Brief Report: A Novel Sodium/Iodide Symporter Mutation, S356F, Causing Congenital Hypothyroidism

Abstract

The sodium-iodide symporter (NIS, SLC5A5) is expressed at the basolateral membrane of the thyroid follicular cell, and facilitates the thyroidal iodide uptake required for thyroid hormone biosynthesis. Biallelic loss-of-function mutations in NIS are a rare cause of dyshormonogenic congenital hypothyroidism. Affected individuals typically exhibit a normally sited, often goitrous thyroid gland, with absent uptake of radioiodine in the thyroid and other NIS-expressing tissues. We report a novel homozygous NIS mutation (c.1067 C>T, p.S356F) in four siblings from a consanguineous Indian kindred, presenting with significant hypothyroidism. Functional characterization of the mutant protein demonstrated impaired plasma membrane localization and cellular iodide transport.

Keywords: SLC5A5; congenital hypothyroidism; dyshormonogenesis; iodide transport.

FIG. 1.

(A) Pedigree showing homozygosity (black) and heterozygosity (black and white) for the NIS p.S356F mutation, in a consanguineous family from India. Corresponding thyroid hormones (measured on ADVIA Centaur XP Immunoassay System) and TG (measured on IMMULITE 1000 Immunoassay System) measured while on suboptimal levothyroxine replacement (100 mcg daily), aged 16, 14, 12, and 8 years, respectively, are aligned to the respective individual. Representative Tc-99m pertechnetate scan images for thyroid and stomach are shown hereunder; there is no uptake of tracer into either organ. (B) Representative sequencing chromatogram for each of the four siblings (all homozygous for p.S356F NIS) and their heterozygous parents, showing the relevant homo- or heterozygous nucleotide substitution (c.1067C>T). (C) Graph showing mean normalized ¹²⁵I uptake in COS-7 cells transfected with pcDNA, WT, or p.S356F NIS, in the presence (white) or absence (black) of 100 μM perchlorate. Data are expressed in counts per minute standardized to transfection efficiency as assessed by B-galactosidase assay and represent the mean and SEM values obtained of three independent experiments performed in triplicate. p-Values were calculated using ANOVA and Tukey's post hoc test ***p < 0.0005. (D) Representative Western blot analysis of whole cell lysates of COS-7 cells transiently transfected with HA-WT NIS (WT), HA-S356F NIS (S356F), or empty vector (pcDNA) and probed with anti-HA and anti-beta actin antibodies. (A) Fully glycosylated mature NIS monomer (∼75–90 kDa), (B) unglycosylated immature NIS monomer (∼55 kDa). (E) Representative immunofluorescence analysis of COS-7 cells transiently transfected with HA-WT NIS (WT), HA-S356F NIS (S356F), and probed with anti-HA antibodies in the nonpermeabilized and permeabilized state (green). The nuclei of nonpermeabilized cells are stained with DAPI (blue). (F) Schematic of the secondary structure of NIS with TMS numbered adjacent to each domain. The structure is annotated with previously reported NIS missense mutations and the position of S356 is depicted in blue. This figure was created in Biorender.com. (G, H) SLC5A5 3D structure homology models generated using PHYRE2 Protein Fold Recognition Server showing a detailed view of WT NIS (G) with the position of Ser356 (green) in transmembrane helix 9 (blue) orientation toward Na+ atoms (orange) and other amino acids reported to be involved in Na⁺ binding (3). (H) Model of Ser356Phe NIS: the mutation is predicted to impede Na⁺ binding due to steric hindrance. Moreover, Ser356 is polar, and the apolar and larger Phe will disrupt the local structure, potentially destabilizing the overall structure of NIS. ANOVA, analysis of variance; COS-7, CV-1 (simian) in Origin, carrying SV40 genetic material; DAPI, 4′,6-diamidino-2-phenylindole; HA, human influenza hemagglutinin; NIS, sodium-iodide symporter; SEM, Standard error of the mean; TG, thyroglobulin; TMS, transmembrane segments; WT, wild-type. Color images are available online.