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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Gene Expression Omnibus


  1. 1.

    & The incidence of congenital heart disease. J. Am. Coll. Cardiol. 39, 1890–1900 (2002).

  2. 2.

    , , & Patterns of recurrence of congenital heart disease: an analysis of 6,640 consecutive pregnancies evaluated by detailed fetal echocardiography. J. Am. Coll. Cardiol. 42, 923–929 (2003).

  3. 3.

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

  4. 4.

    , & Genetics of hypoplastic left heart syndrome. J. Pediatr. 173, 25–31 (2016).

  5. 5.

    et al. Hypoplastic left heart syndrome links to chromosomes 10q and 6q and is genetically related to bicuspid aortic valve. J. Am. Coll. Cardiol. 53, 1065–1071 (2009).

  6. 6.

    et al. Linkage analysis of left ventricular outflow tract malformations (aortic valve stenosis, coarctation of the aorta, and hypoplastic left heart syndrome). Eur. J. Hum. Genet. 17, 811–819 (2009).

  7. 7.

    et al. Evidence in favor of linkage to human chromosomal regions 18q, 5q and 13q for bicuspid aortic valve and associated cardiovascular malformations. Hum. Genet. 121, 275–284 (2007).

  8. 8.

    et al. Global genetic analysis in mice unveils central role for cilia in congenital heart disease. Nature 521, 520–524 (2015).

  9. 9.

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

  10. 10.

    et al. Identification of de novo mutations and rare variants in hypoplastic left heart syndrome. Clin. Genet. 81, 542–554 (2012).

  11. 11.

    et al. Compound heterozygous NOTCH1 mutations underlie impaired cardiogenesis in a patient with hypoplastic left heart syndrome. Hum. Genet. 134, 1003–1011 (2015).

  12. 12.

    , , , & Automated network analysis identifies core pathways in glioblastoma. PLoS One 5, e8918 (2010).

  13. 13.

    , & Identification and characterization of three new components of the mSin3A corepressor complex. Mol. Cell. Biol. 23, 3456–3467 (2003).

  14. 14.

    , , , & Sin3: insight into its transcription regulatory functions. Eur. J. Cell Biol. 92, 237–246 (2013).

  15. 15.

    , , , & Intrauterine pulmonary venous flow and restrictive foramen ovale in fetal hypoplastic left heart syndrome. J. Am. Coll. Cardiol. 43, 1902–1907 (2004).

  16. 16.

    et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

  17. 17.

    et al. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev. Biol. 393, 3–9 (2014).

  18. 18.

    , , & Pervasive genotypic mosaicism in founder mice derived from genome editing through pronuclear injection. PLoS One 10, e0129457 (2015).

  19. 19.

    et al. Fetal reprogramming and senescence in hypoplastic left heart syndrome and in human pluripotent stem cells during cardiac differentiation. Am. J. Pathol. 183, 720–734 (2013).

  20. 20.

    et al. Role of Meis1 in mitochondrial gene transcription of pancreatic cancer cells. Biochem. Biophys. Res. Commun. 410, 798–802 (2011).

  21. 21.

    et al. Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature 497, 249–253 (2013).

  22. 22.

    et al. Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy. Nat. Med. 19, 193–201 (2013).

  23. 23.

    , & Notch signaling in cardiac valve development and disease. Birth Defects Res. A Clin. Mol. Teratol. 91, 449–459 (2011).

  24. 24.

    , , & ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res. 37, W305–W311 (2009).

  25. 25.

    , , , & ToppCluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems. Nucleic Acids Res. 38, W96–W102 (2010).

  26. 26.

    & Twist1 directly regulates genes that promote cell proliferation and migration in developing heart valves. PLoS One 6, e29758 (2011).

  27. 27.

    et al. Development of heart valves requires Gata4 expression in endothelial-derived cells. Development 133, 3607–3618 (2006).

  28. 28.

    et al. Haemogenic endocardium contributes to transient definitive haematopoiesis. Nat. Commun. 4, 1564 (2013).

  29. 29.

    et al. Transcription factor Sp3 knockout mice display serious cardiac malformations. Mol. Cell. Biol. 27, 8571–8582 (2007).

  30. 30.

    & Cardiac expression of Tnnt1 requires the GATA4-FOG2 transcription complex. ScientificWorldJournal 9, 575–587 (2009).

  31. 31.

    et al. Differential gene expression profiles during embryonic heart development in diabetic mice pregnancy. Gene 516, 218–227 (2013).

  32. 32.

    , , , & Natural cardiogenesis-based template predicts cardiogenic potential of induced pluripotent stem cell lines. Circ Cardiovasc Genet 6, 462–471 (2013).

  33. 33.

    , , & Transcriptional profiling of regenerating embryonic mouse hearts. Genom. Data 9, 145–147 (2016).

  34. 34.

    , , , & Shared gene expression profiles in developing heart valves and osteoblast progenitor cells. Physiol. Genomics 35, 75–85 (2008).

  35. 35.

    et al. Lineage and morphogenetic analysis of the cardiac valves. Circ. Res. 95, 645–654 (2004).

  36. 36.

    , & Neural crest cells retain multipotential characteristics in the developing valves and label the cardiac conduction system. Circ. Res. 98, 1547–1554 (2006).

  37. 37.

    , & Transcriptional regulation of cadherins during development and carcinogenesis. Int. J. Dev. Biol. 48, 365–375 (2004).

