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

Abstract

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.

Keywords: congenital hypothyroidism; iodide transport defect; pathogenic synonymous variant; pre-mRNA splicing defect; sodium/iodide symporter.

Figure 1

Identification of synonymous SLC5A5 variants in patients with dyshormonogenic congenital hypothyroidism. Partial sequence chromatograms covering the region of the variants (underlined) in exons 11 (c.1326A>C) (A) and 13 (c.1626C>T) (B) of the SLC5A5 gene. The reference sequence (Refseq) is indicated in gray.

Figure 2

The variant c.1326A>C causes NIS pre-mRNA splicing defect. (A) Scheme of pSPL3-based minigene reporter constructs used in functional assays. The genomic fragment containing exons 11 and 12 along with a fragment flanking introns 10 and 12, and the spacing intron 11 was cloned between pSPL3 vector exons SD and SA using XhoI and BamHI restriction sites. Arrows show pSPL3 vector SD and SA exon-specific primers (SD6 and SA2) used in RT-PCR analysis. Canonical and aberrant splicing products are indicated by dashed lines above and below the construct, respectively. The variant c.1326A>C is indicated. (B) Agarose gel electrophoresis of RT-PCR products from empty, WT or c.1326A>C pSPL3 minigene reporter transiently transfected into HeLa cells. The empty pSPL3 vector, where only SD-SA exons splicing occurred, lead to a 263 bp PCR product (α splicing). The WT pSPL3 minigene mainly yielded a 547 bp PCR product including SD-SA exons (263 bp) flanking the exons 11 (87 bp) and 12 (197 bp) of the SLC5A5 gene (β splicing). The c.1326A>C pSPL3 minigene leads to a main PCR product of 263 bp including only SD-SA exons and secondary PCR products of 457 and 573 bp including SD-SA exons flanking exon 12 alone (γ splicing) or including an artificial pseudoexon derived from the intronic sequence of the reporter vector ( Supplementary Figure 1 ), respectively. Schemes represent the α, β and γ splicing RT-PCR products. (C) Sequencing analysis confirmed the identity of α, β and γ splicing RT-PCR products.