Summary Cerebral cavernous malformations (CCMs) are vascular disorders that affect up to 0.5% of the total population. About 20% of CCMs are inherited because of familial mutations in CCM genes, including CCM1 / KRIT1 , CCM2 / MGC4607 , and CCM3 / PDCD10 , whereas the etiology of a majority of simplex CCM-affected individuals remains unclear. Here, we report somatic mutations of MAP3K3 , PIK3CA , MAP2K7 , and CCM genes in CCM lesions. In particular, somatic hotspot mutations of PIK3CA are found in 11 of 38 individuals with CCMs, and a MAP3K3 somatic mutation (c.1323C>G [p.Ile441Met]) is detected in 37.0% (34 of 92) of the simplex CCM-affected individuals. Strikingly, the MAP3K3 c.1323C>G mutation presents in 95.7% (22 of 23) of the popcorn-like lesions but only 2.5% (1 of 40) of the subacute-bleeding or multifocal lesions that are predominantly attributed to mutations in the CCM1/2/3 signaling complex. Leveraging mini-bulk sequencing, we demonstrate the enrichment of MAP3K3 c.1323C>G mutation in CCM endothelium. Mechanistically, beyond the activation of CCM1/2/3-inhibited ERK5 signaling, MEKK3 p.Ile441Met ( MAP3K3 encodes MEKK3) also activates ERK1/2, JNK, and p38 pathways because of mutation-induced MEKK3 kinase activity enhancement. Collectively, we identified several somatic activating mutations in CCM endothelium, and the MAP3K3 c.1323C>G mutation defines a primary CCM subtype with distinct characteristics in signaling activation and magnetic resonance imaging appearance.

Rationale: Brain arteriovenous malformations (bAVMs) are abnormal entanglement of blood vessels in brain, with direct connections from arteries to veins, lacking functional capillary bed. Although several somatic mutations were reported, the molecular mechanism and genetic disposition of bAVM remain poorly understood. Objective: We aim to identify transcriptional anomalies and critical functional pathways in bAVM lesions, and explore their association with key de novo germline and somatic variants in bAVM patients. Methods and Results: We established a comprehensive bAVM dataset from 269 patients, by performing single-cell sequencing of 17 bAVM lesions, whole-exome sequencing of germline DNA from 60 case-unaffected-parental trios, and genomic/transcriptomic sequencing of 231 bAVM lesions. We found abnormal expression of endothelial and mesenchymal markers in bAVM at both bulk and single-cell level, which was validated by flow-cytometric analysis and immunofluorescence staining, suggesting an involvement of Endothelial-to-Mesenchymal transition (EndMT) process in AVM. Using data from the 60 trios we identified non-synonymous de novo germline mutations (DNMs) affecting 46 genes, including EXPH5 (detected in two independent cases), and vessel-related genes such as EPAS1 and ENG. Interestingly, knockdown of epas1 in zebrafish embryo showed AVM-like phenotype exclusively in brain. Subsequent computational and experimental analyses demonstrated that expression of genes affected by DNMs was enriched in vascular cell types and was involved in EndMT-relevant behaviours including cell migration, angiogenesis and cell-marker transition. Moreover, we detected somatic KRAS mutations in 129 of 179 (72%) cases, and showed that KRAS mutations were associated with bleeding as the first symptom (p=0.0072). Following experimental studies demonstrated that KRAS mutations independently regulated EndMT features, consolidating the involvement of EndMT in this disease. Lastly, we showed that Lovastatin reversed EndMT features in vitro and ex vivo. Conclusions: Our results suggest the convergent role of DNMs and somatic mutations in regulating EndMT in bAVM and provided a potential therapeutic option.