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Hypoplastic left heart syndrome (HLHS) is a relatively rare severe congenital heart defect (CHD) closely linked to other left ventricular outflow tract (LVOT) lesions including bicuspid aortic valve (BAV), one of the most common heart defects. While HLHS, BAV, and other LVOT lesions have a strong genetic underpinning, their genetic etiology remains poorly understood. Findings from a large-scale mouse mutagenesis screen showed HLHS has a multigenic etiology and is genetically heterogenous, explaining difficulties in identifying the genetic causes of HLHS. In Ohia mice, HLHS shows incomplete penetrance. Some mice exhibited small LV with normal aorta, and others a normal LV with hypoplastic aorta, indicating the LV hypoplasia is not hemodynamically driven. In Ohia mutants, HLHS was found to have a digenic modular construction, with mutation in a chromatin modifier causing the small LV phenotype and mutation in Pcdha9 causing the aorta/aortic valve hypoplasia. The Pcdha9 mutation alone can cause BAV, and in the human genome two common deletion copy number variants spanning PCDHA7-10 are associated with BAV. Hence the digenic etiology of HLHS can account for the close association of HLHS, a rare CHD, with BAV, one of the most common CHD. Functional analysis of Ohia HLHS heart tissue showed severe mitochondrial dysfunction in the small LV, while the normal size RV is also affected but milder, suggesting possible role in vulnerability of surgically palliated HLHS patients to heart failure. These findings suggest insights into the genetics of HLHS may yield new therapies for improving outcome for patients with HLHS.

An essential role for cilia in the pathogenesis of congenital heart disease (CHD) has emerged from findings of a large-scale mouse forward genetic screen. High throughput screening with fetal ultrasound imaging followed by whole exome sequencing analysis recovered a preponderance of cilia related genes and cilia transduced cell signaling genes among mutations identified to cause CHD. The perturbation of left-right patterning in CHD pathogenesis is suggested by the association of CHD with heterotaxy, but also by the finding of the co-occurrence of laterality defects with CHD in birth defect registries. Many of the cilia and cilia cell signaling genes recovered were found to be related to Hedgehog signaling. Studies in mice showed cilia transduced hedgehog signaling coordinates left-right patterning with heart looping and differentiation of the heart tube. Cilia transduced Shh signaling also regulates later events in heart development, including outflow tract septation and formation of the atrioventricular septum. More recent work has shown mutations in cilia related genes may also contribute to valve disease that largely manifest in adult life. Overall, these and other findings show cilia play an important role in CHD and also in more common valve diseases.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) affecting 1 in 5000 newborns. We constructed the interactome of 74 HLHS-associated genes identified from a large-scale mouse mutagenesis screen, augmenting it with 408 novel protein–protein interactions (PPIs) using our High-Precision Protein–Protein Interaction Prediction (HiPPIP) model. The interactome is available on a webserver with advanced search capabilities. A total of 364 genes including 73 novel interactors were differentially regulated in tissue/iPSC-derived cardiomyocytes of HLHS patients. Novel PPIs facilitated the identification of TOR signaling and endoplasmic reticulum stress modules. We found that 60.5% of the interactome consisted of housekeeping genes that may harbor large-effect mutations and drive HLHS etiology but show limited transmission. Network proximity of diabetes, Alzheimer’s disease, and liver carcinoma-associated genes to HLHS genes suggested a mechanistic basis for their comorbidity with HLHS. Interactome genes showed tissue-specificity for sites of extracardiac anomalies (placenta, liver and brain). The HLHS interactome shared significant overlaps with the interactomes of ciliopathy- and microcephaly-associated genes, with the shared genes enriched for genes involved in intellectual disability and/or developmental delay, and neuronal death pathways, respectively. This supported the increased burden of ciliopathy variants and prevalence of neurological abnormalities observed among HLHS patients with developmental delay and microcephaly, respectively.

Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney failure in children. Despite growing knowledge of the genetic causes of CAKUT, the majority of cases remain etiologically unsolved. Genetic alterations in roundabout guidance receptor 1 (ROBO1) have been associated with neuronal and cardiac developmental defects in living individuals. Although Slit-Robo signaling is pivotal for kidney development, diagnostic ROBO1 variants have not been reported in viable CAKUT to date. By next-generation-sequencing methods, we identified six unrelated individuals and two non-viable fetuses with biallelic truncating or combined missense and truncating variants in ROBO1. Kidney and genitourinary manifestation included unilateral or bilateral kidney agenesis, vesicoureteral junction obstruction, vesicoureteral reflux, posterior urethral valve, genital malformation, and increased kidney echogenicity. Further clinical characteristics were remarkably heterogeneous, including neurodevelopmental defects, intellectual impairment, cerebral malformations, eye anomalies, and cardiac defects. By in silico analysis, we determined the functional significance of identified missense variants and observed absence of kidney ROBO1 expression in both human and murine mutant tissues. While its expression in multiple tissues may explain heterogeneous organ involvement, variability of the kidney disease suggests gene dosage effects due to a combination of null alleles with mild hypomorphic alleles. Thus, comprehensive genetic analysis in CAKUT should include ROBO1 as a new cause of recessively inherited disease. Hence, in patients with already established ROBO1-associated cardiac or neuronal disorders, screening for kidney involvement is indicated.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) affecting 1 in 5,000 newborns. We constructed the interactome of 74 HLHS-associated genes identified from a large-scale mouse mutagenesis screen, augmenting it with 408 novel protein-protein interactions (PPIs) using our High-precision Protein-Protein Interaction Prediction (HiPPIP) model. The interactome is available on a webserver with advanced search capabilities (http://severus.dbmi.pitt.edu/wiki-HLHS). 364 genes including 73 novel interactors were differentially regulated in tissues/iPSC-derived cardiomyocytes of HLHS patients. Novel PPIs facilitated the identification of TOR signaling and endoplasmic reticulum stress modules. 60.5% of the interactome consisted of housekeeping genes that may harbor large-effect mutations and drive HLHS etiology but show limited transmission. Network proximity of diabetes, Alzheimer’s disease, and liver carcinoma-associated genes to HLHS genes suggested a mechanistic basis for their comorbidity with HLHS. Interactome genes showed tissue-specificity for sites of extracardiac anomalies (placenta, liver and brain). The HLHS interactome shared significant overlaps with the interactomes of ciliopathy and microcephaly-associated genes, with the shared genes respectively enriched for genes involved in intellectual disability and/or developmental delay, and neuronal death pathways. This supported the increased burden of ciliopathy variants and prevalence of neurological abnormalities observed among HLHS patients with developmental delay and microcephaly respectively.

Summary Bicuspid aortic valve (BAV) with ∼1%–2% prevalence is the most common congenital heart defect (CHD). It frequently results in valve disease and aorta dilation and is a major cause of adult cardiac surgery. BAV is genetically linked to rare left-heart obstructions (left ventricular outflow tract obstructions [LVOTOs]), including hypoplastic left heart syndrome (HLHS) and coarctation of the aorta (CoA). Mouse and human studies indicate LVOTO is genetically heterogeneous with a complex genetic etiology. Homozygous mutation in the Pcdha protocadherin gene cluster in mice can cause BAV, and also HLHS and other LVOTO phenotypes when accompanied by a second mutation. Here we show two common deletion copy number variants (delCNVs) within the PCDHA gene cluster are associated with LVOTO. Analysis of 1,218 white individuals with LVOTO versus 463 disease-free local control individuals yielded odds ratios (ORs) at 1.47 (95% confidence interval [CI], 1.13–1.92; p = 4.2 × 10−3) for LVOTO, 1.47 (95% CI, 1.10–1.97; p = 0.01) for BAV, 6.13 (95% CI, 2.75–13.7; p = 9.7 × 10−6) for CoA, and 1.49 (95% CI, 1.07–2.08; p = 0.019) for HLHS. Increased OR was observed for all LVOTO phenotypes in homozygous or compound heterozygous PCDHA delCNV genotype comparison versus wild type. Analysis of an independent white cohort (381 affected individuals, 1,352 control individuals) replicated the PCDHA delCNV association with LVOTO. Generalizability of these findings is suggested by similar observations in Black and Chinese individuals with LVOTO. Analysis of Pcdha mutant mice showed reduced PCDHA expression at regions of cell-cell contact in aortic smooth muscle and cushion mesenchyme, suggesting potential mechanisms for BAV pathogenesis and aortopathy. Together, these findings indicate common variants causing PCDHA deficiency play a significant role in the genetic etiology of common and rare LVOTO-CHD.

