Caterina Di Cosmo

Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion

The mechanism of thyroid hormone (TH) secretion from the thyroid gland into blood is unknown. Humans and mice deficient in monocarboxylate transporter 8 (MCT8) have low serum thyroxine (T4) levels that cannot be fully explained by increased deiodination. Here, we have shown that Mct8 is localized at the basolateral membrane of thyrocytes and that the serum TH concentration is reduced in Mct8-KO mice early after being taken off a treatment that almost completely depleted the thyroid gland of TH. Thyroid glands in Mct8-KO mice contained more non-thyroglobulin-associated T4 and triiodothyronine than did those in wild-type mice, independent of deiodination. In addition, depletion of thyroidal TH content was slower during iodine deficiency. After administration of 125I, the rate of both its secretion from the thyroid gland and its appearance in the serum as trichloroacetic acid-precipitable radioactivity was greatly reduced in Mct8-KO mice. Similarly, the secretion of T4 induced by injection of thyrotropin was reduced in Mct8-KO in which endogenous TSH and T4 were suppressed by administration of triiodothyronine. To our knowledge, this study is the first to demonstrate that Mct8 is involved in the secretion of TH from the thyroid gland and contributes, in part, to the low serum T4 level observed in MCT8-deficient patients.

Thyroid Hormone-Regulated Mouse Cerebral Cortex Genes Are Differentially Dependent on the Source of the Hormone: A Study in Monocarboxylate Transporter-8- and Deiodinase-2-Deficient Mice

Thyroid hormones influence brain development through the control of gene expression. The concentration of the active hormone T(3) in the brain depends on T(3) transport through the blood-brain barrier, mediated in part by the monocarboxylate transporter 8 (Mct8/MCT8) and the activity of type 2 deiodinase (D2) generating T(3) from T(4). The relative roles of each of these pathways in the regulation of brain gene expression is not known. To shed light on this question, we analyzed thyroid hormone-dependent gene expression in the cerebral cortex of mice with inactivated Mct8 (Slc16a2) and Dio2 genes, alone or in combination. We used 34 target genes identified to be controlled by thyroid hormone in microarray comparisons of cerebral cortex from wild-type control and hypothyroid mice on postnatal d 21. Inactivation of the Mct8 gene (Mct8KO) was without effect on the expression of 31 of these genes. Normal gene expression in the absence of the transporter was mostly due to D2 activity because the combined disruption of Mct8 and Dio2 led to similar effects as hypothyroidism on the expression of 24 genes. Dio2 disruption alone did not affect the expression of positively regulated genes, but, as in hypothyroidism, it increased that of negatively regulated genes. We conclude that gene expression in the Mct8KO cerebral cortex is compensated in part by D2-dependent mechanisms. Intriguingly, positive or negative regulation of genes by thyroid hormone is sensitive to the source of T(3) because Dio2 inactivation selectively affects the expression of negatively regulated genes.

A Thyroid Hormone Analog with Reduced Dependence on the Monocarboxylate Transporter 8 for Tissue Transport

Mutations of the thyroid hormone (TH) cell membrane transporter MCT8, on chromosome-X, produce severe mental and neurological impairment in men. We generated a Mct8-deficient mouse (Mct8KO) manifesting the human thyroid phenotype. Although these mice have no neurological manifestations, they have decreased brain T(3) content and high deiodinase 2 (D2) activity, reflecting TH deprivation. In contrast and as in serum, liver T(3) content is high, resulting in increased deiodinase 1 (D1), suggesting that in this tissue TH entry is Mct8 independent. We tested the effect of 3,5-diiodothyropropionic acid (DITPA), a TH receptor agonist, for its dependence on Mct8 in Mct8KO and wild-type (Wt) mice tissues. After depletion of endogenous TH, mice were given three different doses of DITPA. Effects were compared with treatment with two doses of l-T(4). As expected, physiological doses of l-T(4) normalized serum TSH, brain D2, and liver D1 in Wt mice but not the Mct8KO mice. The higher dose of T(4) suppressed TSH in the Wt mice, normalized TSH and brain D2 in Mct8KO mice, but produced a thyrotoxic effect on liver D1 in both genotypes. In contrast DITPA produced similar effects on TSH, D2, and D1 in both Wt and Mct8KO mice. The higher dose fully normalized all measurements and other parameters of TH action. Thus, DITPA is relatively MCT8 independent for entry into the brain and corrects the TH deficit in Mct8KO mice without causing thyrotoxic effect in liver. The potential clinical utility of this analog to patients with MCT8 mutations requires further studies.

