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Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion

Cerebral cavernous malformations are clusters of aberrant vessels that can lead to severe neurological complications. Pathogenic loss-of-function variants in the CCM1, CCM2, or CCM3 gene are associated with the autosomal dominant form of the disease. While interpretation of variants in protein-codin...

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Autores principales: Pilz, Robin A., Skowronek, Dariush, Hamed, Motaz, Weise, Anja, Mangold, Elisabeth, Radbruch, Alexander, Pietsch, Torsten, Felbor, Ute, Rath, Matthias
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9453596/
https://www.ncbi.nlm.nih.gov/pubmed/36090026
http://dx.doi.org/10.3389/fmolb.2022.953048
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author Pilz, Robin A.
Skowronek, Dariush
Hamed, Motaz
Weise, Anja
Mangold, Elisabeth
Radbruch, Alexander
Pietsch, Torsten
Felbor, Ute
Rath, Matthias
author_facet Pilz, Robin A.
Skowronek, Dariush
Hamed, Motaz
Weise, Anja
Mangold, Elisabeth
Radbruch, Alexander
Pietsch, Torsten
Felbor, Ute
Rath, Matthias
author_sort Pilz, Robin A.
collection PubMed
description Cerebral cavernous malformations are clusters of aberrant vessels that can lead to severe neurological complications. Pathogenic loss-of-function variants in the CCM1, CCM2, or CCM3 gene are associated with the autosomal dominant form of the disease. While interpretation of variants in protein-coding regions of the genes is relatively straightforward, functional analyses are often required to evaluate the impact of non-coding variants. Because of multiple alternatively spliced transcripts and different transcription start points, interpretation of variants in the 5′ untranslated and upstream regions of CCM1 is particularly challenging. Here, we identified a novel deletion of the non-coding exon 1 of CCM1 in a proband with multiple CCMs which was initially classified as a variant of unknown clinical significance. Using CRISPR/Cas9 genome editing in human iPSCs, we show that the deletion leads to loss of CCM1 protein and deregulation of KLF2, THBS1, NOS3, and HEY2 expression in iPSC-derived endothelial cells. Based on these results, the variant could be reclassified as likely pathogenic. Taken together, variants in regulatory regions need to be considered in genetic CCM analyses. Our study also demonstrates that modeling variants of unknown clinical significance in an iPSC-based system can help to come to a final diagnosis.
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spelling pubmed-94535962022-09-09 Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion Pilz, Robin A. Skowronek, Dariush Hamed, Motaz Weise, Anja Mangold, Elisabeth Radbruch, Alexander Pietsch, Torsten Felbor, Ute Rath, Matthias Front Mol Biosci Molecular Biosciences Cerebral cavernous malformations are clusters of aberrant vessels that can lead to severe neurological complications. Pathogenic loss-of-function variants in the CCM1, CCM2, or CCM3 gene are associated with the autosomal dominant form of the disease. While interpretation of variants in protein-coding regions of the genes is relatively straightforward, functional analyses are often required to evaluate the impact of non-coding variants. Because of multiple alternatively spliced transcripts and different transcription start points, interpretation of variants in the 5′ untranslated and upstream regions of CCM1 is particularly challenging. Here, we identified a novel deletion of the non-coding exon 1 of CCM1 in a proband with multiple CCMs which was initially classified as a variant of unknown clinical significance. Using CRISPR/Cas9 genome editing in human iPSCs, we show that the deletion leads to loss of CCM1 protein and deregulation of KLF2, THBS1, NOS3, and HEY2 expression in iPSC-derived endothelial cells. Based on these results, the variant could be reclassified as likely pathogenic. Taken together, variants in regulatory regions need to be considered in genetic CCM analyses. Our study also demonstrates that modeling variants of unknown clinical significance in an iPSC-based system can help to come to a final diagnosis. Frontiers Media S.A. 2022-08-25 /pmc/articles/PMC9453596/ /pubmed/36090026 http://dx.doi.org/10.3389/fmolb.2022.953048 Text en Copyright © 2022 Pilz, Skowronek, Hamed, Weise, Mangold, Radbruch, Pietsch, Felbor and Rath. 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 Molecular Biosciences
Pilz, Robin A.
Skowronek, Dariush
Hamed, Motaz
Weise, Anja
Mangold, Elisabeth
Radbruch, Alexander
Pietsch, Torsten
Felbor, Ute
Rath, Matthias
Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion
title Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion
title_full Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion
title_fullStr Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion
title_full_unstemmed Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion
title_short Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion
title_sort using crispr/cas9 genome editing in human ipscs for deciphering the pathogenicity of a novel ccm1 transcription start site deletion
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9453596/
https://www.ncbi.nlm.nih.gov/pubmed/36090026
http://dx.doi.org/10.3389/fmolb.2022.953048
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