Letter | Published:

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

Nature Geneticsvolume 51pages4250 (2019) | Download Citation

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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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

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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

Author notes

  1. These authors contributed equally: Russell A. Gould, Hamza Aziz, Courtney E. Woods.

  2. These authors jointly supervised this work: Andrew S. McCallion, Harry C. Dietz.

Affiliations

  1. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • Russell A. Gould
    • , Hamza Aziz
    • , Courtney E. Woods
    • , Manuel Alejandro Seman-Senderos
    • , Elizabeth Sparks
    • , Rebecca Rose
    • , Nara Sobreira
    • , Gretchen MacCarrick
    • , Christopher L. Bennett
    • , David Valle
    • , Harry C. Dietz
    • , Andrew S. McCallion
    • , Andrew S. McCallion
    •  & Harry C. Dietz
  2. Howard Hughes Medical Institute, Baltimore, MD, USA

    • Russell A. Gould
    • , Hamza Aziz
    • , Christopher L. Bennett
    • , Harry C. Dietz
    •  & Harry C. Dietz
  3. Cardiovascular Genetics, Department of Pediatrics, Centre Hospitalier Universitaire Sainte-Justine Research Centre, Université de Montréal, Montreal, Quebec, Canada

    • Christoph Preuss
    •  & Florian Wünnemann
  4. The Jackson Laboratory, Bar Harbor, ME, USA

    • Christoph Preuss
    • , Gregor Andelfinger
    •  & Gregor Andelfinger
  5. Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • Djahida Bedja
    • , Cassandra R. Moats
    • , Andrew S. McCallion
    •  & Andrew S. McCallion
  6. Heart and Vascular Institute, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • Djahida Bedja
  7. Oregon National Primate Research Center, Portland, OR, USA

    • Cassandra R. Moats
  8. Center for Inherited Disease Research, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • Sarah A. McClymont
    • , Hua Ling
    •  & Hua Ling
  9. Center for Medical Genetics, Faculty of Medicine and Health Sciences, Antwerp University Hospital and University of Antwerp, Antwerp, Belgium

    • Ajay Anand Kumar
    • , Ilse Luyckx
    • , Elyssa Cannaerts
    • , Aline Verstraeten
    • , Bart L. Loeys
    • , Lut Van Laer
    • , Lut Van Laer
    •  & Bart L. Loeys
  10. Center for Molecular Medicine, Department of Medicine Solna, University Hospital Solna, Karolinska Institutet, Stockholm, Sweden

    • Hanna M. Björk
    • , Per Eriksson
    •  & Per Eriksson
  11. Department of Cardiac and Thoracic Vascular Surgery, University Hospital Lübeck, Lübeck, Germany

    • Ann-Cathrin Lehsau
    • , Salah A. Mohamed
    •  & Salah A. Mohamed
  12. Wilmer Eye Institute in the Department of Ophthalmology at the Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • Vinod Jaskula-Ranga
  13. The Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA

    • Henrik Lauridsen
    •  & Jonathan T. Butcher
  14. Rex Hospital, Raleigh, NC, USA

    • Asad A. Shah
  15. The Broad Institute of MIT and Harvard, Cambridge, MA, USA

    • Patrick T. Ellinor
  16. Cardiovascular Research Institute, Massachussets General Hospital, Charlestown, MA, USA

    • Patrick T. Ellinor
  17. Section of Computational Biomedicine, Department of Medicine, Boston University School of Medicine, Boston, MA, USA

    • Honghuang Lin
  18. Thoracic Aortic Center, Division of Cardiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    • Eric M. Isselbacher
  19. Cardiovascular Research Center, Division of Cardiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    • Christian Lacks Lino Cardenas
  20. Division of Cardiovascular and Thoracic Surgery, Duke University Medical Center, Durham, NC, USA

    • G. Chad Hughes
  21. Thoracic Aortic Center and Cardiovascular Genetics Program, Division of Cardiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    • Mark E. Lindsay
  22. Division of Cardiology, The Hospital for Sick Children, Labatt Family Heart Centre, Toronto, Ontario, Canada

    • Luc Mertens
    •  & Luc Mertens
  23. Department of Molecular Medicine and Surgery, University Hospital Solna, Karolinska Institutet, Stockholm, Sweden

    • Anders Franco-Cereceda
    •  & Anders Franco-Cereceda
  24. Department of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

    • Judith M. A. Verhagen
  25. Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

    • Seema Mital
    • , Marja Wessels
    •  & Seema Mital
  26. Department of Human Genetics, Radboud University Medical Centre, Nijmegen, The Netherlands

    • Bart L. Loeys
    •  & Bart L. Loeys
  27. Department of Pediatrics, Université de Montréal, Montreal, Quebec, Canada

    • Gregor Andelfinger
    •  & Gregor Andelfinger
  28. Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • Harry C. Dietz
    • , Andrew S. McCallion
    • , Andrew S. McCallion
    •  & Harry C. Dietz
  29. Department of Pediatrics, Division of Pediatric Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • Harry C. Dietz
    •  & Harry C. Dietz
  30. Baylor College of Medicine, Houston, TX, USA

    • James Lupski

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Consortia

  1. Baylor-Hopkins Center for Mendelian Genomics

  1. MIBAVA Leducq Consortium

Contributions

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.

Corresponding authors

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).

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Tables 1–7

  2. Reporting Summary

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DOI

https://doi.org/10.1038/s41588-018-0265-y