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Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss

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) impl...

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Autores principales: Ascari, Giulia, Rendtorff, Nanna D., De Bruyne, Marieke, De Zaeytijd, Julie, Van Lint, Michel, Bauwens, Miriam, Van Heetvelde, Mattias, Arno, Gavin, Jacob, Julie, Creytens, David, Van Dorpe, Jo, Van Laethem, Thalia, Rosseel, Toon, De Pooter, Tim, De Rijk, Peter, De Coster, Wouter, Menten, Björn, Rey, Alfredo Dueñas, Strazisar, Mojca, Bertelsen, Mette, Tranebjaerg, Lisbeth, De Baere, Elfride
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097100/
https://www.ncbi.nlm.nih.gov/pubmed/33968938
http://dx.doi.org/10.3389/fcell.2021.664317
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author Ascari, Giulia
Rendtorff, Nanna D.
De Bruyne, Marieke
De Zaeytijd, Julie
Van Lint, Michel
Bauwens, Miriam
Van Heetvelde, Mattias
Arno, Gavin
Jacob, Julie
Creytens, David
Van Dorpe, Jo
Van Laethem, Thalia
Rosseel, Toon
De Pooter, Tim
De Rijk, Peter
De Coster, Wouter
Menten, Björn
Rey, Alfredo Dueñas
Strazisar, Mojca
Bertelsen, Mette
Tranebjaerg, Lisbeth
De Baere, Elfride
author_facet Ascari, Giulia
Rendtorff, Nanna D.
De Bruyne, Marieke
De Zaeytijd, Julie
Van Lint, Michel
Bauwens, Miriam
Van Heetvelde, Mattias
Arno, Gavin
Jacob, Julie
Creytens, David
Van Dorpe, Jo
Van Laethem, Thalia
Rosseel, Toon
De Pooter, Tim
De Rijk, Peter
De Coster, Wouter
Menten, Björn
Rey, Alfredo Dueñas
Strazisar, Mojca
Bertelsen, Mette
Tranebjaerg, Lisbeth
De Baere, Elfride
author_sort Ascari, Giulia
collection PubMed
description 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 polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) 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 quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), 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 of 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 workup 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.
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spelling pubmed-80971002021-05-06 Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss Ascari, Giulia Rendtorff, Nanna D. De Bruyne, Marieke De Zaeytijd, Julie Van Lint, Michel Bauwens, Miriam Van Heetvelde, Mattias Arno, Gavin Jacob, Julie Creytens, David Van Dorpe, Jo Van Laethem, Thalia Rosseel, Toon De Pooter, Tim De Rijk, Peter De Coster, Wouter Menten, Björn Rey, Alfredo Dueñas Strazisar, Mojca Bertelsen, Mette Tranebjaerg, Lisbeth De Baere, Elfride Front Cell Dev Biol Cell and Developmental Biology 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 polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) 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 quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), 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 of 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 workup 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. Frontiers Media S.A. 2021-04-21 /pmc/articles/PMC8097100/ /pubmed/33968938 http://dx.doi.org/10.3389/fcell.2021.664317 Text en 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, Rey, Strazisar, Bertelsen, Tranebjaerg and De Baere. https://creativecommons.org/licenses/by/4.0/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.
spellingShingle Cell and Developmental Biology
Ascari, Giulia
Rendtorff, Nanna D.
De Bruyne, Marieke
De Zaeytijd, Julie
Van Lint, Michel
Bauwens, Miriam
Van Heetvelde, Mattias
Arno, Gavin
Jacob, Julie
Creytens, David
Van Dorpe, Jo
Van Laethem, Thalia
Rosseel, Toon
De Pooter, Tim
De Rijk, Peter
De Coster, Wouter
Menten, Björn
Rey, Alfredo Dueñas
Strazisar, Mojca
Bertelsen, Mette
Tranebjaerg, Lisbeth
De Baere, Elfride
Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss
title Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss
title_full Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss
title_fullStr Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss
title_full_unstemmed Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss
title_short Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss
title_sort long-read sequencing to unravel complex structural variants of cep78 leading to cone-rod dystrophy and hearing loss
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097100/
https://www.ncbi.nlm.nih.gov/pubmed/33968938
http://dx.doi.org/10.3389/fcell.2021.664317
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