Iodide transport defect (ITD) is an autosomal recessive disorder caused by the inability of thyrocytes to actively accumulate iodide, the first and rate-limiting step in the thyroid hormone biosynthesis. The Na+/I- symporter (NIS), an intrinsic plasma membrane glycoprotein, represents a highly specialized transport system mediating the active iodide uptake from the bloodstream into the thyroid cell. Up to day, 12 different NIS molecular defects have been characterized as causes of ITD.
We aimed to analyze the presence of NIS gene mutations in patients diagnosed with congenital hypothyroidism (CH) due to a possible ITD.
We studied 6 non-related patients identified in a CH neonatal screening. The diagnosis of ITD was suspected because of the presence of variable goiter, increased TSH and reduced T4 levels as well as reduced or absent thyroidal technetium-99m (99mTc) uptake. Patients were classified in two groups: reduced (1-10%, n=4) or absent (less than 1%, n=2) 99mTc uptake.
Genomic DNA was extracted from peripheral blood mononuclear cells. The 15 exons encoding NIS sequence including untranslated regions and exon-intron boundaries were evaluated for PCR and sequencing. The analysis revealed the presence of an undescribed homozygous transition from C to T at position -54 (exon 1) located in the 5’ untranslated region (5’UTR) in one patient with reduced thyroid 99mTc uptake.
In order to understand the molecular basis and consequences of the discovered mutation functional analysis was performed. Human NIS cDNA wild-type (WT) and mutated (-54C→T) were stably transfected into COS-7 cells. Notably, cells transfected with the mutated NIS sequence only concentrated 5 to 10% of WT level (125Iodide), suggesting that the mutation could be the direct cause of the ITD. The analysis of total and plasma membrane NIS protein expression showed that while the WT sequence was present and properly located in the cell membrane, an almost absent protein expression of the mutant was evidenced. Surprisingly, NIS mRNA levels (RT/qPCR) were similar in the carrying NIS mutated sequence and WT cells. These results raised the possibility that the mutation may exert a translational deficiency.
Since the 5’UTR sequence has a very important role in mRNA stability and translation efficiency, we further analyzed the NIS mRNA polysomal profile of transfected COS-7 cells. Compatible with an active translation, WT NIS mRNA was associated with the polisomal fraction. Meanwhile, mutant NIS mRNA was significantly reduced in polysomes, consistent with a reduced translation rate and a subsequent reduced protein expression.
We describe a new mutation in the NIS gene (-54C→T) as a possible cause of CH. The present work represents the first description of an ITD conducted by a mutation outside of the coding region of NIS. The functional evaluation of the molecular mechanism responsible for the impaired NIS iodide concentration might indicate that the mutation reduced NIS translation initiation. Moreover, our results support the possibility that alterations of proteins related to NIS folding or targeting to the plasma membrane could also account as potential causes of CH since in this study no mutations on NIS sequence were found in the rest of patients with altered thyroid uptake.
