ESPE Abstracts (2016) 86 FC15.1

aCenter for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium; bDepartment of Pathology, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands; cFlanders Institute for Biotechnology (VIB), Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium;
dMolecular Oncology and Pathology, Institut Jacques Monod, France; Université Paris Diderot, Paris, France; eDepartment of Pediatrics, Division of Pediatric Endocrinology, Ghent University Hospital and Ghent University, Ghent, Belgium

Background: While many causes of 46,XY disorders of sex development (DSD) have been elucidated, the mechanisms leading to 46,XX testicular or ovotesticular DSD are poorly understood. It has been hypothesized that both conditions may represent a phenotypic spectrum and that they might be caused by (different) mutations in the same genes.

Methods: Whole exome sequencing was used to identify the molecular cause in ten unrelated patients with 46,XX (ovo)testicular DSD. Transcriptional activation of the variant of interest was assessed using luciferase assays and RNA-seq was performed on patient-derived lymphocytes. Immunohistochemistry was done on gonadal specimens for markers of gonadal development.

Results: We identified a novel heterozygous NR5A1 variant c.274C>T p.(Arg92Trp) in three unrelated 46,XX (ovo)testicular DSD cases. This variant is absent in genomic databases and is predicted to be deleterious. The Arg92 residue is conserved up to zebrafish and located in the RGGR motif in the loop before the C-terminus helix in the conserved Ftz-F1 box. This domain was previously reported to be important for DNA binding specificity and stability. Structural remodeling indeed shows that Trp cannot be introduced without steric clashes. Immunohistochemistry confirmed SRY-independent SOX9 expression and absent FOXL2 in testicular parts of XX gonads and vice versa in ovarian regions. No consistent changes in transcriptional activation were seen. RNA-seq showed upregulation of MAMLD1, a direct target of NR5A1, that has already been associated with hypospadias and 46,XY DSD.

Conclusions: Previously, loss-of-function mutations in NR5A1 were found in a spectrum of male undervirilization and premature ovarian insufficiency in 46,XX females. Here, a novel NR5A1 mutation was found in three unrelated cases with 46,XX testicular or ovotesticular DSD, an ultra-rare condition. We hypothesize that this variant results in upregulation of specific target genes, thereby triggering testicular differentiation in 46,XX individuals. We propose NR5A1 mutation p.(Arg92Trp) as a new cause for 46,XX (ovo)testicular DSD.

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