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Background: Congenital hypothyroidism screening traditionally relies on detecting elevated thyroid-stimulating hormone levels, yet this approach may not detect a specific type of congenital hypothyroidism caused by iodotyrosine dehalogenase-1 (Dehal1) deficiency. The deficiency of this enzyme prevents the deiodination of mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) in the process of iodine recycling. This underscores the potential use of iodotyrosine or its metabolites as non-invasive urinary biomarkers for early diagnosis of congenital hypothyroidism. However, the urinary metabolites of MIT/DIT have not yet been discovered. Thus, this study aimed to identify the urinary metabolites of iodotyrosine in experimental models., Method: Gas chromatography mass spectrometry was used to identify the urinary metabolites of iodotyrosine following intraperitoneal injection of MIT in rats. An isotope dilution mass spectrometric assay was developed for assessment of identified metabolites. Urine samples from Dehal1 knockout mice were used to confirm the results., Results: We identified novel iodotyrosine metabolites, 3-iodo-4-hydroxyphenylacetic acid (IHPA), and 3,5-diiodo-4-hydroxyphenylacetic acid (Di-IHPA) as the primary urinary metabolites of MIT and DIT respectively. The concentrations of urinary IHPA and Di-IHPA were significantly higher in Dehal1 knockout mice., Conclusion: Our findings suggest that IHPA is detected in larger quantities and may hold more clinical significance than previously identified biomarkers like MIT and DIT, making it a promising candidate for diagnosing congenital hypothyroidism or other conditions associated with iodine recycling inhibition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Many cellular processes are governed by protein-protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein-protein interactions with time resolution at an organelle-wide scale, we developed a quantitative mass spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology, such as Hsp70-/90-assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein-folding diseases and cellular processes., (© 2024. The Author(s).)

Thyroid hormones are crucial for brain development and their deficiency during fetal and postnatal periods can lead to mood and cognitive disorders. We aimed to examine the consequences of thyroid hormone deficiency on anxiety-related behaviors and protein expression of hippocampal glutamate transporters in congenital hypothyroid male offspring rats. Possible beneficial effects of treadmill exercise have also been examined. Congenital hypothyroidism was induced by adding propylthiouracil (PTU) to drinking water of pregnant Wistar rats from gestational day 6 until the end of the weaning period (postnatal day 28). Next, following 4 weeks of treadmill exercise (5 days per week), anxiety-related behaviors were examined using elevated plus maze (EPM) and light/dark box tests. Thereafter, protein expression of astrocytic (GLAST and GLT-1) and neuronal (EAAC1) glutamate transporters were measured in the hippocampus by immunoblotting. Hypothyroid rats showed decreased anxiety-like behavior, as measured by longer time spent in the open arms of the EPM and in the light area of the light/dark box, compared to control rats. Hypothyroid rats had significantly higher GLAST and GLT-1 and lower EAAC1 protein levels in the hippocampus than did the euthyroid rats. Following exercise, anxiety levels decreased in the euthyroid group while protein expression of EAAC1 increased and returned to normal levels in the hypothyroid group. Our findings indicate that thyroid hormone deficiency was associated with alterations in protein expression of glutamate transporters in the hippocampus. Up-regulation of hippocampal GLAST and GLT-1 could be at least one of the mechanisms associated with the anxiolytic effects of congenital hypothyroidism., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) ( p  < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

Thyroid hormones (THs) regulate gene expression by binding to nuclear TH receptors (TRs) in the cell. THs are indispensable for brain development. However, we have little knowledge about how congenital hypothyroidism in neurons affects functions of the central nervous system in adulthood. Here, we report specific TH effects on functional development of the cerebellum by using transgenic mice overexpressing a dominant-negative TR (Mf-1) specifically in cerebellar Purkinje cells (PCs). Adult Mf-1 mice displayed impairments in motor coordination and motor learning. Surprisingly, long-term depression (LTD)-inductive stimulation caused long-term potentiation (LTP) at parallel fiber (PF)-PC synapses in adult Mf-1 mice, although there was no abnormality in morphology or basal properties of PF-PC synapses. The LTP phenotype was turned to LTD in Mf-1 mice when the inductive stimulation was applied in an extracellular high-Ca 2+ condition. Confocal calcium imaging revealed that dendritic Ca 2+ elevation evoked by LTD-inductive stimulation is significantly reduced in Mf-1 PCs but not by PC depolarization only. Single PC messenger RNA quantitative analysis showed reduced expression of SERCA2 and IP 3 receptor type 1 in Mf-1 PCs, which are essential for mGluR1-mediated internal calcium release from endoplasmic reticulum in cerebellar PCs. These abnormal changes were not observed in adult-onset PC-specific TH deficiency mice created by adeno-associated virus vectors. Thus, we propose the importance of TH action during neural development in establishing proper cerebellar function in adulthood, independent of its morphology. The present study gives insight into the cellular and molecular mechanisms underlying congenital hypothyroidism-induced dysfunctions of central nervous system and cerebellum.

Thyroid hormones are critical for the development of opsins involved in color vision. Hypothyroid mice show delayed M-opsin development and expanded distribution of S-opsin on the retina. However, the effects of maternal hypothyroidism on opsin development remain unknown. This study investigates the effects of congenital central hypothyroidism and maternal hypothyroidism on opsin development in thyrotropin-releasing hormone knockout (TRH-/-) mice. We examined the mRNA expression and protein distribution of S/M-opsin on postnatal days (P)12 and 17, as well as mRNA expression of type 2 and 3 iodothyronine deiodinase (DIO2 and DIO3, respectively) in the retina and type 1 iodothyronine deiodinase (DIO1) in the liver at P12 in TRH+/- mice born to TRH+/- or TRH-/- dams, and conducted S/M-opsin analysis in TRH+/+ or TRH-/- mice born to TRH+/- dams at P12, P17, and P30. M-opsin expression was lower in TRH+/- mice born to TRH-/- dams than in those born to TRH+/- dams, whereas S-opsin expression did not significantly differ between them. DIO1, DIO2, and DIO3 mRNA expression levels were not significantly different between the two groups; therefore, thyroid function in peripheral tissues in the pups was similar. S/M-opsin expression did not significantly differ between the TRH+/+ and TRH-/- mice born to TRH+/- dams on any postnatal day. These results demonstrate that maternal hypothyroidism causes M-opsin developmental delay during the early developmental stages of neonatal mice, and TRH-/- mice, a model of congenital central hypothyroidism, born to a euthyroid dam do not have delayed opsin development.

Background: The sodium/iodide symporter (NIS) mediates active iodide accumulation in the thyroid follicular cell. Autosomal recessive iodide transport defect (ITD)-causing loss-of-function NIS variants lead to dyshormonogenic congenital hypothyroidism due to deficient iodide accumulation for thyroid hormonogenesis. Here, we aimed to identify, and if so to functionally characterize, novel ITD-causing NIS pathogenic variants in a patient diagnosed with severe dyshormonogenic congenital hypothyroidism due to a defect in iodide accumulation in the thyroid follicular cell, as suggested by nondetectable radioiodide accumulation in a normally located thyroid gland, as well as in salivary glands. Methods: The proposita NIS-coding SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of the novel NIS variants were performed. Results: Sanger sequencing revealed novel compound heterozygous SLC5A5 gene variants (c.970-3C>A and c.1106A>T, p.D369V). In silico analysis suggested that c.970-3C>A disrupts the canonical splice acceptor site located in intron 7. Splicing minigene reporter assay revealed that c.970-3C>A causes exon 8 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variant p.Y324H fs *148. Moreover, in silico analysis indicated p.D369V as pathogenic. Functional in vitro studies demonstrated that p.D369V NIS does not mediate iodide accumulation, as p.D369V causes NIS to be retained in the endoplasmic reticulum. Mechanistically, we propose an intramolecular ionic interaction involving the β carboxyl group of D369 and the guanidinium group of R130, located in transmembrane segment 4. Of note, an Asp residue at position 369-which is highly conserved in SLC5A family members-is required for functional NIS expression at the plasma membrane. Conclusions: We uncovered a critical intramolecular interaction between R130 and D369 required for NIS maturation and plasma membrane expression. Moreover, we identified the first intronic variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape in the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma., (© 2021 Federation of American Societies for Experimental Biology.)

