Oral Presentation The Annual Scientific Meeting of the Endocrine Society of Australia and the Society for Reproductive Biology 2014

Identification of SOX9 enhancers disrupted in isolated disorders of sex development (#169)

Rajini Sreenivasan 1 2 , Sabina Benko 3 , Chris Gordon 3 , Stefan Bagheri-Fam 1 , Stanislas Lyonnet 3 , Vincent Harley 1
  1. MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia
  2. Department of Anatomy and Neuroscience, University of Melbourne, Parkville, VIC, Australia
  3. INSERM U-781, Hôpital Necker-Enfants Malades, Paris, France
Disorders of sex development (DSDs) are congenital conditions resulting in atypical chromosomal, gonadal or anatomic sex. Around 80% of 46,XY DSD cases still remain unexplained genetically. Phenotypes include gonadal dysgenesis, male-to-female sex reversal and hypospadias, where the urethra is abnormally positioned along the penis. Mutations in SOX9, a gene essential for testicular development, have been implicated in DSDs, but its tissue-specific regulation is not fully understood. SOX9 tissue-specific enhancer mutations can cause isolated clinical phenotypes. Through genome wide mapping, mutations around SOX9 have been identified in isolated DSD patients. All patients had gonadal defects and hypospadias. We hypothesised that a 78 kb region encompassing these mutations contains a novel gonadal enhancer that we aimed to identify. Comparative genomic analysis identified seven highly conserved sub-regions within TES2. Unexpectedly, transgenic mouse analysis revealed that the most highly conserved sub-region (SR4) was expressed in the genital tubercle (GT; penis primordium) instead of the gonad. Immunofluorescence analysis on mouse GT sections confirmed a novel domain of SOX9 expression within the urethral plate epithelium. These data suggest that loss of SOX9 expression due to SR4 mutations may have contributed to the hypospadias phenotype. To investigate whether SOX9 plays a direct role in hypospadias, we conditionally ablated Sox9 in the GT using mice transgenic for Shh-Cre. Immunofluorescence analysis confirmed loss of SOX9 expression in the urethra of Shh-Cre/+;Sox9flox/flox mice. Abnormal tail and limb development were observed, consistent with the role of Sox9 in bone development. However, preliminary analysis at stages before masculinisation revealed no abnormalities in the GT. GT development in these mice is currently being analysed at later stages. This study may reveal a novel cause of hypospadias. In conclusion, we have identified putative SOX9 enhancers that may be required for human testicular and GT development, the disruption of which cause DSDs.