Diiodothyropropionic Acid (DITPA) in the Treatment of MCT8 Deficiency

CONTEXT Monocarboxylate transporter 8 (MCT8) is a thyroid hormone-specific cell membrane transporter. MCT8 deficiency causes severe psychomotor retardation and abnormal thyroid tests. The great majority of affected children cannot walk or talk, and all have elevated serum T(3) levels, causing peripheral tissue hypermetabolism and inability to maintain weight. Treatment with thyroid hormone is ineffective. In Mct8-deficient mice, the thyroid hormone analog, diiodothyropropionic acid (DITPA), does not require MCT8 to enter tissues and could be an effective alternative to thyroid hormone treatment in humans. OBJECTIVE The objective of the study was to evaluate the effect and efficacy of DITPA in children with MCT8 deficiency. METHODS This was a multicenter report of four affected children given DITPA on compassionate grounds for 26-40 months. Treatment was initiated at ages 8.5-25 months, beginning with a small dose of 1.8 mg, increasing to a maximal 30 mg/d (2.1-2.4 mg/kg · d), given in three divided doses. RESULTS DITPA normalized the elevated serum T(3) and TSH when the dose reached 1 mg/kg · d and T(4) and rT(3) increased to the lower normal range. The following significant changes were also observed: decline in SHBG (in all subjects), heart rate (in three of four), and ferritin (in one of four). Cholesterol increased in two subjects. There was no weight loss and weight gain occurred in two. None of the treated children required a gastric feeding tube or developed seizures. No adverse effects were observed. CONCLUSION DITPA (1-2 mg/kg · d) almost completely normalizes thyroid tests and reduces the hypermetabolism and the tendency for weight loss. The effects of earlier commencement and long-term therapy remain to be determined.

Clinical and molecular characterization of a novel selenocysteine insertion sequence-binding protein 2 (SBP2) gene mutation (R128X)

CONTEXT Although acquired abnormalities of thyroid hormone metabolism are common, inherited defects in humans involving the synthesis of selenoproteins, including iodothyronine deiodinases, have been described in only one recent publication. OBJECTIVE We report the study of a novel selenocysteine insertion sequence-binding protein 2 (SBP2) gene mutation (R128X) and its clinical and molecular characterization. SUBJECTS AND METHODS A family of African origin was studied. The proband presented with growth retardation, low serum selenium level, and thyroid test abnormalities consisting of high serum total and free T(4) concentrations associated with low T(3), high rT(3), and normal TSH. The entire coding region of the SBP2 gene was sequenced and minigenes constructed to explain the nature of the defect. RESULTS The proband was homozygous for a nonsense gene mutation that produces an early stop codon (R128X). Both parents and a sister were heterozygous but showed no growth or thyroid test abnormalities. Despite the severity of the defect, the patient had a relatively mild phenotype, similar to that associated with partial SBP2 deficiency. In vitro analysis showed that the mutant minigene synthesized SBP2 from at least three downstream ATGs capable of generating molecules containing the essential functional domains. Treatment with l-T(3) accelerated the growth velocity and advanced the bone age. CONCLUSIONS We identified a novel SBP2 gene mutation producing an early arrest in the synthesis of a full-length molecule. The demonstration that SBP2 isoforms containing all functional domains could be synthesized from three downstream ATGs explains the relatively mild phenotype caused by this defect.

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.

The Syndrome of Inherited Partial SBP2 Deficiency in Humans

Selenium (Se) is an essential trace element required for the biosynthesis of selenoproteins. Selenocysteine insertion sequence (SECIS) binding protein 2 (SBP2) represents a key trans-acting factor for the co-translational insertion of selenocysteine into selenoproteins. In 2005, we reported the first mutations in the SBP2 gene in two families in which the probands presented with transient growth retardation associated with abnormal thyroid function tests. Intracellular metabolism of thyroid hormone (TH) and availability of the active hormone, triiodothyronine, is regulated by three selenoprotein iodothyronine deiodinases (Ds). While acquired changes in D activities are common, inherited defects in humans were not known. Affected children were either homozygous or compound heterozygous for SBP2 mutations. Other selenoproteins, glutathione peroxidase, and selenoprotein P were also reduced in affected subjects. Since our initial report, another family manifesting a similar phenotype was found to harbor a novel SBP2 mutation. In vivo studies of these subjects have explored the effects of Se and TH supplementation. In vitro experiments have provided new insights into the effect of SBP2 mutations. In this review we discuss the clinical presentation of SBP2 mutations, their effect on protein function, consequence for selenoproteins, and the clinical course of subjects with SBP2 defects.

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.