Research in Disorders of Sexual Development (DSD) has played a major role in understanding the genetic control of sex determination and the opposing pathways controlling gonadal development. In the testis pathway, SOX9 expression is critical to testis induction. Conversely, extinguishing SOX9 expression appears essential for normal ovarian development. For example, in XX DSD patients with SOX9 duplications, testes develop in XX individuals lacking the Y-linked testis-determining gene, SRY. Recent studies of the ovary pathway have not yet identified the long sought mechanism by which SOX9 transcription is normally extinguished in XX gonads. This could be a key step in the ovary pathway that opposes the testis pathway. Our goal is to identify a causative mutation in the subtype of XX DSD in which XX individuals develop testes while their XX siblings develop ovotestes. Studies in these patients have been impeded because the disorder is uncommon, family sizes are small, and there are no rodent models. The canine model is the only model of this XX DSD subtype. In the canine research pedigree, XX DSD is an autosomal recessive trait with expression limited to XX siblings, which develop testes or ovotestes. Early studies suggested that the testis pathway is incompletely suppressed or inappropriately activated in these gonads. Using GWAS, we have identified and resequenced a region significantly associated with canine XX DSD in the model pedigree. This region overlaps the regulatory region of SOX9.
Our Specific Aims are:
Specific Aim 1 Hypothesis: The causative mutation for canine XX DSD lies within a genetic switch that normally extinguishes SOX9 transcription in XX gonads. To test this hypothesis, we will produce transcriptomes [RNA-seq] to complete a pilot study comparing gonadal gene expression in XX DSD embryos to that of normal XX and XY controls. If our hypothesis is correct, SOX9 expression will be greater in XX DSD gonads than those of XX controls, perhaps approaching levels observed in XY gonads. If our hypothesis is incorrect, the gonadal gene expression data from this project will be valuable for constructing alternative hypotheses.
Specific Aim 2 Hypothesis: The causative mutation for canine XX DSD lies within a genetic switch that is conserved in mammals. To test this hypothesis, we will use a combined comparative genomics and bioinformatics approach to compare two existing datasets, a microarray dataset from XX DSD patients and resequencing data from the canine XX DSD model. If our hypothesis is correct, we will identify orthologs in XX DSD individuals (humans and dogs) that are evolutionarily conserved, yet contain nucleotides that are different from the reference genomes. Those nucleotide differences will be our candidate mutations. Results from this project will remove two major obstacles to the study of XX DSD by identifying candidate mutations in human patients and the canine model, and confirming that the canine XX DSD model is an appropriate model in which to further characterize key elements in the testis and ovary pathways.
The mechanism by which 46,XX patients develop testes while their 46,XX siblings develop ovotestes is unknown. The canine model is the only model of this XX DSD subtype. Identification of candidate mutations and altered gonadal gene expression in the model could change our understanding of sex determination and improve diagnostic testing for XX DSD patients.