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Original Research ARTICLE

Front. Cell Dev. Biol. | doi: 10.3389/fcell.2021.664317

Long-read sequencing to unravel complex structural variants of CEP78 leading to Cone‐Rod Dystrophy and Hearing Loss Provisionally accepted The final, formatted version of the article will be published soon. Notify me

 Giulia Ascari1, 2*,  Nanna D. Rendtorff3,  Marieke De Bruyne2, 4,  Julie De Zaeytijd5, Michel Van Lint6, Miriam Bauwens2, 4,  Mattias Van Heetvelde2, 4,  Gavin Arno7, 8, 9,  Julie Jacob10,  David Creytens11, 12, Jo Van Dorpe11, 12, Thalia Van Laethem2, 4, Toon Rosseel2, 4, Tim De Pooter13, 14, Peter De Rijk13, 14, Wouter De Coster15, 16,  Björn Menten2, 4, Alfredo Duenas Rey2, 4, Mojca Strazisar13, 14,  Mette Bertelsen3, 17, Lisbeth Tranebjarg3, 18 and  Elfride De Baere2, 4*
  • 1Department of Biomolecular Medicine, Ghent University, Belgium
  • 2Center for Medical Genetics, Ghent University Hospital, Belgium
  • 3The Kennedy Center, Department of Clinical Genetics, Copenhagen University Hospital, Rigshospitalet, Denmark
  • 4Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University, Belgium
  • 5Department of Ophthalmology, Ghent University Hospital, Belgium
  • 6Department of Ophthalmology, Antwerp University Hospital, Belgium
  • 7NIHR Great Ormond Street Hospital Biomedical Research Centre, United Kingdom
  • 8Moorfields Eye Hospital, NHS Foundation Trust, United Kingdom
  • 9Institute of Ophthalmology, Faculty of Brain Sciences, University College London, United Kingdom
  • 10Department of Ophthalmology, University Hospitals Leuven, Belgium
  • 11Department of Pathology, Ghent University Hospital, Belgium
  • 12Department of Diagnostic Sciences, Ghent University, Belgium
  • 13Neuromics Support Facility, VIB Center for Molecular Neurology, VIB, Belgium
  • 14Neuromics Support Facility, Department of Biomedical Sciences, University of Antwerp, Belgium
  • 15Applied and Translational Neurogenomics Group, VIB Center for Molecular Neurology, VIB, Belgium
  • 16Applied and Translational Neurogenomics Group, Department of Biomedical Sciences, University of Antwerp, Belgium
  • 17Department of Ophthalmology, Rigshospitalet-Glostrup, University of Copenhagen, Denmark
  • 18Institute of Clinical Medicine, University of Copenhagen, Denmark, Denmark

Inactivating variants as well as a missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive Cone-Rod Dystrophy with Hearing Loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole genome sequencing (sWGS) combined with quantitative PCR and long-range PCR, or ExomeDepth analysis on whole exome sequencing data. Targeted or whole genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on CEP78 expression was assessed using qPCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single nucleotide variant, two SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a heterozygous and homozygous state, respectively. Assessment of the molecular consequences of the SVs on patient’s materials displayed a loss-of-function effect. Delineation and characterization of the 15 kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive for a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235 kb deletion was delineated using whole genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic work-up of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole genome LRS in identifying and characterizing complex SVs in patients with ocular diseases.

Keywords: CEP78, inherited retinal disease (IRD), cone-rod dystrophy with hearing loss (CRDHL), Long-read sequencing, Structural variants, Single-cell gene expression analysis

Received: 05 Feb 2021; Accepted: 08 Mar 2021.

Copyright: © 2021 Ascari, Rendtorff, De Bruyne, De Zaeytijd, Van Lint, Bauwens, Van Heetvelde, Arno, Jacob, Creytens, Van Dorpe, Van Laethem, Rosseel, De Pooter, De Rijk, De Coster, Menten, Duenas Rey, Strazisar, Bertelsen, Tranebjarg and De Baere. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Mx. Giulia Ascari, Ghent University, Department of Biomolecular Medicine, Ghent, Belgium,
Mx. Elfride De Baere, Center for Medical Genetics, Ghent University Hospital, Ghent, 9000, East Flanders, Belgium,