Introduction: I^? transportdefect (ITD) is an autosomal recessive disorder whose hallmark is the inabilityof the thyroid follicular cell to mediate active I^? accumulation. ITD is an uncommoncause of dyshormonogenetic congenital hypothyroidism due to loss-of-function mutationsin the Na⁺/I^? Symporter(NIS)-coding slc5a5 gene. Here, weaimed to determine whether a pediatric patient with a clinical phenotype of ITDharbors an inactivating mutation in the slc5a5gene, and if so, to ascertain the molecular mechanisms of the effect of themutation on NIS function.

Methods: The wholecoding region of the slc5a5 gene wasPCR-amplified and subjected to Sanger sequencing, and in silico computational and invitro functional studies of a newly identified NIS mutation were performed.

Results: We reporta novel homozygous missense and loss-of-function mutation in the slc5a5 gene as a cause of dyshormonogenic congenitalhypothyroidism. The proband carries a G>A transition at position +1.682 inexon 14 resulting in a Gly to Glu substitution at residue 561 (G561E) locatedin the intracellularly facing NIS carboxy-terminus. We show that G561E markedlyreduces I^? uptakewhen the protein is heterologously expressed in MDCK-II cells, because thetransport of G561E NIS to the plasma membrane is severely impaired. Significantly,G561Q NIS, like G561E, is severely retained in the endoplasmic reticulum. Bioinformaticsreveal fully conserved tryptophan-acidic (WD) motif whose disruption(W565A/D566A NIS) leads to NIS retention in the endoplasmic reticulum. Computationaland biochemical analysis indicates that G561E impair the recognition of the adjacentWD motif by the kinesin light chain (KLC) 2, thus impairing mutant NIS exitfrom the endoplasmic reticulum. Moreover, short-hairpin RNA-mediated KLC2knock-down in FRTL-5 cells reduces NIS expression at the plasma membrane, andconsequently minimal NIS-mediated I^? accumulation.

Conclusions: Altogether,our data indicate that G561E shifts the equilibrium of the unstructured WDmotif towards a more structured and rigid conformation unable to interact with KLC2,thus severely affecting NIS maturation beyond the endoplasmic reticulum and reducingI^?accumulation in the thyroid follicular cell.