Bicuspid aortic valve (BAV) is a common congenital heart defect (population incidence, 1–2%)1,2,3 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 AscAA5–8, 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.
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ROBO4 variants have been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and have the following accession codes: SCV000804228, SCV000804229, SCV000804230, SCV000804231, SCV000804232, SCV000804233, SCV000804234, SCV000804235, SCV000804236, SCV000804237, SCV000804238, SCV000804239. Exome sequencing data are not publicly available owing to consent restrictions.
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We gratefully acknowledge support from the Leducq Foundation to A.S.M. and H.C.D., from the National Human Genome Research Institute (NHGRI) (1U54HG006542) to D.V. and J.L., from the National Heart, Lung, and Blood Institute (NHLBI) (HL110328, HL128745) and the NIH (S10OD012287) to J.T.B. We also thank the American Philosophical Society for support of H.A. through the Daland Fellowship. In addition, we thank Johns Hopkins University School of Medicine, McKusick Nathans Institute of Genetic Medicine Center for Functional Investigation in Zebrafish (FINZ) for their technical support and Corinne Boehm for her assistance in depositing variant information to ClinVar. B.L.L. is senior clinical investigator of the Fund for Scientific Research, Flanders, and holds a starting grant from the European Research Council (ERC-StG-2012-30972-BRAVE). A.V. is a postdoctoral researcher supported by the Fund for Scientific Research Flanders. I.L. is supported by a PhD grant from the Agency for Innovation by Science and Technology (IWT). M.E.L. is supported by the Toomey Fund for Aortic Dissection Research and the Fredman Fellowship in Aortic Disease. G.A. is a FQRS Senior Clinical Research Fellow.
The authors declare no competing interests.
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Integrated supplementary information
AscAA, ascending aortic aneurysm; AoRA, aortic root aneurysm; AoDiss, aortic dissection; AoSurg, underwent aortic repair surgery; BAV, bicuspid aortic valve.
(a), Sanger sequencing verification of the heterozygous obligate splice-site mutation (g.124757628C>A, c.2056+1G>T) in family 1. (b), H&E staining of aortic valve from the affected patient (1:II:1), n = 1. c, Sanger sequencing verification of the missense mutation (c.190C>T, p.Arg64Cys) in family 2.
Supplementary Figure 3 Cellular phenotypes observed in HAECs transfected with ROBO4 mutant alleles or siRNA to silence ROBO4 expression.
(a), HAECs were transfected with co-plasmid (control GFP plasmid), co-siRNA (control siRNAs), 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 mRNA expression levels for ACTA2 (encoding ⍺-smooth muscle actin; ⍺-SMA) were quantified via qRT–PCR and immunofluorescence, respectively (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). (b), HAECs were transfected with either co-siRNA or siRNA and incubated for 72 h; afterward, mRNA levels for the indicated genes were quantified via qRT–PCR (n = 3 and 6, respectively). Asterisks signify significant differences per a two-sided t test (**P < 0.01). (c), Cellular morphology was captured via bright-field microscopy and quantified as a circularity index using ImageJ; n ≥ 200 cells were assessed per condition. (d), Endothelial invasion was assessed using an endothelial aggregate invasion assay on collagen gels. (e), Migration and proliferation were measured using a scratch assay and 5-bromo-2′-deoxyuridine (BrDU) proliferation assay, respectively. For (c–e), n = 3. Asterisks signify significant differences per a one-way ANOVA with Tukey’s post-hoc (d.f. = 7, 7, and 2, respectively; *P < 0.05). For all graphs, the mean is used as the measure of center.
Supplementary Figure 4 Immunofluorescent staining for ROBO4 in the developing mouse outflow tract (OFT) and human adult ascending aorta.
(a), Mouse ROBO4 expression in the endothelial layer of the embryonic endocardial OFT cushions and the primordial aortic valve leaflets at E11.5 and E17, respectively. (b), Mouse ROBO4 expression in the endothelium of the OFT (E17) and ascending aorta (5 weeks). (c), Human control ROBO4 expression in an ascending aorta. The arrow indicates ROBO4-expressing cells in the intima.
