title: 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: 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.
  
  
  METHODS
  This was designed as an in vitro study.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Clinical Endocrinology Branch, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Coppotelli
authors.forename: Aaron
authors.initials: A
authors.lastname: Summers
authors.forename: Aaron
authors.initials: A
authors.lastname: Chidakel
authors.forename: Jaime M
authors.initials: JM
authors.lastname: Ross
authors.forename: Francesco S
authors.initials: FS
authors.lastname: Celi
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D000483:Alleles; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011110:Polymorphism, Genetic; D013961:Thyroid Gland / Q000378:metabolism*; D014158:Transcription, Genetic; D000097564:Iodothyronine Deiodinase Type II
chemical_list: D020121:5' Untranslated Regions; D007453:Iodide Peroxidase; D008156:Luciferases; D000097564:Iodothyronine Deiodinase Type II