Figure 2.

$\mathit{C}\mathit{C}\mathit{M}3$ gene disruption promotes clonal expansion of endothelial cells. (A) In patients with a CCM3 germline mutation (${\mathit{CCM}\mathit{3}}^{+\mathrm{/}-}$), a second somatic CCM3 mutation in an endothelial cell (${\mathit{CCM}\mathit{3}}^{-\mathrm{/}-}$) initiates CCM formation. A ${\mathit{CCM}\mathit{3}}^{-\mathrm{/}-}$ mutant endothelial cell undergoes clonal expansion and forms a CCM that is characterized by endothelial mosaicism of ${\mathit{CCM}\mathit{3}}^{-\mathrm{/}-}$ and ${\mathit{CCM}\mathit{3}}^{+\mathrm{/}-}$ endothelial cells. The impaired endothelial barrier function can lead to bleeding into the surrounding brain tissue. (B) CCM3 knockout endothelial cells were generated with CRISPR/Cas9 genome editing. Biallelic loss-of-function variants were introduced into the first coding exon of CCM3 (knockout [KO] clones 1 and 2: c.[87_88insAG];[87_88insAG] [p.[Phe30Serfs*5];[Phe30Serfs*5]]; KO3: c.[90dupT];[87_88insAGTTGGATAAACATGTTTATCCAACT] [p.[Asn31*];[Phe30Serfs*13]]). (C) ${\mathit{CCM}\mathit{3}}^{-\mathrm{/}-}$ CI-huVECs demonstrated significant expansion in co-culture with ${\mathit{CCM}\mathit{3}}^{+\mathrm{/}+}$ CI-huVECs. Knockout and wild-type (WT) allele frequencies were determined by amplicon deep sequencing after six days of co-culture.