Oral Presentation 40th Annual Lorne Genome Conference 2019

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9 (#15)

Brittany Croft 1 2 3 , Thomas Ohnesorg 1 4 , Jacky Hewitt 1 5 , Josephine Bowles 6 7 , Alexander Quinn 7 , Jacqueline Tan 1 , Vincent Corbin 8 , Emanuele Pelosi 7 , Jocelyn van den Bergen 1 , Rajini Sreenivasan 1 3 , Ingrid Knarston 1 , Gorjana Robevska 1 , Dung Chi Vu 9 , John Hutson 1 4 , Vincent Harley 3 , Katie Ayers 1 4 , Peter Koopman 7 , Andrew Sinclair 1 4
  1. Murdoch Childrens Research Institute , Melbourne, VIC, Australia
  2. Department of Molecular and Translational Science, Monash University, Melbourne, VIC, Australia
  3. Hudson Institute of Medical Research, Melbourne, VIC, Australia
  4. Department of Paediatrics , University of Melbourne, Melbourne, VIC, Australia
  5. Department of Paediatric Urology, Monash Childrens Hospital, Melbourne, VIC, Australia
  6. School of Biomedical Sciences, University of Queensland, Brisbane, QLD, Australia
  7. Institute for Molecular Bioscience , The University of Queensland, Brisbane, QLD, Australia
  8. Bioinformatics Division, Walter & Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia
  9. Department of Medical Genetics, Metabolism & Endocrinology, National Children’s Hospital, Hanoi, Vietnam

Disorders of sex development (DSDs) are conditions affecting the development of the gonads or genitalia. Variants in two key genes directing the early stages of gonadal development — testis-determining gene SRY and its direct target SOX9 — are an established cause of 46,XY DSD, but the genetic basis of many DSDs remains unknown. SRY-mediated SOX9 upregulation in the early gonad is crucial for testis development, yet the elements that regulate SOX9 have not been identified in humans.

We screened a cohort of 44 DSD patients for Copy Number Variations (CNVs) in the upstream regulatory region of SOX9, using CGH-array technology. Leading to the identification of four DSD patients with duplications or deletions upstream of SOX9 that allowed the precise mapping of the minimal critical regions required for sex-reversal. Bioinformatic analysis of the overlapping CNVs identified three putative enhancers for SOX9, and luciferase tiling assays confirmed their potential as novel SOX9 human testis enhancers. In cell-based reporter assays, these enhancers responded to different combinations of testis-specific regulators including SRY, NR5A1 (SF1) and SOX9 itself, and were repressed by a female sex-determining factor FOXL2.

The three enhancers play different roles in SOX9 initiation, upregulation and maintenance. Importantly, all three enhancers showed synergistic activity and together co-operate to drive SOX9 up-regulation over two-fold in the male gonad. One enhancer is human-specific, the second has a conserved function in humans and mice resulting in complete sex reversal when deleted, and mice null for the third enhancer showed reduced Sox9 transcription without sex reversal.

This is the first study to identify SOX9 core enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. Together, these three enhancers provide a hitherto missing link of how SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.