Two cases of thyroid dysgenesis caused by different novel PAX8 mutations in the DNA-binding region: in vitro studies reveal different pathogenic mechanisms

Abstract

Background: Mutations in PAX8, a transcription factor gene, cause thyroid dysgenesis (TD). The extreme variability of the thyroid phenotype makes it difficult to identify individuals harboring PAX8 gene mutations. Here we describe two patients with TD and report two novel PAX8 gene mutations (S54R and R133Q). We performed in vitro studies to functionally characterize these mutations.

Methods: Using PAX8 expression vectors, we investigated whether the PAX8 mutants localized correctly to the nucleus. To analyze the DNA-binding properties of S54R and R133Q, electrophoretic mobility shift assays were performed. Furthermore, we measured whether the mutant PAX8 proteins were able to activate the thyroglobulin (TG)- and the thyroperoxidase (TPO)-promoters.

Results: S54R had an impaired binding to DNA and a negligible activity on the TG- and the TPO-promoters. The DNA-binding property of R133Q, which is located in the highly conserved terminal portion of the PAX8 DNA-binding domain, was normal. Interestingly, it also exhibited dramatically impaired activation of the TG- and TPO-promoters. However, R133Q has no dominant negative effect on the WT protein in vitro. Thus, the underlying molecular mechanism by which the function of R133Q is impaired remains to be elucidated.

Conclusions: We identified and functionally characterized two novel mutations of the PAX8 gene that lead to TD by distinct mechanisms. A structural defect of the mutant R133Q leading to a reduced capability for induced fit upon DNA interaction might explain the disparity between its apparently normal binding to DNA, but lack of promoter activation.

FIG. 1.

Pedigree of the two families. Individuals with thyroid dysgenesis are indicated with a filled black box.

FIG. 2.

Nuclear localization of the two identified PAX8 gene mutations. HeLa cells were transiently transfected with plasmids expressing a PAX8-EGFP-WT or PAX8-EGFP-mutant (S54R, R133Q) and TTF1-EGFP-WT as a control. The PAX8 and TTF1 proteins were visualized by immunofluorescence microscopy. All proteins (WT and S54R, R133Q) are localized in the nucleus.

FIG. 3.

Electrophoresis mobility shift assay with the thyroid peroxidase (TPO) promoter-binding site and the thyroglobulin (TG) promoter-binding site. Oligonucleotides containing the PAX8-binding sequences of the TPO and TG gene promoters, respectively, were synthesized and labeled with an infra red dye (IRD700). The in vitro synthesized PAX8 proteins were incubated with labeled oligonucleotides, separated via PAG, and visualized (for details see Materials and Methods). (A) PAX8 WT as well as the mutant R133Q binds normally to the promoter sequence. In contrast, S54R lost its binding ability to the TPO response element. Furthermore, the binding to the TG promoter sequence is shown. Compared to WT and R133Q, S54R binds weakly to the PAX8-binding sites of the TG promoter. (B) The DNA binding of WT, R133Q, and S54R is specific, since a decreased shift can be observed after adding increasing amounts of unlabeled oligos.

FIG. 4.

Transient transfection of HeLa cells utilizing a TG-luciferase reporter gene. HeLa cells were transiently transfected with 0.25 μg TG-luciferase and various amounts of WT or mutant PAX8 expression constructs. At all DNA concentrations tested, the mutations S54R (gray bars) and R133Q (white bars) have a significantly reduced transcriptional activity on the reporter gene.

FIG. 5.

S54R exerts a dominant negative effect, whereas R133Q has no dominant negative effect. HeLa cells were transiently transfected with 0.25 μg TG-luciferase reporter constructs as well as 0.02 μg or 0.04 μg of PAX8 WT or R133Q or S54R expression constructs, respectively. In addition, WT and mutants were transfected together (0.02 μg WT+0.02 μg mutant, or 0.02 μg WT+0.04 μg mutant). pcDNA was utilized to keep equal amounts of DNA in each transfection.