Differentiated thyroid carcinoma (DTC) combined with congenital hypothyroidism (CH) is a rare situation, and there is no well-established causal relationship. CH is a common congenital endocrine, while DTC occurring in childhood represents 0.4-3% of all malignancies at this stage of life. The association of CH with DTC could be related to dyshormonogenetic goiter (DHG) or developmental abnormalities. This review will explore the clinical features and the molecular mechanisms potentially associated with the appearance of DTC in CH: sporadic somatic driver mutations, chronic increase of thyroid-stimulating hormone (TSH) levels, higher concentrations of hydrogen peroxide (H2O2), cell division cycle associated 8 (Borelain/CDC8) gene mutations, and in others genes associated with CH - either alone or associated with the mechanisms involved in dyshormonogenesis. There are some pitfalls in the diagnosis of thyroid cancer in patients with CH with nodular goiter, as the proper cytological diagnosis of nodules of patients with dyshormonogenesis might be demanding due to the specific architectural and cytological appearance, which may lead to an erroneous interpretation of malignancy. The purpose of this article is to suggest an analytical framework that embraces the fundamental relationships between the various aspects of CH and CDT. In face of this scenario, the entire genetic and epigenetic context, the complex functioning, and cross talk of cell signaling may determine cellular mechanisms promoting both the maintenance of the differentiated state of the thyroid follicular cell and the disruption of its homeostasis leading to cancer. Whereas, the exact mechanisms for thyroid cancer development in CH remain to be elucidated., (Society for Endocrinology 2021.)

Background: Loss-of-function mutations of thyrotropin receptor (TSHR) are one of the main causes of congenital hypothyroidism. As for many disease-associated G-protein coupled receptors (GPCRs), these mutations often affect the correct trafficking and maturation of the receptor, thus impairing the expression on the cell surface. Several retained GPCR mutants are able to effectively bind their ligands and to transduce signals when they are forced to the cell surface by degradation inhibition or by treatment with chaperones. Despite the large number of well-characterized retained TSHR mutants, no attempts have been made for rescue. Further, little is known about TSHR degradation pathways. We hypothesize that, similar to other GPCRs, TSHR retained mutants may be at least partially functional if their maturation and membrane expression is facilitated by chaperones or degradation inhibitors. Methods: We performed in silico predictions of the functionality of known TSHR variants and compared the results with available in vitro data. Western blot, confocal microscopy, enzyme-linked immunosorbent assays, and dual luciferase assays were used to investigate the effects of degradation pathways inhibition and of chemical chaperone treatments on TSHR variants' maturation and functionality. Results: We found a high discordance rate between in silico predictions and in vitro data for retained TSHR variants, a fact indicative of a conserved potential to initiate signal transduction if these mutants were expressed on the cell surface. We show experimentally that some maturation defective TSHR mutants are able to effectively transduce Gs/cAMP signaling if their maturation and expression are enhanced by using chemical chaperones. Further, through the characterization of the intracellular retained p.N432D variant, we provide new insights on the TSHR degradation mechanism, as our results suggest that aggregation-prone mutant can be directed toward the autophagosomal pathway instead of the canonical proteasome system. Conclusions: Our study reveals alternative pathways for TSHR degradation. Retained TSHR variants can be functional when expressed on the cell surface membrane, thus opening the possibility of further studies on the pharmacological modulation of TSHR expression and functionality in patients in whom TSHR signaling is disrupted.

Complete absence of thyroid hormone is incompatible with life in vertebrates. Thyroxine is synthesized within thyroid follicles upon iodination of thyroglobulin conveyed from the endoplasmic reticulum (ER), via the Golgi complex, to the extracellular follicular lumen. In congenital hypothyroidism from biallelic thyroglobulin mutation, thyroglobulin is misfolded and cannot advance from the ER, eliminating its secretion and triggering ER stress. Nevertheless, untreated patients somehow continue to synthesize sufficient thyroxine to yield measurable serum levels that sustain life. Here, we demonstrate that TGW2346R/W2346R humans, TGcog/cog mice, and TGrdw/rdw rats exhibited no detectable ER export of thyroglobulin, accompanied by severe thyroidal ER stress and thyroid cell death. Nevertheless, thyroxine was synthesized, and brief treatment of TGrdw/rdw rats with antithyroid drug was lethal to the animals. When untreated, remarkably, thyroxine was synthesized on the mutant thyroglobulin protein, delivered via dead thyrocytes that decompose within the follicle lumen, where they were iodinated and cannibalized by surrounding live thyrocytes. As the animals continued to grow goiters, circulating thyroxine increased. However, when TGrdw/rdw rats age, they cannot sustain goiter growth that provided the dying cells needed for ongoing thyroxine synthesis, resulting in profound hypothyroidism. These results establish a disease mechanism wherein dead thyrocytes support organismal survival.

Background: The fetal hypothalamic-pituitary-adrenal (HPA) axis plays a key role in the control of parturition and maturation of organ systems in preparation for birth. In hypothyroid fetuses, gestational length may be prolonged and maturational processes delayed. The extent to which the effects of thyroid hormone deficiency in utero on the timing of fetal maturation and parturition are mediated by changes to the structure and function of the fetal HPA axis is unknown. Methods: In twin sheep pregnancies where one fetus was thyroidectomized and the other sham-operated, this study investigated the effect of hypothyroidism on circulating concentrations of adrenocorticotrophic hormone (ACTH) and cortisol, and the structure and secretory capacity of the anterior pituitary and adrenal glands. The relative population of pituitary corticotrophs and the masses of the adrenal zones were assessed by immunohistochemical and stereological techniques. Adrenal mRNA abundances of key steroidogenic enzymes and growth factors were examined by quantitative polymerase chain reaction. Results: Hypothyroidism in utero reduced plasma concentrations of ACTH and cortisol. In thyroid-deficient fetuses, the mass of corticotrophs in the anterior pituitary gland was unexpectedly increased, while the mass of the zona fasciculata and its proportion of the adrenal gland were decreased. These structural changes were associated with lower adrenocortical mRNA abundances of insulin-like growth factor (IGF)-I and its receptor, and key steroidogenic enzymes responsible for glucocorticoid synthesis. The relative mass of the adrenal medulla and its proportion of the adrenal gland were increased by thyroid hormone deficiency in utero , without any change in expression of phenylethanolamine N -methyltransferase or the IGF system. Conclusions: Thyroid hormones are important regulators of the structure and secretory capacity of the pituitary-adrenal axis before birth. In hypothyroid fetuses, low plasma cortisol may be due to impaired adrenocortical growth and steroidogenic enzyme expression, secondary to low circulating ACTH concentration. Greater corticotroph population in the anterior pituitary gland of the hypothyroid fetus indicates compensatory cell proliferation and that there may be abnormal corticotroph capacity for ACTH synthesis and/or impaired hypothalamic input. Suppression of the development of the fetal HPA axis by thyroid hormone deficiency may contribute to the delay in fetal maturation and delivery observed in hypothyroid offspring.

