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Novel disease loci
Original research
New locus underlying auriculocondylar syndrome (ARCND): 430 kb duplication involving TWIST1 regulatory elements
  1. Vanessa Luiza Romanelli Tavares1,
  2. Sofia Ligia Guimarães-Ramos1,
  3. Yan Zhou2,
  4. Cibele Masotti1,3,
  5. Suzana Ezquina1,4,
  6. Danielle de Paula Moreira1,
  7. Henk Buermans5,
  8. Renato S Freitas6,
  9. Johan T Den Dunnen5,
  10. Stephen R F Twigg2,
  11. Maria Rita Passos-Bueno1
  1. 1 Genética e Biologia Evolutiva, Universidade de São Paulo Instituto de Biociências, Sao Paulo, Brazil
  2. 2 Clinical Genetics Group, MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK
  3. 3 Molecular Oncology Center, Hospital Sírio-Libanês, Sao Paulo, Brazil
  4. 4 Centre for Cancer Genetic Epidemiology, University of Cambridge, Cambridge, UK
  5. 5 Leiden Genome Technology Center, Leiden University Medical Center, Leiden, The Netherlands
  6. 6 Centro de Atendimento Integral ao Fissurado Lábio Palatal, Curitiba, Brazil
  1. Correspondence to Dr Maria Rita Passos-Bueno, Genética e Biologia Evolutiva, Universidade de São Paulo Instituto de Biociências, Sao Paulo, São Paulo, Brazil; passos{at}ib.usp.br; Dr Stephen R F Twigg, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK; stephen.twigg{at}imm.ox.ac.uk

Abstract

Background Auriculocondylar syndrome (ARCND) is a rare genetic disease that affects structures derived from the first and second pharyngeal arches, mainly resulting in micrognathia and auricular malformations. To date, pathogenic variants have been identified in three genes involved in the EDN1-DLX5/6 pathway (PLCB4, GNAI3 and EDN1) and some cases remain unsolved. Here we studied a large unsolved four-generation family.

Methods We performed linkage analysis, resequencing and Capture-C to investigate the causative variant of this family. To test the pathogenicity of the CNV found, we modelled the disease in patient craniofacial progenitor cells, including induced pluripotent cell (iPSC)-derived neural crest and mesenchymal cells.

Results This study highlights a fourth locus causative of ARCND, represented by a tandem duplication of 430 kb in a candidate region on chromosome 7 defined by linkage analysis. This duplication segregates with the disease in the family (LOD score=2.88) and includes HDAC9, which is located over 200 kb telomeric to the top candidate gene TWIST1. Notably, Capture-C analysis revealed multiple cis interactions between the TWIST1 promoter and possible regulatory elements within the duplicated region. Modelling of the disease revealed an increased expression of HDAC9 and its neighbouring gene, TWIST1, in neural crest cells. We also identified decreased migration of iPSC-derived neural crest cells together with dysregulation of osteogenic differentiation in iPSC-affected mesenchymal stem cells.

Conclusion Our findings support the hypothesis that the 430 kb duplication is causative of the ARCND phenotype in this family and that deregulation of TWIST1 expression during craniofacial development can contribute to the phenotype.

  • gene duplication
  • congenital
  • hereditary
  • and neonatal diseases and abnormalities
  • genetic variation
  • high-throughput nucleotide sequencing
  • human genetics

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information. Data availability statement All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

https://creativecommons.org/licenses/by/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/licenses/by/4.0/.

https://doi.org/10.1136/jmedgenet-2021-107825

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    Data availability statement

    All data relevant to the study are included in the article or uploaded as supplementary information. Data availability statement All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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    Footnotes

    • VLRT and SLG-R contributed equally.

    • Contributors VLRT conducted Sanger sequencing, linkage analysis, whole exome sequencing analysis, gene prioritisation and CNV analysis. SLG-R performed in vitro cellular studies and real-time QPCR. YZ conducted targeted sequencing and Capture-C. CM performed linkage analysis. SE conducted whole exome sequencing analysis. DPM performed microsatellite experiment. HB conducted exome data processing. RSF identified patients, collected and analysed clinical data, and provided the biological specimens. JTDD conducted whole exome sequencing. SRFT conducted targeted sequencing and Capture-C. VLRT, SLG-R, SRFT and MRP-B wrote the manuscript. SRFT and MRP-B supervised and conceived the study. MRP-B is responsible for the overall content of the manuscript acting as guarantor. All the authors revised the manuscript for important intellectual content and approved the final version.

    • Funding Work was supported by CEPID/FAPESP (2013/08028-1) and CNPq (MRPB/303712/2016-3) in Brazil and in Oxford: Action Medical Research (GN2483 to SRFT), VTCT Foundation Fellowship (SRFT, Andrew Wilkie), the MRC through the WIMM Strategic Alliance (G0902418 and MC_UU_12025) and Wellcome (Investigator Award 102731 to Andrew Wilkie and Project Grant 093329 to Andrew Wilkie and SRFT). Funding for the DECIPHER project was provided by Wellcome.

    • Competing interests None declared.

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

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.