Global genetic analysis in mice unveils central role for cilia in congenital heart disease

Journal name:
Nature
Volume:
521,
Pages:
520–524
Date published:
DOI:
doi:10.1038/nature14269
Received
Accepted
Published online

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.

At a glance

Figures

  1. Ultrasound diagnoses of CHD and cilia defects in CHD mutants.
    Figure 1: Ultrasound diagnoses of CHD and cilia defects in CHD mutants.

    a, b, Vevo 2100 colour flow imaging showed criss-crossing of blood flow indicating normal aorta (Ao) and pulmonary artery (PA) alignment (a; Supplementary Video 1), confirmed by histopathology (b). Cd, caudal; Cr, cranial; L, left; LV, left ventricle; R, right; RV, right ventricle. c, d, Embryonic day (E)16.5 mutant mouse (line b2b327) exhibited a blood flow pattern indicating single great artery (pulmonary artery) and ventricular septal defect (VSD) (c), suggesting aortic atresia with ventricular septal defect, confirmed by histopathology (d). eh, Colour flow imaging of E15.5 mutant mouse (line b2b2025) with heterotaxy (stomach on right; Supplementary Video 4) showed side by side aorta and pulmonary artery, with the aorta emerging from the right ventricle, indicating DORV/ventricular septal defect (e, f; Supplementary Video 2) and the presence of AVSD (g, h; Supplementary Videos 3,4). LA, left atrium; RA, right atrium. ik, Histopathology also showed a bicuspid aortic valve (BAV) (i), interrupted aortic arch (IAA) (j), and common atrioventricular (AV) valve (k). ln, Cc2d2a-mutant mouse exhibits dextrocardia with ventricular inversion (dextroversion) (m), and AVSD (l) with malformed atrioventricular cushions (n), but normal outflow cushions. Atr, atrium; mLV, morphologic left ventricle; m/m, Cc2d2a-mutant mouse; mRV, morphologic right ventricle. ox, Confocal imaging of E12.5 Cc2d2a-mutant mouse (m/m) versus wild-type (+/+) embryo sections showed no cilia in the atrioventrcular cushion (o, p), but normal ciliation in the outflow cushion (OFT cushion) (q, r). sx, Fewer and shorter cilia were observed in other mutant embryo tissues. Red, acetylated tubulin (Acet-tub.); green, IFT88.

  2. CHD genes recovered from mouse mutagenesis screen.
    Figure 2: CHD genes recovered from mouse mutagenesis screen.

    Diagrams illustrate biological context of CHD gene function (colour highlighting indicates CHD genes recovered; asterisks denote CHD genes recovered from previous screen26). For clarity, ciliome genes (Dctn5, Fuz) with endocytic function and/or involved in cilia-transduced signalling are not shown in the ciliogenesis panel. AP, adaptor protein complex; R, receptor; MVB, multi-vesicular body; TGN, trans-Golgi network; Ub, ubiquitination.

  3. Interactome network of CHD genes with known and predicted interactors.
    Figure 3: Interactome network of CHD genes with known and predicted interactors.

    a, An interactome network was constructed comprising the 61 CHD genes (magenta squares) and their known (blue edges) and predicted (magenta edges) interactions. b, The CHD interactome yielded significant enrichment of GO terms related to developmental processes. Circle size is proportional to the number of genes associated with each GO term.

  4. Breeding, phenotyping and mutation recovery pipeline for mouse forward genetic screen.
    Extended Data Fig. 1: Breeding, phenotyping and mutation recovery pipeline for mouse forward genetic screen.

    a, Two generation backcross breeding scheme used to generate G3 mutants with recessive mutations causing congenital heart defects, with all offspring from a single G1 male defined as a distinct pedigree or mutant line. b, Pipeline for recovery and curation and cryopreservation of CHD mutant mouse models and the recovery of pathogenic CHD causing mutations.

