Rationale: We previously identified somatic activating mutations in the KRAS (Kirsten rat sarcoma viral oncogene homologue) gene in the endothelium of the majority of human sporadic brain arteriove...

Somatic Gain of KRAS Function in the Endothelium Is Sufficient to Cause Vascular Malformations That Require MEK but Not PI3K Signaling.

Background Intracranial arteriovenous malformation (AVM) is a common cause of primary intracerebral hemorrhage in young adults. Lesions typically are sporadic and contain somatic mutations in KRAS or BRAF. The purpose of this study was to identify somatic mutations in a cohort of participants with brain AVM and to determine if any genotype-phenotype associations exist. Methods Human brain AVM specimens (n = 16) were collected during a clinically-indicated procedure and subjected to multiplex targeted sequencing using molecular inversion probe (MIP-seq) for mutations in KRAS, BRAF, HRAS, NRAS, and MAP2K1. Endothelial cells (ECs) were separated from non-ECs by immune-affinity purification. Droplet digital PCR (ddPCR) was used to confirm mutations and to screen for mutations that may have been missed by MIP-seq. Patient and AVM characteristics were recorded. Results We detected somatic mutations in 10 of 16 specimens (63%). Eight had KRAS mutations [G12D (n = 5), G12V (n = 3)] and two had BRAF mutations [V600E (n = 1), Q636X (n = 1)]. We found no difference in age, sex, presenting symptom, AVM location, or AVM size between patients with a confirmed mutation and those without. Nor did we observe differences in these features between patients with KRAS or BRAF mutations. However, two patients with BRAF mutations presented at an older age than other study participants. Conclusions Somatic mutations in KRAS and, less commonly in BRAF, are found in many but not all intracranial AVM samples. Currently, there are no obvious genotype-phenotype correlations that can be used to predict whether a somatic mutation will be detected and, if so, which gene will be mutated.

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Somatic mutations in intracranial arteriovenous malformations.

Cavernous malformations (CMs) affecting the central nervous system occur in approximately 0.16% to 0.4% of the general population. The majority (85%) of the CMs are in a sporadic form, but the genetic background of sporadic CMs remains enigmatic. Of the 81 patients, 73 (90.1%) patients were detected carrying somatic missense variants in 2 genes: MAP3K3 and PIK3CA by whole-exome sequencing (WES). The mutation spectrum correlated with lesion size (P = 0.001), anatomical distribution (P < 0.001), MRI appearance (P = 0.004) and haemorrhage events (P = 0.006). PIK3CA mutation was a significant predictor of overt haemorrhage events (P = 0.003, OR = 11.252, 95% CI = 2.275-55.648). Enrichment of endothelial cell (EC) population was associated with a higher fractional abundance of the somatic mutations. Overexpression of the MAP3K3 mutation perturbed angiogenesis of EC models in vitro and zebrafish embryos in vivo. Distinct transcriptional signatures between different genetic subgroups of sporadic CMs were identified by single-cell RNA-sequencing (scRNA-seq) and verified by pathological staining. Significant apoptosis in MAP3K3 mutation carriers and overexpression of GDF15 and SERPINA5 in PIK3CA mutation carriers contributed to their phenotype. We identified activating MAP3K3 and PIK3CA somatic mutations in the majority (90.1%) of sporadic CMs and PIK3CA mutations could confer a higher risk for overt haemorrhage. Our data provide insights into genomic landscapes, propose a mechanistic explanation and underscore the possibility of a molecular classification for sporadic CMs.

Somatic MAP3K3 and PIK3CA mutations in sporadic cerebral and spinal cord cavernous malformations.

Arteriovenous malformations (AVMs) are abnormal connections of vessels that shunt blood directly from arteries into veins. Rupture of brain AVMs (bAVMs) can cause life-threatening intracranial bleeding. Even though the majority of bAVM cases are sporadic without a family history, some cases are familial. Most of the familial cases of bAVMs are associated with a genetic disorder called hereditary hemorrhagic telangiectasia (HHT). The mechanism of bAVM formation is not fully understood. The most important advances in bAVM basic science research is the identification of somatic mutations of genes in RAS-MAPK pathways. However, the mechanisms by which mutations of these genes lead to AVM formation are largely unknown. In this review, we summarized the latest advance in bAVM studies and discussed some pathways that play important roles in bAVM pathogenesis. We also discussed the therapeutic implications of these pathways.

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Recent Advances in Basic Research for Brain Arteriovenous Malformation.

Lymphatic malformations (LMs) are congenital, nonneoplastic vascular malformations associated with postzygotic activating PIK3CA mutations. The mutation spectrum within LMs is narrow, with the majority having 1 of 3 hotspot mutations. Despite this relative genetic homogeneity, clinical presentations differ dramatically. We used molecular inversion probes and droplet digital polymerase chain reaction to perform deep, targeted sequencing of PIK3CA in 271 affected and unaffected tissue samples from 81 individuals with isolated LMs and retrospectively collected clinical data. Pathogenic PIK3CA mutations were identified in affected LM tissue in 64 individuals (79%) with isolated LMs, with variant allele fractions (VAFs) ranging from 0.1% to 13%. Initial analyses revealed no correlation between VAF and phenotype variables. Recognizing that different mutations activate PI3K to varying degrees, we developed a metric, the genotype-adjusted VAF (GVAF), to account for differences in mutation strength, and found significantly higher GVAFs in LMs with more severe clinical characteristics including orofacial location or microcystic structure. In addition to providing insight into LM pathogenesis, we believe GVAF may have broad applicability for genotype-phenotype analyses in mosaic disorders.

Genotype correlates with clinical severity in PIK3CA-associated lymphatic malformations.

Brain and spinal arteriovenous malformations are congenital lesions causing intracranial haemorrhage or permanent disability especially in young people. We investigated whether the vast majority or all brain and spinal arteriovenous malformations are associated with detectable tumour-related somatic mutations. In a cohort of 31 patients (21 with brain and 10 with spinal arteriovenous malformations), tissue and paired blood samples were analysed with ultradeep next generation sequencing of a panel of 422 common tumour genes to identify the somatic mutations. We used droplet digital polymerase chain reaction to confirm the panel sequenced mutations and identify the additional low variant frequency mutations. The association of mutation variant frequencies and clinical features were analysed. The average sequencing depth was 1077 ± 298×. High prevalence (87.1%) of KRAS/BRAF somatic mutations was found in brain and spinal arteriovenous malformations with no other replicated tumour-related mutations. The prevalence of KRAS/BRAF mutation was 81.0% (17 of 21) in brain and 100% (10 of 10) in spinal arteriovenous malformations. We detected activating BRAF mutations and two novel mutations in KRAS (p.G12A and p.S65_A66insDS) in CNS arteriovenous malformations for the first time. The mutation variant frequencies were negatively correlated with nidus volumes of brain (P = 0.038) and spinal (P = 0.028) arteriovenous malformations but not ages. Our findings support a causative role of somatic tumour-related mutations of KRAS/BRAF in the overwhelming majority of brain and spinal arteriovenous malformations. This pathway homogeneity and high prevalence implies the development of targeted therapies with RAS/RAF pathway inhibitors without the necessity of tissue genetic diagnosis.10.1093/brain/awy307_video1awy307media15978667388001.

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Xiangyuan Ma

Papers

High prevalence of KRAS/BRAF somatic mutations in brain and spinal cord arteriovenous malformations.