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H2AFY promoter deletion causes PITX1 endoactivation and Liebenberg syndrome
  1. Bjørt K Kragesteen1,2,
  2. Francesco Brancati3,4,
  3. Maria Cristina Digilio5,
  4. Stefan Mundlos6,7,
  5. Malte Spielmann1,6
  1. 1 Human Molecular Genomics Group, Max Planck Institute for Molecular Genetics, Berlin, Germany
  2. 2 Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
  3. 3 Istituto Dermopatico dell’Immacolata, IDI-IRCCS, Rome, Italy
  4. 4 Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy
  5. 5 Medical Genetics, Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, Rome, Italy
  6. 6 Institute for Medical and Human Genetics, University Medicine Berlin, Berlin, Germany
  7. 7 Development and Disease Group, Max Planck Institute for Molecular Genetics, Berlin, Germany
  1. Correspondence to Dr Malte Spielmann, Max-Planck-Institute for Molecular Genetics, Berlin 14195, Germany; spielmann{at}


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.

  • pitx1
  • cnvs
  • liebenberg
  • non coding mutations

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  • Contributors BKK and MS: designed the study, collected the data and performed the genetic investigations. BKK performed mouse experiments. BKK and MS: analysed the data and wrote the manuscript. FB, MCD, SM and MS: performed the clinical evaluation of the patients. All authors revised and approved the final version.

  • Funding This study was supported by a grant from the Deutsche Forschungsgemeinschaft (SP1532/2-1). FB received funding from grant GR-2013-02356227.

  • Competing interests None declared.

  • Patient consent Obtained.

  • Provenance and peer review Not commissioned; externally peer reviewed.