Identification of a novel pax8 gene sequence variant in four members of the same family: from congenital hypothyroidism with thyroid hypoplasia to mild subclinical hypothyroidism.

Abstract Background: Congenital hypothyroidism is often secondary to thyroid dysgenesis, including thyroid agenesis, hypoplasia, ectopic thyroid tissue or cysts. Loss of function mutations in TSHR, PAX8, NKX2.1, NKX2.5 and FOXE1 genes are responsible for some forms of inherited congenital hypothyroidism, with or without hypoplastic thyroid. The aim of this study was to analyse the PAX8 gene sequence in several members of the same family in order to understand whether the variable phenotypic expression, ranging from congenital hypothyroidism with thyroid hypoplasia to mild subclinical hypothyroidism, could be associated to the genetic variant in the PAX8 gene, detected in the proband. Methods: We screened a hypothyroid child with thyroid hypoplasia for mutations in PAX8, TSHR, NKX2.1, NKX2.5 and FOXE1 genes. We studied the inheritance of the new variant R133W detected in the PAX8 gene in the proband’s family, and we looked for the same substitution in 115 Caucasian European subjects and in 26 hypothyroid children. Functional studies were performed to assess the in vitro effect of the newly identified PAX8 gene variant. Results: A new heterozygous nucleotide substitution was detected in the PAX8 DNA-binding motif (c.397C/T, R133W) in the proband, affected by congenital hypothyroidism with thyroid hypoplasia, in his older sister, displaying a subclinical hypothyroidism associated with thyroid hypoplasia and thyroid nodules, in his father, affected by hypothyroidism with thyroid hypoplasia and thyroid nodules, and his first cousin as well, who revealed only a subclinical hypothyroidism. Functional studies of R133W-PAX8 in the HEK293 cells showed activation of the TG promoter comparable to the wild-type PAX8. Conclusions: In vitro data do not prove that R133W-PAX8 is directly involved in the development of the thyroid phenotypes reported for family members carrying the substitution. However, it is reasonable to conceive that, in the cases of transcriptions factors, such as Pax8, which establish several interactions in different protein complexes, genetic variants could have an impact in vivo. Keywords: PAX8 gene, Thyroid, Congenital hypothyroidism, Variable phenotypic expressivity, R133W-PAX

In vitro study of new DUOX2 mutants

Context: Some cases of congenital hypothyroidism (CH) are associated with a gland of normal size. Objective: To explore the cause of organification defect in one child with CH and a eutopic thyroid gland, genetic analyses of TPO, DUOX2, and DUOXA2 genes were performed. Patient: One child with CH, a eutopic thyroid gland, and a partial organification defect was shown after 123I scintigraphy and perchlorate test. Methods: In the child with the organification defect, TPO, DUOX2, and DUOXA2 genes were analyzed. The functional activity of the DUOX2 mutants was studied after expression in eukaryotic cells. Results: No TPO or DUOXA2 gene mutations were identified. Direct sequencing of the DUOX2 gene revealed a compound heterozygous genotype for S911L and C1052Y substitutions. S911L and C1052Y caused a partial defect in H2O2 production after transient expression in HeLa cells. Conclusions: We performed a genetic analysis in one child with CH and a eutopic thyroid gland. Two new mutations in DUOX2 gene responsible for the partial deficit in the organification process were identified. A new case of congenital hypothyroidism with a normally located thyroid gland and two new mutations in DUOX2 protein partially impairing H2O2 generation is described

Ipotiroidismo congenito causato da una nuova mutazione omozigote del gene della tireoglobulina

