I- TRANSPORT DEFECT-CAUSING NIS MUTANTS UNCOVER A CRITICAL TRYPTOPHAN-ACID MOTIF REQUIRED FOR PLASMA MEMBRANE TRANSPORT

Buenos Aires

Congreso; XVII Latin American Thyroid Congress; 2019

Sociedad Latinoamericana de Tiroides

Introduction: I- transport defect (ITD) is an autosomal recessive disorder characterized by impaired thyroidal I- accumulation dueto loss-of-function mutations in the Na+/I- symporter (NIS)-coding SLC5A5 gene. Objectives: To characterize novel homozygous(p.G561E) and compound heterozygous (p.G543R and p.L562M) SLC5A5 mutants found in two ITD-suspected patients on thebasis of non-detectable I- accumulation in an eutopic thyroid gland. Methods: SLC5A5 gene coding region was PCR-amplified andsubjected to Sanger sequencing. In silico and functional in vitro studies of novel NIS variants were performed. Results: Functionalstudies revealed that G561E and L562M markedly reduces I- uptake, when the proteins are heterologously expressed in non-thyroidepithelial cells, because their targeting to the plasma membrane is severely impaired. G543R NIS was previously reported as nonfunctional. G561Q NIS, like G561E, is mainly retained in the endoplasmic reticulum (ER). Bioinformatics reveal a fully conservedtryptophan-acidic (WD) motif whose disruption leads to NIS retention in the ER. Computational and biochemical analysis indicatethat G561E and L562M impair the recognition of the flanking WD motif by the kinesin light chain (KLC) 2, thus impairing mutantNIS exit from the ER. Moreover, short-hairpin RNA-mediated KLC2 knock-down in FRTL-5 cells reduces NIS expression at theplasma membrane, and consequently NIS-mediated I- accumulation. Conclusion: Newly identified NIS variants negatively impact onthe three-dimensional structure of its flanking WD motif severely reducing the interaction with the ER-to-Golgi transport adaptorKLC2, thus impairing NIS maturation beyond the ER and reducing I- accumulation in thyroid follicular cells. Conflict of interest:None declared.