Global genetic analysis in mice unveils central role for cilia in congenital heart disease (original) (raw)

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Acknowledgements

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.

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Author notes

  1. You Li, Nikolai T. Klena, George C. Gabriel and Xiaoqin Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, 15201, Pennsylvania, 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, 15261, Pennsylvania, USA
    William Devine
  3. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, 01605, Massachusetts, USA
    Jovenal T. San Agustin & Gregory J. Pazour
  4. Department of Biomedical Informatics, University of Pittsburgh School of Medicine, Pittsburgh, 15206, Pennsylvania, USA
    Mohamed Thahir & Madhavi K. Ganapathiraju
  5. Intelligent Systems Program, School of Arts and Sciences, University of Pittsburgh, Pittsburgh, 16260, Pennsylvania, USA
    Mohamed Thahir & Madhavi K. Ganapathiraju
  6. The Jackson Laboratory, Bar Harbor, 04609, Maine, USA
    Herbert Pratt, Judy Morgan, Leslie Haynes, Cynthia L. Smith, Janan T. Eppig & Laura Reinholdt
  7. The Heart Center, Children’s National Medical Center, 20010, Washington DC, USA
    Linda Leatherbury

Authors

  1. You Li
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  2. Nikolai T. Klena
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  3. George C. Gabriel
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  4. Xiaoqin Liu
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  5. Andrew J. Kim
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  6. Kristi Lemke
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  7. Yu Chen
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  8. Bishwanath Chatterjee
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  9. William Devine
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  10. Rama Rao Damerla
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  11. Chienfu Chang
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  12. Hisato Yagi
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  13. Jovenal T. San Agustin
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  14. Mohamed Thahir
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  15. Shane Anderton
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  16. Caroline Lawhead
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  17. Anita Vescovi
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  18. Herbert Pratt
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  19. Judy Morgan
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  20. Leslie Haynes
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  21. Cynthia L. Smith
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  22. Janan T. Eppig
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  23. Laura Reinholdt
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  24. Richard Francis
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  25. Linda Leatherbury
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  26. Madhavi K. Ganapathiraju
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  27. Kimimasa Tobita
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  28. Gregory J. Pazour
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  29. Cecilia W. Lo
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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.

Corresponding author

Correspondence toCecilia W. Lo.

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Competing interests

The authors declare no competing financial interests.

Additional information

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).

Extended data figures and tables

Extended Data Figure 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.

Extended Data Figure 2 Situs anomalies and congenital heart defects in Ap1b1 b2b1660 mutants.

a–c, 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. d–g, 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.

Extended Data Figure 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.

Extended Data Figure 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 _Dnah5_c.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 _Dnah5_c.133290-10T>A mutant transcript versus that of wild type. d, PCR amplification of _Dnah5_c.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.

Extended Data Figure 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.

Extended Data Figure 6 CHD phenotypes associated with mutations affecting endocytic trafficking.

a–c, 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.

Extended Data Figure 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).

Extended Data Table 1 CHD genes with multiple alleles

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Extended Data Table 2 Ciliome mutations in laterality and non-laterality lines

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Extended Data Table 3 Mouse CHD genes and associated human diseases

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Li, Y., Klena, N., Gabriel, G. et al. Global genetic analysis in mice unveils central role for cilia in congenital heart disease.Nature 521, 520–524 (2015). https://doi.org/10.1038/nature14269

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