BACKGROUND: Structural variants (SVs) affecting non-coding cis-regulatory 
elements are a common cause of congenital limb malformation. Yet, the functional 
interpretation of these non-coding variants remains challenging. The human 
Liebenberg syndrome is characterised by a partial transformation of the arms 
into legs and has been shown to be caused by SVs at the PITX1 locus leading to 
its misregulation in the forelimb by its native enhancer element Pen. This study 
aims to elucidate the genetic cause of an unsolved family with a mild form of 
Liebenberg syndrome and investigate the role of promoters in long-range gene 
regulation.
METHODS: Here, we identify SVs by whole genome sequencing (WGS) and use 
CRISPR-Cas9 genome editing in transgenic mice to assign pathogenicity to the 
SVs.
RESULTS: In this study, we used WGS in a family with three mildly affected 
individuals with Liebenberg syndrome and identified the smallest deletion 
described so far including the first non-coding exon of H2AFY. To functionally 
characterise the variant, we re-engineered the 8.5 kb deletion using CRISPR-Cas9 
technology in the mouse and showed that the promoter of the housekeeping gene 
H2afy insulates the Pen enhancer from Pitx1 in forelimbs; its loss leads to 
misexpression of Pitx1 by the pan-limb activity of the Pen enhancer causing 
Liebenberg syndrome.
CONCLUSION: Our data indicate that housekeeping promoters may titrate 
promiscuous enhancer activity to ensure normal morphogenesis. The deletion of 
the H2AFY promoter as a cause of Liebenberg syndrome highlights this new 
mutational mechanism and its role in congenital disease.