  5. Situs anomalies and congenital heart defects in Ap1b1b2b1660 mutants.
    Extended Data Fig. 2: Situs anomalies and congenital heart defects in Ap1b1b2b1660 mutants.

    ac, Mutants from line 1660, identified with an Ap1b1 mutation, exhibit situs solitus (a), situs inversus (b) or heterotaxy (c). Situs solitus, characterized by normal left–right visceral organ positioning, the heart apex (arrow) points to the left (levocardia), four lung lobes are on the right and one on the left, stomach is to the left, and the dominant liver lobe is on the right. With situs inversus, there is complete mirror reversal of organ situs, while with heterotaxy, visceral organ situs is randomized, such as dextrocardia with levogastria shown in c. dg, The heterotaxy mutant in c exhibits complex CHD with atrioventricular septal defect (AVSD) (d), ventricular septal defect (VSD) (e), duplicated inferior vena cava (IVC) (f) and left pulmonary isomerism with bilateral single lung lobes (g). Ao, aorta; L1–5, lung lobes 1–5; Lv1–3, live lobes 1–3; mLA, morphologic left atrium; mLV, morphologic left ventricle; mRA, morphologic right atrium; mRV, morphologic right ventricle; PA, pulmonary artery; Stm, stomach.

  6. Distribution of pathogenic mutations recovered from the forward genetic screen.
    Extended Data Fig. 3: Distribution of pathogenic mutations recovered from the forward genetic screen.

    a, Distribution of all incidental coding mutations (left), pathogenic mutations (middle) and ciliome CHD genes (right) recovered from 113 CHD mouse mutant lines. b, Recovery of pathogenic mutations and associated CHD phenotypes. Grey-filled boxes indicate CHD mutations in genes not previously identified to cause CHD. Ao, aorta; AVSD, atrioventricular septal defect; BVH, biventricular hypertrophy; DORV, double outlet right ventricle; IAA, interrupted aortic arch; MAPCA, major aortopulmonary collateral artery; PA, pulmonary artery; PTA, persistent truncus arteriosus; RAA, right aortic arch; TGA, transposition of the great arteries; VSD, ventricular septal defect; VS, vascular sling.

  7. Pathogenic splicing mutations causing CHD.
    Extended Data Fig. 4: Pathogenic splicing mutations causing CHD.

    a, The 19 pathogenic splicing mutations recovered are shown, with mutations located beyond the 2-base canonical splice junction highlighted in grey. b, Schematic diagram showing the anomalous Dnah5c.133290-10T>A mutant transcript observed, with the polymerase chain reaction (PCR) primer location and anomalous PCR product size indicated. c, Sanger sequencing profile showing point mutation in Dnah5c.133290-10T>A mutant transcript versus that of wild type. d, PCR amplification of Dnah5c.133290-10T>A heterozygous mutant (m/+) showed the expected 514 bp wild-type and 271 bp mutant PCR product, while only the 271 bp mutant product was observed in the homozygous mutant (m/m) sample.

  8. Ciliome mutations causing CHD with and without laterality defects.
    Extended Data Fig. 5: Ciliome mutations causing CHD with and without laterality defects.

    A flow chart showing the distribution of ciliome versus non-ciliome CHD genes among laterality versus non-laterality CHD lines, and further stratification of ciliome CHD genes affecting primary versus motile cilia function.

  9. CHD phenotypes associated with mutations affecting endocytic trafficking.
    Extended Data Fig. 6: CHD phenotypes associated with mutations affecting endocytic trafficking.

    ac, Outflow tract malalignment defects with double outlet right ventricle (DORV) and overriding aorta (Ao) were observed in Ap2b1 (a), Dnm2 (b) and Snx17 (c) mutants, with Ap2b1 (a) mutant also showing anterior positioning of the aorta (Taussig–Bing type DORV). Snx17 mutant also has AVSD. d, e, Lrp2 (d) and Lrp1 (e) mutants both exhibited outflow tract septation defect with persistent truncus arteriosus (PTA). Scale bar, 0.5 mm.