L\u2019ipotiroidismo congenito dovuto a deficit di tireoglobulina (TG) \ue8 una malattia autosomica recessiva con una prevalenza di 1:4000091:100000 nati vivi e caratterizzata da gozzo, bassi livelli sierici di TG, elevati livelli di TSH con bassi livelli di ormoni tiroidei e test al perclorato negativo. La TG umana, una proteina di 2768 aminoacidi, \ue8 codificata da un grosso gene con 48 esoni localizzato su cromosoma 8q24. Mutazioni del gene della TG sono associate a gozzo congenito con ipotiroidismo o eutiroidismo. Ad oggi sono state descritte e caratterizzate 50 mutazioni del gene della TG umana. Lo scopo del nostro studio \ue8 stato quello di eseguire l\u2019analisi del gene della TG in due sorelle nate da genitori consanguinei affette da ipotiroidismo congenito. Le due sorelle sono state identificate allo screening neonatale e trattate dopo la conferma di ipotiroidismo con L9Tiroxina. Alla et\ue0 di 7 anni, una sorella, dopo sospensione della L9tiroxina presentava un franco ipotiroidismo, TG indosabile; alla scintigrafia era presente una tiroide in sede con captazione del 12% dopo 24 ore; la ecografia dimostrava la presenza di una ghiandola in sede di normali dimensioni. La seconda sorella, nonostante il precoce trattamento con L9 tiroxina, era anche affetta da ritardo mentale. La ecografia confermava la presenza di una tiroide in sede di normali dimensioni, e la tireoglobulina era indosabile. Il DNA genomico \ue8 stato estratto dal sangue delle pazienti e da quello del padre, unico genitore in vita, utilizzando un kit commerciale. Tutti i 48 esoni che compongono il gene della TG sono stati amplificati per PCR, sequenziati con BigDye Terminator Kit e analizzati su 3130xl genetic analyzer. Nel sangue di entrambe le pazienti \ue8 stata identificata una mutazione puntiforme omozigote a livello dell\u2019esone 10 del gene della TG (CGA/TGA) che determina la formazione di un codone di stop in posizione 768 (R768X). Il risultato \ue8 quindi la presenza di una proteina precocemente troncata e quindi non funzionante. Erano inoltre presenti varianti alleliche gi\ue0 descritte in letteratura. In conclusione, abbiamo identificato due sorelle con ipotiroidismo congenito e tiroide in sede di normali dimensioni e TG indosabile. L\u2019analisi genetica ha dimostrato una alterazione della Tireoglobulina come causa dell\u2019ipotiroidismo

Relative potencies and additivity of perchlorate, thiocyanate, nitrate, and iodide on the inhibition of radioactive iodide uptake by the human sodium iodide symporter

The presence of perchlorate (ClO(4) (-)) in some U.S. drinking water supplies has raised concern about potential adverse thyroidal health effects, because ClO(4) (-) is known to competitively inhibit iodide uptake at the sodium iodide symporter (NIS). Humans are nutritionally and environmentally exposed to other competitive inhibitors of iodide uptake, including thiocyanate (SCN(-)) and nitrate (NO(3) (-)). The joint inhibiting effects of these three anions was studied by exposing Chinese hamster ovary cells stably expressing human NIS to varying concentrations of each anion separately, and in combination, and conducting measurements of (125)I(-) uptake. The entire data set was fit to a single Hill equation using maximum likelihood. The relative potency of ClO(4) (-) to inhibit (125)I(-) uptake at the NIS was found to be 15, 30 and 240 times that of SCN(-), I(-), and NO(3) (-) respectively on a molar concentration basis, with no evidence of synergism. These results are consistent with a common mode of action by these anions of simple competitive interaction, in which a concentration of any one of ClO(4) (-) SCN(-), and NO(3) (-), occurring either individually or as part of a mixture of the three anions, is indistinguishable from a concentration or dilution of either one of the remaining two ions in inhibiting iodine uptake at the NIS

STUDY OF POTENTIAL INHIBITORS OF THYROID IODIDE UPTAKE BY USING CHO CELLS STABLY EXPRESSING THE HUMAN SODIUM/IODIDE SYMPORTER (hNIS) PROTEIN

