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.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Mice are well suited for modeling human congenital heart disease (CHD), given their 4-chamber cardiac anatomy. However, mice with CHD invariably die prenatally/neonatally, causing CHD phenotypes to ...be missed. Therefore, we investigated the efficacy of noninvasive microcomputed tomography (micro-CT) to screen for CHD in stillborn/fetal mice. These studies were performed using chemically mutagenized mice expected to be enriched for birth defects, including CHD.
Stillborn/fetal mice obtained from the breeding of N-ethyl-N-nitrosourea mutagenized mice were formalin-fixed and stained with iodine, then micro-CT scanned. Those diagnosed with CHD and some CHD-negative pups were necropsied. A subset of these were further analyzed by histopathology to confirm the CHD/no-CHD diagnosis. Micro-CT scanning of 2105 fetal/newborn mice revealed an abundance of ventricular septal defects (n=307). Overall, we observed an accuracy of 89.8% for ventricular septal defect diagnosis. Outflow tract anomalies identified by micro-CT included double outlet right ventricle (n=36), transposition of the great arteries (n=14), and persistent truncus arteriosus (n=3). These were diagnosed with a 97.4% accuracy. Aortic arch anomalies also were readily detected with an overall 99.6% accuracy. This included right aortic arch (n=28) and coarctation/interrupted aortic arch (n=12). Also detected by micro-CT were atrioventricular septal defects (n=22), tricuspid hypoplasia/atresia (n=13), and coronary artery fistulas (n=16). They yielded accuracies of 98.9%, 100%, and 97.8%, respectively.
Contrast enhanced micro-CT imaging in neonatal/fetal mice can reliably detect a wide spectrum of CHD. We conclude that micro-CT imaging can be used for routine rapid assessments of structural heart defects in fetal/newborn mice.
Congenital heart disease (CHD) has a multifactorial pathogenesis, but a genetic contribution is indicated by heritability studies. To investigate the spectrum of CHD with a genetic pathogenesis, we ...conducted a forward genetic screen in inbred mice using fetal echocardiography to recover mutants with CHD. Mice are ideally suited for these studies given that they have the same four-chamber cardiac anatomy that is the substrate for CHD.
Ethylnitrosourea mutagenized mice were ultrasound-interrogated by fetal echocardiography using a clinical ultrasound system, and fetuses suspected to have cardiac abnormalities were further interrogated with an ultrahigh-frequency ultrasound biomicroscopy. Scanning of 46 270 fetuses revealed 1722 with cardiac anomalies, with 27.9% dying prenatally. Most of the structural heart defects can be diagnosed using ultrasound biomicroscopy but not with the clinical ultrasound system. Confirmation with analysis by necropsy and histopathology showed excellent diagnostic capability of ultrasound biomicroscopy for most CHDs. Ventricular septal defect was the most common CHD observed, whereas outflow tract and atrioventricular septal defects were the most prevalent complex CHD. Cardiac/visceral organ situs defects were observed at surprisingly high incidence. The rarest CHD found was hypoplastic left heart syndrome, a phenotype never seen in mice previously.
We developed a high-throughput, 2-tier ultrasound phenotyping strategy for efficient recovery of even rare CHD phenotypes, including the first mouse models of hypoplastic left heart syndrome. Our findings support a genetic pathogenesis for a wide spectrum of CHDs and suggest that the disruption of left-right patterning may play an important role in CHD.
Abstract only Introduction: Large scale forward genetic screens with ethyl-nitroso-urea (ENU) mutagenesis can provide a non-gene biased approach to elucidate the genetic etiology of congenital heart ...disease (CHD). We are using noninvasive fetal echocardiography to elucidate the genetic etiology of CHD with scanning of 100,000 mouse fetuses to achieve 5 fold genome coverage. Methods: C57BL/6J mice (G0) are ENU mutagenized and the resulting G1 males and their G2 daughters are backcrossed to generate G3 fetuses for ultrasound scanning. Primary screening used the clinical Acuson Sequoia ultrasound system with a 15 MHz transducer. Fetuses with CHD were further scanned at 40 MHz with the Vevo 2100 ultra-high frequency biomicroscope (UBM). CHD diagnoses were susbsequently confirmed by necropsy, microCT, and/or histopathology. Results: Ultrasound scanning of >20,000 G3 fetuses from >600 G1 pedigrees revealed 4% of fetuses with cardiac defects. A wide spectrum of CHD was observed, including DORV, TGA, PTA, pulmonary/aortic atresia, coarctation, arch anomalies, atrioventricular septal defects (AVSD) and other defects. The most common CHD found was ventricular septal defect. In contrast, hypoplastic left heart syndrome was found in only one mutant line (Figure). We also recovered 10 lines with laterality defects. Heterotaxy mutants exhibited complex CHD, but no heart disease was seen with situs inversus totalis. Overall, the UBM had higher detection sensitivity for cardiac situs (P<0.01), AVSD (P<0.01) and outflow tract malalignment (P<0.01), with 98.3% of the cardiac situs, 91% of the AVSD and 70% of the outflow defects identified by UBM. Conclusions: Our studies showed the integration of conventional ultrasound with the UBM for fetal mouse cardiovascular phenotyping can maximize the recovery of CHD mutants. The wide spectrum of CHD observed in our large scale screen indicates the importance of genetic contribution in all varieties of structural heart disease.
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