Patrizia Agretti

In vitro assay of thyroid disruptors affecting TSH-stimulated adenylate cyclase activity

Several natural or synthetic chemicals have been indicated as potential thyroid disruptors. The development of in vitro assays has been recommended to comprehensively assess the potential thyroid disrupting activity of a substance or a complex mixture. In this study, 12 substances suspected for acting as thyroid disruptors were tested for their ability to inhibit TSH-stimulated cAMP production in vitro. Those substances producing an inhibition were further studied to establish the level at which they interfere with this step of thyroid cell function. Using Chinese hamster ovary cells (CHO) transfected with the recombinant human TSH receptor, a dose-dependent inhibition of TSH-stimulated adenylate cyclase activity was produced by 1,1-bis-(4-chlorphenyl)-2,2,2-trichloroethan (DDT), Aroclor 1254 and Melissa Officinalis. All three substances also inhibited the cAMP production stimulated by TSH receptor antibody. Melissa Officinalis produced a significant inhibition of TSH binding to its receptor and of antibody binding to TSH, while no significant changes were produced by Aroclor 1254 or DDT in these assays. These data suggest that principles contained in Melissa Officinalis may block the binding of TSH to its receptor by acting both on the hormone and the receptor itself, while DDT and Aroclor 1254 affect cAMP production mainly at post-receptor step. In conclusion, we have developed a set of in vitro assays that allow investigation into the effect of thyroid disruptors on the TSH-mediated activation of the cAMP cascade. These assays may be useful to identify the mechanism of action of thyroid disruptors, coming beside and supporting animal studies or epidemiological surveys.

Thyroid Hormone Action in the Adult Brain: Gene Expression Profiling of the Effects of Single and Multiple Doses of Triiodo-L-Thyronine in the Rat Striatum

Thyroid hormones have profound effects on mood and behavior, but the molecular basis of thyroid hormone action in the adult brain is relatively unknown. In particular, few thyroid hormone-dependent genes have been identified in the adult brain despite extensive work carried out on the developing brain. In this work we performed global analysis of gene expression in the adult rat striatum in search for genomic changes taking place after administration of T(3) to hypothyroid rats. The hormone was administered in two different schedules: 1) a single, large dose of 25 microg per 100 g body weight (SD) or 2) 1.5 microg per 100 g body weight once daily for 5 d (RD). Twenty-four hours after the single or last of multiple doses, gene expression in the striatum was analyzed using Codelink microarrays. SD caused up-regulation of 149 genes and down-regulation of 88 genes. RD caused up-regulation of 18 genes and down-regulation of one gene. The results were confirmed by hybridization to Affymetrix microarrays and by TaqMan PCR. Among the genes identified are genes involved in circadian regulation and the regulation of signaling pathways in the striatum. These results suggest that thyroid hormone is involved in regulation of striatal physiology at multiple control points. In addition, they may explain the beneficial effects of large doses of thyroid hormone in bipolar disorders.

Thyrotropin-Stimulating Hormone Receptor Gene Analysis in Pediatric Patients with Non-Autoimmune Subclinical Hypothyroidism

CONTEXT Mutations in TSH receptor (TSHR) are notoriously associated with congenital hypothyroidism as well as with non-autoimmune subclinical hypothyroidism, a mild form of TSH resistance that is not as well characterized by diagnostic procedures. OBJECTIVE The genetic analysis of the TSHR gene was performed to determine the prevalence of TSHR gene mutations in non-autoimmune subclinical hypothyroidism during the pediatric age. The new mutations were studied for genotypic-phenotypic correlation. PATIENTS Thirty-eight children (ages 0.5-18.0 yr) affected by non-autoimmune subclinical hypothyroidism diagnosed in our center (follow-up from 1 to 11.5 yr) and normal at neonatal screening were enrolled in the genetic study. In 11 cases, the relatives were included in the genetic analysis. RESULTS Eleven different mutations of the TSHR gene were identified in 11 of the 38 patients. Two are new: the nonsense mutation C31X and the missense mutation P68S, which shows a decrease in TSH binding capacity but not in biological activity. In all cases the carrier parent was identified. CONCLUSIONS To date, this study demonstrates the highest prevalence (29%) of TSHR gene mutations in children and adolescents with non-autoimmune subclinical hypothyroidism not selected by neonatal screening. Functional studies show that some mutations cause a slight inactivation of the TSHR. This reveals a possible limit of the in vitro study or of the knowledge of mechanisms involving TSHR, or that other candidate genes must be considered.

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

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.

