A conserved regulatory atlas reveals enhancers critical for mouse and human gonadal development

Differences of Sex Development (DSD) are congenital conditions where sex development is atypical. Like many other rare conditions, genetic diagnostic rates for DSD still sit below 50%. Unexplained cases may harbour disruptions in non-coding regulatory regions, which remain largely unexplored. Here, we integrate chromatin accessibility datasets from mouse and human embryonic gonads to construct a complete comparative regulatory atlas of gonad development. Using this resource, we identified a conserved upstream enhancer of the key gene, Wt1. We identify rare variants in this enhancer in eight individuals with DSD and demonstrate that its deletion in mice results in XY male-to-female sex reversal and XX sub-fertility, thereby establishing it as critical for gonadal development. We build on this atlas to create a tissue specific computational filtration pipeline to prioritise non-coding variants in whole-genome sequencing datasets from 74 undiagnosed individuals with DSD. This dramatically reduces millions of potential candidates to a small set of candidate variants. This led to identification of a variant that disrupts the activity a conserved enhancer upstream of NR5A1 which segregates with gonadal dysgenesis in a family. Our study reveals regulatory elements essential for gonadal development and provides a framework for uncovering pathogenic non-coding variants in other rare developmental conditions.