Congenital heart defect is one of the most common structural birth defects in the human population. It is highly associated with heterotaxy, a birth defect involving randomized left-right patterning of visceral organ situs. Large scale mouse forward genetics have led to the finding of a central role for cilia in CHD pathogenesis, with some cilia and non-cilia mutations causing CHD with heterotaxy. Interestingly, many of the mutations causing CHD with heterotaxy can give rise to three laterality outcomes comprising normal situs solitus, mirror symmetric situs inversus totalis, or randomized situs with heterotaxy. Given CHD is largely observed only with heterotaxy, this suggests a new paradigm is needed for investigating the genetics of CHD associated with heterotaxy. Furthermore, analysis of data from multiple large birth cohorts have independently confirmed a broader involvement of laterality disturbance in CHD. This was demonstrated by the common cooccurrence of rare laterality defects with CHD lesions of a wide spectrum. These findings suggest left-right patterning is tightly intertwined with the developmental processes that regulate cardiac morphogenesis and its disturbance may contribute to all types of CHD even in the absence of laterality defects.

We recently conducted a large-scale mouse mutagenesis screen and uncovered a central role for cilia in the pathogenesis of congenital heart disease (CHD). Though our screen was phenotype based, most of the genes recovered were cilia-related, including genes encoding proteins important for ciliogenesis, cilia-transduced cell signaling, and vesicular trafficking. Also unexpected, many of the cilia related genes recovered are known direct protein-protein interactors, even though each gene was recovered independently in unrelated mouse lines. These findings suggest a cilia-based protein-protein interactome network may provide the context for congenital heart disease pathogenesis. This could explain the incomplete penetrance and variable expressivity of human CHD, and account for its complex non-Mendelian etiology. Supporting these findings in mice, a preponderance of cilia and cilia related cell signaling genes were observed among de novo pathogenic variants identified in a CHD patient cohort. Further clinical relevance unfolded with the observation of a high prevalence of respiratory cilia dysfunction in CHD patients. This was associated with increased postsurgical respiratory complications. Together these findings highlight the importance of cilia in CHD pathogenesis and suggest possible clinical translation with instituting pulmonary therapy to improve outcome for CHD patients undergoing congenital cardiac surgeries.

Background Modeling cardiovascular diseases in mice has provided invaluable insights into the cause of congenital heart disease. However, the small size of the mouse heart has precluded translational studies. Given current high‐efficiency gene editing, congenital heart disease modeling in other species is possible. The pig is advantageous given its cardiac anatomy, physiology, and size are similar to human infants. We profiled pig cardiovascular development and generated genetically edited pigs with congenital heart defects. Methods and Results Pig conceptuses and fetuses were collected spanning 7 stages (day 20 to birth at day 115), with at least 3 embryos analyzed per stage. A combination of magnetic resonance imaging and 3‐dimensional histological reconstructions with episcopic confocal microscopy were conducted. Gross dissections were performed in late‐stage or term fetuses by using sequential segmental analysis of the atrial, ventricular, and arterial segments. At day 20, the heart has looped, forming a common atria and ventricle and an undivided outflow tract. Cardiac morphogenesis progressed rapidly, with atrial and outflow septation evident by day 26 and ventricular septation completed by day 30. The outflow and atrioventricular cushions seen at day 20 undergo remodeling to form mature valves, a process continuing beyond day 42. Genetically edited pigs generated with mutation in chromatin modifier SAP130 exhibited tricuspid dysplasia, with tricuspid atresia associated with early embryonic lethality. Conclusions The major events in pig cardiac morphogenesis are largely complete by day 30. The developmental profile is similar to human and mouse, indicating gene edited pigs may provide new opportunities for preclinical studies focused on outcome improvements for congenital heart disease.

