Whole-exome sequencing in a Japanese multiplex family identifies new susceptibility genes for intracranial aneurysms

Abstract

Background: Intracranial aneurysms (IAs) cause subarachnoid hemorrhage, which has high rates of mortality and morbidity when ruptured. Recently, the role of rare variants in the genetic background of complex diseases has been increasingly recognized. The aim of this study was to identify rare variants for susceptibility to IA.

Methods: Whole-exome sequencing was performed on seven members of a Japanese pedigree with highly aggregated IA. Candidate genes harboring co-segregating rare variants with IA were re-sequenced and tested for association with IA using additional 500 probands and 323 non-IA controls. Functional analysis of rare variants detected in the pedigree was also conducted.

Results: We identified two gene variants shared among all four affected participants in the pedigree. One was the splicing donor c.1515+1G>A variant in NPNT (Nephronectin), which was confirmed to cause aberrant splicing by a minigene assay. The other was the missense p.P83T variant in CBY2 (Chibby family member 2). Overexpression of p.P83T CBY2 fused with red fluorescent protein tended to aggregate in the cytoplasm. Although Nephronectin has been previously reported to be involved in endothelial angiogenic functions, CBY2 is a novel molecule in terms of vascular pathophysiology. We confirmed that CBY2 was expressed in cerebrovascular smooth muscle cells in an isoform2-specific manner. Targeted CBY2 re-sequencing in additional case-control samples identified three deleterious rare variants (p.R46H, p.P83T, and p.L183R) in seven probands, showing a significant enrichment in the overall probands (8/501) compared to the controls (0/323) (p = 0.026, Fisher's extract test).

Conclusions: NPNT and CBY2 were identified as novel susceptibility genes for IA. The highly heterogeneous and polygenic architecture of IA susceptibility can be uncovered by accumulating extensive analyses that focus on each pedigree with a high incidence of IA.

Fig 1. Japanese multiplex families with IA.

Pedigree charts of the exome-sequenced family (A) and the Sanger-sequenced affected siblings (B, C) who carried pathogenic variants in CBY2 and NPNT. Colored plus and minus signs (+ and −) indicate mutated and wild-type alleles, respectively. CBY2 and NPNT were identified from the exome data using genome-wide linkage analysis in F2054 (D). Abbreviations: HLOD, maximum heterogeneity logarithm of odds.

Fig 2. In silico splicing analysis of the NPNT c.1515+1G>A variant.

A functional assessment of the splice-site variant was performed using three web-based and one locally installed programs. All of the programs consistently predict that this variant causes loss of the splice donor site. Abbreviation: N/A, not applicable.

Fig 3. Minigene splicing assay of the c.1515+1G>A variant in NPNT.

(A) The pET01 construct used in this study. The sequences containing the c.1515+1G>A variant in NPNT intron 10 (A allele) or those that did not (G allele) were subcloned into the multiple cloning site of the pET01 vector. The arrows under the 3’ and 5’ exons indicate the primer pair used in RT-PCR after transfection. The primer sequences are provided in S2-1 Table in S1 File. (B) RT-PCR analysis using HeLa cells transfected with the wild-type, mutant, or empty (mock) pET01 vector. The RT-PCR products were separated in a 3% agarose gel electrophoresis and were stained with ethidium bromide. (C) Sequencing chromatograms confirmed the exon 10 was totally skipped in the mutant RT-PCR product.

Fig 4. Immunohistochemistry of cerebral arterial specimens.

The upper six panels show immunohistochemical staining of a surgically resected peripheral cerebral artery from a patient with a brain tumor: (A) hematoxylin-eosin staining, (B) Elastica van Gieson staining (EVG) of elastic fibers, (C) anti-GFAP antibody staining of the glia around the vessel, (D) anti-CBY2 antibody staining, (E) anti-SMA antibody staining of vascular smooth muscle cells, and (F) anti-CD34 antibody staining of vascular endothelial cells. CBY2 is expressed in vascular smooth muscle cells in the tunica media. (G and H) The lower four panels show anti-CBY2 antibody staining of the surgically resected AVM and IA walls. CBY2 was expressed in smooth muscle cells in the arteriolar wall (G) and was also confirmed in residual smooth muscle cells in the IA wall (H).

Fig 5. Cell imaging of CBY2-expressing COS7 cells.

(A) Wild-type or p.P83T CBY2-DsRed was transiently expressed in COS7 cells. The upper three panels represent mock-transfected cells. p.P83T CBY2-DsRed exhibited dot-like aggregations in the cytoplasm. (B) These aggregates were observed more frequently in the cells expressing p.P83T CBY2 than that expressing wild-type CBY2 (p = 0.034, unpaired Student’s t-test). DsRed-positive cells were counted in three independent fields of view.