  10. CHD genes associated with axon guidance.
    Extended Data Fig. 7: CHD genes associated with axon guidance.

    Diagram illustrating the biological context of several CHD genes known to be involved in axonal guidance (colour highlighting indicates CHD genes recovered from the present screen). Adapted from QIAGEN’s Ingenuity Pathway Analysis (http://www.qiagen.com/ingenuity).

Tables

  1. CHD genes with multiple alleles
    Extended Data Table 1: CHD genes with multiple alleles
  2. Ciliome mutations in laterality and non-laterality lines
    Extended Data Table 2: Ciliome mutations in laterality and non-laterality lines
  3. Mouse CHD genes and associated human diseases
    Extended Data Table 3: Mouse CHD genes and associated human diseases

Videos

  1. Ultrasound imaging of normal fetus shown in Figure 1a.
    Video 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. Ultrasound imaging of mutant fetus from line b2b2025 shows DORV and presence of a VSD
    Video 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. Ultrasound imaging of mutant fetus from line b2b2025 shows AVSD and muscular VSD
    Video 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. Ultrasound imaging of mutant fetus from line b2b2025 shows heterotaxy
    Video 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. Videomicroscopy of ciliary motion of tracheal epithelia of newborn Foxj1 mutant mice
    Video 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. Videomicroscopy of ciliary motion in embryonic node of Foxj1 mutant embryo
    Video 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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Author information

  1. These authors contributed equally to this work.

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

Affiliations

  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

Contributions

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 financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

All mutant mouse lines recovered in this mouse mutagenesis screen and their phenotype description and causative mutations are curated in the MGI database (http://www.informatics.jax.org) 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 (http://benchtobassinet.com/ForResearchers/BasicScienceDataResourceSharing/GeneDiscoveryinMouseModels.aspx) The mouse exome datasets are available from the GNomEx Cardiovascular Development Consortium Datahub (https://b2b.hci.utah.edu/gnomex/gnomexGuestFlex.jsp?topicNumber=67).

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Breeding, phenotyping and mutation recovery pipeline for mouse forward genetic screen. (233 KB)

    a, Two generation backcross breeding scheme used to generate G3 mutants with recessive mutations causing congenital heart defects, with all offspring from a single G1 male defined as a distinct pedigree or mutant line. b, Pipeline for recovery and curation and cryopreservation of CHD mutant mouse models and the recovery of pathogenic CHD causing mutations.

  2. Extended Data Figure 2: Situs anomalies and congenital heart defects in Ap1b1b2b1660 mutants. (156 KB)

    ac, Mutants from line 1660, identified with an Ap1b1 mutation, exhibit situs solitus (a), situs inversus (b) or heterotaxy (c). Situs solitus, characterized by normal left–right visceral organ positioning, the heart apex (arrow) points to the left (levocardia), four lung lobes are on the right and one on the left, stomach is to the left, and the dominant liver lobe is on the right. With situs inversus, there is complete mirror reversal of organ situs, while with heterotaxy, visceral organ situs is randomized, such as dextrocardia with levogastria shown in c. dg, The heterotaxy mutant in c exhibits complex CHD with atrioventricular septal defect (AVSD) (d), ventricular septal defect (VSD) (e), duplicated inferior vena cava (IVC) (f) and left pulmonary isomerism with bilateral single lung lobes (g). Ao, aorta; L1–5, lung lobes 1–5; Lv1–3, live lobes 1–3; mLA, morphologic left atrium; mLV, morphologic left ventricle; mRA, morphologic right atrium; mRV, morphologic right ventricle; PA, pulmonary artery; Stm, stomach.

  3. Extended Data Figure 3: Distribution of pathogenic mutations recovered from the forward genetic screen. (310 KB)

    a, Distribution of all incidental coding mutations (left), pathogenic mutations (middle) and ciliome CHD genes (right) recovered from 113 CHD mouse mutant lines. b, Recovery of pathogenic mutations and associated CHD phenotypes. Grey-filled boxes indicate CHD mutations in genes not previously identified to cause CHD. Ao, aorta; AVSD, atrioventricular septal defect; BVH, biventricular hypertrophy; DORV, double outlet right ventricle; IAA, interrupted aortic arch; MAPCA, major aortopulmonary collateral artery; PA, pulmonary artery; PTA, persistent truncus arteriosus; RAA, right aortic arch; TGA, transposition of the great arteries; VSD, ventricular septal defect; VS, vascular sling.

