Electrophoretic mobility shift assay of NR5A1 mutants.

<p>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 <i>Amh</i> 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.</p

New <i>NR5A1</i> mutations and phenotypic variations of gonadal dysgenesis

<div><p>Mutations in <i>NR5A1</i> have been reported as a frequent cause of 46,XY disorders of sex development (DSD) associated to a broad phenotypic spectrum ranging from infertility, ambiguous genitalia, anorchia to gonadal dygenesis and female genitalia. Here we present the clinical follow up of four 46,XY DSD patients with three novel heterozygous mutations in the <i>NR5A1</i> gene leading to a p.T40P missense mutation and a p.¹⁸DKVSG²²del nonframeshift deletion in the DNA-binding domain and a familiar p.Y211Tfs*83 frameshift mutation. Functional analysis of the missense and nonframeshift mutation revealed a deleterious character with loss of DNA-binding and transactivation capacity. Both, the mutations in the DNA-binding domain, as well as the familiar frameshift mutation are associated with highly variable endocrine values and phenotypic appearance. Phenotypes vary from males with spontaneous puberty, substantial testosterone production and possible fertility to females with and without Müllerian structures and primary amenorrhea. Exome sequencing of the sibling’s family revealed <i>TBX2</i> as a possible modifier of gonadal development in patients with <i>NR5A1</i> mutations.</p></div

Transactivation of the human <i>AMH</i>- and <i>STAR</i>-promoter.

<p>The transactivation capacity of mutant NR5A1-T40P and NR5A1-¹⁸DKVSG²²del were compared to wt-NR5A1 using human <i>AMH-</i> (A) and <i>STAR</i>-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).</p

Hormonal and clinical data of patients.

<p>Hormonal and clinical data of patients.</p

Electropherogram of <i>NR5A1</i> mutations.

<p>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.</p