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Autosomal dominant cerebral cavernous malformation (CCM) represents a genetic disorder with a high mutation detection rate given that stringent inclusion criteria are used and copy number variation analyses are part of the diagnostic workflow. Pathogenic variants in either CCM1 (KRIT1), CCM2 or CCM3 (PDCD10) can be identified in 87–98% of CCM families with at least two affected individuals. However, the interpretation of novel sequence variants in the 5′-region of CCM2 remains challenging as there are various alternatively spliced transcripts and different transcription start sites. Comprehensive genetic and clinical data of CCM2 patients with variants in cassette exons that are either skipped or included into alternative CCM2 transcripts in the splicing process can significantly facilitate clinical variant interpretation. We here report novel pathogenic CCM2 variants in exon 3 and the adjacent donor splice site, describe the natural history of CCM disease in mutation carriers and provide further evidence for the classification of the amino acids encoded by the nucleotides of this cassette exon as a critical region within CCM2. Finally, we illustrate the advantage of a combined single nucleotide and copy number variation detection approach in NGS-based CCM1/CCM2/CCM3 gene panel analyses which can significantly reduce diagnostic turnaround time.
Cerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1
−/− BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1
−/−
BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1
−/−
BOECs which was observed in CCM1
+/− BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells.
Cerebral cavernous malformations (CCM) are low-flow vascular lesions prone to cause severe hemorrhage-associated neurological complications. Pathogenic germline variants in CCM1, CCM2, or CCM3 can be identified in nearly 100% of CCM patients with a positive family history. In line with the concept that tumor-like mechanisms are involved in CCM formation and growth, we here demonstrate an abnormally increased proliferation rate of CCM3-deficient endothelial cells in co-culture with wild-type cells and in mosaic human iPSC-derived vascular organoids. The observation that NSC59984, an anticancer drug, blocked the abnormal proliferation of mutant endothelial cells further supports this intriguing concept. Fluorescence-activated cell sorting and RNA sequencing revealed that co-culture induces upregulation of proangiogenic chemokine genes in wild-type endothelial cells. Furthermore, genes known to be significantly downregulated in CCM3−/− endothelial cell mono-cultures were upregulated back to normal levels in co-culture with wild-type cells. These results support the hypothesis that wild-type ECs facilitate the formation of a niche that promotes abnormal proliferation of mutant ECs. Thus, targeting the cancer-like features of CCMs is a promising new direction for drug development.