Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births1; the incidence of CHD is up to tenfold higher in human fetuses2,3. A genetic contribution is strongly suggested by the association of CHD with chromosome abnormalities and high recurrence risk4. Here we report findings from a recessive forward genetic screen in fetal mice, showing that cilia and cilia-transduced cell signalling have important roles in the pathogenesis of CHD. The cilium is an evolutionarily conserved organelle projecting from the cell surface with essential roles in diverse cellular processes. Using echocardiography, we ultrasound scanned 87,355 chemically mutagenized C57BL/6J fetal mice and recovered 218 CHD mouse models. Whole-exome sequencing identified 91 recessive CHD mutations in 61 genes. This included 34 cilia-related genes, 16 genes involved in cilia-transduced cell signalling, and 10 genes regulating vesicular trafficking, a pathway important for ciliogenesis and cell signalling. Surprisingly, many CHD genes encoded interacting proteins, suggesting that an interactome protein network may provide a larger genomic context for CHD pathogenesis. These findings provide novel insights into the potential Mendelian genetic contribution to CHD in the fetal population, a segment of the human population not well studied. We note that the pathways identified show overlap with CHD candidate genes recovered in CHD patients5, suggesting that they may have relevance to the more complex genetics of CHD overall. These CHD mouse models and >8,000 incidental mutations have been sperm archived, creating a rich public resource for human disease modelling.

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  1. 1.

    , , , & Prevalence of congenital heart defects in metropolitan Atlanta, 1998–2005. J. Pediatr. 153, 807–813 (2008)

  2. 2.

    Incidence of congenital heart disease: II. Prenatal incidence. Pediatr. Cardiol. 16, 103–113 (1995)

  3. 3.

    , , & Spectrum of congenital heart defects and extracardiac malformations associated with chromosomal abnormalities: results of a seven year necropsy study. Heart 82, 34–39 (1999)

  4. 4.

    et al. Recurrence of congenital heart defects in families. Circulation 120, 295–301 (2009)

  5. 5.

    et al. De novo mutations in histone-modifying genes in congenital heart disease. Nature 498, 220–223 (2013)

  6. 6.

    et al. Interrogating congenital heart defects with noninvasive fetal echocardiography in a mouse forward genetic screen. Circ Cardiovasc Imaging 7, 31–42 (2014)

  7. 7.

    & Diagnosis of heterotaxy syndrome by fetal echocardiography. Am. J. Cardiol. 82, 1147–1149 (1998)

  8. 8.

    et al. Prenatal diagnosis of cardiosplenic syndromes: a 10-year experience. Ultrasound Obstet. Gynecol. 22, 451–459 (2003)

  9. 9.

    , , , & Spectrum of cardiovascular disease, accuracy of diagnosis, and outcome in fetal heterotaxy syndrome. Am. J. Cardiol. 97, 720–724 (2006)

  10. 10.

    et al. Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia. J. Clin. Invest. 117, 3742–3752 (2007)

  11. 11.

    et al. Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome. Genet. Med. 11, 473–487 (2009)

  12. 12.

    & The left–right axis in the mouse: from origin to morphology. Development 133, 2095–2104 (2006)

  13. 13.

    et al. Ion torrent sequencing for conducting genome-wide scans for mutation mapping analysis. Mamm. Genome 25, 120–128 (2014)

  14. 14.

    et al. A gene-driven approach to the identification of ENU mutants in the mouse. Nature Genet. 30, 255–256 (2002)

  15. 15.

    et al. A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Mol. Cell 6, 1287–1295 (2000)

  16. 16.

    , & A phenotype-based screen for embryonic lethal mutations in the mouse. Proc. Natl Acad. Sci. USA 95, 7485–7490 (1998)

  17. 17.

    , , & Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left–right asymmetry. J. Clin. Invest. 102, 1077–1082 (1998)

  18. 18.

    & The roles of evolutionarily conserved functional modules in cilia-related trafficking. Nature Cell Biol. 15, 1387–1397 (2013)

  19. 19.

    , & Spectrum of clinical diseases caused by disorders of primary cilia. Proc. Am. Thorac. Soc. 8, 444–450 (2011)

  20. 20.

