A mutation in FGF9 identified in 46,XY Sex Reversal — ASN Events
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A mutation in FGF9 identified in 46,XY Sex Reversal (#158)

Brittany Croft 1 2 , Anthony Bird 1 , Makoto Ono 1 3 , Stefanie Eggers 2 4 , Stefan Bagheri-Fam 1 , Janelle Ryan 1 , Patrick Western 1 , Andrew Kueh 5 , Elizabeth Thompson 6 , Tim Thomas 5 , Peter Stanton 1 , Masayo Harada 7 , Andrew Sinclair 2 , Vincent Harley 1
  1. Hudson Institute of Medical Research, Melbourne, VIC, Australia
  2. Murdoch Childrens Research Institute, Parkville, Melbourne, Victoria, Australia
  3. Department of Paediatrics, Tokyo Bay Urayasu Ichikawa Medical Centre, Chiba, Japan
  4. Victorian Clinical Genetics Services, Royal Children's Hospital, Melbourne, Victoria, Australia
  5. Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria, Australia
  6. SA Clinical Genetics Service, Women’s and Children’s Hospital, Adelaide, South Australia, Australia
  7. Department of Clinical Anatomy, Tokyo Medical and Dental University, Tokyo, Japan

Background: Disorders of sex development (DSDs) are a range of congenital conditions, including 46,XY gonadal dysgenesis (GD), where only 30% of cases receive a specific molecular diagnosis. Improving our understanding of the genetic causes of 46,XY GD is critical to improve clinical diagnosis and management of these conditions. Among the genes promoting male sex determination, induced by upstream SRY/SOX9 signalling, is fibroblast growth factor (FGF) 9. Expressed within the pre-Sertoli cell lineage, FGF9 suppresses female gonadal development via its receptor FGFR2. In mice, both are critical for testis determination as FGF9/FGFR2 knockouts show XY sex reversal. Despite this, to date no FGF9 gene mutations/deletions/insertions have been identified in human DSD patients.

Results: A 1032 gene panel specific to DSD has identified an FGF9 variant, a maternally derived heterozygous single nucleotide substitution c.583G>A (p.D195N), in a 46,XY GD female. Who presented with delayed puberty, primary amenorrhea, clitromegaly, Müllerian duct remnants, and raised testosterone levels. In silico analysis predicted the D195N variant to be deleterious for FGF9 protein function. In vitro studies indicated the D195 residue lies at the homodimerisation interface, which is an essential residue for FGF9 structure and function.  

Purified recombinant FGF9-D195N protein showed reduced affinity for heparin, a property necessary for stable FGF-FGFR complexes. In vitro analysis has shown a reduced ability to induce Sertoli cell differentiation and proliferation, a vital requirement for the formation of a healthy testis in utero. To model the patients’ D195N mutation in vivo, Fgf9D195N/+ knockin mice were generated via CRISPR/Cas9 gene-editing. E15.5 Fgf9D195N/D195N embryonic XY gonads exhibit a truncated male-specific coelomic blood vessel. Immunofluorescence analysis revealed ectopic expression of the female meiotic marker γH2AX, indicative of XY sex reversal. A second FGF9 mutation has been described previously in a family with multiple synostosis, but no gonadal defects. Analysis of an established mouse model of multiple synostosis, bearing the Fgf9N143T/N143T mutation, also revealed a truncated coelomic blood vessel and partial sex reversal in the embryonic XY gonads.

Conclusion: Our results suggest that FGF9 homodimerisation and heparin binding are required for FGF9 function during testis development. A disruption in one or both of these pathways has led to sex reversal in a 46,XY GD patient. As such, human FGF9 mutations may underlie a proportion of isolated DSD cases.

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