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

Objective: 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. Design: This was designed as an in vitro study. Main outcome: 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. Conclusion: 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.