Abstract
Disorders of sex development (DSDs) are conditions affecting development of the gonads or genitalia. 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. Here, we identified four DSD patients with overlapping duplications or deletions upstream of SOX9. Bioinformatic analysis identified three putative enhancers for SOX9 that responded to different combinations of testis-specific regulators. All three enhancers showed synergistic activity and together drive SOX9 in the testis. 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 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.
Highlights
In this study we redefine the upstream regulatory landscape of human SOX9
We focused on genetic intervals in the 2 Mb SOX9 upstream regulatory region that were previously associated with 46,XY and 46,XX DSD16–22
CGH-array showed these duplications overlapped with each other and the XYSR region, defining a minimum critical region of 5.2 kb (Fig. 1b). We anticipated that this redefined region would include a core gonadal enhancer for SOX9 implicated in both 46,XY and 46,XX Disorders of sex development (DSDs) (Fig. 1b)
Summary
In this study we redefine the upstream regulatory landscape of human SOX9. We refined the 32.5 kb XYSR and 24 kb RevSex intervals and analysed the 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. Deletion of these three enhancers in mice resulted in different outcomes ranging from: no apparent effect to reduced Sox[9] transcription and complete sex reversal. Given that duplication or deletion of these sequences results in sex reversal in SRY-negative 46,XX males and 46,XY females, respectively, our results suggest a mechanism by which these enhancers have crucial roles in human sex development and DSD
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