Somatic Variants in SVIL in Cerebral Aneurysms

Abstract

Background and objectives: While somatic mutations have been well-studied in cancer, their roles in other complex traits are much less understood. Our goal is to identify somatic variants that may contribute to the formation of saccular cerebral aneurysms.

Methods: We performed whole-exome sequencing on aneurysm tissues and paired peripheral blood. RNA sequencing and the CRISPR/Cas9 system were then used to perform functional validation of our results.

Results: Somatic variants involved in supervillin (SVIL) or its regulation were found in 17% of aneurysm tissues. In the presence of a mutation in the SVIL gene, the expression level of SVIL was downregulated in the aneurysm tissue compared with normal control vessels. Downstream signaling pathways that were induced by knockdown of SVIL via the CRISPR/Cas9 system in vascular smooth muscle cells (vSMCs) were determined by evaluating changes in gene expression and protein kinase phosphorylation. We found that SVIL regulated the phenotypic modulation of vSMCs to the synthetic phenotype via Krüppel-like factor 4 and platelet-derived growth factor and affected cell migration of vSMCs via the RhoA/ROCK pathway.

Discussion: We propose that somatic variants form a novel mechanism for the development of cerebral aneurysms. Specifically, somatic variants in SVIL result in the phenotypic modulation of vSMCs, which increases the susceptibility to aneurysm formation. This finding suggests a new avenue for the therapeutic intervention and prevention of cerebral aneurysms.

Figure 1. Somatic Variants From Whole-Exome Sequencing

(A) The top 25 genes with somatic variants observed in more than 2 samples from aneurysm tissues in 30 patients. The gene, SVIL, was found to have the highest number of somatic mutations (N = 4 missense mutations, N = 1 synonymous mutation [not shown] in the transcription factor binding site for EP300 on SVIL). (B) Variant classification and single-nucleotide variant class in the patients. There were 9.4 mutations per patient sample (median 7, range 1–29) with the most common variant being a missense mutation. (C) Overall distribution of 6 different conversions of all variants. SVIL = supervillin.

Figure 2. GO Analysis

GO analysis of genes where somatic mutations were found in aneurysm tissue. (A) Top 10 GO terms for molecular function. (B) Genes related to the 5 significant GO terms. GO = gene ontology.

Figure 3. Differential Expression of Phenotypic Switching Genes in Cerebral Aneurysm Tissue

Differential expression in genes related to phenotypic switching of vascular smooth muscle cells from RNA sequencing of 10 aneurysms and 5 middle cerebral artery controls. ACTA2 = α-SMA, TAGLN = SM22α (*p < 0.05, **p < 0.01). MCA = middle cerebral arteries

Figure 4. SVIL Knockdown in Vascular Smooth Muscle Cells Affects Genes Involved in Phenotypic Modulation

(A) The mRNA levels of SVIL in aneurysm tissues where somatic mutations were found in the SVIL gene compared with control middle cerebral artery by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). (B) The mRNA and protein levels of SVIL using SVIL sgRNA (sequence 5) by RT-qPCR and Western blot. (C) Comparison of mRNA and protein levels of markers related to phenotypic switching after SVIL sgRNA delivery by RT-qPCR and Western blot. Error bars represent means ± SD. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001). SVIL = supervillin.

Figure 5. SVIL Stimulates Cell Migration

vSMCs were treated with scramble sgRNA or SVIL sgRNA, and then Y27632 (20 μM, a ROCK inhibitor) or phosphate-buffered saline (as control) was added to each. (A) Comparison of protein levels of markers related to RhoA/ROCK pathway. (B and D) Representative confocal images of the cells after immunostaining for F-actin (green) and 4’,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars represent 100 µm. (C and E) Analysis of migration assay using the wound healing assay. LIMK = LIM kinase; ROCK = Rho-associated, coiled-coil-containing protein kinase; SVIL = supervillin; VSMC = vascular smooth muscle cells.