Introduction

Dyshormonogenic congenitalhypothyroidism is caused by loss-of-function mutations in genes requiredfor normal thyroid hormonogenesis.Particularly, mutations in the Na⁺/I⁻ symporter(NIS)-coding SLC5A5 gene cause an uncommonautosomal recessive disorder known as I⁻ transport defect (ITD), asa consequence of impaired I⁻ accumulation in the thyroid follicularcell.

Methods

Genomic DNA was isolated fromfrozen whole blood and the entire exonic nucleotide sequence encoding the SLC5A5 gene was studied using Sangersequencing. Genetic testing wasapproved by the institutional review board at Hospital de Niños Dr. Ricardo Gutiérrez. In silico computational and in vitro functional studies of newlyidentified NIS mutants were performed.

Results

Three unrelated female pediatricpatients were diagnosed by neonatal screening with severe dyshormonogeniccongenital hypothyroidism. ITD was suspected on the basis of non-detectable radioiodideaccumulation in a normally located, non-goitrous thyroid gland, as well as in salivaryglands.

SLC5A5 gene analysis revealed six non-synonymous compoundheterozygous variants (p.Q136L/p.D369V, p.D331N/p.S547R, and p.G543K/p.L562M)in association with an ITD phenotype.Notably, none of them were reported in the Exome Aggregation Consortiumdatabase.

The pathologic significance of allNIS mutants was investigated in silicousing prediction softwares (i.e. SIFT, Polyphen-2, and MutationTaster). Predictionprograms indicated that all NIS mutants might be disease associated.

Functional studies in vitro demonstrated that the NIS mutants p.D369V, p.S547R, andP.G543K were associated with non-detectable I⁻ accumulation when transientlytransfected into HEK-293T cells, which do not express NIS endogenously. The NISmutants p.Q136L, p.D331N, and L562M have not been tested yet. Moreover, flowcytometry, immunofluorescence and immunoblot analysis revealed that thesemutant proteins are completely retained in the endoplasmic reticulum and,therefore do not reach the plasma membrane.

Conclusions

Although themechanism by which these mutants impair NIS transport to the plasma membraneremains unknown, here we provide a brief characterization of novel molecularevents leading to defective I^- accumulation in the thyroidfollicular cell.