The length of FOXE1 polyalanine tract in congenital hypothyroidism: Evidence for a pathogenic role from familial, molecular and cohort studies.

Introduction: FOXE1 is required for thyroid function and its homozygous mutations cause a rare syndromic form of congenital hypothyroidism (CH). FOXE1 has a polymorphic polyalanine tract whose involvement in thyroid pathology is controversial. Starting from genetic studies in a CH family, we explored the functional role and involvement of FOXE1 variations in a large CH population. Methods: We applied NGS screening to a large CH family and a cohort of 1752 individuals and validated these results by in silico modeling and in vitro experiments. Results: A new heterozygous FOXE1 variant segregated with 14-Alanine tract homozygosity in 5 CH siblings with athyreosis. The p.L107V variant demonstrated to significantly reduce the FOXE1 transcriptional activity. The 14-Alanine-FOXE1 displayed altered subcellular localization and significantly impaired synergy with other transcription factors, when compared with the more common 16-Alanine-FOXE1. The CH group with thyroid dysgenesis was largely and significantly enriched with the 14-Alanine-FOXE1 homozygosity. Discussion: We provide new evidence that disentangle the pathophysiological role of FOXE1 polyalanine tract, thereby significantly broadening the perspective on the role of FOXE1 in the complex pathogenesis of CH. FOXE1 should be therefore added to the group of polyalanine disease-associated transcription factors.

Short-Term Exposure Effects of the Environmental Endocrine Disruptor Benzo(a)Pyrene on Thyroid Axis Function in Zebrafish.

Benzo(a)Pyrene (BaP) is one of the most widespread polycyclic aromatic hydrocarbons (PAHs) with endocrine disrupting properties and carcinogenic effects. In the present study, we tested the effect of BaP on thyroid development and function, using zebrafish as a model system. Zebrafish embryos were treated with 50 nM BaP from 2.5 to 72 h post fertilization (hpf) and compared to 1.2% DMSO controls. The expression profiles of markers of thyroid primordium specification, thyroid hormone (TH) synthesis, hypothalamus-pituitary-thyroid (HPT) axis, TH transport and metabolism, and TH action were analyzed in pools of treated and control embryos at different developmental stages. BaP treatment did not affect early markers of thyroid differentiation but resulted in a significant decrease of markers of TH synthesis (tg and nis) likely secondary to defective expression of the central stimulatory hormones of thyroid axis (trh, tshba) and of TH metabolism (dio2). Consequently, immunofluorescence of BaP treated larvae showed a low number of follicles immunoreactive to T4. In conclusion, our results revealed that the short-term exposure to BaP significantly affects thyroid function in zebrafish, but the primary toxic effects would be exerted at the hypothalamic-pituitary level thus creating a model of central hypothyroidism.

Clinical Consequences of Variable Results in the Measurement of Free Thyroid Hormones: Unusual Presentation of a Family with a Novel Variant in the THRB Gene Causing Resistance to Thyroid Hormone Syndrome.

INTRODUCTION: Resistance to thyroid hormone ß (RTHß) is an inherited syndrome caused by dominant negative variants in the THRB gene (NM_000461.5). The clinical picture of RTHß is variable, and patients harboring the same variant may display different degrees of disease severity. CASE PRESENTATION: A 30-year-old man presented with thyrotoxicosis and central hyperthyroidism and was found to have a novel variant in the exon 10 of THRB gene (c.C1282G, p.L428V), located within the third hot spot region of the C-terminal of the receptor. Surprisingly, the same variant was found in two other relatives with an apparent normal thyroid function at initial screening. After exclusion of a TSH-secreting adenoma and serum interference in the proband, and the finding that exogenous levothyroxine failed to suppress the TSH in the brother affected by nodular goiter, relatives' thyroid function tests (TFTs) were reassessed with additional analytical method revealing biochemical features consistent with RTHß in all carriers of the p.L428V variant. Functional studies showed a slightly impaired in vitro transcriptional activity of p.L428V. Interestingly' the expression of the human p.L428V thyroid hormone receptor beta in the zebrafish embryo background generated a phenotype consistent with RTHß. CONCLUSION: Variable results of TFTs on some immunoassays can be a cause of RTHß diagnostic delay, but the genotype-phenotype correlation in this family and functional studies support p.L428V as a novel THRB variant expanding the spectrum of gene variants causing RTHß. In vivo, rather than in vitro, functional assays may be required to demonstrate the dominant negative action of THRB variants.