(a), A mutant line for robo4 was generated using CRISPR–Cas9. A 7-bp deletion was induced in exon 6 (robo4∆7). (b), robo4 expression was analyzed by qRT–PCR on four adult hearts per genotype (three technical replicates averaged, both male and female) and then biological replicates were averaged (horizontal line). Results are shown normalized to β-actin mRNA expression. The graph shows the averaged technical replicates for four (n = 4) biological replicates per genotype run on the same plate. Asterisks signify significant differences to wild-type per a two-tailed Welch’s t test, P = 0.024 for wild-type and heterozygous mutants, P = 0.0082 for wild-type and homozygous mutants, *P < 0.05; **P < 0.01. (c), Representative color Doppler echocardiograms for wild-type zebrafish show blood flow during systole (blue) and during diastole (red). A representative pulsed-wave Doppler image of a wild-type zebrafish shows normal flow pattern. Representative color Doppler echocardiograms for robo4∆7/∆7 mutant zebrafish show blood flow during systole (blue) and regurgitation (red) during diastole from the bulbus arteriosus (BA) into the ventricle (V). The white dotted line marks the ventricle from the bulbus arteriosus. A representative pulsed-wave Doppler image of robo4∆7/∆7 zebrafish shows regurgitant flow through the ventriculo-bulbar valve (orange arrow). The yellow arrow marks normal flow through the ventriculo-bulbar valve. (d), Approximately 11 of 41 (26.8%) of robo4∆7 adult mutants (heterozygous and homozygous, both male and female) exhibit aberrant echocardiograms with regurgitation or turbulence, while only 4 of 45 (8.88%) of wild-type adult fish (clutch mate and AB fish, both male and female) showed this phenotype. Statistical differences were calculated per Fisher’s exact test, one-tailed P value, *P = 0.028 (P < 0.05 is considered statistically significant). The tester was blinded to genotype during the echocardiography procedure and analysis. (e), Histological staining (H&E and Masson’s trichrome) of the ventricle and bulbus arteriosus for wild-type and robo4∆7/∆7 zebrafish. V, ventricle; BA, bulbus arteriosus.
At all stages, mutants did not show gross cardiac, craniofacial, or trunk defects (n = 100 embryos). The tester was blinded to genotype during phenotyping. h.p.f., hours post-fertilization; d.p.f., days post-fertilization.
(a), Robo4 mRNA expression, normalized to Gapdh, was analyzed by pooling four samples per genotype and performing qPCR. Error bars show mean +/− s.d., n = 4 per genotype pooled, one experiment. Asterisks signify statistical differences per a two-tailed Student’s t test relative to control (**P = 0.0007). (b), H&E histology of a quadricuspid valve in a regurgitant Robo4tm1Lex/tm1Lex mouse. WT, wild-type mice; KO, knockout mice.
Supplementary Figure 8 A knock-in line harboring a mutation (RoboSkip13) at the splice acceptor site in exon 13 (c.2089+1G>T).
(a), Sanger sequencing of the splice-site mutation. (b,c), In-frame skipping of exon 13 (Ex12/14) or inclusion of exon 13 with activation of a cryptic splice donor in intron 13 that adds 15 bp to the mature mRNA (Ex12/13+15bp/14), which is predicted to add five extra amino acids between those encoded by exons 13 and 14. (d), TaqMan probes were used to directly compare exon 12/13 and exon 2/3 expression as this would provide a relative ratio of exon 13 skipping versus wild-type. Error bars show mean +/− s.d. (n = 4). Asterisks signify statistical differences per a one-way ANOVA with Tukey’s post hoc (d.f. = 2, *P < 0.05, **P < 0.01).
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Gould, R.A., Aziz, H., Woods, C.E. et al. ROBO4 variants predispose individuals to bicuspid aortic valve and thoracic aortic aneurysm. Nat Genet 51, 42–50 (2019). https://doi.org/10.1038/s41588-018-0265-y
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