Fig. 1. An ETS-A site in the ZRS enhancer contains two human variants that are associated with polydactyly, both of which subtly increase ETS binding affinity.

a, The human ZRS contains five known and functionally validated ETS sites, ETS1–ETS5, all of which have suboptimal affinity. We identify a new site, ETS-A, which has a relative affinity of 0.15. Six HOX sites (yellow) and one HAND2 site (pink) have also been previously identified. Thirty-one SNVs associated with polydactyly are found in humans (black bars), and SNVs are also found in other species such as cats, mice and chicks (green bars). b, Two human SNVs associated with polydactyly, denoted French 2 and Indian 2, occur within the ETS-A site. Both SNVs lead to a subtle increase in relative affinity of ETS-A to 0.24 and 0.26 respectively. c–f, The ETS-A sequence in a reference (Ref) mouse (c) drives the expression of Shh (d) and Ptch1 (e) restricted to the posterior domain of the developing limb bud in E11.75 and E12.0 embryos, respectively, as shown by in situ hybridization. f. Skeletal staining shows a WT mouse hindlimb with normal digit morphology. g–j, The French 2 SNV (g) drives the ectopic expression of Shh (h) and Ptch1 (i) in the anterior limb bud of homozygous embryos (arrow) in addition to the normal domain of posterior expression. j, A mouse hindlimb homozygous for French 2 has an extra triphalangeal thumb. k–n, The Indian 2 SNV (k) drives the ectopic expression of Shh (l) and Ptch1 (m) in homozygous embryos (arrow). n, A hindlimb from an Indian 2 homozygous mouse has an extra triphalangeal thumb. We did not calculate n for Shh because the expression is highly dynamic and thus hard to accurately capture; instead, we calculate the n of Ptch1 as a readout of Shh.