Analysis of large-scale human genomic data has yielded unexplained mutations known to cause severe disease in healthy individuals. Here, we report the unexpected recovery of a rare dominant lethal mutation in

Analysis of large scale human genomic data has yielded unexplained mutations known to cause severe disease in healthy individuals. Here we report the unexpected recovery of a rare dominant lethal mutation in TPM1, a sarcomeric actin-binding protein, in 8 individuals with large atrial septal defect (ASD) in a 5-generation pedigree. Mice with TPM1 mutation exhibited early embryonic lethality with disrupted myofibril assembly and no heartbeat. However, patient induced pluripotent stem cell derived cardiomyocytes showed normal beating with mild myofilament defect, indicating disease suppression. A variant in TLN2, another myofilament actin-binding protein, was identified as a candidate suppressor. Mouse CRISRP knockin of both the TLN2/TPM1 variants rescued heart beating, with near term fetuses exhibiting large ASD. Thus, the role of TPM1 in ASD pathogenesis unfolded with the suppression of its embryonic lethality by a TLN2 protective variant. These findings provide evidence that genetic resiliency can arise with genetic suppression of a deleterious mutation.

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.

Discovery of most autosomal recessive disease-associated genes has involved analysis of large, often consanguineous multiplex families or small cohorts of unrelated individuals with a well-defined clinical condition. Discovery of new dominant causes of rare, genetically heterogeneous developmental disorders has been revolutionized by exome analysis of large cohorts of phenotypically diverse parent-offspring trios. Here we analyzed 4,125 families with diverse, rare and genetically heterogeneous developmental disorders and identified four new autosomal recessive disorders. These four disorders were identified by integrating Mendelian filtering (selecting probands with rare, biallelic and putatively damaging variants in the same gene) with statistical assessments of (i) the likelihood of sampling the observed genotypes from the general population and (ii) the phenotypic similarity of patients with recessive variants in the same candidate gene. This new paradigm promises to catalyze the discovery of novel recessive disorders, especially those with less consistent or nonspecific clinical presentations and those caused predominantly by compound heterozygous genotypes.

Heterotaxy results from a failure to establish normal left-right asymmetry early in embryonic development. By whole-exome sequencing, whole-genome sequencing and high-throughput cohort resequencing, we identified recessive mutations in MMP21 (encoding matrix metallopeptidase 21) in nine index cases with heterotaxy. In addition, Mmp21-mutant mice and mmp21-morphant zebrafish displayed heterotaxy and abnormal cardiac looping, respectively, suggesting a new role for extracellular matrix remodeling in the establishment of laterality in vertebrates.

Hypoplastic left heart syndrome (HLHS) is one of the most lethal congenital heart defects, and remains clinically challenging. While surgical palliation allows most HLHS patients to survive their critical heart disease with a single-ventricle physiology, many will suffer heart failure, requiring heart transplantation as the only therapeutic course. Current paradigm suggests HLHS is largely of hemodynamic origin, but recent findings from analysis of the first mouse model of HLHS showed intrinsic cardiomyocyte proliferation and differentiation defects underlying the left ventricular (LV) hypoplasia. The findings of similar defects of lesser severity in the right ventricle suggest this could contribute to the heart failure risks in surgically palliated HLHS patients. Analysis of 8 independent HLHS mouse lines showed HLHS is genetically heterogeneous and multigenic in etiology. Detailed analysis of the Ohia mouse line accompanied by validation studies in CRISPR gene-targeted mice revealed a digenic etiology for HLHS. Mutation in Sap130, a component of the HDAC repressor complex, was demonstrated to drive the LV hypoplasia, while mutation in Pcdha9, a protocadherin cell adhesion molecule played a pivotal role in the valvular defects associated with HLHS. Based on these findings, we propose a new paradigm in which complex CHD such as HLHS may arise in a modular fashion, mediated by multiple mutations. The finding of intrinsic cardiomyocyte defects would suggest hemodynamic intervention may not rescue LV growth. The profound genetic heterogeneity and oligogenic etiology indicated for HLHS would suggest that the genetic landscape of HLHS may be complex and more accessible in clinical studies built on a familial study design.