From Endoderm to Progenitors: An Update on the Early Steps of Thyroid Morphogenesis in the Zebrafish.

The mechanisms underlying thyroid gland development have a central interest in biology and this review is aimed to provide an update on the recent advancements on the early steps of thyroid differentiation that were obtained in the zebrafish, because this teleost fish revealed to be a suitable organism to study the early developmental stages. Physiologically, the thyroid precursors fate is delineated by the appearance among the endoderm cells of the foregut of a restricted cell population expressing specific transcription factors, including pax2a, nkx2.4b, and hhex. The committed thyroid primordium first appears as a thickening of the pharyngeal floor of the anterior endoderm, that subsequently detaches from the floor and migrates to its final location where it gives rise to the thyroid hormone-producing follicles. At variance with mammalian models, thyroid precursor differentiation in zebrafish occurs early during the developmental process before the dislocation to the eutopic positioning of thyroid follicles. Several pathways have been implicated in these early events and nowadays there is evidence of a complex crosstalk between intrinsic (coming from the endoderm and thyroid precursors) and extrinsic factors (coming from surrounding tissues, as the cardiac mesoderm) whose organization in time and space is probably required for the proper thyroid development. In particular, Notch, Shh, Fgf, Bmp, and Wnt signaling seems to be required for the commitment of endodermal cells to a thyroid fate at specific developmental windows of zebrafish embryo. Here, we summarize the recent findings produced in the various zebrafish experimental models with the aim to define a comprehensive picture of such complicated puzzle.

Glis3 as a Critical Regulator of Thyroid Primordium Specification.

Background: GLIS3 (GLI-Similar protein 3) is a transcription factor involved in several cellular processes. Homozygous mutations in the GLIS3 gene have been typically associated with neonatal diabetes and congenital hypothyroidism (CH) in a syndrome called NDH. NDH patients present developmental abnormalities including endocrine pancreas defects and a spectrum of thyroid abnormalities, mainly including thyroid dysgenesis (TD). The mouse models revealed a key role of Glis3 in pancreatic islets but not in early thyroid development, as Glis3 was described to retain a role in regulating thyroid hormone synthesis downstream the thyrotropin (TSH)/TSHR signaling pathway and in postnatal follicle proliferation. Hence, in this study, we have been taking advantage of the zebrafish model to gain insights on the Glis3 activity during thyroid organogenesis. Methods: Transient glis3-knockdown zebrafish embryos (called glis3 morphants) were generated by the microinjection of specific glis3 morpholinos at one- to two-cell stage to analyze the thyroid phenotype in vivo. Several additional analyses (in situ hybridization, immunohistochemistry, and pharmacological treatments) were performed for further molecular characterization. Results: The analysis of thyroid embryonic development revealed that Glis3 is involved in early steps of thyroid specification. glis3 morphants exhibited a reduced expression of the early transcription factors nkx2.4 and pax2a at the thyroid primordium level, which is not caused by changes in proliferation or apoptosis of the pharyngeal endoderm. As a result, the differentiated thyroid tissue in morphants appeared reduced in size with decreased expression of tg and slc5a5, a low number of thyroxine (T4)-producing follicles, associated with an elevation of tshba (homologous of the human TSHß), thus resembling the clinical and biochemical manifestations of patients with TD. Interestingly, glis3 morphants have pancreatic ß-cell defects, but not liver defects. In vitro and in vivo data also demonstrated that Glis3 is an effector of the Sonic Hedgehog (SHH) pathway. Molecular and pharmacological inhibition of SHH reproduced the thyroid defects observed in glis3 morphant. Conclusions: Our results demonstrate that glis3, within the SHH pathway, appears to determine the number of endodermal cells committed to a thyroid fate. This is the first evidence of the involvement of Glis3 in TD, thereby expanding the understanding of the genetic basis of thyroid development and CH.

Thyroid Hormone Hyposensitivity: From Genotype to Phenotype and Back.

