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

Affiliation
Department of Human Genetics ,University Medicine Greifswald, and Interfaculty Institute of Genetics and Functional Genomics ,University of Greifswald ,Greifswald ,Germany
Pilz, Robin A.;
Affiliation
Department of Human Genetics ,University Medicine Greifswald, and Interfaculty Institute of Genetics and Functional Genomics ,University of Greifswald ,Greifswald ,Germany
Skowronek, Dariush;
Affiliation
Department of Neurosurgery ,University Hospital Bonn ,Bonn ,Germany
Hamed, Motaz;
GND
12954275X
Affiliation
Jena University Hospital ,Friedrich Schiller University
Weise, Anja;
Affiliation
Institute of Human Genetics ,Medical Faculty and University Hospital Bonn ,University of Bonn ,Bonn ,Germany
Mangold, Elisabeth;
Affiliation
Department of Neuroradiology ,University Hospital Bonn ,Bonn ,Germany
Radbruch, Alexander;
Affiliation
Institute of Neuropathology ,DGNN Brain Tumor Reference Center ,University of Bonn ,Bonn ,Germany
Pietsch, Torsten;
Affiliation
Department of Human Genetics ,University Medicine Greifswald, and Interfaculty Institute of Genetics and Functional Genomics ,University of Greifswald ,Greifswald ,Germany
Felbor, Ute;
Affiliation
Department of Human Genetics ,University Medicine Greifswald, and Interfaculty Institute of Genetics and Functional Genomics ,University of Greifswald ,Greifswald ,Germany
Rath, Matthias

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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License Holder: Copyright © 2022 Pilz, Skowronek, Hamed, Weise, Mangold, Radbruch, Pietsch, Felbor and Rath.

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