CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.
Autosomal dominant cerebral cavernous malformations (ccM) are leaky vascular lesions that can cause epileptic seizures and stroke-like symptoms. Germline mutations in either CCM1, CCM2 or CCM3 are found in the majority of patients with multiple CCMs or a positive family history. Recently, the first copy number neutral inversion in CCM2 has been identified by whole genome sequencing in an apparently mutation-negative CCM family. We here asked the question whether further structural genomic rearrangements can be detected within nGS gene panel data of unsolved ccM cases. Hybrid capture nGS data of eight index patients without a pathogenic single nucleotide, indel or copy number variant were analyzed using two bioinformatics pipelines. In a 58-year-old male with multiple CCMs in his brain and spinal cord, we identified a 294 kb insertion within the coding sequence of CCM2. fine mapping of the breakpoints, molecular cytogenetic studies, and multiplex ligation-dependent probe amplification verified that the structural variation was an inverted unbalanced insertion that originated from 1p12-p11.2. As this rearrangement disrupts exon 6 of CCM2 on 7p13, it was classified as pathogenic. Our study demonstrates that efforts to detect structural variations in known disease genes increase the diagnostic sensitivity of genetic analyses for well-defined Mendelian disorders. Cerebral cavernous malformations (CCM; MIM: 116860, 603284, 603285) are irregular clusters of enlarged and thin-walled vessels that can present as sporadic or autosomal dominant cerebrovascular disease. Aside from epileptic seizures and headaches, CCM patients may present with stroke-like symptoms due to chronic or acute bleeding events 1. Pathogenic germline variants have been identified in CCM1 (also known as KRIT1) 2,3 , CCM2 4,5 , and CCM3 (PDCD10) 6. The mutational spectrum primarily includes nonsense, frameshift, splice, and copy number variants (CNVs). 282 unique CCM1, 84 CCM2 and 75 CCM3 variants are classified as disease-causing in the Human Gene Mutation Database (HGMD Professional 2019.3) 7. Depending on the inclusion criteria for genetic analyses, mutation detection rates of 87 to 98% have been reported for familial CCM cases and up to 60% for sporadic ones 8-11. The mutation detection rate in the latter group could even be higher if patients with associated developmental venous anomalies or a history of radiotherapy to the brain were excluded 1,12. Although pathogenic variants in a yet unknown CCM4 candidate gene have been discussed for unresolved cases 13 , we recently were able to identify the first copy number neutral inversion in CCM2 in an apparently mutation-negative CCM family by whole genome sequencing (WGS) 14. Disease-causing structural variants (SVs) may explain a part of the "missing heritability" in rare diseases 15. SVs are defined as structural and quantitative chromosomal rearrangements that compromise cytogenetically visible and submicroscopic variants 16,17. They contribute to phenotypic variation but can also cause human diseas...
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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