Congenital heart disease (CHD) affects up to 1% of live births1. Although a genetic etiology is indicated by an increased recurrence risk2,3, sporadic occurrence suggests that CHD genetics is complex4. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS5,6,7. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR–Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.

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We thank T. Yagi (Osaka University) for providing plasmid for Pcdha9 in situ probes and A. Handen (University of Pittsburgh) for assisting with programming. We also thank C.G Burns (Harvard Medical School) for providing the transgene marker, Tg (5.7myl7: nDsRed2). This work was supported by funding from the NIH (U01-HL098180 (C.W.L.), R01-HL132024 (C.W.L.), R01-GM104412 (C.W.L.), S10-OD010340 (C.W.L.), R01-MH094564 (M.K.G.), and OD011185 (S.A.M.)), the Children's Heart Foundation (L.J.M. and D.W.B.), and the Junior Cooperative Society (D.W.B.).

Author information


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

    • Xiaoqin Liu
    • , Hisato Yagi
    • , Shazina Saeed
    • , Abha S Bais
    • , George C Gabriel
    • , Zhaohan Chen
    • , You Li
    • , Molly C Schwartz
    • , William T Reynolds
    • , Manush Saydmohammed
    • , Brian Gibbs
    • , Yijen Wu
    • , William Devine
    • , Bishwanath Chatterjee
    • , Nikolai T Klena
    • , Dennis Kostka
    • , Omar Khalifa
    • , Anchit Bhagat
    • , Maliha Zahid
    • , Michael Tsang
    •  & Cecilia W Lo
  2. The Jackson Laboratory, Bar Harbor, Maine, USA.

    • Kevin A Peterson
    •  & Stephen A Murray
  3. Pathology & Laboratory Medicine and the Electron Microscope Research Core, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.

    • Karen L de Mesy Bentley
  4. Department of Biomedical Informatics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

    • Madhavi K Ganapathiraju
  5. Department of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

    • Phillip Dexheimer
    •  & Bruce J Aronow
  6. Division of Cardiology, Children's National Medical Center, Washington, D.C., USA.

    • Linda Leatherbury
  7. Department of Cardiology, Boston Children's Hospital, Boston, Massachusetts, USA.

    • William Pu
  8. Department of Cell Biology, Center for Biological Imaging, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

    • Simon Watkins
  9. Division of Cardiology, University of San Diego School of Medicine, San Diego, California, USA.

    • Paul Grossfeld
  10. Department of Pediatrics, Division of Cardiology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.

    • George A Porter Jr
  11. Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

    • Lisa J Martin
  12. Pediatric Cardiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.

    • D Woodrow Benson


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Study design, C.W.L.; fetal ultrasound imaging and mutant recovery, X.L.; mouse phenotype analysis, X.L., S.S., Z.C., W.D., L.L., G.C.G. Y.W., M.C.S., W.T.R., and B.G.; mouse breeding and genotyping, X.L., S.S., Z.C., H.Y., and B.C.; mouse exome sequencing analysis, Y.L. and N.T.K.; network analysis, B.J.A. and M.K.G.; cardiomyocyte proliferation and apoptosis, S.S., Z.C., and X.L.; in situ hybridization, Z.C., X.L., and H.Y.; mitochondria function analysis and electron microscopy, Z.C., X.L., G.A.P., and K.L.d.M.B.; Sap130 gene and protein expression analysis, H.Y. and S.S.; LV and RV cardiac-tissue harvesting, RNA extraction, and RNA-seq, X.L., B.G., C.W.L., Z.C., and H.Y.; RNA-seq bioinformatics and ToppGene analysis, A.S.B., D.K., and B.J.A.; Sap130 ChIP–seq, H.Y. and A.S.B.; zebrafish morpholino experiments, M.S., M.T., S.W., and B.G.; zebrafish CRISPR mutant generation and analysis, M.S. and M.T.; CRISPR Sap130 guide-RNA design, W.P.; CRISPR–Cas9 F0 mouse embryo production and analysis, K.A.P., S.A.M., Z.C., G.C.G., M.C.S., and X.L.; CRISPR–Cas9 F0 founder-mouse production and F1 offspring propagation, K.A.P., S.A.M., and X.L.; mouse microarray analysis, B.J.A., C.W.L., and H.Y.; recruitment of subjects with HLHS and sample collection, C.W.L., O.K., L.J.M., D.W.B., and P.G.; human exome sequencing analysis and multigene human and mouse comparisons, C.W.L., X.L., B.J.A., A.B., A.S.B., L.J.M., and P.D.; statistics, L.J.M., M.Z., and X.L.; manuscript preparation, C.W.L., X.L., L.J.M., D.W.B., G.A.P., K.A.P., M.T., B.J.A., D.K., A.S.B., P.D., and H.Y.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Cecilia W Lo.

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–22 and Supplementary Tables 1–7

Excel files

  1. 1.

    Supplementary Data 1

    Mutations recovered in HLHS Mutant Mouse Lines.

