Letter | Published:

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%)1,2,3 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 AscAA58, 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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Data availability

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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Change history

  • 07 December 2018

    In the version of this article initially published, the name of author Christian Lacks Lino Cardenas was incorrect in the XML such that the surname was coded as Cardenas whereas it should have been coded as Lino Cardenas. The error has been corrected in the XML of the article.

References

  1. 1.

    Fedak, P. W. M. et al. Clinical and pathophysiological implications of a bicuspid aortic valve. Circulation 106, 900–904 (2002).

  2. 2.

    Mack, G. & Silberbach, M. Aortic and pulmonary stenosis. Pediatr. Rev. 21, 79–85 (2000).

  3. 3.

    Ward, C. Clinical significance of the bicuspid aortic valve. Heart 83, 81–85 (2000).

  4. 4.

    Tadros, T. M., Klein, M. D. & Shapira, O. M. Ascending aortic dilatation associated with bicuspid aortic valve: pathophysiology, molecular biology, and clinical implications. Circulation 119, 880–890 (2009).

  5. 5.

    Cripe, L., Andelfinger, G., Martin, L. J., Shooner, K. & Benson, D. W. Bicuspid aortic valve is heritable. J. Am. Coll. Cardiol. 44, 138–143 (2004).

  6. 6.

    Garg, V. et al. Mutations in NOTCH1 cause aortic valve disease. Nature 437, 270–274 (2005).

  7. 7.

    McKellar, S. H. et al. Novel NOTCH1 mutations in patients with bicuspid aortic valve disease and thoracic aortic aneurysms. J. Thorac. Cardiovasc. Surg. 134, 290–296 (2007).

  8. 8.

    Tan, H. L. et al. Nonsynonymous variants in the SMAD6 gene predispose to congenital cardiovascular malformation. Hum. Mutat. 33, 720–727 (2012).

  9. 9.

    Clementi, M., Notari, L., Borghi, A. & Tenconi, R. Familial congenital bicuspid aortic valve: a disorder of uncertain inheritance. Am. J. Med. Genet. 62, 336–338 (1996).

  10. 10.

    Huntington, K., Hunter, A. G. W. & Chan, K. L. A prospective study to assess the frequency of familial clustering of congenital bicuspid aortic valve. J. Am. Coll. Cardiol. 30, 1809–1812 (1997).

  11. 11.

    Mckusick, V. A., Logue, R. B. & Bahnson, H. T. Association of aortic valvular disease and cystic medial necrosis of the ascending aorta; report of four instances. Circulation 16, 188–194 (1957).

  12. 12.

    Loscalzo, M. L. et al. Familial thoracic aortic dilation and bicommissural aortic valve: a prospective analysis of natural history and inheritance. Am. J. Med. Genet. A 143, 1960–1967 (2007).

  13. 13.

    Isselbacher, E. M. Thoracic and abdominal aortic aneurysms. Circulation 111, 816–828 (2005).

  14. 14.

    Williams, J. A. et al. Early surgical experience with Loeys-Dietz: a new syndrome of aggressive thoracic aortic aneurysm disease. Ann. Thorac. Surg. 83, S785–S790 (2007).

  15. 15.

    Van Hemelrijk, C., Renard, M. & Loeys, B. The Loeys-Dietz syndrome: an update for the clinician. Curr. Opin. Cardiol. 25, 546–551 (2010).

  16. 16.

    Guo, D.-C. et al. Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections. Nat. Genet. 39, 1488–1493 (2007).

  17. 17.

    Pereira, L. et al. Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome. Nat. Genet. 17, 218–222 (1997).

  18. 18.

    Zhu, L. et al. Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus. Nat. Genet. 38, 343–349 (2006).

  19. 19.

    Wang, L. et al. Mutations in myosin light chain kinase cause familial aortic dissections. Am. J. Hum. Genet. 87, 701–707 (2010).

  20. 20.

    van de Laar, I. M. B. H. et al. Phenotypic spectrum of the SMAD3-related aneurysms-osteoarthritis syndrome. J. Med. Genet. 49, 47–57 (2012).