    , & When cilia go bad: cilia defects and ciliopathies. Nature Rev. Mol. Cell Biol. 8, 880–893 (2007)

  21. 21.

    et al. TGF-β signaling is associated with endocytosis at the pocket region of the primary cilium. Cell Rep. 3, 1806–1814 (2013)

  22. 22.

    et al. Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways. Cell 145, 513–528 (2011)

  23. 23.

    , , & The polycystic kidney disease-related proteins Bicc1 and SamCystin interact. Biochem. Biophys. Res. Commun. 383, 16–21 (2009)

  24. 24.

    et al. ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3. Nature Genet. 45, 951–956 (2013)

  25. 25.

    et al. Polarized traffic of LRP1 involves AP1B and SNX17 operating on Y-dependent sorting motifs in different pathways. Mol. Biol. Cell 20, 481–497 (2009)

  26. 26.

    et al. Cardiovascular phenotyping of fetal mice by noninvasive high-frequency ultrasound facilitates recovery of ENU-induced mutations causing congenital cardiac and extracardiac defects. Physiol. Genomics 24, 23–36 (2005)

  27. 27.

    et al. Microcomputed tomography provides high accuracy congenital heart disease diagnosis in neonatal and fetal mice. Circ Cardiovasc Imaging 6, 551–559 (2013)

  28. 28.

    et al. The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance. J. Cell Biol. 157, 103–114 (2002)

  29. 29.

    , & Piecing together a ciliome. Trends Genet. 22, 491–500 (2006)

  30. 30.

    , & BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21, 3448–3449 (2005)

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We thank R. Ramirez for early assistance with the mutagenesis breeding pipeline, R. Subramanian and D. Farkas for early assistance with necropsy and pathology examination of mutants, A. Srinivasan for early assistance with exome sequencing, S. Fatakia for assistance with sequencing data maintenance, M. Wong and C. Krise for assistance with mouse curation, B. Beutler for advice on mapping mutations using intercrosses with the C57BL/10J strain and whole-mouse exome sequencing analysis, D. Weeks and Y. Shan for assistance in statistical modelling of target gene size estimates, E. Goldmuntz for helpful discussions and critical review of the manuscript, and the New England Research Institutes (NERI) for constructing the CHD Mouse Mutation Database. The project was supported by award numbers U01HL098180 (to C.W.L.) and U01HL098188 (to NERI) from the National Heart, Lung, and Blood Institute, R01MH094564 (to M.K.G.) from the National Institute of Mental Health, and HG000330 (to J.E.) from the National Human Genome Research Institute. Funding was also provided by the University of Pittsburgh School of Medicine. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute, the National Human Genome Research Institute or the National Institutes of Health.

Author information

Author notes

    • You Li
    • , Nikolai T. Klena
    • , George C. Gabriel
    •  & Xiaoqin Liu

    These authors contributed equally to this work.


  1. Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15201, USA

    • You Li
    • , Nikolai T. Klena
    • , George C. Gabriel
    • , Xiaoqin Liu
    • , Andrew J. Kim
    • , Kristi Lemke
    • , Yu Chen
    • , Bishwanath Chatterjee
    • , Rama Rao Damerla
    • , Chienfu Chang
    • , Hisato Yagi
    • , Shane Anderton
    • , Caroline Lawhead
    • , Anita Vescovi
    • , Richard Francis
    • , Kimimasa Tobita
    •  & Cecilia W. Lo
  2. Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA

    • William Devine
  3. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA

    • Jovenal T. San Agustin
    •  & Gregory J. Pazour
  4. Department of Biomedical Informatics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15206, USA

    • Mohamed Thahir
    •  & Madhavi K. Ganapathiraju
  5. Intelligent Systems Program, School of Arts and Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 16260, USA

    • Mohamed Thahir
    •  & Madhavi K. Ganapathiraju
  6. The Jackson Laboratory, Bar Harbor, Maine 04609, USA

    • Herbert Pratt
    • , Judy Morgan
    • , Leslie Haynes
    • , Cynthia L. Smith
    • , Janan T. Eppig
    •  & Laura Reinholdt
  7. The Heart Center, Children’s National Medical Center, Washington DC 20010, USA

