Disorders of sex development (DSDs) are conditions affecting development of the gonads or genitalia. Until recently only 13% of DSD patients received a diagnosis. Using genomic sequencing approaches we have improved diagnostic rates for DSD to 43%. However, nearly 60% of patients still lack a diagnosis. Consequently, we explored whether disruption of the regulatory regions of gonad genes may account for a significant proportion of these undiagnosed patients.
Variants in two key genes, SRY and its 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 regulatory elements underlying this have not been identified in humans. We analysed copy number variations (CNVs) in the upstream regulatory region of SOX9 in DNA from patients with DSD, allowing us to define several minimal critical regions for sex-reversal. We redefined the upstream regulatory landscape of human SOX9. Using new patient data, we refined the 32.5 kb XYSR and 24 kb RevSex intervals and analysed these genomic regions using bioinformatic and luciferase tiling approaches, to identify three putative enhancers 5′ of SOX9. In cell-based reporter assays these enhancers responded to different combinations of testis-specific regulators including SRY, SF1 and SOX9 itself. When combined, all three enhancers show synergistic activity, significantly increasing their individual enhancer activity. In vivo, deletion of these three enhancers in mice resulted in different outcomes ranging from: no apparent effect to reduced Sox9 transcription and complete sex reversal.
This is the first study to identify SOX9 enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. These enhancers provide a hitherto missing link by which SRY activates SOX9 initiation, upregulation and maintenance in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.