  21. 21.

    Loeys, B. L. et al. Aneurysm syndromes caused by mutations in the TGF-β receptor. N. Engl. J. Med. 355, 788–798 (2006).

  22. 22.

    Park, K. W. et al. Robo4 is a vascular-specific receptor that inhibits endothelial migration. Dev. Biol. 261, 251–267 (2003).

  23. 23.

    Jones, C. A. et al. Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability. Nat. Med. 14, 448–453 (2008).

  24. 24.

    Cai, H. et al. Roundabout 4 regulates blood-tumor barrier permeability through the modulation of ZO-1, occludin, and claudin-5 expression. J. Neuropathol. Exp. Neurol. 74, 25–37 (2015).

  25. 25.

    Mommersteeg, M. T. M., Yeh, M. L., Parnavelas, J. G. & Andrews, W. D. Disrupted Slit-Robo signalling results in membranous ventricular septum defects and bicuspid aortic valves. Cardiovasc. Res. 106, 55–66 (2015).

  26. 26.

    Bedell, V. M. et al. Roundabout4 is essential for angiogenesis in vivo. Proc. Natl Acad. Sci. USA 102, 6373–6378 (2005).

  27. 27.

    Carmeliet, P. et al. Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat. Genet. 17, 439–444 (1997).

  28. 28.

    Borges, L. F. et al. Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta. Histopathology 57, 917–932 (2010).

  29. 29.

    Maleki, S. et al. Mesenchymal state of intimal cells may explain higher propensity to ascending aortic aneurysm in bicuspid aortic valves. Sci. Rep. 6, 35712 (2016).

  30. 30.

    Kostina, A. S. et al. Notch-dependent EMT is attenuated in patients with aortic aneurysm and bicuspid aortic valve. Biochim. Biophys. Acta 1862, 733–740 (2016).

  31. 31.

    Brooke, B. S. et al. Angiotensin II blockade and aortic-root dilation in Marfan’s syndrome. N. Engl. J. Med. 358, 2787–2795 (2008).

  32. 32.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  33. 33.

    Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

  34. 34.

    Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).

  35. 35.

    McKenna, A. et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

  36. 36.

    DePristo, M. A. et al. A framework for variation discovery and genotyping using next- generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).

  37. 37.

    Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

  38. 38.

    Anderl, J., Ma, J. & Armstrong, L. Improved assays for quantification of in vitro vascular permeability (https://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8623.pdf).

  39. 39.

    Gould, R. A. et al. Multi-scale biomechanical remodeling in aging and genetic mutant murine mitral valve leaflets: insights into Marfan syndrome. PLoS ONE 7, e44639 (2012).

  40. 40.

    Chiu, Y.-N., Norris, R. A., Mahler, G., Recknagel, A. & Butcher, J. T. Transforming growth factor β, bone morphogenetic protein, and vascular endothelial growth factor mediate phenotype maturation and tissue remodeling by embryonic valve progenitor cells: relevance for heart valve tissue engineering. Tissue. Eng. Part A 16, 3375–3383 (2010).

  41. 41.

    Gould, R. A. et al. Cyclic strain anisotropy regulates valvular interstitial cell phenotype and tissue remodeling in three-dimensional culture. Acta Biomater. 8, 1710–1719 (2012).

  42. 42.

    Liang, C.-C. C., Park, A. Y. & Guan, J.-L. L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2, 329–333 (2007).

  43. 43.

    Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio). 5th edn, (Univ. Oregon Press, Eugene, 2007).

  44. 44.

    Sander, J. D., Zaback, P., Joung, J. K., Voytas, D. F. & Dobbs, D. Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res. 35, W599–W605 (2007).

  45. 45.

    Sander, J. D. et al. ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res. 38, W462–W468 (2010).

  46. 46.

    Jao, L.-E., Wente, S. R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl Acad. Sci. USA 110, 13904–13909 (2013).

  47. 47.

