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.
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Extended data figures and tables
Extended Data Figures
- 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.
- Extended Data Figure 2: Situs anomalies and congenital heart defects in Ap1b1b2b1660 mutants. (156 KB)
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. (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.
- 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.
- 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.
- Extended Data Figure 6: CHD phenotypes associated with mutations affecting endocytic trafficking. (437 KB)
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. (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
- 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.
- 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).
- 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.
- 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.
- 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.
- 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.
- Supplementary Data 1 (1.4 MB)
This data file contains CHD mutations and comparison to other mutant alleles and human diseases.
- Supplementary Data 2 (19.4 MB)
This data file contains position and sequence conservation of CHD mutations.
- Supplementary Data 3 (167 KB)
This data file contains gene ontology and pathway analyses of CHD interactome genes.
- 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.