BACKGROUND: Thyroid gland is highly dependent on dietary intake of iodine for normal function, so it is particularly subjected to "endocrine disruptor" action. The human sodium/iodide symporter (hNIS) is an integral plasma membrane glycoprotein mediating the active transport of iodide into thyroid follicular cells, a crucial step for thyroid hormone biosynthesis. Beyond to perchlorate and thyocianate ions a few other inhibitors of iodide uptake have been described. AIM: The aim of this study was to investigate if 10 substances usually used as drugs in clinical practice were able to inhibit NIS-mediated iodide uptake in vitro. MATERIALS AND METHODS: A CHO cell line stably expressing hNIS was used to test any inhibition of NIS-mediated iodide uptake exerted by drugs. Perchlorate and thyocianate ions were used as positive controls. RESULTS: None of the analyzed substances was able to significantly inhibit iodide uptake in our system. As we expected, perchlorate and thyocianate ions were able to inhibit iodide uptake in a dose-dependent manner. CONCLUSIONS: In conclusion, we carried out an in vitro assay to evaluate the potential inhibitory effect of common drugs on NISmediated iodide uptake by using CHO-hNIS cells. None of the analyzed substances was able to inhibit iodide uptake; only perchlorate and thyocianate were able to inhibit iodide uptake in a dose-dependent manner

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 mutation

Ipotiroidismo congenito causato da una mutazione omozigote del gene della tireoperossidasi

L\u2019ipotiroidismo congenito con gozzo dovuto a difetti di organificazione dello iodio \ue8 ereditato come tratto recessivo ed \ue8 spesso dovuto a mutazioni del gene della tireoperossidasi (TPO). Il gene umano della TPO contiene 17 esoni ed \ue8 localizzato sul cromosoma 2p25. Il gene codifica per un enzima contenente un gruppo eme di 933 aminoacidi molto simile alla mieloperossidasi umana. Ad oggi sono state descritte pi\uf9 di 50 mutazioni del gene della TPO che determinano una alterata attivit\ue0 funzionale. Lo scopo del nostro studio \ue8 stato quello di eseguire l\u2019analisi del gene della TPO in una paziente affetta da ipotiroidismo congenito trattato tardivamente associati a gozzo nodulare. La paziente presentava un alterato sviluppo psicosomatico ed era in terapia con L9T4 dall\u2019et\ue0 di circa 3 anni. All\u2019esame ecografico era presente una tiroide di dimensioni superiori alla norma con nodulo destro di 5 cm. Gli esami di laboratorio mostravano valori di FT3, FT4 e TG nella norma, TSH inferiore alla norma ed assenza di anticorpi anti tiroide in terapia con L9 T4. A completamento dell\u2019iter diagnostico \ue8 stato effettuato test al perclorato dopo somministrazione di TSH ricombinante che si rivelava positivo con dismissione del radioiodio del 91% a 1 ora dalla somministrazione di perclorato di potassio. Il DNA genomico \ue8 stato estratto dal sangue della paziente e di 50 soggetti normali di controllo utilizzando un kit commerciale. Tutti i 17 esoni che compongono il gene della TPO sono stati amplificati per PCR, sequenziati con BigDye Terminator Kit e analizzati su 3130xl genetic analyzer. Nel sangue della paziente \ue8 stata identificata una nuova mutazione puntiforme omozigote a livello dell\u2019esone 10 del gene della TPO (ACG/ATG) che determina la sostituzione in posizione 561 dell\u2019aminoacido treonina con l\u2019aminoacido metionina (T561M). Erano inoltre presenti varianti alleliche gi\ue0 descritte in letteratura. La mutazione non \ue8 stata identificata nel DNA da sangue dei 50 soggetti normali. In conclusione, abbiamo identificato una nuova mutazione omozigote del gene della TPO in una paziente affetta da ipotiroidismo congenito trattato tardivamente. La mutazione \ue8 responsabile del difetto di organificazione dello iodio che ha determinato l\u2019ipotiroidismo

The thyroid disruptor 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane appears to be an uncompetitive inverse agonist for the thyrotropin receptor

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