The transcription factor GLIS3 is an important factor in hormone biosynthesis and thyroid development, and mutations in GLIS3 are relatively rare. Deletions of more than one of the 11 exons of GLIS3 occur in most patients with various extrathyroidal abnormalities and congenital hypothyroidism (CH), and only 18 missense variants of GLIS3 related to thyroid disease have been reported. The aim of this study was to report the family history and molecular basis of patients with CH who carry GLIS3 variants. Three hundred and fifty-three non-consanguineous infants with CH were recruited and subjected to targeted exome sequencing of CH-related genes. The transcriptional activity and cellular localization of the variants in GLIS3 were investigated in vitro. We identified 20 heterozygous GLIS3 exonic missense variants, including eight novel sites, in 19 patients with CH. One patient carried compound heterozygous GLIS3 variants (p.His34Arg and p.Pro835Leu). None of the variants affected the nuclear localization. However, three variants (p.His34Arg, p.Pro835Leu, and p.Ser893Phe) located in the N-terminal and C-terminal regions of the GLIS3 protein downregulated the transcriptional activation of several genes required for thyroid hormone (TH) biosynthesis. This study of patients with CH extends the current knowledge surrounding the spectrum of GLIS3 variants and the mechanisms by which they cause TH biosynthesis defects., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The contribution of PAX8 genetic variants to congenital hypothyroidism (CH) is not well understood. We aimed to study the genetic variability of exons 3 and 5 of PAX8 gene among a cohort of children with congenital hypothyroidism in correspondence to their clinical aspect. Blood samples were collected from 117 children (63 girls and 54 boys) with CH and enrolled as cases (Group I). All cases underwent biochemical confirmation with low FT4 and high TSH levels and thyroid gland imaging, along with equal number of matched apparently healthy individuals who served as controls (Group II). Genomic materials for exons 3 and 5 of PAX8 gene were extracted, amplified by PCR, detected by electrophoresis, purified, and sequenced by the Sanger technique through the application of ABI 3730x1 DNA Sequencer. Out of 117 cases, eight different effective PAX8 mutations were detected in exon 3 (G23D, V35I, I34T, Q40P, p.R31C, p.R31H, p.R31A, and p.I47T) in 14 patients with their sonographic findings ranged from normal, hypoplastic to thyroid agenesis. Besides the reported mutations, one novel mutation; R31A was detected in 1 euotopic case. Exon 5 analysis revealed no detected mutations elsewhere. In contrast, all healthy control children showed no mutation and normal sonographic findings. Mutations in exon 3 of PAX8 gene, implies its important role in thyroid development and function, as a first estimate of PA8 mutation rate in Egyptian patients with CH having normal and dysgenetic gland. Using ultrasound is mandatory for diagnosis and guiding the treatment of children with CH., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Background: Congenital hypothyroidism due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the majority of cases. This situation reflects, in part, our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification. Methods: We treated zebrafish embryos during different developmental periods with small-molecule compounds known to manipulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. We used the antisense morpholino (MO) technique to create a zebrafish acardiac model. For thyroid rescue experiments, bone morphogenetic protein (BMP) pathway induction in zebrafish embryos was obtained by manipulation of heat-shock inducible transgenic lines. Results: Combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of MO-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development. Conclusion: Collectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.

Context: Inactivating variants of thyrotropin (thyroid-stimulating hormone; TSH) receptor (TSHR) cause congenital hypothyroidism. More than 60 such variants have been reported so far, most of which were located in the extracellular or transmembrane domain., Objective: We report the identification and characterization of a frameshift TSHR variant in the intracytoplasmic C-tail region., Methods: Sequencing of TSHR was performed in a patient with congenital hypothyroidism. The functionality of the identified variants was assessed by expressing TSHR in HEK293 cells and measuring TSH-dependent activation of the cAMP-response element-luciferase reporter. A series of systematic mutagenesis experiments were performed to characterize the frameshifted amino acid sequence., Results: The proband was heterozygous for a known TSHR variant (p.Arg519His) and a novel frameshift TSHR variant (p.Val711Phefs*18), which removed 54 C-terminal residues and added a 17-amino acid frameshifted sequence. The loss of function of Val711Phefs*18-TSHR was confirmed in vitro, but the function of Val711*-TSHR was found to be normal. Western blotting showed the low protein expression of Val711Phefs*18-TSHR. Fusion of the frameshift sequence to green fluorescent protein or luciferase induced inactivation of them, indicating that the sequence acted as a degron. A systematic mutagenesis study revealed that the density of hydrophobic residues in the frameshift sequence determined the stability. Eight additional frameshift TSHR variants that covered all possible shifted frames in C-tail were created, and another frameshift variant (Thr748Profs*27) with similar effect was found., Conclusions: We characterized a naturally occurring frameshift TSHR variant located in C-tail, and provided a unique evidence that hydrophobicity in the C-terminal region of the receptor affects protein stability., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To analyze the genetic causes of congenital hypothyroidism through the targeted exome sequencing of pediatric patients with congenital hypothyroidism with thyroid gland in situ. METHOD: The study population included 20 patients diagnosed with congenital hypothyroidism with thyroid gland in situ at the Pediatric Endocrinology Clinic of Pusan National University Hospital. Targeted exome sequencing was performed on eight causative genes, including thyroid stimulating hormone receptor ( TSHR ), mutation in which can cause hypothyroidism with a small or normal sized thyroid gland, and thyroglobulin ( TG ), thyroid peroxidase ( TPO ), dual oxidase 2 ( DUOX2 ), dual oxidase maturation factor 2 ( DUOXA2 ), iodotyrosine deiodinase ( IYD ), solute carrier family 26 member 4 (SLC26A4), and solute carrier family 5 member 5 ( SLC5A5 ), mutations in which are known to cause thyroid dyshormonogenesis., Results: Permanent, subclinical, and transient hypothyroidism were diagnosed in 15 (75%), three (15%), and two (10%) patients, respectively. Genetic mutations were identified in 16 patients (80% positivity rate). Targeted exome sequencing of eight genes identified 24 variants in these patients: 11 DUOX2 variants in eight patients; six TSHR variants in five patients; five TG variants in three patients; and two DUOXA2 variants in two patients. Of these 24 variants, 10 (41.6%) were novel. No variants were identified in TPO, IYD, SLC5A5 , or SLC26A4 . Two patients displayed triallelic (digenic) mutations (in TG and TSHR in one patient and DUOX2 and TSHR in the other). No variants were identified in three patients with permanent hypothyroidism and one patient with transient hypothyroidism. Genetic variations that could explain the congenital hypothyroidism phenotypes were identified in 12/15 cases (80%)., Conclusions: Targeted exome sequencing identified the genetic causes of congenital hypothyroidism with thyroid gland in situ in 80% of the patients studied, with DUOX2 and TSHR mutations being the most common. As many of the identified variants were novel, additional studies on the genetic causes of congenital hypothyroidism are warranted., (Copyright © 2021 by the Association of Clinical Scientists, Inc.)