Structural birth defect (SBD) is a major cause of morbidity and mortality in the newborn period. Although the etiology of SBD is diverse, a wide spectrum of SBD associated with ciliopathies points to the cilium as having a central role in the pathogenesis of SBDs. Ciliopathies are human diseases arising from disruption of cilia structure and/or function. They are associated with developmental anomalies in one or more organ systems and can involve defects in motile cilia, such as those in the airway epithelia or from defects in nonmotile (primary cilia) that have sensory and cell signaling function. Availability of low cost next generation sequencing has allowed for explosion of new knowledge in genetic etiology of ciliopathies. This has led to the appreciation that many genes are shared in common between otherwise clinically distinct ciliopathies. Further insights into the relevance of the cilium in SBD has come from recovery of pathogenic mutations in cilia-related genes from many large-scale mouse forward genetic screens with differing developmental phenotyping focus. Our mouse mutagenesis screen for congenital heart disease (CHD) using noninvasive fetal echocardiography has yielded a marked enrichment for pathogenic mutations in genes required for motile or primary cilia function. These novel mutant mouse models will be invaluable for modeling human ciliopathies and further interrogating the role of the cilium in the pathogenesis of SBD and CHD. Overall, these findings suggest a central role for the cilium in the pathogenesis of a wide spectrum of developmental anomalies associated with CHD and SBDs.

Ciliary motion defects cause defective mucociliary transport (MCT) in primary ciliary dyskinesia (PCD). Current diagnostic tests do not assess how MCT is affected by perturbation of ciliary motion. In this study, we sought to use micro-optical coherence tomography (μOCT) to delineate the mechanistic basis of cilia motion defects of PCD genes by functional categorization of cilia motion. Tracheae from three PCD mouse models were analyzed using μOCT to characterize ciliary motion and measure MCT. We developed multiple measures of ciliary activity, integrated these measures, and quantified dyskinesia by the angular range of the cilia effective stroke (ARC). Ccdc39–/– mice, with a known severe PCD mutation of ciliary axonemal organization, had absent motile ciliary regions, resulting in abrogated MCT. In contrast, Dnah5–/– mice, with a missense mutation of the outer dynein arms, had reduced ciliary beat frequency (CBF) but preserved motile area and ciliary stroke, maintaining some MCT. Wdr69–/– PCD mice exhibited normal motile area and CBF and partially delayed MCT due to abnormalities of ciliary ARC. Visualization of ciliary motion using μOCT provides quantitative assessment of ciliary motion and MCT. Comprehensive ciliary motion investigation in situ classifies ciliary motion defects and quantifies their contribution to delayed mucociliary clearance.

Left-right patterning is among the least well understood of the three axes defining the body plan, and yet it is of no less importance, with left-right patterning defects causing structural birth defects with high morbidity and mortality, such as in complex congenital heart disease, biliary atresia, or intestinal malrotation. The cell signaling pathways that govern left-right asymmetry are highly conserved and involved multiple components of the transforming growth factor beta (TGFβ) superfamily of cell signaling molecules. Central to left-right patterning is the differential activation of Nodal on the left and bone morphogenetic protein (BMP) signaling on the right. In addition, a plethora of other cell signaling pathways including sonic hedgehog (Shh), fibroblast growth factor (FGF), and Notch also contribute to the regulation of left-right patterning. In vertebrate embryos such as the mouse, frog, or zebrafish, the specification of left-right identity requires the left-right organizer (LRO) containing cells with motile and primary cilia. Cilia-generated flow plays an important role in the left-sided propagation of Nodal signaling. Ultimately, it is the left-sided expression of the transcription factor paired-like homeodomain 2 (Pitx2) that drives visceral organ asymmetry. Interestingly, while this overall scheme for left-right patterning is well conserved evolutionarily, are striking differences that suggests caution in broadly generalizing conclusions on the molecular pathways regulating left-right patterning.

