Neanderthal-derived variants shape craniofacial enhancer activity at a human disease locus

Facial appearance is one of the most variable morphological features between humans. There is a major genetic component to facial shape with contributions from both rare and common genetic variants. Deletion of an enhancer cluster 1.45 megabases upstream of the SOX9 gene (EC1.45) results in Pierre Robin sequence, a human craniofacial disorder characterised by underdevelopment of the lower jaw and frequently associated with cleft palate. We reasoned that single nucleotide variants in EC1.45 may cause more subtle alterations to facial morphology. Here, we took advantage of recent human evolution, and the distinct morphology of the Neanderthal lower jaw, to explore the impact of three Neanderthal-derived single nucleotide variants on EC1.45 function and jaw development. Utilising a dual enhancer-reporter system in zebrafish, we observed enhanced Neanderthal regulatory activity relative to the human orthologue during a specific developmental window. We show that EC1.45 is selectively active in neural crest-like progenitor cells which lie in close apposition with and are transcriptionally related to precartilaginous condensations that contribute to craniofacial skeletal development. To test the potential consequences of increased SOX9 expression in this specific cellular population during jaw development, we overexpressed human SOX9 specifically in EC1.45-active cells and observed increased volume of developing cartilaginous precursors. Together our work implicates Neanderthal-derived variants in altering the activity of a disease-associated enhancer with potential to impact craniofacial skeletal development and jaw morphology across recent hominin evolution.