Background and Objective: The effects of hypothyroidism during pregnancy and lactation on carbohydrate metabolism have been mostly studied in male animals. The aim of this study is, therefore, to investigate the effect of fetal and neonatal hypothyroidism (FH and NH) on glucose tolerance in middle-aged female rat offsprings., Methods: Pregnant female rats were divided into three groups: Rats in the control group consumed tap water, while those in the FH and NH groups consumed 250 mg/L of 6-propyl-2-thiouracil (PTU) in their drinking water during gestation or lactation periods, respectively. After weaning, the female offspring were separated and divided into 3 groups (n=8/group): Control, FH, and NH. Bodyweight was recorded monthly and an intravenous glucose tolerance test (IVGTT) was performed at month 12., Results: Compared to controls, female rats in the FH group had significantly higher plasma glucose levels than controls throughout the IVGTT except at min 60. Values at min 5 of the FH and control group were 196.1±1.9 and 155.3±5.9 mg/dL, respectively (P<0.05). In the NH group, plasma glucose levels were significantly higher only at min 5 (185.7±14.1 vs. 155.3±5.9 mg/dL, P<0.05)., Conclusion: Hypothyroidism during fetal or neonatal periods caused glucose intolerance in middle- aged female offspring rats., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for triiodothyronine and thyroxine hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism. Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification-mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several congenital hypothyroidism variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and engagement by targeting factors for endoplasmic reticulum-associated degradation. Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for 1 Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis., Competing Interests: Conflict of interest Authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Thyroid hormone deficiency during crucial stages of development causes congenital hypothyroidism. This syndrome alters hypothalamic pathways involved in long-term bodyweight regulation as ObRb-STAT3 leptin signaling pathway, which is associated with metabolic syndrome. This study aimed to determine if thyroxine treatment during pregnancy and lactation in hypothyroid mothers avoids, in the congenital hypothyroid offspring, the alterations in metabolic programming related to metabolic syndrome and the ObRb-STAT3 leptin signaling pathway in hypothalamus. Twenty-four virgin female Wistar rats were divided into euthyroid, hypothyroid, and hypothyroid with thyroxine treatment (20 μg/kg/day T 4 since pregnancy until lactation). The bodyweight and energy intake, insulin resistance, glucose tolerance, metabolic and hormonal parameters were determined in offspring at 28 weeks after birth. Then, the rats were euthanized to obtain adipose tissue reserves and hypothalamus to measure the expression of ObRb, STAT3, pSTAT3, and SOCS3. Congenital hypothyroidism presented metabolic syndrome such as insulin resistance, glucose tolerance, dyslipidemias, an increase in cardiovascular risk (Castelli I males:166.67%, females: 173.56%; Castelli II males: 375.51%, females: 546.67%), and hypothalamic leptin resistance (SOCS3, Males: 10.96%, females: 25.85%). Meanwhile, the thyroxine treatment in the mothers during pregnancy and lactation prevents the metabolic disturbance. In conclusion, thyroxine treatment during the critical perinatal stage for metabolic programming prevents congenital hypothyroidism-caused metabolic syndrome and hypothalamic leptin resistance., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Increased intestinal permeability (IIP) precedes several autoimmune disorders. Although Hashimoto’s thyroiditis (HT) is the most common autoimmune disorder, the role of IIP in its pathogenesis had received little attention. Zonulin plays a critical role in IIP by modulating intracellular tight junctions. Rise of serum zonulin levels were shown to indicate IIP in human subjects. In this case-control study, we examined the hypothesis that patients with HT have IIP. We studied 30 children and adolescents with HT, and 30 patients with congenital hypothyroidism (CH) matched for age, gender and body mass index (BMI). Serum zonulin levels, free thyroxine (fT4), thyroid stimulating hormone (TSH), anti-thyroglobulin antibody and anti-thyroid peroxidase antibody were measured. Zonulin levels were significantly higher in patients with HT than patients with CH (59.1±22.9 ng/mL vs. 43.3±32.9 ng/mL, p=0.035). In patients with HT, zonulin levels were positively correlated with weight (r=0.406, p=0.03), BMI (r=0.486, p=0.006) and levothyroxine dose (r=0.463, p=0.02). In patients with CH, zonulin levels were positively correlated with age (r=0.475, p=0.008), weight (r=0.707, p<0.001), BMI (r=0.872, p<0.001) and levothyroxine dose (r=0.485, p=0.007). After adjusting for age, weight, TSH and fT4 levels, serum zonulin was only associated with levothyroxine dose in patients with HT (R 2 =0.36, p=0.05). In patients with CH, only weight was associated with zonulin levels (R 2 =0.62, p<0.001). In conclusion, higher zonulin levels in children and adolescents with HT suggested IIP in these patients. Additionally, the association between zonulin levels and levothyroxine dose might imply a relationship between serum zonulin and disease severity.

Introduction: The objective of this study is to investigate the role of thyroid hormone (TH) in the pathogenesis of intestinal dysganglionosis (ID)., Methods: A zebrafish model of congenital hypothyroidism (CH) was created by exposing the larvae to the 6-propyl-2-thiouracil (PTU). The enteric neurons were labeled with anti-HuC/D antibodies. The number of enteric neurons was counted. The larval intestine was dissociated and stained with anti-p75 and anti-α4 integrin antibodies. Mitosis and apoptosis of the p75 + α4 integrin + enteric neural crest cells (ENCCs) were studied using flow cytometry. Intestinal motility was studied by analyzing the transit of fluorescent tracers., Results: PTU (25 mg/L) significantly reduced TH production at 6- and 9-days post fertilization without changing the body length, body weight, and intestinal length of the larvae. Furthermore, PTU inhibited mitosis of ENCCs and reduced the number of enteric neurons throughout the larval zebrafish intestine. Importantly, PTU inhibited intestinal transit of fluorescent tracers. Finally, thyroxine supplementation restored ENCC mitosis, increased the number of enteric neurons, and recovered intestinal motility in the PTU-treated larvae., Conclusions: PTU inhibited TH production, reduced the number of enteric neurons, impaired intestinal motility, and impeded ENCC mitosis in zebrafish, suggesting a possible role of CH in the pathogenesis of ID.

Background: Development of adipose tissue before birth is essential for energy storage and thermoregulation in the neonate and for cardiometabolic health in later life. Thyroid hormones are important regulators of growth and maturation in fetal tissues. Offspring hypothyroid in utero are poorly adapted to regulate body temperature at birth and are at risk of becoming obese and insulin resistant in childhood. The mechanisms by which thyroid hormones regulate the growth and development of adipose tissue in the fetus, however, are unclear. Methods: This study examined the structure, transcriptome, and protein expression of perirenal adipose tissue (PAT) in a fetal sheep model of thyroid hormone deficiency during late gestation. Proportions of unilocular (UL) (white) and multilocular (ML) (brown) adipocytes, and UL adipocyte size, were assessed by histological and stereological techniques. Changes to the adipose transcriptome were investigated by RNA sequencing and bioinformatic analysis, and proteins of interest were quantified by Western blotting. Results: Hypothyroidism in utero resulted in elevated plasma insulin and leptin concentrations and overgrowth of PAT in the fetus, specifically due to hyperplasia and hypertrophy of UL adipocytes with no change in ML adipocyte mass. RNA sequencing and genomic analyses showed that thyroid deficiency affected 34% of the genes identified in fetal adipose tissue. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathways were associated with adipogenic, metabolic, and thermoregulatory processes, insulin resistance, and a range of endocrine and adipocytokine signaling pathways. Adipose protein levels of signaling molecules, including phosphorylated S6-kinase (pS6K), glucose transporter isoform 4 (GLUT4), and peroxisome proliferator-activated receptor γ (PPARγ), were increased by fetal hypothyroidism. Fetal thyroid deficiency decreased uncoupling protein 1 (UCP1) protein and mRNA content, and UCP1 thermogenic capacity without any change in ML adipocyte mass. Conclusions: Growth and development of adipose tissue before birth is sensitive to thyroid hormone status in utero . Changes to the adipose transcriptome and phenotype observed in the hypothyroid fetus may have consequences for neonatal survival and the risk of obesity and metabolic dysfunction in later life.

