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.

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.