Targeted Next-Generation Sequencing of Congenital Hypothyroidism-Causative Genes Reveals Unexpected Thyroglobulin Gene Variants in Patients with Iodide Transport Defect.

Congenital iodide transport defect is an uncommon autosomal recessive disorder caused by loss-of-function variants in the sodium iodide symporter (NIS)-coding SLC5A5 gene and leading to dyshormonogenic congenital hypothyroidism. Here, we conducted a targeted next-generation sequencing assessment of congenital hypothyroidism-causative genes in a cohort of nine unrelated pediatric patients suspected of having a congenital iodide transport defect based on the absence of 99mTc-pertechnetate accumulation in a eutopic thyroid gland. Although, unexpectedly, we could not detect pathogenic SLC5A5 gene variants, we identified two novel compound heterozygous TG gene variants (p.Q29* and c.177-2A>C), three novel heterozygous TG gene variants (p.F1542Vfs*20, p.Y2563C, and p.S523P), and a novel heterozygous DUOX2 gene variant (p.E1496Dfs*51). Splicing minigene reporter-based in vitro assays revealed that the variant c.177-2A>C affected normal TG pre-mRNA splicing, leading to the frameshift variant p.T59Sfs*17. The frameshift TG variants p.T59Sfs*17 and p.F1542Vfs*20, but not the DUOX2 variant p.E1496Dfs*51, were predicted to undergo nonsense-mediated decay. Moreover, functional in vitro expression assays revealed that the variant p.Y2563C reduced the secretion of the TG protein. Our investigation revealed unexpected findings regarding the genetics of congenital iodide transport defects, supporting the existence of yet to be discovered mechanisms involved in thyroid hormonogenesis.

Silent but Not Harmless: A Synonymous SLC5A5 Gene Variant Leading to Dyshormonogenic Congenital Hypothyroidism.

Background: Congenital iodide transport defect (ITD) is an uncommon cause of dyshormonogenic congenital hypothyroidism characterized by the absence of active iodide accumulation in the thyroid gland. ITD is an autosomal recessive disorder caused by loss-of-function variants in the sodium/iodide symporter (NIS)-coding SLC5A5 gene. Objective: We aimed to identify, and if so to functionally characterize, novel ITD-causing SLC5A5 gene variants in a cohort of five unrelated pediatric patients diagnosed with dyshormonogenic congenital hypothyroidism with minimal to absent 99mTc-pertechnetate accumulation in the thyroid gland. Methods: The coding region of the SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of a novel synonymous variant were performed. Results: Sanger sequencing revealed a novel homozygous synonymous SLC5A5 gene variant (c.1326A>C in exon 11). In silico analysis revealed that the c.1326A>C variant is potentially deleterious for NIS pre-mRNA splicing. The c.1326A>C variant was predicted to lie within a putative exonic splicing enhancer reducing the binding of splicing regulatory trans-acting protein SRSF5. Splicing minigene reporter assay revealed that c.1326A>C causes exon 11 or exon 11 and 12 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variants p.G415_P443del and p.G415Lfs*32, respectively. Significantly, the frameshift variant p.G415Lfs*32 is predicted to be subjected to degradation by nonsense-mediated decay. Conclusions: We identified the first exonic synonymous SLC5A5 gene variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape of the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.

An Intramolecular Ionic Interaction Linking Defective Sodium/Iodide Symporter Transport to the Plasma Membrane and Dyshormonogenic Congenital Hypothyroidism.

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.Y324Hfs*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 ß 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 PDZ protein SCRIB regulates sodium/iodide symporter (NIS) expression at the basolateral plasma membrane.

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.

A Novel SLC5A5 Variant Reveals the Crucial Role of Kinesin Light Chain 2 in Thyroid Hormonogenesis.

CONTEXT: Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. OBJECTIVE: This work aims to determine the molecular basis of a patient's ITD clinical phenotype. METHODS: The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. The propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. RESULTS: Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptophan-acidic motif by the kinesin-1 subunit kinesin light chain 2 (KLC2), interfering with NIS maturation beyond the endoplasmic reticulum, and reducing iodide accumulation. Structural bioinformatic analysis suggests that G561E shifts the equilibrium of the unstructured tryptophan-acidic motif toward a more structured conformation unrecognizable to KLC2. Consistently, knockdown of Klc2 causes defective NIS maturation and consequently decreases iodide accumulation in rat thyroid cells. Morpholino knockdown of klc2 reduces thyroid hormone synthesis in zebrafish larvae leading to a hypothyroid state as revealed by expression profiling of key genes related to the hypothalamic-pituitary-thyroid axis. CONCLUSION: We report a novel NIS pathogenic variant associated with dyshormonogenic congenital hypothyroidism. Detailed molecular characterization of G561E NIS uncovered the significance of KLC2 in thyroid physiology.

