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Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births1, but the incidence of CHD is up to ten fold 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 the cilium and cilia transduced cell signaling play 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 signaling, and 10 genes regulating vesicular trafficking, a pathway important for ciliogenesis and cell signaling. Surprisingly, many CHD genes encoded interacting proteins, suggesting 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 pathways identified show overlap with CHD candidate genes recovered in CHD patients5, suggesting they may have relevance to the more complex genetics of CHD overall. These CHD mouse models and >8,000 incidental mutations are sperm archived, creating a rich public resource for human disease modeling.

Noonan syndrome is characterized by short stature, facial dysmorphia and a wide spectrum of congenital heart defects. Mutations of PTPN11, KRAS and SOS1 in the RAS-MAPK pathway cause approximately 60% of cases of Noonan syndrome. However, the gene(s) responsible for the remainder are unknown. We have identified five different mutations in RAF1 in ten individuals with Noonan syndrome; those with any of four mutations causing changes in the CR2 domain of RAF1 had hypertrophic cardiomyopathy (HCM), whereas affected individuals with mutations leading to changes in the CR3 domain did not. Cells transfected with constructs containing Noonan syndrome-associated RAF1 mutations showed increased in vitro kinase and ERK activation, and zebrafish embryos with morpholino knockdown of raf1 demonstrated the need for raf1 for the development of normal myocardial structure and function. Thus, our findings implicate RAF1 gain-of-function mutations as a causative agent of a human developmental disorder, representing a new genetic mechanism for the activation of the MAPK pathway.

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 HLHS5–7. 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.

We describe the first mutation in SMAD8 in a patient with IPAH. Our findings suggest the involvement of SMAD8 in the pathogenesis of PAH.

Background Patients with congenital heart disease (CHD) and heterotaxy show high postsurgical morbidity/mortality, with some developing respiratory complications. Although this finding is often attributed to the CHD, airway clearance and left-right patterning both require motile cilia function. Thus, airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD) may contribute to increased respiratory complications in heterotaxy patients. Methods and Results We assessed 43 CHD patients with heterotaxy for airway CD. Videomicrocopy was used to examine ciliary motion in nasal tissue, and nasal nitric oxide (nNO) was measured; nNO level is typically low with PCD. Eighteen patients exhibited CD characterized by abnormal ciliary motion and nNO levels below or near the PCD cutoff values. Patients with CD aged >6 years show increased respiratory symptoms similar to those seen in PCD. Sequencing of all 14 known PCD genes in 13 heterotaxy patients with CD, 12 without CD, 10 PCD disease controls, and 13 healthy controls yielded 0.769, 0.417, 1.0, and 0.077 novel variants per patient, respectively. One heterotaxy patient with CD had the PCD causing DNAI1 founder mutation. Another with hyperkinetic ciliary beat had 2 mutations in DNAH11, the only PCD gene known to cause hyperkinetic beat. Among PCD patients, 2 had known PCD causing CCDC39 and CCDC40 mutations. Conclusions Our studies show that CHD patients with heterotaxy have substantial risk for CD and increased respiratory disease. Heterotaxy patients with CD were enriched for mutations in PCD genes. Future studies are needed to assess the potential benefit of prescreening and prophylactically treating heterotaxy patients for CD.

Wdpcp, a protein required for both planar cell polarity and ciliogenesis, regulates cell polarity and alignment via direct modulation of the actin cytoskeleton.

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