    Jaskula-Ranga, V. & Zack, D. J. grID: A CRISPR-Cas9 guide RNA database and resource for genome-editing. Preprint at bioRxiv https://doi.org/10.1101/097352 (2016).

  48. 48.

    Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

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Acknowledgements

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.

Author information

H.C.D., B.L.L., G.A., E.S., G.M., and the MIBAVA Leducq Consortium recruited participants for the study. A.S.M., H.C.D., G.A., and B.L.L. were instrumental in the experimental design and interpretation of the data. C.P., N.S., H.Ling, A.A.K., I.L., E.C., A.V., H.M.B., A.-C.L., V.J.-R., H.J., A.A.S., C.L.B., P.T.E., H.Lin, E.M.I., C.L.L.C., J.T.B., G.C.H., M.E.L., Baylor-Hopkins Center for Mendelian Genomics, L.M., A.F.-C., J.M.A.V., M.W., S.M., P.E., S.A.Mohamed., L.V.L., and F.W. were instrumental in analyses of portions of the sequencing data and clinical descriptions. R.A.G. and M.A.S.-S. performed in vitro experiments, and R.A.G. performed mouse experiments with assistance from D.B. under the supervision of H.C.D. C.E.W. performed all zebrafish experiments with assistance from C.R.M., R.R., and S.A.McClymont under the supervision of A.S.M. The initial mouse studies, genetic analysis, and identification of the gene of interest were performed by H.A. under the supervision of H.C.D. H.C.D., A.S.M., C.E.W., R.A.G., H.A., and M.A.S.-S. wrote the manuscript with contributions from all remaining authors.

Competing interests

The authors declare no competing interests.

Correspondence to Andrew S. McCallion or Harry C. Dietz.

Integrated supplementary information

  1. Supplementary Figure 1 Pedigrees of the seven other families enrolled into our WES initiative.

    AscAA, ascending aortic aneurysm; AoRA, aortic root aneurysm; AoDiss, aortic dissection; AoSurg, underwent aortic repair surgery; BAV, bicuspid aortic valve.

  2. Supplementary Figure 2 ROBO4 variants and pathology identified in the WES family cohort.

    (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.

  3. 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 (ce), 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.

  4. 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.

  5. Supplementary Figure 5 robo4 deficiency results in aberrant blood flow in the adult zebrafish.

    (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.

  6. Supplementary Figure 6 robo4 loss-of-function mutants do not show an overt embryonic phenotype.

    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.

  7. Supplementary Figure 7 Phenotype data for Robo4tm1Lex knockout mice with AscAA.

    (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.

  8. 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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Further reading

Fig. 1: Identification of ROBO4 variants segregating in families with BAV and aortic aneurysms.
Fig. 2: Evaluation of ascending aortic aneurysm tissue resected from patient 1.II:1, compared to an age- and sex-matched control.
Fig. 3: ROBO4 mutant alleles impair endothelial barrier function.
Fig. 4: Robo4 knockout causes aortic valve defects and aortic aneurysm in mice.
Fig. 5: Knock-in splice-site mutation (c.2089+1G>T; Robo4Skip13) causes aortic valve defects and aortic aneurysm in mice.
Supplementary Figure 1: Pedigrees of the seven other families enrolled into our WES initiative.
Supplementary Figure 2: ROBO4 variants and pathology identified in the WES family cohort.
Supplementary Figure 3: Cellular phenotypes observed in HAECs transfected with ROBO4 mutant alleles or siRNA to silence ROBO4 expression.
Supplementary Figure 4: Immunofluorescent staining for ROBO4 in the developing mouse outflow tract (OFT) and human adult ascending aorta.
Supplementary Figure 5: robo4 deficiency results in aberrant blood flow in the adult zebrafish.
Supplementary Figure 6: robo4 loss-of-function mutants do not show an overt embryonic phenotype.
Supplementary Figure 7: Phenotype data for Robo4tm1Lex knockout mice with AscAA.
Supplementary Figure 8: A knock-in line harboring a mutation (RoboSkip13) at the splice acceptor site in exon 13 (c.2089+1G>T).