Identification of a limb enhancer that is removed by pathogenic deletions downstream of the SHOX gene

Haploinsufficiency of the human SHOX gene causes Léri-Weill dyschondrosteosis (LWD), characterized by shortening of the middle segments of the limbs and Madelung deformity of the wrist. As many as 35% of LWD cases are caused by deletions of non-coding sequences downstream of SHOX that presumably remove an enhancer or enhancers necessary for SHOX expression in developing limbs. We searched for these active sequences using a transgenic mouse assay and identified a 563 basepair (bp) enhancer with specific activity in the limb regions where SHOX functions. This enhancer has previously escaped notice because of its poor evolutionary conservation, although it does contain 100 bp that are conserved in non-rodent mammals. A primary cell luciferase assay confirmed the enhancer activity of the conserved core sequence and demonstrated that putative HOX binding sites are required for its activity. This enhancer is removed in most non-coding deletions that cause LWD. However, we did not identify any likely pathogenic variants of the enhancer in a screen of 124 LWD individuals for whom no causative mutation had been found, suggesting that only larger deletions in the region commonly cause LWD. We hypothesize that loss of this enhancer contributes to the pathogenicity of deletions downstream of SHOX.