits signal transduction from Gas protein. In this study we investigated whether Rhes can interfere with stimulation of cAMP/protein kinase A (PKA) pathway of TSH, FSH and LH receptors (TSHr, FSHr, LHr) and of activated TSHr mutants. Receptors were transiently transfected in COS-7 cells with or without Rhes; cAMP was evaluated in basal conditions and after hormone stimulation. Constitutive and bovine TSH (bTSH)-stimulated activity of wild type (wt) and mutated TSHr was inhibited after Rhes co-transfection. Rhes decreased cAMP after FSH and hCG β-subunit (βhCG) stimulation in cells expressing the cognate receptors. In binding experiments Rhes, as another membrane protein, sodium/iodide symporter (NIS), reduced membrane expression of wt TSHr (wtTSHr). In conclusion, Rhes can interfere with the functional activity of wt and mutated TSHr and with the respective hormone-stimulated cAMP production of FSHr and LHr. This interference is not specific and due to the co-expression of two membrane proteins.

Markers of cell proliferation, apoptosis, and angiogenesis in thyroid adenomas: a comparative immunohistochemical and genetic investigation of functioning and nonfunctioning nodules.

Abstract: Objective: To perform (i) an immunohistochemical investigation of cell proliferation, apoptosis, angiogenesis, and malignancy markers in 15 functioning and 15 nonfunctioning thyroid adenomas, and in normal adjacent tissue, and (ii) a genetic analysis of thyroid-stimulating hormone receptor (TSH-r), Gsα, and RAS mutations in the same group of adenomas, in order to describe their expression within tissues and to correlate them with the hormonal functioning. Design: Thirty patients who underwent surgery for a solitary thyroid nodule were included in the study. Adenomas and normal adjacent tissues were evaluated by immunohistochemistry using the following antibodies: MIB-1 for proliferative activity, bcl-2 and mutant p53 for apoptosis control, vascular endothelial growth factor-A (VEGF-A) for angiogenic activity, and galectin-3 as a marker for malignancy. To calculate microvascular density, “hot spots” were selected and defined by cells positive for CD34 staining. Genetic analysis for TSH-r, Gsα, and H-, K-, ...

A fast method to detect cell surface expression of thyrotropin receptor (TSHr): the microchip flow cytometry analysis.

Abstract: Loss-of-function mutations of the thyrotropin receptor (TSHr) may be responsible for congenital hypothyroidism or isolated hyperthyreotropinemia. To study cell surface expression of inactivating TSHr mutations detected in patients with isolated hyperthyreotropinemia (L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T), we used the Agilent 2100 bioanalyzer to perform microchip flow cytometry analysis. The previously described TSHr inactivating mutation T477I was used as control. The level of receptor expression in COS-7 cells transfected with the T477I measured by binding assay was four times lower with respect to the wild-type TSHr. The very low expression of T477I was confirmed by fluorescence-activated cell sorting (FACS) analysis and by microchip flow cytometry analysis, suggesting that this method can be a reliable system to measure receptor cell surface expression. Other inactivating TSHr mutations were expressed in COS-7 cells for binding studies, FACS analysis, and microchip flow cytometry analysis. Binding studies showed that L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T mutants had a low expression at the cell surface, as demonstrated by Bmax values. Data obtained by binding studies were in good agreement with data obtained by FACS analysis and microchip flow cytometry analysis. In conclusion, the low number of cells required for analysis and the ease of use make the microchip flow cytometry analysis a very reliable and favorable system to study cell surface expression of TSHr mutations.