Table 1.
Hormonal and clinical data of patients.
Fig 1.
Electropherogram of NR5A1 mutations.
Electropherogram of the heterozygous c.118A>C mutation in patient 1 (A), the nonframeshift deletion c.51_65delGGACAAGGTGTCCGG in patient 2 (B), the frame-shift deletion c.630_636delGTACGGC in patient 3 (C) and the pedigree of the family of patient 3 and 4 (D). Circles denote phenotypic females, squares denote phenotypic males. Filled squares and circles correspond to a DSD condition, dots to a carrier status.
Fig 2.
Scheme of the DNA-binding domain containing the zinc finger domain (ZnF) and the Ftz-F1 box, the hinge region (HR) and the ligand binding domain (LBD) with activation function 2 (AF2) of NR5A1. Positions of the mutation p.T40P, p.D18_G22del and amino acid Y211 are indicated.
Fig 3.
Transactivation of the human AMH- and STAR-promoter.
The transactivation capacity of mutant NR5A1-T40P and NR5A1-18DKVSG22del were compared to wt-NR5A1 using human AMH- (A) and STAR-promoter (B) containing reporter genes. The empty pCMV-Myc vector represents background activity. Wild type (WT) activity was set 100%. RLU = relative luciferase activity. Error bars represent standard deviations, ***P = <0.001, t-test comparison of WT and mutant. C: Immunoblot detection of myc-tagged NR5A1 proteins. Equal loadings were verified by detection of total proteins using the TGX Stain Free system (BioRad). D: statistical analysis of 3 western blots of three different experiments showing an approximately equal protein accumulation of NR5A1-WT and mutant 18DKVSG22del, while mutant T40P showed an 2–3 fold enhanced accumulation. Images were quantified and normalized to total protein using Image Lab 5.2.1 (BioRad).
Fig 4.
Electrophoretic mobility shift assay of NR5A1 mutants.
A) DNA-binding capability of mutant NR5A1-T40P and NR5A1-18DKVSG22del were compared to wt-NR5A1 using a SF-1 responsive element of the mouse Amh promoter. Nuclear extracts containing WT-SF1 shifted the labelled probe (arrow). A 200 fold excess of unlabelled competitor abolished the shifted signal, while a 200 fold excess of unlabelled mutated competitor rescues the shifted signal. Both mutant proteins have lost their DNA-binding capability. B) Aliquots of the nuclear extracts used in EMSA were separated by SDS gel electrophoresis, transferred to a nitrocellulose membrane and stained with Ponceau S and anti-myc antibodies to verify equal SF-1 protein concentrations.