The study investigates the role of affinity-optimizing single-nucleotide variants (SNVs) in the ZRS enhancer, which is crucial for limb and digit development in vertebrates. The ZRS contains 31 human SNVs associated with polydactyly, a condition characterized by an extra digit. The researchers found that the ETS sites within the ZRS have low affinity, with one site, ETS-A, having an extremely low affinity of 0.15. Two human SNVs, French 2 and Indian 2, and a synthetic variant, Syn 0.25, subtly increase the affinity of ETS-A to around 0.24 to 0.26. These variants cause polydactyly in mice, with similar penetrance and severity. The study also found that a greater increase in affinity results in more severe and penetrant phenotypes. Affinity-optimizing SNVs in other ETS, IRF, HOX, and AP-1 sites across various enhancers cause gain-of-function (GOF) gene expression. The prevalence of suboptimal-affinity sites in enhancers suggests that SNVs optimizing these sites can be pathogenic. The findings highlight the importance of identifying and predicting causal enhancer variants by focusing on affinity-optimizing SNVs, which could improve the understanding and treatment of enhanceropathies.The study investigates the role of affinity-optimizing single-nucleotide variants (SNVs) in the ZRS enhancer, which is crucial for limb and digit development in vertebrates. The ZRS contains 31 human SNVs associated with polydactyly, a condition characterized by an extra digit. The researchers found that the ETS sites within the ZRS have low affinity, with one site, ETS-A, having an extremely low affinity of 0.15. Two human SNVs, French 2 and Indian 2, and a synthetic variant, Syn 0.25, subtly increase the affinity of ETS-A to around 0.24 to 0.26. These variants cause polydactyly in mice, with similar penetrance and severity. The study also found that a greater increase in affinity results in more severe and penetrant phenotypes. Affinity-optimizing SNVs in other ETS, IRF, HOX, and AP-1 sites across various enhancers cause gain-of-function (GOF) gene expression. The prevalence of suboptimal-affinity sites in enhancers suggests that SNVs optimizing these sites can be pathogenic. The findings highlight the importance of identifying and predicting causal enhancer variants by focusing on affinity-optimizing SNVs, which could improve the understanding and treatment of enhanceropathies.