A 4-day-old female neonate with raised cord blood thyroid-stimulating hormone (127 μIU/mL) underwent Tc thyroid scan to rule out thyroid dysgenesis. The images revealed midline focus of lingual thyroid as the only functioning thyroid tissue. In addition, bilateral focal and symmetrical breast uptake was seen without clinically palpable breast nodule on either side. Transplacental transfer of maternal hormones leading to stimulation of neonatal breasts explains this unusual scan finding. One should be aware of this rare pattern of focal breast uptake in Tc-pertechnetate scan in neonates with congenital hypothyroidism to avoid scan misinterpretation.

Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and one of the most common preventable causes of both cognitive and motor deficits. CH is a heterogeneous group of thyroid disorders in which inadequate production of thyroid hormone occurs due to defects in proteins involved in the gland organogenesis (dysembryogenesis) or in multiple steps of thyroid hormone biosynthesis (dyshormonogenesis). Dysembryogenesis is associated with genes responsible for the development or growth of thyroid cells: such as NKX2-1, FOXE1, PAX8, NKX2-5, TSHR, TBX1, CDCA8, HOXD3 and HOXB3 resulting in agenesis, hypoplasia or ectopia of thyroid gland. Nevertheless, the etiology of the dysembryogenesis remains unknown for most cases. In contrast, the majority of patients with dyshormonogenesis has been linked to mutations in the SLC5A5, SLC26A4, SLC26A7, TPO, DUOX1, DUOX2, DUOXA1, DUOXA2, IYD or TG genes, which usually originate goiter. About 800 genetic mutations have been reported to cause CH in patients so far, including missense, nonsense, in-frame deletion and splice-site variations. Many of these mutations are implicated in specific domains, cysteine residues or glycosylation sites, affecting the maturation of nascent proteins that go through the secretory pathway. Consequently, misfolded proteins are permanently entrapped in the endoplasmic reticulum (ER) and are translocated to the cytosol for proteasomal degradation by the ER-associated degradation (ERAD) machinery. Despite of all these remarkable advances in the field of the CH pathogenesis, several points on the development of this disease remain to be elucidated. The continuous study of thyroid gene mutations with the application of new technologies will be useful for the understanding of the intrinsic mechanisms related to CH. In this review we summarize the present status of knowledge on the disorders in the protein folding caused by thyroid genes mutations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Thyroid hormones are essential during infancy and childhood for growth and brain development. The formation and maturation of the newborn's hypothalamic-pituitary-thyroid axis begin in utero with fetal dependence on maternal thyroid hormones early in the pregnancy. As the fetal thyroid gland begins to produce thyroid hormones in the second trimester, the reliance decreases and remains at lower levels until birth. After birth, the detachment from the placenta and the change in thermal environment lead to a rapid increase in circulating thyroid-stimulating hormone in the neonate within hours, resulting in subsequent increases in thyroxine and triiodothyronine concentrations. Preterm infants may have lower thyroxine concentrations because of an immature hypothalamic-pituitary-thyroid axis at the time of birth and premature discontinuation of transference of maternal thyroid hormones. Similarly, infants with critical illness unrelated to the thyroid gland may have lower thyroxine levels. Infants born to mothers with Graves' disease are at risk for hypothyroidism and hyperthyroidism, which is related to the placental transfer of maternal autoantibodies, as well as antithyroid medications. An understanding of the normal embryology and physiology of the fetal and neonatal thyroid will help in evaluating a newborn for thyroid disorders., (Copyright © 2020 by the American Academy of Pediatrics.)

1) Background: Central congenital hypothyroidism (CCH) is a rare endocrine disorder that can be caused by mutations in the β-subunit of thyrotropin ( TSH B ). The TSHB mutation C105Vfs114X leads to isolated thyroid-stimulating-hormone-(TSH)-deficiency and results in a severe phenotype. The aim of this study was to gain more insight into the underlying molecular mechanism and the functional effects of this mutation based on two assumptions: a) the three-dimensional (3D) structure of TSH should be modified with the C105V substitution, and/or b) whether the C-terminal modifications lead to signaling differences. 2) Methods: wild-type (WT) and different mutants of hTSH were generated in human embryonic kidney 293 cells (HEK293 cells) and TSH preparations were used to stimulate thyrotropin receptor (TSHR) stably transfected into follicular thyroid cancer cells (FTC133-TSHR cells) and transiently transfected into HEK293 cells. Functional characterization was performed by determination of Gs, mitogen activated protein kinase (MAPK) and Gq/11 activation. 3) Results: The patient mutation C105Vfs114X and further designed TSH mutants diminished cyclic adenosine monophosphate (cAMP) signaling activity. Surprisingly, MAPK signaling for all mutants was comparable to WT, while none of the mutants induced PLC activation. 4) Conclusion: We characterized the patient mutation C105Vfs114X concerning different signaling pathways. We identified a strong decrease of cAMP signaling induction and speculate that this could, in combination with diverse signaling regarding the other pathways, accounting for the patient's severe phenotype., Competing Interests: The authors declare no conflict of interest.

Objective: Determine the milk quality effect during lactation on the metabolic and thyroid programming of hypothyroid offspring., Materials and Methods: Ten-week-old female Wistar rats were divided into two groups: euthyroid and thyroidectomy-caused hypothyroidism. The rats were matted and, one day after birth, the pups were divided into three groups: euthyroid offspring (EO), hypothyroid offspring (HO) and hypothyroid with a euthyroid replacement wet nurse (HRO). During lactation, the milk quality and offspring body length were evaluated. The body weight and energy intake were determined on a weekly basis, as well as the metabolic profile at the prepubertal (P35-36) and postpubertal (P55-56) ages. At P56, the animals were sacrificed, the adipose tissues were weighed and the thyroid glands were dissected for histological processing., Results: The milk of the hypothyroid wet nurse decreases proteins (16-26%), lipids (22-29%) and lactate (22-37%) with respect to euthyroid. The HO has a lower body weight gain (23-33%), length (11-13%) and energy intake (15-21%). In addition, HO presents impaired fasting glucose and dyslipidemia, as well as a reduction in seric thyroid hormone (18-34%), adipose reserves (26-68%) and thyroid gland weight (25-34%). The HO present thyroid gland cytoarchitecture alteration. The HRO develop the same metabolic alterations as the HO. However, the thyroid gland dysfunction was partially prevented because the HRO improved under about 10% of the serum thyroid hormone concentration, the thyroid gland weight although histological glandular changes presented., Conclusions: The replacement of hypothyroid offspring with a euthyroid wet nurse during lactation can improve the thyroid programming without modifying metabolic programming.

