Global genetic analysis in mice unveils central role for cilia in congenital heart disease

Abstract

Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births; the incidence of CHD is up to tenfold higher in human fetuses. A genetic contribution is strongly suggested by the association of CHD with chromosome abnormalities and high recurrence risk. 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 patients, 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.

Figure 1. Ultrasound diagnoses of CHD and cilia defects in CHD mutants

Vevo 2100 color flow imaging showed criss-crossing of blood flow indicating normal aorta (Ao) and pulmonary artery (PA) alignment (a, Supplemental Movie S1), confirmed by histopathology (b). E16.5 mutant (line b2b327) exhibited blood flow pattern indicating single great artery (PA) and ventricular septal defect (VSD) (c, Supplemental Movie S2), suggesting aortic atresia with VSD, confirmed by histopathology (d). Color flow imaging of E15.5 mutant (line b2b2025) with heterotaxy (stomach on right; Supplemental Movie S3c) had side-by-side Ao/PA with Ao emerging from right ventricle (RV), indicating DORV/VSD (e,f,Supplemental Movie S3a), and presence of AVSD (g,h,Supplemental Movie S3b,S3c). Histopathology also showed bicuspid aortic valve (BAV,i), interrupted aortic arch (IAA,j), and common AV valve (k). (l-n). Cc2d2a mutant exhibits dextrocardia with ventricular inversion (dextroversion) (m), and AVSD (l) with malformed AV cushions (n), but normal outflow cushions. (o-x). Confocal imaging of E12.5 Cc2d2a mutant (m/m) vs. wildtype (+/+) embryo sections showed no cilia in AV cushion (o,p), but normal ciliation in outflow cushion (q,r). Fewer and shorter cilia were observed in other mutant embryo tissues (s-x). Red:acetylated tubulin;green:IFT88.

Figure 2. Congenital heart disease genes recovered from mouse mutagenesis screen

Diagrams illustrate biological context of CHD gene function (color-highlighting indicate CHD genes recovered; asterisk denote CHD genes recovered from previous screen). For clarity, ciliome genes (Dctn5,Fuz) with endocytic function and/or involved in cilia-transduced signaling were not shown in the ciliogenesis panel.

Figure 3. Interactome network of CHD genes with known and predicted interactors

An interactome network was constructed (a) comprising the 61 CHD genes (magenta squares) and their known (blue edges) and predicted (red edges) interactions. The CHD interactome yielded significant enrichment of GO terms related to developmental processes. Circle size proportional to number of genes associated with GO terms.