Identification and Functional Analysis of Novel Dual Oxidase 2 (DUOX2) Mutations in Children with Congenital or Subclinical Hypothyroidism

CONTEXT Congenital hypothyroidism (CH) associated with goiter or a gland of normal size has been linked to dual oxidase 2 (DUOX2) mutations in the presence of iodide organification defect. OBJECTIVE Thirty unrelated children with CH or subclinical hypothyroidism (SH) identified during infancy with a eutopic thyroid gland, coming from our Screening Centre for CH or referred from other regions of Italy, were studied with the perchlorate discharge test to identify organification defects. Eleven children with iodide organification defect were considered for the genetic analysis of TPO, DUOX2, and dual oxidase maturation factor 2 (DUOXA2) genes. PATIENTS Eight children with CH and three with SH and eutopic thyroid gland were included in the study. After discontinuation of therapy, a partial or complete organification defect was shown after ¹²³I scintigraphy and perchlorate test. METHODS TPO, DUOX2, and DUOXA2 genes were analyzed, and functional activity of DUOX2 variants was studied in HeLa cells. RESULTS Sequencing of the DUOX2 gene revealed a deletion S965fsX994 in three children; two were euthyroid after 1 month of L-T₄ discontinuation but developed SH after 5 and 18 months, respectively, whereas the other child had SH. One child with SH showed H678R, R701Q, and P982A substitutions, and another child with SH showed only the P982A. One child with SH showed the Y1150C mutation, and another euthyroid child showed the A728T mutation. Functional studies confirmed that S965fsX994, Y1150C, and A728T mutations were responsible for the defect in H₂O₂ production, whereas H678R, R701Q, and P982A did not alter H₂O₂ production in vitro. CONCLUSIONS Genetic analysis of the DUOX2 gene was performed in 11 children with organification defect. Two new mutations (Y1150C and A728T) and the deletion S965FsX994 were responsible for the deficit in the organification process and the phenotypes. Three polymorphisms (H678R, P982A, and R701Q) were identified.

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

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.

Genetic analysis of TTF-2 gene in children with congenital hypothyroidism and cleft palate, congenital hypothyroidism, or isolated cleft palate.

Homozygous null mice for thyroid transcription factor (TTF)-2 gene exhibit cleft palate and thyroid malformation. We performed a genetic analysis of the TTF-2 gene in 2 children with congenital hypothyroidism (CH) and cleft palate, 45 children with thyroid dysgenesis, 19 children with isolated cleft palate or cleft lip, 4 patients with thyroid hemiagenesis. The entire coding-region of the TTF-2 gene was analyzed by direct sequencing. Direct sequencing of the TTF-2 gene revealed polymorphisms in the length of the polyalanine tract. The most frequent stretch length was 14 residues and it was found in 50 of 70 (71%) and in 45 of 53 (85%) normal healthy controls. A polyalanine tract of 16 residues in the heterozygous state was seen in 18 of 70 (26%) cases and in 4 of 53 (7%) normal subjects. In 1 of 4 (25%) case of hemiagenesis a polyalanine tract of 16 residues in the homozygous state was observed. In 1 of 26 agenesis the polyalanine tract consisted of 12 residues in the heterozygous state. Direct sequencing also revealed the presence of two silent polymorphisms. No mutations were identified in the TTF-2 gene. In conclusion, our results show that no genetic alteration was present in the TTF-2 gene of these patients, suggesting that defects in the TTF-2 gene are a rare event.

The sodium-iodide symporter expression in placental tissue at different gestational age: an immunohistochemical study

Background  Iodide (I−) is crucial for foetal thyroid function. Foetal iodide results from maternal circulating iodide and from deiodination of iodothyronines within the placenta. The Na+/I− symporter (NIS) localized in placental cells appears to be involved in iodide exchange. Low NIS expression has been reported in trophoblast cells from the first trimester and pregnancy at term.

Congenital hypothyroidism due to a new deletion in the sodium/iodide symporter protein

objective  Iodide transport defect (ITD) is a rare disorder characterised by an inability of the thyroid to maintain an iodide gradient across the basolateral membrane of thyroid follicular cells, that often results in congenital hypothyroidism. When present the defect is also found in the salivary glands and gastric mucosa and it has been shown to arise from abnormalities of the sodium/iodide symporter (NIS).

Functional studies of new TSH receptor (TSHr) mutations identified in patients affected by hypothyroidism or isolated hyperthyrotrophinaemia.

Objectives  To establish the role of new TSH receptor (TSHr) variants (P27T, E34 K, R46P, D403N, W488R and M527T) recently identified in children with congenital hypothyroidism (CH) or subclinical hypothyroidism (SH) with a thyroid gland of normal size.