Oral Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

First FGF9 mutation in a patient with a disorder of sex development (#256)

Daniel Bird 1 , Makoto Ono 1 , Stefanie Eggers 2 , Brittany Croft 1 , Stefan Bagheri-Fam 1 , Janelle Ryan 1 , Andrew Kueh 3 , Peter Stanton 1 , Tim Thomas 3 , Andrew Sinclair 2 , Masayo Harada 4 , Vincent Harley 1
  1. Hudson Institute of Medical Research, Clayton, VIC, Australia
  2. Murdoch Childrens Research Institute, Parkville, VIC, Australia
  3. Walter and Eliza Hall Institute, Parkville, VIC, Australia
  4. Tokyo Medical and Dental University, Tokyo, Japan

Disorders of sex development (DSDs) include 46,XY gonadal dysgenesis (GD), where a specific genetic diagnosis is made in only ~30% of patients. Improved understanding of the genetic causes of 46,XY GD is therefore required to better inform clinical diagnosis and management. Among the genes induced by upstream SRY-SOX9 signalling to promote male sex determination is fibroblast growth factor (FGF) 9. Expressed within the pre-Sertoli cell lineage, FGF9 suppresses the female program of development via its receptor FGFR2c. In mouse both are critical for testis determination as FGF9/FGFR2c knockout mice show XY sex reversal. Despite this, to date no FGF9 gene deletions/insertions have been identified in humans. However, patient screening of a 46,XY GD female patient with a 1032 DSD gene panel identified a missense variant in FGF9: D195N. In silico analysis predicted the D195N variant to be deleterious for FGF9 protein function, and in vitro studies indicated the D195 residue lies at the homodimerisation interface. Purified FGF9 D195N protein showed a reduced affinity for heparin, a property necessary for stable FGF-FGFR complexes, and reduced ability to induce Sertoli cell proliferation in vitro. To model the D195N mutation in vivo, Fgf9D195N/+ knockin mice were generated via CRISPR/Cas9 gene-editing. Pilot studies showed that E15.5 Fgf9D195N/D195N embryonic XY gonads exhibit a truncated male-specific coelomic blood vessel, and immunofluorescence analysis revealed ectopic expression of the female meiotic marker gH2AX, indicative of sex reversal. We also investigated gonadal development in Fgf9N143T/N143T mice which also carry a mutation in the FGF9 homodimerisation domain. Likewise, E15.5 Fgf9N143T/N143T embryonic XY gonads show a truncated coelomic blood vessel and partial sex reversal. Together, these results suggest that FGF9 homodimerisation and heparin binding are required for FGF9 function in testes determination. In addition, human FGF9 mutations may be the cause of a subset of hitherto undiagnosed human DSD patients.