The Transcription Factor NF-κB Mediates Thyrotropin-Stimulated Expression of Thyroid Differentiation Markers.

Background: The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor is a key regulator of cell survival, proliferation, and gene expression. Although activation of NF-κB signaling in thyroid follicular cells after thyrotropin (TSH) receptor (TSHR) engagement has been reported, the downstream signaling leading to NF-κB activation remains unexplored. Here, we sought to elucidate the mechanisms that regulate NF-κB signaling activation in response to TSH stimulation. Methods: Fisher rat-derived thyroid cell lines and primary cultures of NF-κB essential modulator (NEMO)-deficient mice thyrocytes were used as models. Signaling pathways leading to the activation of NF-κB were investigated by using chemical inhibitors and phospho-specific antibodies. Luciferase reporter gene assays and site-directed mutagenesis were used to monitor NF-κB-dependent gene transcriptional activity and the expression of thyroid differentiation markers was assessed by reverse transcription quantitative polymerase chain reaction and Western blot, respectively. Chromatin immunoprecipitation (ChIP) was carried out to investigate NF-κB subunit p65 DNA binding, and small interfering RNA (siRNA)-mediated gene knockdown approaches were used for studying gene function. Results: Using thyroid cell lines, we observed that TSH treatment leads to protein kinase C (PKC)-mediated canonical NF-κB p65 subunit nuclear expression. Moreover, TSH stimulation phosphorylated the kinase TAK-1, and its knockdown abolished TSH-induced NF-κB transcriptional activity. TSH induced the transcriptional activity of the NF-κB subunit p65 in a protein kinase A (PKA)-dependent phosphorylation at Ser-276. In addition, p65 phosphorylation at Ser-276 induced acetyl transferase p300 recruitment, leading to its acetylation on Lys-310 and thereby enhancing its transcriptional activity. Evaluation of the role played by NF-κB in thyroid physiology demonstrated that the canonical NF-κB inhibitor BAY 11-7082 reduced TSH-induced expression of thyroid differentiation markers. The involvement of NF-κB signaling in thyroid physiology was confirmed by assessing the TSH-induced gene expression in primary cultures of NEMO-deficient mice thyrocytes. ChIP and the knockdown experiments revealed that p65 is a nuclear effector of TSH actions, inducing the transcripcional expression of thyroid differentiation markers. Conclusions: Taken together, our results point to NF-κB being a pivotal mediator in the TSH-induced thyroid follicular cell differentiation, a relevant finding with potential physiological and pathophysiological implications.

Revisión de las alteraciones en la expresión génica y vías apoptóticas provocadas en la nefrotoxicidad inducida por cisplatino / Review of alterations in gene expression and apoptotic pathways caused in nephrotoxicity induced by cisplatin

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Novel Sodium/Iodide Symporter Compound Heterozygous Pathogenic Variants Causing Dyshormonogenic Congenital Hypothyroidism.

Iodide transport defect (ITD) is an autosomal recessive disorder caused by deficient iodide accumulation into the thyroid follicular cell. ITD is an uncommon cause of dyshormonogenetic congenital hypothyroidism that results from inactivating mutations in the sodium/iodide symporter (NIS)-coding SLC5A5 gene. NIS is a key basolateral plasma membrane glycoprotein that efficiently mediates active iodide uptake in the thyroid-constituting the first step in the biosynthesis of the iodine-containing thyroid hormones-and other tissues, including salivary glands, lactating breast, and small intestine. The proposita, a 20-day-old female born in 1992, was diagnosed with congenital hypothyroidism through newborn screening. ITD was suspected on the basis of nondetectable radioiodide accumulation in a normally located nongoitrous thyroid gland, as well as in salivary glands. Sanger sequencing revealed nonpreviously reported compound heterozygous missense SLC5A5 gene variants (c.991G>A, p.D331N and c.1.641C>A, p.S547R). Notably, these variants have not been reported in public databases (i.e., Exome Aggregation Consortium, 1000 Genomes, and Single Nucleotide Polymorphism). In silico analysis using prediction softwares (i.e., SIFT, Polyphen-2, and MutationTaster2) support the pathologic significance of p.D331N and p.S547R NIS. Moreover, functional in vitro studies demonstrate that D331N and S547R NIS severely reduce iodide uptake when the proteins are heterologously expressed in HEK-293T cells because of a pronounced impairment of D331N and S547R NIS targeting to the plasma membrane. Of note, a charged residue at position 331 and a serine residue at position 547-which are highly conserved in SLC5A family members-are required for NIS plasma membrane targeting. We report two novel missense pathogenic variants in a compound heterozygous state in the SLC5A5 gene, detected through Sanger sequencing, in a pediatric female patient with dyshormonogenic congenital hypothyroidism.