Thyroid hormone action defects (THADs) have been classically considered conditions of impaired sensitivity to thyroid hormone (TH). They were originally referring to alterations in TH receptor genes (THRA and THRB), but the discovery of genetic mutations and polymorphisms causing alterations in cell membrane transport (e.g., MCT8) and metabolism (e.g., SECISBP2, DIO2) led recently to a new and broader definition of TH hyposensitivity (THH), including not only THADs but all defects that could interfere with the activity of TH. Due to the different functions and tissue-specific expression of these genes, affected patients exhibit highly variable phenotypes. Some of them are characterized by a tissue hypothyroidism or well-recognizable alterations in the thyroid function tests (TFTs), whereas others display a combination of hypo- and hyperthyroid manifestations with normal or only subtle biochemical defects. The huge effort of basic research has greatly aided the comprehension of the molecular mechanisms underlying THADs, dissecting the morphological and functional alterations on target tissues, and defining the related-changes in the biochemical profile. In this review, we describe different pictures in which a specific alteration in the TFTs (TSH, T4, and T3 levels) is caused by defects in a specific gene. Altogether these findings can help clinicians to early recognize and diagnose THH and to perform a more precise genetic screening and therapeutic intervention. On the other hand, the identification of new genetic variants will allow the generation of cell-based and animal models to give novel insight into thyroid physiology and establish new therapeutic interventions.

GLIS3 and Thyroid: A Pleiotropic Candidate Gene for Congenital Hypothyroidism.

Variations in the transcription factor Gli-similar 3 (GLIS3) gene have been associated to variable congenital endocrine defects, including both morphogenetic and functional thyroid alterations. Evidence from Glis3 knockout mice indicates a relevant role for GLIS3 in thyroid hormone biosynthesis and postnatal thyroid gland growth, with a mechanism of action downstream of the TSH/TSHR interaction. However, the pathophysiological role of this transcription factor during the embryonic thyroid development remains unexplored. In this manuscript, we will provide an overview of the current knowledge on GLIS3 function during development. As a perspective, we will present preliminary evidence in the zebrafish model in support of a potential role for this pleiotropic transcription factor in the early stages of thyroid gland development.

Genetics and management of congenital hypothyroidism.

Several evidences support a relevant genetic origin for Congenital Hypothyroidism (CH), however familial forms are uncommon. CH can be due to morphogenetic or functional defects and several genes have been originally associated either with thyroid dysgenesis or dyshormonogenesis, with a highly variable expressivity and a frequently incomplete penetrance of the genetic defects. The phenotype-driven genetic analyses rarely yielded positive results in more than 10% of cases, thus raising doubts on the genetic origin of CH. However, more recent unsupervised approaches with systematic Next Generation Sequencing (NGS) analysis revealed the existence of hypomorphic alleles of these candidate genes whose combination can explain a significant portion of CH cases. The co-segregation studies of the hypothyroid phenotype with multiple gene variants in pedigrees confirmed the potential oligogenic origin of CH, which finally represents a suitable explanation for the frequent sporadic occurrence of this disease.

In vivo Functional Consequences of Human THRA Variants Expressed in the Zebrafish.

BACKGROUND: Heterozygous mutations in the thyroid hormone receptor alpha (THRA) gene cause resistance to thyroid hormone alpha (RTHα), a disease characterized by variable manifestations reminiscent of untreated congenital hypothyroidism but a raised triiodothyronine/thyroxine ratio and normal thyrotropin levels. It was recently described that zebrafish embryos expressing a dominant negative (DN) form of thraa recapitulate the key features of RTHα, and that zebrafish and human receptors are functionally interchangeable. METHODS: This study expressed several human thyroid hormone receptor alpha (hTRα) variants in zebrafish embryos and analyzed the resulting phenotypes. RESULTS: All hTRα-injected embryos showed variable defects, including cerebral and cardiac edema likely caused by an aberrant looping during heart development, anemia, and an incomplete formation of the vascular network. Moreover, the hTRα-injected embryos presented severe defects of motorneurons and craniofacial development, thus affecting their autonomous feeding and swimming behaviors. Surprisingly, expression of all hTRα mutants had no detectable effect on thyrotropin beta and thyrotropin-releasing hormone transcripts, indicating that their DN action is limited on the thyroid hormone reception beta 2 targets at the hypothalamic/pituitary level in vivo. As previously described in vitro, treatment with high triiodothyronine doses can efficiently revert the observed defects only in embryos injected with missense hTRα variants. CONCLUSION: Injection of human THRA variants in zebrafish embryos causes tissue-specific defects recapitulating most of the RTHα clinical and biochemical manifestations. The described manipulation of zebrafish embryos represents a novel in vivo model to screen the functional consequences of THRA variants and the rescue potential of new therapeutic compounds.