Individuals with bicuspid aortic valve (BAV) frequently develop other cardiovascular complications, such as ascending aortic aneurysm (AscAA). A study provides new insights into the pathogenesis of this congenital heart defect by reporting the identification of ROBO4 variants associated with both BAV and AscAA.

Whole-exome sequencing of samples from families with BAV and AscAA showed that in one family, eight members who had aortic root aneurysm, including two individuals with associated BAV, had a mutation at the splice acceptor site of ROBO4 exon 13, resulting in the expression of a shorter form of the ROBO4 protein. Targeted sequencing of 441 additional individual probands with BAV and/or AscAA showed that seven probands were also carrying ROBO4 missense variants that altered ROBO4 amino acid sequence.

Immunofluorescence staining and histological analysis of aortic tissue from one of the two family members with BAV and AscAA and from a control individual without structural heart disease showed that ROBO4 mutations led to a disruption of endothelial barrier function, via decreased ROBO4 expression in endothelial cells, and induction of pathological remodelling in the aortic media. In cultured aortic endothelial cells, ROBO4 knockdown or overexpression of ROBO4 variants resulted in a decrease in VE-cadherin levels, a component of the tight junctions between endothelial cells, whereas the expression of aortic smooth muscle actin, a marker of mesenchymal transition, was induced. Knock-in mice with a mutation altering ROBO4 splicing had a complex cardiovascular phenotype, with aortic valve defect and AscAA. These findings show that altered endothelial function can cause both BAV and AscAA as primary manifestations of the same underlying gene defect.