Nephroprotective Effect of Embryonic Stem Cells Reducing Lipid Peroxidation in Kidney Injury Induced by Cisplatin.

INTRODUCTION: The acute kidney injury (AKI) is characterized by a sudden glomerular filtration reduction. Renal or intrinsic causes of AKI include nephrotoxicity induced by exogenous agents like cisplatin, which causes oxidative stress altering the biochemical process and leading to apoptosis. Therefore, this research is aimed at analyzing the embryonic stem cells (ESC) nephroprotective effect in AKI induced by cisplatin, employing genetic, phenotypic, and microspectroscopic techniques. METHODS: Thirty mice were randomly divided into three groups (n = 10): the healthy, isotonic salt solution (ISS), and mouse embryonic stem cells (mESC) groups. The ISS and mESC groups were subjected to AKI using cisplatin; 24 h post-AKI received an intraperitoneal injection of ISS or 1 × 106 mESC, respectively. At days 4 and 8 post-AKI, five mice of each group were sacrificed to analyze the histopathological, genetic (PDK4 and HO-1), protein (p53), and vibrational microspectroscopic changes. RESULTS: Histopathologically, interstitial nephritis and acute tubular necrosis were observed; however, the mESC group showed a more preserved microarchitecture with high cellularity. Additionally, the PDK4 and HO-1 gene expression only increased in the ISS group on day 4 post-AKI. Likewise, p53 was more immunoexpressed at day 8 post-AKI in the ISS group. About biomolecular analysis by microspectroscopy, bands associated with lipids, proteins, and nucleic acids were evidenced. Besides, ratios related to membrane function (protein/lipid), unsaturated lipid content (olefinic/total lipid, olefinic/total CH2, and CH2/CH3), and lipid peroxidation demonstrated oxidative stress induction and lipid peroxidation increase mainly in the ISS group. Finally, the principal component analysis discriminated against each group; nonetheless, some data of the healthy and mESC groups at day 8 were correlated. CONCLUSIONS: The mESC implant diminishes cisplatin nephrotoxicity, once the protective effect in the reduction of lipid peroxidation was demonstrated, reflecting a functional and histological restoration.

Implications of Na+/I- Symporter Transport to the Plasma Membrane for Thyroid Hormonogenesis and Radioiodide Therapy.

Iodine is a crucial component of thyroid hormones; therefore, a key requirement for thyroid hormone biosynthesis is that iodide (I-) be actively accumulated in the thyroid follicular cell. The ability of the thyroid epithelia to concentrate I- is ultimately dependent on functional Na+/ I- symporter (NIS) expression at the plasma membrane. Underscoring the significance of NIS for thyroid physiology, loss-of-function mutations in the NIS-coding SLC5A5 gene cause an I- transport defect, resulting in dyshormonogenic congenital hypothyroidism. Moreover, I- accumulation in the thyroid cell constitutes the cornerstone for radioiodide ablation therapy for differentiated thyroid carcinoma. However, differentiated thyroid tumors often exhibit reduced (or even undetectable) I- transport compared with normal thyroid tissue, and they are diagnosed as cold nodules on thyroid scintigraphy. Paradoxically, immunohistochemistry analysis revealed that cold thyroid nodules do not express NIS or express normal, or even higher NIS levels compared with adjacent normal tissue, but NIS is frequently intracellularly retained, suggesting the presence of posttranslational abnormalities in the transport of the protein to the plasma membrane. Ultimately, a thorough comprehension of the mechanisms that regulate NIS transport to the plasma membrane would have multiple implications for radioiodide therapy, opening the possibility to identify new molecular targets to treat radioiodide-refractory thyroid tumors. Therefore, in this review, we discuss the current knowledge regarding posttranslational mechanisms that regulate NIS transport to the plasma membrane under physiological and pathological conditions affecting the thyroid follicular cell, a topic of great interest in the thyroid cancer field.