Enhancers control gene expression and are linked to many diseases. The ZRS enhancer, crucial for limb development, contains 31 SNVs associated with polydactyly. This study shows that low-affinity ETS sites in the ZRS, particularly ETS-A, can be optimized by SNVs to increase binding affinity, leading to polydactyly. Two human SNVs, French 2 and Indian 2, subtly increase ETS-A affinity, causing similar phenotypes. A greater affinity increase results in more severe phenotypes. Affinity-optimizing SNVs in other enhancer sites also cause gain-of-function gene expression. The prevalence of low-affinity sites in enhancers makes them vulnerable to pathogenic SNVs that slightly increase affinity. This suggests that identifying such SNVs could help find causal variants for enhanceropathies. The ZRS enhancer is highly conserved and regulated by multiple transcription factors. The study shows that ETS-A, a low-affinity site, is functional and that SNVs in it can cause ectopic expression and polydactyly. Synthetic variants with increased affinity also cause similar effects. The study also shows that affinity-optimizing SNVs in other enhancer sites, such as HOX, IRF, and AP-1, can cause GOF expression. These findings suggest that low-affinity sites are important for enhancer specificity and that SNVs optimizing these sites can be pathogenic. The study highlights the importance of understanding enhancer mechanisms to identify causal variants. The results demonstrate that subtle changes in enhancer affinity can significantly affect gene expression and development. The study also shows that affinity-optimizing SNVs can be used to predict causal variants. The findings suggest that enhancer variants with increased affinity can cause developmental defects, and that understanding these variants is crucial for identifying disease-causing mutations. The study provides insights into the role of enhancer affinity in gene regulation and disease.Enhancers control gene expression and are linked to many diseases. The ZRS enhancer, crucial for limb development, contains 31 SNVs associated with polydactyly. This study shows that low-affinity ETS sites in the ZRS, particularly ETS-A, can be optimized by SNVs to increase binding affinity, leading to polydactyly. Two human SNVs, French 2 and Indian 2, subtly increase ETS-A affinity, causing similar phenotypes. A greater affinity increase results in more severe phenotypes. Affinity-optimizing SNVs in other enhancer sites also cause gain-of-function gene expression. The prevalence of low-affinity sites in enhancers makes them vulnerable to pathogenic SNVs that slightly increase affinity. This suggests that identifying such SNVs could help find causal variants for enhanceropathies. The ZRS enhancer is highly conserved and regulated by multiple transcription factors. The study shows that ETS-A, a low-affinity site, is functional and that SNVs in it can cause ectopic expression and polydactyly. Synthetic variants with increased affinity also cause similar effects. The study also shows that affinity-optimizing SNVs in other enhancer sites, such as HOX, IRF, and AP-1, can cause GOF expression. These findings suggest that low-affinity sites are important for enhancer specificity and that SNVs optimizing these sites can be pathogenic. The study highlights the importance of understanding enhancer mechanisms to identify causal variants. The results demonstrate that subtle changes in enhancer affinity can significantly affect gene expression and development. The study also shows that affinity-optimizing SNVs can be used to predict causal variants. The findings suggest that enhancer variants with increased affinity can cause developmental defects, and that understanding these variants is crucial for identifying disease-causing mutations. The study provides insights into the role of enhancer affinity in gene regulation and disease.