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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Gene Expression Omnibus
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.).
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.
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.
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.
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.
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.
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.
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.
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.
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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Mammalian Genome (2017)