Affinity-optimizing variants within the ZRS enhancer disrupt limb development

SummaryAn emerging regulatory principle governing enhancers is the use of suboptimal affinity binding sites to encode tissue-specific gene expression. Here we investigate if optimizing single-nucleotide variants that violate this principle can disrupt tissue-specific gene expression and development. The ZRS enhancer mediates expression of Shh in the posterior of the developing limb buds and is critical for limb and digit development. We find that the ZRS contains suboptimal-affinity ETS binding sites. Two human mutations and a synthetic mutation that optimize the affinity of the ETS-A site from 0.15 to 0.25 relative binding affinity cause polydactyly with the same penetrance and severity. Further increasing the affinity of the ETS-A site results in more penetrant and severe phenotypes. The prevalent use of suboptimal affinity binding sites within enhancers to encode tissue-specificity creates a vulnerability within genomes whereby variants that optimize affinity, even subtly, can be pathogenic. This provides a generalizable approach to identify causal variants that underlie enhanceropathies. In BriefSubtle increases in low-affinity sites underlie human limb defects, while greater increases in affinity lead to more severe and penetrant phenotypes. HighlightsPrediction and validation of pathogenic enhancer variants Very subtle increases in affinity of low-affinity sites are pathogenic Penetrance and severity of phenotype scales with increase in affinity