  38. 38.

    et al. Polycomb complex 2 is required for E-cadherin repression by the Snail1 transcription factor. Mol. Cell. Biol. 28, 4772–4781 (2008).

  39. 39.

    et al. The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1. EMBO J. 29, 1803–1816 (2010).

  40. 40.

    , & Epigenetic regulation and role of metastasis suppressor genes in pancreatic ductal adenocarcinoma. BMC Cancer 13, 264 (2013).

  41. 41.

    et al. Extensive linkage disequilibrium, a common 16.7-kilobase deletion, and evidence of balancing selection in the human protocadherin alpha cluster. Am. J. Hum. Genet. 72, 621–635 (2003).

  42. 42.

    et al. Bicuspid aortic valve: inter-racial difference in frequency and aortic dimensions. JACC Cardiovasc. Imaging 5, 981–989 (2012).

  43. 43.

    . et al. Total expression and dual gene-regulatory mechanisms maintained in deletions and duplications of the Pcdha cluster. J. Biol. Chem. 284, 32002–32014 (2009).

  44. 44.

    1000 Genomes Project Consortium. et al. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 56–65 (2012).

  45. 45.

    et al. Inheritance analysis of congenital left ventricular outflow tract obstruction malformations: segregation, multiplex relative risk, and heritability. Am. J. Med. Genet. A. 134A, 180–186 (2005).

  46. 46.

    et al. Hypoplastic left heart syndrome is heritable. J. Am. Coll. Cardiol. 50, 1590–1595 (2007).

  47. 47.

    et al. Fetal aortic valvuloplasty for evolving hypoplastic left heart syndrome: postnatal outcomes of the first 100 patients. Circulation 130, 638–645 (2014).

  48. 48.

    Snail1 links transcriptional control with epigenetic regulation. EMBO J. 29, 1787–1789 (2010).

  49. 49.

    , & Epigenetic regulation of EMT: the Snail story. Curr. Pharm. Des. 20, 1698–1705 (2014).

  50. 50.

    , , & Imaging techniques for visualizing and phenotyping congenital heart defects in murine models. Birth Defects Res. C Embryo Today 99, 93–105 (2013).

  51. 51.

    et al. The BioGRID interaction database: 2015 update. Nucleic Acids Res. 43, D470–D478 (2015).

  52. 52.

    et al. Human Protein Reference Database: 2009 update. Nucleic Acids Res. 37, D767–D772 (2009).

  53. 53.

    et al. Schizophrenia interactome with 504 novel protein-protein interactions. NPJ Schizophr. 2, 16012 (2016).

  54. 54.

    & In search of the biological significance of modular structures in protein networks. PLOS Comput. Biol. 3, e107 (2007).

  55. 55.

    , , , & Heart of glass regulates the concentric growth of the heart in zebrafish. Curr. Biol. 13, 2138–2147 (2003).

  56. 56.

    et al. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One 9, e98186 (2014).

  57. 57.

    , & Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl. Acad. Sci. USA 110, 13904–13909 (2013).

  58. 58.

    et al. The permeability transition pore controls cardiac mitochondrial maturation and myocyte differentiation. Dev. Cell 21, 469–478 (2011).

  59. 59.

    , , , & Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes. Biochim. Biophys. Acta 1797, 913–921 (2010).

  60. 60.

    , & Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study. J. Appl. Physiol (1985) 114, 161–171 (2013).

  61. 61.

    et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

  62. 62.

    , & HTSeq: a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

  63. 63.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  64. 64.

    et al. Myocardial alternative RNA splicing and gene expression profiling in early stage hypoplastic left heart syndrome. PLoS One 7, e29784 (2012).

  65. 65.

    , , & Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

  66. 66.

    et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

  67. 67.

    , , & Computational approaches for human disease gene prediction and ranking. Adv. Exp. Med. Biol. 799, 69–84 (2014).

  68. 68.

    et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

Download references


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


  1. Search for Xiaoqin Liu in:

  2. Search for Hisato Yagi in:

  3. Search for Shazina Saeed in:

  4. Search for Abha S Bais in:

  5. Search for George C Gabriel in:

  6. Search for Zhaohan Chen in:

  7. Search for Kevin A Peterson in:

  8. Search for You Li in:

  9. Search for Molly C Schwartz in:

  10. Search for William T Reynolds in:

  11. Search for Manush Saydmohammed in:

  12. Search for Brian Gibbs in:

  13. Search for Yijen Wu in:

  14. Search for William Devine in:

  15. Search for Bishwanath Chatterjee in:

  16. Search for Nikolai T Klena in:

  17. Search for Dennis Kostka in:

  18. Search for Karen L de Mesy Bentley in:

  19. Search for Madhavi K Ganapathiraju in:

  20. Search for Phillip Dexheimer in:

  21. Search for Linda Leatherbury in:

  22. Search for Omar Khalifa in:

  23. Search for Anchit Bhagat in:

  24. Search for Maliha Zahid in:

  25. Search for William Pu in:

  26. Search for Simon Watkins in:

  27. Search for Paul Grossfeld in:

  28. Search for Stephen A Murray in:

  29. Search for George A Porter in:

  30. Search for Michael Tsang in:

  31. Search for Lisa J Martin in:

  32. Search for D Woodrow Benson in:

  33. Search for Bruce J Aronow in:

  34. Search for Cecilia W Lo in:


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

PDF files

  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.

About this article

Publication history