A three nucleotide difference in the HNF-1 binding site of the thyroxine-binding globulin (TBG) promoter causes low TBG expression in dog

Objective: Thyroxine-binding globulin (TBG) is the main thyroid hormone transport protein in blood and accounts for over 80% of T4 binding in most mammals. Dogs have been reported to have reduced T4 binding capacity in blood, supposedly due to TBG deficiency. We present a study on thyroid function tests and TBG expression in dogs. Methods: Thyroid function tests (TSH, free T4 [FT4] and total T4 [TT4]) were measured in serum from 658 dogs by automated chemiluminescence immunoassays. T4 binding capacity was measured by a TBG-specific T4 binding assay. To study dog TBG expression, we sequenced the gene and its promoter and determined its ability to drive luciferase expression after transfection in HepG2 cells. Results: The ratio of free T4 to total T4 was higher in dog sera (1:2000; FT4 10.75 pmol/l: TT4 20.31 nmol/l) than in human sera (1:6400; FT4 15.89 pmol/l: TT4 101.41 nmol/l), indicating a lower T4 binding capacity in dog sera. This could be verified by the T4 binding assay: compared to human serum set as 100%, dog sera had a greatly reduced T4 binding capacity of 7%±3% (n=20), similar to other carnivorous mammals not expressing TBG. In the dog TBG promoter, three adjacent nucleotides within the 13 nucleotide binding site for its most relevant transcription factor, hepatocyte nuclear factor (HNF)-1, differ from the HNF-1 consensus and the human sequence. We compared the transcriptional activity of the human TBG promoter and a mutant matching the dog HNF-1 binding site sequence. With the dog HNF-1 sequence, the TBG promoter completely lost the ability to drive luciferase expression (human: 317±34 vs. dog: 0.37±0.01; P<0.001; expressed as relative light units, RLU). In contrast, the mouse HNF-1 binding site differs by only one nucleotide from the human TBG promoter and had similar transcriptional activity (human: 317±34 vs. mouse: 302±2.4 RLU; n.s.). Conclusion: A three nucleotide difference in the HNF-1 binding site of the dog TBG promoter severely impairs its transcriptional activity and leads to low T4 binding capacity in dogs.