    • Linda Leatherbury


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Study design: C.W.L. ENU mutagenesis, line cryopreservation and JAX strain datasheet construction: H.P., L.R., J.M., L.H. Mouse breeding, sample collection, sample tracking: S.A., C.L., K.L., G.C.G., A.V., C.W.L. Electronic database construction and maintenance: C.C. MGI curation: K.T., G.C.G., L.L., C.W.L., C.L.S., J.T.E. CHD phenotyping: X.L., K.L., Y.C., G.C.G., A.J.K., S.A., W.D., C.W.L., L.L., K.T., R.F. Cilia immunostain and histology: J.T.S.A., G.J.P., R.F. Analysis of airway and node cilia motility: R.F., K.L., G.C.G., A.J.K. Exome sequencing analysis: Y.L. Mutation validation: N.T.K., B.C., R.R.D., H.Y., Y.L. Mutation mapping: R.R.D., N.T.K., B.C., Y.L. Interactome analysis: M.K.G., M.T. Ciliome and pathway annotation: C.W.L., G.J.P., G.C.G., N.T.K., Y.L. Manuscript preparation: C.W.L., Y.L., N.T.K., G.C.G.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Cecilia W. Lo.

All mutant mouse lines recovered in this mouse mutagenesis screen and their phenotype description and causative mutations are curated in the MGI database ( and can be retrieved by entering “b2b” in the search box. All mutant mouse lines curated in MGI can be reanimated from sperm cryopreserved in the Jackson Laboratory (JAXMice) Repository. All mutations recovered by mouse exome sequencing analysis are searchable together with phenotype information via the public Bench to Bassinet Congenital Heart Disease Mouse Mutation Database ( The mouse exome datasets are available from the GNomEx Cardiovascular Development Consortium Datahub (

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Data 1

    This data file contains CHD mutations and comparison to other mutant alleles and human diseases.

  2. 2.

    Supplementary Data 2

    This data file contains position and sequence conservation of CHD mutations.

  3. 3.

    Supplementary Data 3

    This data file contains gene ontology and pathway analyses of CHD interactome genes.

  4. 4.

    Supplementary Data 4

    This data file contains ciliome and pathway annotation for 28 CHD candidate genes recovered from Pediatric Cardiac Genomics Consortium human CHD patient exome sequencing analysis.


  1. 1.

    Ultrasound imaging of normal fetus shown in Figure 1a.

    Vevo 2100 color flow Doppler imaging in coronal view show normal alignment of the two great arteries with normal connection to the two ventricles.

  2. 2.

    Ultrasound imaging of mutant fetus from line b2b2025 shows DORV and presence of a VSD

    Vevo2100 color flow imaging in coronal view of fetus in Figure 1e-k showed aorta and pulmonary artery side-by side, both emerging from the right ventricle (RV) indicating DORV, and a shunting of blood between the two ventricles indicating ventricular septal defect (VSD).

  3. 3.

    Ultrasound imaging of mutant fetus from line b2b2025 shows AVSD and muscular VSD

    Vevo 2100 color flow imaging in transverse view of fetus in Figure 1e-k detected forward blood flow and regurgitation from a common atrioventricular valve suggesting atrioventricular septal defect. Also observed was a muscular ventricular septal defect.

  4. 4.

    Ultrasound imaging of mutant fetus from line b2b2025 shows heterotaxy

    Vevo2100 2D imaging in coronal view of fetus in Figure 1e-k detected heart apex pointing to left suggesting levocardia, but stomach (Stom) located on right, which together indicated this fetus has heterotaxy.

  5. 5.

    Videomicroscopy of ciliary motion of tracheal epithelia of newborn Foxj1 mutant mice

    Tracheal airway epithelium in a newborn homozygous Foxj1b2b774 mutant mouse shows normal ciliary motion.

  6. 6.

    Videomicroscopy of ciliary motion in embryonic node of Foxj1 mutant embryo

    Cilia in the embryonic node of a homozygous Foxj1b2b774 mutant embryo shows dyskinetic ciliary motion and no nodal flow.

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