The goal of this study is to investigate whether congenital hypothyroidism induced by MMI during gestation (G) or gestation plus lactation (GL) would affect the leptin action upon body weight control on hypothalamus. Six to eight pups per group were killed at 90 days of age. For statistical analysis one-way ANOVA followed by the Holm-Sìdak post hoc test was used. Hypothyroidism resulted in a significant increase in leptin serum levels in G 20% and GL 25% (p<0.04). There was a significant expression decrease of OBR in G 45% and GL 63%; pSTAT3 in G 56% and GL 51%; pERK in G 50% and GL 48%; POMC in G 41% and GL 46% (p<0.04), while a significant increase was assigned to SOCS3 in G 52% and GL 170% (p<0.04) protein expression. We can conclude that hypothyroxinemia condition in rats on adulthood results in impairment of the leptin signaling pathway via ObRb-STAT3 in the hypothalamus, which is likely to be involved in the leptin resistance., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: Hypothyroidism has been related to low-weight births, abortion and prematurity, which have been associated with changes in the content of glycogen and vascularization of the placenta. Since hypothyroidism can cause dyslipidemia, it may affect the lipid content in the uterus affecting the development of fetuses., Objective: To investigate the effect of hypothyroidism on the lipid levels in serum and uterus during pregnancy and their possible association with the size of fetuses., Method: Adult female rabbits were grouped in control (n = 6) and hypothyroid (n = 6; treated with methimazole for 29 days before and 19 days after copulation). Food intake and body weight were daily registered. At gestational day 19 (GD19), dams were sacrificed under an overdose of anesthesia. Morphometric measures of fetuses were taken. Total cholesterol (TC), triglyceride (TAG), and glucose concentrations were quantified in blood, uterus and ovaries of dams. The expression of uterine 3β- hydroxysteroid dehydrogenase (3β-HSD) was quantified by Western blot., Results: Hypothyroidism reduced food intake and body weight of dams, as well as promoted low abdominal diameters of fetuses. It did not induce dyslipidemia and hyperglycemia at GD19 and did not modify the content of lipids in the ovary. However, it reduced the content of TAG and TC in the uterus, which was associated with uterine hyperplasia and an increased expression of 3β-HSD in the uterus., Conclusion: Hypothyroidism alters the lipid content in the uterus that might subsequently affect the energy production and lipid signaling important to fetal development., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Extracellular hydrogen peroxide is required for thyroperoxidase-mediated thyroid hormone synthesis in the follicular lumen of the thyroid gland. Among the NADPH oxidases, dual oxidases, DUOX1 and DUOX2, constitute a distinct subfamily initially identified as thyroid oxidases, based on their level of expression in the thyroid. Despite their high sequence similarity, the two isoforms present distinct regulations, tissue expression, and catalytic functions. Inactivating mutations in many of the genes involved in thyroid hormone synthesis cause thyroid dyshormonogenesis associated with iodide organification defect. This chapter provides an overview of the genetic alterations in DUOX2 and its maturation factor, DUOXA2, causing inherited severe hypothyroidism that clearly demonstrate the physiological implication of this oxidase in thyroid hormonogenesis. Mutations in the DUOX2 gene have been described in permanent but also in transient forms of congenital hypothyroidism. Moreover, accumulating evidence demonstrates that the high phenotypic variability associated with altered DUOX2 function is not directly related to the number of inactivated DUOX2 alleles, suggesting the existence of other pathophysiological factors. The presence of two DUOX isoforms and their corresponding maturation factors in the same organ could certainly constitute an efficient redundant mechanism to maintain sufficient H 2 O 2 supply for iodide organification. Many of the reported DUOX2 missense variants have not been functionally characterized, their clinical impact in the observed phenotype remaining unresolved, especially in mild transient congenital hypothyroidism. DUOX2 function should be carefully evaluated using an in vitro assay wherein (1) DUOXA2 is co-expressed, (2) H 2 O 2 production is activated, (3) and DUOX2 membrane expression is precisely analyzed.

Adverse neurodevelopmental consequences remain a primary concern when evaluating the effects of thyroid hormone (TH) disrupting chemicals. Though the developing brain is a known target of TH insufficiency, the relationship between THs in the serum and the central nervous system is not well characterized. To address this issue, dose response experiments were performed in pregnant rats using the goitrogen propylthiouracil (PTU) (dose range 0.1-10 ppm). THs were quantified in the serum and brain of offspring at gestational day 20 (GD20) and postnatal day 14 (PN14), two developmental stages included in OECD and EPA regulatory guideline/guidance studies. From the dose response data, the quantitative relationships between THs in the serum and brain were determined. Next, targeted gene expression analyses were performed in the fetal and neonatal cortex to test the hypothesis that TH action in the developing brain is linked to changes in TH concentrations within the tissue. Results show a significant reduction of T4/T3 in the serum and brain of the GD20 fetus in response to low doses of PTU; interestingly, very few genes were significantly different at any dose tested. In the PN14 pup significant reductions of T4/T3 in the serum and brain were also detected; however, twelve transcriptional targets were identified in the neonatal cortex that correlated well with reduced brain THs. These results show that serum T4 is a good predictor of brain THs, and offer several target genes that could serve as pragmatic readouts of T4/T3 dysfunction within the PN14 cortex.

EphA5 and its ligand ephrin-A5 interaction can trigger synaptogenesis during early hippocampus development. We have previously reported that abnormal EphA5 expression can result in synaptogenesis disorder in congenital hypothyroidism (CH) rats. To better understand its precise molecular mechanism, we further analyzed the characteristics of ephrin-A5 expression in the hippocampus of CH rats. Our study revealed that ephrin-A5 expression was downregulated by thyroid hormone deficiency in the developing hippocampus and hippocampal neurons in rats. Thyroxine treatment for hypothyroid hippocampus and triiodothyronine treatment for hypothyroid hippocampal neurons significantly improved ephrin-A5 expression but could not restore its expression to control levels. Hypothyroid hippocampal neurons in-vitro showed synaptogenesis disorder characterized by a reduction in the number and length of neurites. Furthermore, the synaptogenesis-associated molecular expressions of NMDAR-1 (NR1), PSD95 and CaMKII were all downregulated correspondingly. These results suggest that ephrin-A5 expression may be decreased in CH, and abnormal activation of ephrin-A5/EphA5 signaling affects synaptogenesis during brain development. Such findings provide an important basis for exploring the pathogenesis of CH genetically.

Background: Dual oxidases (DUOX1 and DUOX2) were initially identified as H 2 O 2 sources involved in thyroid hormone synthesis. Congenital hypothyroidism (CH) resulting from inactivating mutations in the DUOX2 gene highlighted that DUOX2 is the major H 2 O 2 provider to thyroperoxidase. The role of DUOX1 in the thyroid remains unknown. A recent study suggests that it could compensate for DUOX2 deficiency in CH. Both DUOX enzymes and their respective maturation factors DUOXA1 and DUOXA2 form a stable complex at the cell surface, which is fundamental for their enzymatic activity. Recently, intra- and intermolecular disulfide bridges were identified that are essential for the structure and the function of the DUOX2-DUOXA2 complex. This study investigated the involvement of cysteine residues conserved in DUOX1 toward the formation of disulfide bridges, which could be important for the function of the DUOX1DUOXA1 complex., Methods: To analyze the role of these cysteine residues in both the targeting and function of dual oxidase, different human DUOX1 mutants were constructed, where the cysteine residues were replaced with glycine. The effect of these mutations on cell surface expression and H 2 O 2 -generating activity of the DUOX1-DUOXA1 complex was analyzed., Results: Mutations of two cysteine residues (C118 and C1165), involved in the formation of the intramolecular disulfide bridge between the N-terminal ectodomain and one of the extracellular loops, mildly altered the function and the targeting of DUOX1, while this bridge is crucial for DUOX2 function. Unlike DUOXA2, with respect to DUOX2, the stability of the maturation factor DUOXA1 is not dependent on the oxidative folding of DUOX1. Only mutation of C579 induced a strong alteration of both targeting and function of the oxidase by preventing the covalent interaction between DUOX1 and DUOXA1., Conclusion: An intermolecular disulfide bridge rather than an intramolecular disulfide bridge is important for both the trafficking and H 2 O 2 -generating activity of the DUOX1-DUOXA1 complex.

PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin. Here we report a stepwise feedback mechanism entailing key residues within distinctive interfacing motifs of EZH2 or EED that are found to be mutated in cancers and/or Weaver syndrome. PRC2 harboring these EZH2 or EED mutants manifested little activity in vivo but, unexpectedly, exhibited similar chromatin association as wild-type PRC2, indicating an uncoupling of PRC2 activity and recruitment. With genetic and chemical tools, we demonstrated that targeting allosteric activation overrode the gain-of-function effect of EZH2 Y646X oncogenic mutations. These results revealed critical implications for the regulation and biology of PRC2 and a vulnerability in tackling PRC2-addicted cancers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The EphA5 receptor is well established as an axon guidance molecule during neural system development and plays an important role in dendritic spine formation and synaptogenesis. Our previous study has showed that EphA5 is decreased in the developing brain of congenital hypothyroidism (CH) and the EphA5 promoter methylation modification participates in its decrease. c-Fos, a well-kown transcription factor, has been considered in association with brain development. Bioinformatics analysis showed that the EphA5 promoter region contained five putative c-fos binding sites. The chromatin immunoprecipitation (ChIP) assays were used to assess the direct binding of c-fos to the EphA5 promoter. Furthermore, dual-luciferase assays showed that these three c-fos protein binding sites were positive regulatory elements for EphA5 expression in PC12 cells. Moreover, We verified c-fos positively regulation for EphA5 expression in CH model. Q-PCR and Western blot showed that c-fos overexpression could upregulate EphA5 expression in hippocampal neurons of rats with CH. Our results suggest that c-fos positively regulates EphA5 expression in CH rat model.

Thyroid stimulating hormone receptor (TSHR), a G-protein-coupled receptor, is important for thyroid development and growth. In several cases, frameshift and/or nonsense mutations in TSHR were found in the patients with congenital hypothyroidism (CH), however they have not been functionally studied in an animal model. In the present work, we generated a unique Tshr Df/Df rat model that recapitulates the phenotypes in TSHR Y444X patient by CRISPR/Cas genome editing technology. In this rat model, TSHR is truncated at the second transmembrane domain, leading to CH phenotypes as what was observed in the patients, including dwarf, thyroid aplasia, infertility, TSH resistant as well as low serum thyroid hormone levels. The phenotypes can be reversed, at least partially, by levothyroxine (L-T4) treatment after weaning. The thyroid development is severely impaired in the Tshr Df/Df rats due to the suppression of the thyroid specific genes, i.e., thyroperoxidase (Tpo), thyroglobulin (Tg) and sodium iodide symporter (Nis), at both mRNA and protein levels. In conclusion, the Tshr Df/Df rat serves as a brand new genetic model to study CH in human, and will greatly help to shed light into the development of terminal organs that are sensitive to thyroid hormones.

Thyroid peroxidase (TPO) deficiency, caused by biallelic TPO mutations, is a well-established genetic form of congenital hypothyroidism (CH). More than 100 patients have been published, and the patients have been diagnosed mostly in the frame of newborn screening (NBS) programs. Correlation between clinical phenotypes and TPO activity remains unclear. Here, we report clinical and molecular findings of two unrelated TPO mutation-carrying mildly hypothyroid patients. The two patients were born at term after an uneventful pregnancy and delivery, and were NBS negative. They sought medical attention due to goiter at age 8 years. Evaluation of the thyroid showed mild elevation of serum TSH levels, normal or slightly low serum T 4 levels, high serum T 3 to T 4 molar ratio, high serum thyroglobulin levels, and high thyroidal 123 I uptake. We performed next-generation sequencing-based genetic screening, and found that one patient was compound heterozygous for two novel TPO mutations (p.Asp224del; c.820-2A>G), and the other was homozygous for a previously known mutation (p.Trp527Cys). In vitro functional analyses using HEK293 cells showed that the two amino acid-altering mutations (p.Asp224del and p.Trp527Cys) caused partial loss of the enzymatic activity. In conclusion, we report that TPO mutations with residual activity are associated with mild TPO deficiency, which is clinically characterized by marked goiter, mild TSH elevation, high serum T 3 to T 4 molar ratio, and high serum thyroglobulin levels. Our findings illuminate the hitherto under-recognized correlation between clinical phenotypes and residual enzymatic activity among patients with TPO deficiency.

Congenital hypothyroidism (CH), the most frequent form of preventable mental retardation, is predicted to have a relevant genetic origin. However, CH is frequently reported to be sporadic and candidate gene variations were found in <10% of the investigated patients. Here, we characterize the involvement of 11 candidate genes through a systematic Next Generation Sequencing (NGS) analysis. The NGS was performed in 177 unrelated CH patients (94 gland-in-situ; 83 dysgenesis) and in 3,538 control subjects. Non-synonymous or splicing rare variants (MAF < 0.01) were accepted, and their functional impact was predicted by a comprehensive bioinformatic approach and co-segregation studies. The frequency of variations in cases and controls was extended to 18 CH-unrelated genes. At least one rare variant was accepted in 103/177 patients. Monogenic recessive forms of the disease were found in five cases, but oligogenic involvement was detected in 39 patients. The 167 variations were found to affect all genes independently of the CH phenotype. These findings were replicated in an independent cohort of additional 145 CH cases. When compared to 3,538 controls, the CH population was significantly enriched with disrupting variants in the candidate genes (P = 5.5 × 10-7), but not with rare variations in CH-unrelated genes. Co-segregation studies of the hypothyroid phenotype with multiple gene variants in several pedigrees confirmed the potential oligogenic origin of CH. The systematic NGS approach reveals the frequent combination of rare variations in morphogenetic or functional candidate genes in CH patients independently of phenotype. The oligogenic origin represents a suitable explanation for the frequent sporadic CH occurrence., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Iodide Handling Disorders lead to defects of the biosynthesis of thyroid hormones (thyroid dyshormonogenesis, TD) and thereafter congenital hypothyroidism (CH), the most common endocrine disease characterized by low levels of circulating thyroid hormones. The prevalence of CH is 1 in 2000-3000 live births. Prevention of CH is based on prenatal diagnosis, carrier identification, and genetic counseling. In neonates a complete diagnosis of TD should include clinical examination, biochemical thyroid tests, thyroid ultrasound, radioiodine or technetium scintigraphy and perchlorate discharge test (PDT). Biosynthesis of thyroid hormones requires the presence of iodide, thyroid peroxidase (TPO), a supply of hydrogen peroxide (DUOX system), an iodine acceptor protein, thyroglobulin (TG), and the rescue and recycling of iodide by the action of iodotyrosine deiodinase or iodotyrosine dehalogenase 1 (IYD or DEHAL1). The iodide transport is a two-step process involving transporters located either in the basolateral or apical membranes, sodium iodide symporter (NIS) and pendrin (PDS), respectively. TD has been linked to mutations in the solute carrier family 5, member 5 transporter (SLC5A5, encoding NIS), solute carrier family 26, member 4 transporter (SLC26A4, encoding PDS), TPO, DUOX2, DUOXA2, TG and IYD genes. These mutations produce a heterogeneous spectrum of CH, with an autosomal recessive inheritance. Thereafter, the patients are usually homozygous or compound heterozygous for the gene mutations and the parents, carriers of one mutation. In the last two decades, considerable progress has been made in identifying the genetic and molecular causes of TD. Recent advances in DNA sequencing technology allow the massive screening and facilitate the studies of phenotype variability. In this article we included the most recent data related to disorders caused by mutations in NIS, TPO, TG and IYD., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