  2. 2.

    Supplementary Data 2

    Netbox analysis of mutations recovered from HLHS mutant lines

  3. 3.

    Supplementary Data 3

    RNAseq analysis of differentially expressed genes in Ohia HLHS mutant heart tissue

  4. 4.

    Supplementary Data 4

    Pathway enrichment analysis of Ohia HLHS RNAseq Data

  5. 5.

    Supplementary Data 5

    ChIPseq analysis of Sap130 targets

  6. 6.

    Supplementary Data 6

    ToppGene pathway enrichment analysis of HLHS RNAseq and Sap130 ChIPseq data

  7. 7.

    Supplementary Data 7

    Gene expression analysis of microarray data from different mouse embryonic cardiac tissues and cell lines for Sap130 and Pcdha9

  8. 8.

    Supplementary Data 8

    Multihit genes in human HLHS patients and Ohia HLHS mice, and interactome analysis

  9. 9.

    Supplementary Data 9

    Multihit genes in 1000 genomes CEU subjects


  1. 1.

    Fetal ultrasound color flow Doppler imaging of Ohia HLHS mutant.

    Color flow Doppler imaging using the Vevo2100 ultrasound system showed much less blood flow into the left ventricle, suggesting mitral valve stenosis. A tiny blood flow (blue blood flow) was observed flowing into the aorta, indicating aortic stenosis. Also note small VSD and foramen ovale opening with left to right shunt.

  2. 2.

    Fetal ultrasound 2D imaging of HLHS Ohia mutant.

    2D imaging using the Vevo2100 ultrasound system showed an Ohia HLHS heart in four-chamber view. Note the muscle-bound hypolastic left ventricle with small lumen and poor contractility.

  3. 3.

    Video microscopy of HLHS Ohia mutant heart.

    Video microscopy of an E14.5 Ohia HLHS fetus showed most of the contractile motion of the heart was associated with the right ventricle, with the left ventricle showing only weak contraction and no visible blood flow. Also note the hypolastic and displaced thymus, severely hypoplastic ascending aorta and interrupted aortic arch and muscle-bound hypoplastic left ventricle.

  4. 4.

    Serial histopathology image stack of HLHS Ohia mutant heart in coronal view.

    A serial histopathology image stack obtained by episcopic confocal microscopy of an E14.5 HLHS heart showed muscle-band hypoplastic LV with almost no lumen, mitral valve stenosis, cushion-like aortic valve and hypoplastic ascending aorta and hypoplastic right aortic arch.

  5. 5.

    Color flow Doppler imaging of homozygous Ohia Pcdha9 mutant.

    Echocardiography of adult Pcdha9m/m mutant and Pcdha9+/+ wildtype mice revealed aortic stenosis with regurgitation in the mutant mouse. This is indicated by high velocity jet flowing across the aortic valve in systole, and regurgitant flow retrograde from the descending aorta to the LV through the aortic valve in diastole.

  6. 6.

    Functional MRI imaging of Ohia Pcdha9m/m mutant in coronal view.

    MRI in coronal view of the same Pcdha9m/m mutant and. Pcdha9+/+ wildtype adult mice examined by echocardiography in Video 5 confirmed aortic stenosis and regurgitation in the mutant mouse. This is indicated by the thickened aortic valve with domeshaped opening and high velocity jet flowing across the aortic valve in systole, and with retrograde regurgitant diastolic flow to the LV.

  7. 7.

    Functional MRI imaging of Ohia Pcdha9m/m mutant in transverse view.

    MRI in short axis view of the same mice shown in Video 6 revealed abnormal bicuspid aortic valve (BAV) in the Pcdha9m/m mutant, while the normal three-leaflet aortic valve is seen in the Pcdha9+/+ wildtype mouse.

  8. 8.

    Color flow Doppler imaging show HLHS in mutant from CRISPR/Cas9 targeted Sap130/Pcdha9 transgenic mice.

    Color flow Doppler imaging using the Vevo2100 ultrasound system revealed HLHS in an F2 embryo double homozygous for the CRISPR/Cas9 generated Sap130/Pcdha9 mutations. Note absence of blood flow into the very small LV with no lumen, suggesting mitral valve atresia with hypoplastic LV. A tiny blood flow (red blood flow) into the aorta originating from the RV was observed, indicating aortic stenosis and double outlet right ventricle (DORV). Also note a regurgitant flow associated with the pulmonary valve.

  9. 9.

    Serial histopathology image stack of CRISPR/CAS Sap130/Pcdha9 targeted HLHS mutant embryo shown in coronal view.

    A serial histopathology image stack obtained by episcopic confocal microscopy of the CRISPR/Cas9 transgenic mutant embryo shown in Video 8 revealed muscle-bound hypoplastic LV with almost no lumen, mitral valve atresia, hyoplastic ascending aorta, and with both the aorta and the pulmonary artery arising from RV, indicating DORV subtype of HLHS.

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