Functional Characterization of the 258 A/G (D2-ORFa-Gly3Asp) Human Type-2 Deiodinase Polymorphism: A Naturally Occurring Variant Increases the Enzymatic Activity by Removing a Putative Repressor Site in the 5′ UTR of the Gene

Abstract

5' deiodination of thyroid hormone represents an important step in the modulation of the hormonal message. Previous studies indicate that the naturally occurring polymorphism located in 5'-untranslated region of the gene, 258 A/G, is associated with a decrease in circulating T4/T3 ratio, suggesting an increased gene expression. The aim of this study was to characterize the gene variant in vitro. This was designed as an in vitro study. The wild-type and mutant promoters were cloned into a reporter vector and transfected into HEK-293, GH3, and H3B cells. Compared to the 258G wild-type allele, the 258A variant had an increased basal activity in all the cell lines (HEK-293 258A 13998 +/- 371.9 RLU vs. 258G 5593 +/- 124.2 RLU, p < 0.0001). Electrophoresis mobility shift assay (EMSA) experiments were performed with nuclear extracts obtained from HEK-293 cells and from human thyroid, muscle, and liver. The EMSA experiments showed that the 258A variant decreased the binding ability of a nuclear protein in HEK-293 cells, thyroid, and muscle. No specific binding was observed in liver nuclei. These data suggest that the increase in gene transcription induced by the 258A polymorphism could be mediated by reduction in the binding ability of a putative nuclear repressor.