Novel Compound Heterozygous Pathogenic Mutations of SLC5A5 in a Chinese Patient With Congenital Hypothyroidism

Abstract

Background and objectives: Defects in the human sodium/iodide symporter (SLC5A5) gene have been reported to be one of the causes of congenital hypothyroidism (CH). We aimed to identify SLC5A5 mutations in Chinese patients with CH and to evaluate the function of the mutation.

Methods: Two hundred and seventy-three patients with primary CH were screened for mutations in SLC5A5 using next-generation sequencing. We investigated the expression and cellular localization of the novel compound heterozygous mutation in SLC5A5. The functional activity of the mutants was further examined in vitro.

Results: In 273 patients with CH, two previously undescribed pathogenic mutations p.Gly51AlafsTer45 (G51fs) and p.Gly421Arg (G421R) in a compound heterozygous state in SLC5A5 were identified in a pediatric patient. G51fs was located in the first intercellular loop connecting transmembrane segment I and II, whereas G421R was in the transmembrane segment (TMS) XI. G51fs and G421R resulted in a truncated NIS and reduced protein expression, respectively. In vitro experiments further showed that the normal function of iodine transport of sodium-iodide symporter (NIS) mutants was markedly impaired.

Conclusion: The undescribed compound heterozygous mutation of SLC5A5 was discovered in a Chinese CH patient. The mutation led to significantly reduced NIS expression and impaired iodide transport function accompanied by the impaired location of the NIS on the plasma membrane. Our study thus provides further insights into the roles of SLC5A5 in CH pathogenesis.

Keywords: SLC5A5; congenital hypothyroidism; iodine transport; mutation; next-generation sequencing.

Figure 1

Sodium-iodide symporter (NIS) secondary structure model. Blue cylinders represent the 13 transmembrane segments (TMS). The amino-terminus faces the extracellular space and the carboxy-terminus is in the cytosol. The novel pathogenic variants p.G51fs and p.G421R are highlighted with blue lines.

Figure 2

Identification and characterization of the compound heterozygous NIS mutation p.G51fs/p.G421R. (A) Partial sequence chromatograms cover the region of the variants in exon 1 (c.152del, p.G51fs) and 11 (c.1261G>A, p.G421R) of SLC5A5 gene. The nucleotide and corresponding amino acid sequences using the three-letter code are indicated. The proband was compound heterozygous for the mutant alleles p.G51fs and p.G421R NIS. Arrowheads denote the mutated sites. P, proband; F, father; M, mother. (B) Pedigree analysis of the members of the proband’s family. The mutated alleles for p.G51fs and p.G421R NIS are noted below. (C) Amino acid sequences of NIS from other metazoan species were aligned with those of humans by PSI/TM-Coffee (http://tcoffee.crg.cat/apps/tcoffee/index.html). The mutated amino acids in all NIS homologs are indicated by dotted boxes.

Figure 3

Electrostatic surface and inter-atomic distances of WT NIS and G421R. (A) Local solid surface and electrostatic potentials are greatly changed by G421R mutation. Positive and negative potentials are shown in blue and red, respectively. (B) The cartoon representation of the crystal structure of NIS based on vSGLT. Those previously reported residues, which are particularly close to the mutation site, are shown in the magnified regions. Dotted lines connect atoms for which inter-atomic distances are given in angstroms. The specific values are shown in the figure. The left part of figure represents WT NIS, and the right G421R.

Figure 4

(A) Representative Western blot analysis of whole-cell lysates from transiently transfected 293T cells probed with anti-FLAG and GAPDH antibodies. The proteins of WT (wild-type), G421R and G51fs were visualized by Odyssey CLx Infra-Red Imaging system. The molecular weight band was on the right (WT, G421R unglycosylated~ 55 kDa, mature glycosylated~ 100 kDa; G51fs monomer~13 kDa, dimer~ 26 kDa). (B) Iodide transport activity for of G421R and G51fs NIS mutants. Mock (p3xFLAG-CMV-10 plasmids), or expressing WT (wild-type) NIS, D331N, G421R and G51fs NIS plasmids were transfected alone or co-transfected into 293T cells. The cells were incubated with with131I at 5 KBq/mL in the absence (black bars) or presence (white bars) of 1 mM perchlorate and the intracellular iodide accumulation was detected using a γ counter. The values are representative of more than three independent experiments and represent the mean ± SD of picomoles ¹³¹I per microgram of DNA; each experiment was performed in triplicate. ***p < 0.001.

Figure 5

Characterization of cellular localization of G421R and G51fs NIS mutants in 293T cells. pEGFP-N2 empty vector was employed as a negative control (Mock). Human WT NIS, D331N, G421R, and G51fs mutants were cloned into pEGFP-N2 plasmids (green). The cell membrane was labeled with a fluorescent probe Dil (red). Mock, WT NIS, D331N, G421R, and G51fs plasmids were transfected into 293T cells 24 h after seeding. Mutant D331N NIS showed slightly decreased membrane fluorescence, compared with the WT. However, G51fs and G421R mutants displayed severely reduced cell membrane localization in 293T cells. Nuclei were stained with DAPI (blue). Scale: 10 μm.