  4. Extended Data Figure 4: Pathogenic splicing mutations causing CHD. (269 KB)

    a, The 19 pathogenic splicing mutations recovered are shown, with mutations located beyond the 2-base canonical splice junction highlighted in grey. b, Schematic diagram showing the anomalous Dnah5c.133290-10T>A mutant transcript observed, with the polymerase chain reaction (PCR) primer location and anomalous PCR product size indicated. c, Sanger sequencing profile showing point mutation in Dnah5c.133290-10T>A mutant transcript versus that of wild type. d, PCR amplification of Dnah5c.133290-10T>A heterozygous mutant (m/+) showed the expected 514 bp wild-type and 271 bp mutant PCR product, while only the 271 bp mutant product was observed in the homozygous mutant (m/m) sample.

  5. Extended Data Figure 5: Ciliome mutations causing CHD with and without laterality defects. (118 KB)

    A flow chart showing the distribution of ciliome versus non-ciliome CHD genes among laterality versus non-laterality CHD lines, and further stratification of ciliome CHD genes affecting primary versus motile cilia function.

  6. Extended Data Figure 6: CHD phenotypes associated with mutations affecting endocytic trafficking. (437 KB)

    ac, Outflow tract malalignment defects with double outlet right ventricle (DORV) and overriding aorta (Ao) were observed in Ap2b1 (a), Dnm2 (b) and Snx17 (c) mutants, with Ap2b1 (a) mutant also showing anterior positioning of the aorta (Taussig–Bing type DORV). Snx17 mutant also has AVSD. d, e, Lrp2 (d) and Lrp1 (e) mutants both exhibited outflow tract septation defect with persistent truncus arteriosus (PTA). Scale bar, 0.5 mm.

  7. Extended Data Figure 7: CHD genes associated with axon guidance. (185 KB)

    Diagram illustrating the biological context of several CHD genes known to be involved in axonal guidance (colour highlighting indicates CHD genes recovered from the present screen). Adapted from QIAGEN’s Ingenuity Pathway Analysis (http://www.qiagen.com/ingenuity).

Extended Data Tables

  1. Extended Data Table 1: CHD genes with multiple alleles (105 KB)
  2. Extended Data Table 2: Ciliome mutations in laterality and non-laterality lines (97 KB)
  3. Extended Data Table 3: Mouse CHD genes and associated human diseases (346 KB)

Supplementary information

Video

  1. Video 1: Ultrasound imaging of normal fetus shown in Figure 1a. (1.79 MB, Download)
    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. Video 2: Ultrasound imaging of mutant fetus from line b2b2025 shows DORV and presence of a VSD (2.25 MB, Download)
    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. Video 3: Ultrasound imaging of mutant fetus from line b2b2025 shows AVSD and muscular VSD (2.28 MB, Download)
    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. Video 4: Ultrasound imaging of mutant fetus from line b2b2025 shows heterotaxy (2.26 MB, Download)
    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. Video 5: Videomicroscopy of ciliary motion of tracheal epithelia of newborn Foxj1 mutant mice (3.22 MB, Download)
    Tracheal airway epithelium in a newborn homozygous Foxj1b2b774 mutant mouse shows normal ciliary motion.
  6. Video 6: Videomicroscopy of ciliary motion in embryonic node of Foxj1 mutant embryo (7.98 MB, Download)
    Cilia in the embryonic node of a homozygous Foxj1b2b774 mutant embryo shows dyskinetic ciliary motion and no nodal flow.

Excel files

  1. Supplementary Data 1 (1.4 MB)

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

  2. Supplementary Data 2 (19.4 MB)

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

  3. Supplementary Data 3 (167 KB)

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

  4. Supplementary Data 4 (74 KB)

    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.

Additional data