ROBO4 variants predispose individuals to bicuspid aortic valve and thoracic aortic aneurysm

Abstract

Bicuspid aortic valve (BAV) is a common congenital heart defect (population incidence, 1-2%)^1-3 that frequently presents with ascending aortic aneurysm (AscAA)⁴. BAV/AscAA shows autosomal dominant inheritance with incomplete penetrance and male predominance. Causative gene mutations (for example, NOTCH1, SMAD6) are known for ≤1% of nonsyndromic BAV cases with and without AscAA^5-8, impeding mechanistic insight and development of therapeutic strategies. Here, we report the identification of variants in ROBO4 (which encodes a factor known to contribute to endothelial performance) that segregate with disease in two families. Targeted sequencing of ROBO4 showed enrichment for rare variants in BAV/AscAA probands compared with controls. Targeted silencing of ROBO4 or mutant ROBO4 expression in endothelial cell lines results in impaired barrier function and a synthetic repertoire suggestive of endothelial-to-mesenchymal transition. This is consistent with BAV/AscAA-associated findings in patients and in animal models deficient for ROBO4. These data identify a novel endothelial etiology for this common human disease phenotype.

Figure 1.

Identification of ROBO4 variants segregating in families with bicuspid aortic valve and aortic aneurysms. (a) Whole exome sequencing (WES) revealed segregation of a heterozygous obligate splice site mutation (g.124757628C>A, c.2056+1G>T) in a multigenerational family. (b) cDNA amplicons spanning exons 11–14 were analyzed from patient (proband 1.II:4) and control fibroblasts. Sanger sequencing confirmed skipping of exon 13 (108 base pair). (c) A missense mutation (p.Arg64Cys) was observed in a small family. The missense mutation resides at the Ig-like C2-type-1 (extracellular domain). (d) Sequencing of 736 individuals with BAV/TAA and 376 controls. Eleven rare mutations (MAF<0.1% with CADD>20 or MAF<0.01%) were identified in 13/736 patients and one in 1/376 controls (p.Asp510Val). AscAA: ascending aortic aneurysm, BAV: bicuspid aortic valve, AoRA: aortic root aneurysm, AVS: aortic valve stenosis, ASD: atrial septal defect, AVR: aortic valve replacement, NP: non-penetrant

Figure 2.

Evaluation of ascending aortic aneurysm tissue resected from patient 1.II:1, compared to an age- and sex-matched control. (a) Immunofluorescent staining of CD31 (green), ROBO4 (red), and DAPI (blue) at the intima-media interface. Arrows indicate ROBO4+ cells deep within the aortic media. (b) Immunofluorescent staining of ⍺-SMA (green), ROBO4 (red), and DAPI (blue) at the intima-media interface. (c) Immunofluorescent staining of CD31 (green), ROBO4 (red), and DAPI (blue) at the endothelial layer. (d) Immunofluorescent staining of albumin (red) and DAPI (blue) at the luminal surface. (e) Histological staining (H&E, Masson’s, and VVG) of patient and control ascending aortic tissue. VVG: Verhoeff-Van Gieson

Figure 3.

ROBO4 mutant alleles impair endothelial barrier function. (a) HAECs were transfected with either co-plasmid (control GFP plasmid), co-siRNA (control siRNA), OE-WT (overexpression of ROBO4 wild-type plasmid), siRNA (global ROBO4 knockdown), siRNA-Ex13 (ROBO4 knockdown through targeting of exon 13), OE-SS (overexpression of ROBO4 cDNA plasmid without exon 13), SS-alone (overexpression of ROBO4 cDNA plasmid without exon 13 plus silencing of endogenous ROBO4 using siRNA targeting exon 13), OE-R64C (overexpression of ROBO4 cDNA plasmid with p.Arg64Cys) and ROBO4 mRNA expression was quantified. (b) HAECs were cultured to confluency and a dextran permeability assay was used to assess the integrity of the endothelial barrier. Error bars show mean ± SE, n = 6. Asterisks signify significant differences per one-way ANOVA with Tukey’s Post-hoc (D.F.=7), *P < 0.05, **P < 0.01. (c,d) Immunofluorescent staining of tight junction protein ZO-1 (red), VE-cadherin (green) and DAPI (blue). TJP1 (encoding tight junction protein ZO-1) expression and CDH5 (encoding VE-cadherin) expression were analyzed via qRT-PCR. For both a c and d, error bars show mean ±SD, n = 6. Asterisks signify significant differences per a one-way ANOVA with Tukey’s Post-hoc (D.F.=7), *P < 0.05, **P < 0.01.

Figure 4.

Robo4 knockout causes aortic valve defects and aortic aneurysm in mice. (a) Wild-type (Robo4^+/+), heterozygous (Robo4^+/tm1Lex), and knockout (Robo4^{tm1Lex/tm1Lex}) mice were aged to 20 weeks and the cardiovascular phenotype was determined. Aortic dilatation was defined as a Z-score greater than two when compared to wild-type male mice by echocardiography. Statistical differences per a one-tailed Fisher’s exact test, n = 112 total male and female mice, P < 0.05 was considered statistically significant. (b) Representative parasternal long axis and pulsed-wave Doppler images. Red arrows indicate a dilated segment of the ascending aorta. The yellow arrow shows increased velocity of outflow across the aortic valve during systole while the orange arrow shows regurgitant flow during diastole in a Robo4^{tm1Lex/tm1Lex} mouse (similar patterns were seen in the seven affected mice). (c) Gross and histological examination of the aortic valve. Numbers refer to individual commissures. AscAA: ascending aortic aneurysm, AoRA: aortic root aneurysm, *abnormal aortic valve morphology including BAV, distal commissure fusion, leaflet thickening, or quadricuspid aortic valve among mutant mice with flow abnormalities by echocardiogram. Comparison is made to a representative sample of wild-type mice. **pure bicommissural aortic valve that was definitively evident by gross inspection. See Supplementary Table 3 for raw data.

Figure 5.

Knock-in splice site mutation (c.2089+1G>T; Robo4^Skip13) causes aortic valve defects and aortic aneurysm in mice. (a) Wild-type (Robo4^+/+), heterozygous (Robo4^+/Skip13), and homozygous (Robo4^{Skip13/Skip13}) mice were aged to 20 weeks and the cardiovascular phenotype was determined. Aortic dilatation was defined as a Z-score greater than two when compared to wild-type male mice by echocardiography. Significant differences per a one-tailed Fisher’s exact test, n = 106 total male mice, P < 0.05 was considered statistically significant. (b) Representative parasternal long axis and pulsed-wave Doppler images. Red arrows indicate a dilated segment of the aortic root. The yellow arrow shows increased velocity of outflow across the aortic valve during systole while the orange arrow shows regurgitant flow during diastole in a Robo4^Skip13 mouse (similar patterns were seen in the five affected mice).. (c) Gross and histological examination of the aortic valve. Numbers refer to individual commissures. AscAA: ascending aortic aneurysm, AoRA: aortic root aneurysm. *abnormal aortic valve morphology including BAV, distal commissure fusion, leaflet thickening, or quadricuspid aortic valve among mutant mice with flow abnormalities by echocardiogram. Comparison is made to a representative sample of wild-type mice. **pure bicommissural aortic valve that was definitively evident by gross inspection. See Supplementary Table 4 for raw data.