After the identification of thyroid H 2 O 2 generation system (DUOX) and of its maturation factors (DUOXA), defects in DUOX2 and/or DUOXA2 were rapidly recognized as the possible cause of congenital hypothyroidism (CH) due to thyroid dyshormonogenesis. The present Review reports data on the prevalence of DUOX2 mutations, which is variable among different series but invariably high, pointing to DUOX2 defects as one of the leading causes of dyshormonogenesis. Differently, DUOXA defects seem to be rarely involved in the pathogenesis of CH. Genotype-phenotype correlations are also reported, highlighting the great intra- and inter-familial phenotype variability which appears to be a constant feature of the defects in the H 2 O 2 generation systems. Finally, the hypotheses to explain the phenotypic variability of the DUOX2/A2 mutations are discussed, such as the existence of other H 2 O 2 generating systems, the age variability in thyroid hormones requirements, the differences in ethnicity, in iodine intake, and in the methodological approaches., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Thyroid scintigraphy holds a key place in the etiologic workup of neonatal hypothyroidism. Routine screening for this disorder in maternity hospitals in industrialized countries, for nearly 40 years, has permitted early treatment and thereby helped to prevent its physical and mental complications. Neonatal hypothyroidism affects approximately 1 in 3000 births. The most common causes are abnormal thyroid gland development and defective hormone synthesis by an eutopic thyroid gland. The incidence of the latter has risen in recent years, for reasons that remain unclear. A thorough etiologic workup helps to determine the disease type. Current guidelines recommend thyroid imaging by means of ultrasound and scintigraphy. Ultrasound should be done by a practitioner trained to examine the cervical region of newborns, as the thyroid is very small and must be distinguished from the particular aspect of the "thyroid empty lodge." Ultrasound lacks sensitivity for detecting small ectopic glands but is the gold standard for measuring thyroid dimensions. Scintigraphy provides an etiologic diagnosis in most cases. The two isotopes used in this setting are technetium-99m and iodine-123. The latter isotope gives more contrast and allows the perchlorate discharge test to be performed to detect abnormal iodide organification in the neonate with an eutopic thyroid. If scintigraphy cannot be performed during the neonatal period, a postponed procedure can be achieved after 3 years of age. Close cooperation between the nuclear medicine physician and the pediatric endocrinologist is crucial for timely and optimized scintigraphy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

Objective: Based on mutations in PAX8 is associated with thyroid dysgenesis. We aim to identify and characterize PAX8 mutations in a large cohort of congenital hypothyroidism(CH) from thyroid dysgenesis in Chinese population., Methods: We screened 453 unrelated Chinese patients with CH from thyroid dysgenesis for PAX8 mutations by sequencing the whole coding regions of PAX8 on genomic DNA isolated from blood. Cell transfection assays using various vector constructs and induced mutagenesis as well as electrophoretic mobility shift assays were used to investigate the effects of selected mutations on the transcribing and binding activities of PAX8 at the promoters of target genes for thyroglobulin (TG) and thyroperoxidase (TPO)., Results: Five PAX8 mutations were found, yielding a mutation prevalence of 5/453 (1.1%). We selected two mutations in the critical paired domain of PAX8 and generated mutants D94N and G41V. We demonstrated G41V was unable to bind the specific sequence in the promoters of TG and TPO and activate them. D94N could bind to TG and TPO promoters and normally activate the TG promoter transcription but not the TPO promoter transcription. We also demonstrated a dominant negative role of the PAX8 mutants in impairing the function of the wild-type PAX8., Conclusion: We for the first time documented the prevalence and characterized the function of PAX8 mutations in CH in Chinese population. The study specifically demonstrated the role of novel mutations D94N and G41V in impairing the function of PAX8, providing further evidence for genetic PAX8 defects as a disease mechanism in CH.

The goal of this study was to evaluate the influence of hypothyroidism induced by MMI, during gestation (G) or gestation plus lactation (GL) on testis and its relation with leptin in rats. Six to eight pups were killed at 90 days of age. For statistical analysis One-way ANOVA followed by the Holm-Sìdak post hoc test was used. Hypothyroidism resulted in a significant reduction in LH, FSH and testosterone and an increase in leptin serum levels (p < 0.04). There was a significant decrease in StAR, AR, FSHR, LHR, pSTAT3 and SOCS3 (p < 0.04) protein expression and in the fertility parameters (p < 0.04). We can conclude that hypothyroidism is associated with reduction of steroidogenesis and spermatogenesis leading to a low fertility potential in these animals. This outcome could be a consequence of low pituitary stimulus and testicular response and probably are not related with leptin hormone since its signaling pathway is down-regulated in the testis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background/aims: Transient congenital hypothyroidism (TCH) could disturb carbohydrate metabolism in adulthood. Aging is associated with increased risk of type 2 diabetes. This study aims to address effects of TCH on mRNA expressions of glucose transporters (GLUTs) and glucokinase (GcK) in islets and insulin target tissues of aged offspring rats., Methods: The TCH group received water containing 0.025% 6-propyl-2-thiouracil during gestation. Offspring from control and TCH groups (n=6 in each group) were followed until month 19. Gene expressions of GLUTs and GcK were measured at months 3 and 19., Results: Compared to controls, aged TCH rats had higher GLUT4 expression in heart (4.88 fold) and soleus (6.91 fold), while expression was lower in epididymal fat (12%). In TCH rats, GLUT2 and GcK expressions in islets were lower in young (12% and 10%, respectively) and higher in aged (10.85 and 8.42 fold, respectively) rats. In addition, liver GLUT2 and GcK expressions were higher in young (13.11 and 21.15 fold, respectively) and lower in aged rats (44% and 5%, respectively)., Conclusion: Thyroid hormone deficiency during fetal period impaired glucose sensing apparatus and changed glucose transporter expression in insulin-sensitive tissues of aged offspring rats. These changes may contribute to impaired carbohydrate metabolism., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Abnormalities in thyroid function are common endocrine disorders that affect 5-10 % of the general population, with hypothyroidism occurring more frequently than hyperthyroidism. Clinical symptoms and signs are often nonspecific, particularly in hypothyroidism. Muscular symptoms (stiffness, myalgias, cramps, easy fatigability) are mentioned by the majority of patients with frank hypothyroidism. Often underestimated is the fact that muscle symptoms may represent the predominant or the only clinical manifestation of hypothyroidism, raising the issue of a differential diagnosis with other causes of myopathy, which sometimes can be difficult. Elevated serum creatine kinase, which not necessarily correlates with the severity of the myopathic symptoms, is certainly suggestive of muscle impairment, though it does not explain the cause. Rare muscular manifestations, associated with hypothyroidism, are rhabdomyolysis, acute compartment syndrome, Hoffman's syndrome and Kocher-Debré-Sémélaigne syndrome. Though the pathogenesis of hypothyroid myopathy is not entirely known, proposed mechanisms include altered glycogenolytic and oxidative metabolism, altered expression of contractile proteins, and neuro-mediated damage. Correlation studies of haplotype, muscle gene expression and protein characterization, could help understanding the pathophysiological mechanisms of this myopathic presentation of hypothyroidism.

Thyroid hormones are of central relevance for growth and development. However, the underlying molecular mechanisms are still not fully understood. Recent studies in humans and mice have demonstrated that serum levels of selenium (Se) and copper (Cu) are positively affected by thyroid hormones. Given the importance of these trace elements for many biochemical processes, we tested whether this interaction is found in children at risk for hypothyroidism, potentially providing a novel factor contributing to the disturbed development observed in congenital hypothyroidism (CH). We conducted a cross-sectional analysis of 84 children diagnosed with CH displaying a wide range of thyroid hormone concentrations. Serum Se and Cu concentrations were measured by total reflection X-ray fluorescence. Data for thyrotropin (TSH) were available in all, thyroxine (T4) and free thyroxine (fT4) in the majority and triiodothyronine (T3) in 29 of the children. Spearman rank analyzes were performed. Cu and thyroid hormones showed a strong positive correlation (Cu/T4, rho=0.5241, P=0.0003; Cu/T3, rho=0.6003, P=0.0006). Unlike in adults, no associations were found between Se and any of the thyroid hormones. Our data highlight that serum Cu and thyroid hormones are strongly associated already in early postnatal life. Severely hypothyroid children are thus at risk of developing a Cu deficiency if not adequately nourished or supplemented. This finding needs to be verified in larger groups of children in order not to miss an easily-avoidable risk factor for poor development., (Copyright © 2016 Elsevier GmbH. All rights reserved.)