The formation of the heart with its complex arterial and venous connections in vertebrates is critically dependent on patterning of the left-right axis during early embryonic development. Abnormalities in left-right patterning can lead to a variety of congenital heart defects, life-threatening birth defects present in nine out of every one thousand infants born each year in the United States. Recent work has uncovered a highly conserved pathway responsible for left-right axis specification. This pathway involves ciliated cells in the embryonic node that induce asymmetric activation of a nodal signaling cascade ultimately driving left-sided expression of the Pitx2 transcription factor. We summarize recent insights gained from studies in animal models, and present a brief overview of the complex processes regulating the specification of cardiac asymmetry, and its contribution to congenital heart defects.

A central role for cilia in the pathogenesis of congenital heart disease was uncovered by our large-scale mouse mutagenesis screen for mutations causing congenital heart disease. This is supported by human clinical studies, which showed a high prevalence of ciliary dysfunction and respiratory symptoms and disease in patients with congenital heart disease. Our mouse studies indicate this involves essential roles for both primary and motile cilia in the pathogenesis of congenital heart disease. As laterality defects were also observed with high prevalence among the congenital heart disease mutants, this further suggested an important role for left-right patterning in the pathogenesis of congenital heart disease. This finding is reminiscent of the high prevalence of heterotaxy among human fetuses with congenital heart disease, indicating the fetal mouse screen may provide a window into the unborn human fetal population. Clinically, congenital heart disease patients with ciliary dysfunction were found to have more respiratory symptoms and disease, a finding with significant clinical implications, as congenital heart disease patients undergoing surgical palliation often have respiratory complications with high morbidity. While this is usually attributed to the heart disease, we propose this may involve intrinsic airway clearance deficits from ciliary dysfunction. Thus the presurgical screening of congenital heart disease patients for respiratory ciliary dysfunction may provide opportunities to provide perioperative pulmonary therapy to enhance airway clearance for at-risk patients. Such change in the standard of care may improve outcome, especially for those congenital heart disease patients who must endure multiple rounds of cardiac surgeries.

Introduction: Mutant mouse models with congenital heart disease (CHD) were observed to have distinct brain dysplasia, prompting an assessment for brain anomalies in CHD patients. CHD infants with brain magnetic resonance imaging (MRI) assessments were recruited and scored using a novel brain dysplasia index. Validation was conducted with parallel brain connectome analysis and neurodevelopment outcome assessments. Methods: The Ohia mutant mouse line and others were analyzed for brain defects using 3D episcopic confocal histopathology. In parallel, we recruited 220 CHD neonates with MRI scans from two large centers. This included pre and postoperative conventional MRI sequences and diffusion tensor imaging (DTI). Brain connectivity measurements with DTI-graph analysis and neurodevelopmental testing at 18 months of age (Battelle Developmental Inventory) were obtained. Results: The CHD mouse mutant analysis revealed high prevalence of brain dysplasia involving the olfactory bulb, cerebellum, hippocampus, corpus callosum and choroid plexus. Based on these findings, a brain dysplasia index (BDI) was developed for clinical assessment of CHD patients, with one point for abnormality in any of these structures right or left and for presence of extra-axial cerebral spinal fluid. Analysis of 220 CHD neonates yielded BDI scores ranging from 1 to 17, with average score of 4.1. An elevated BDI score was seen across a spectrum of cardiac lesions, and was similar pre-op and post-op. DTI graph connectivity measurements (reduced nodal efficiency) correlated with high BDI score (p Conclusion: A brain dysplasia index was developed based on brain anomalies seen in CHD mutant mouse models. Infants with CHD of a broad spectrum with high BDI score showed abnormal brain connectivity and poor neurodevelopmental outcome. These findings suggest the poor neurodevelopmental outcome associated with CHD may involve subtle brain dysplasia with a shared developmental etiology with the structural heart disease.

We present a stratification of the genetic basis of primary ciliary dyskinesia (PCD), based on screening >230 individuals for gene mutations using various approaches including whole exome sequencing. PCD is a genetically heterogeneous recessive ciliopathy, characterized by chronic lung disease and laterality and fertility defects arising from cilia and sperm dysmotility. Most PCD is caused by loss of the ciliary outer dynein arm motors (ODA) essential for motility, arising from mutations in ODA subunits or ODA docking and targeting proteins. Gene panel resequencing of candidate ciliopathy genes in affected children from a consanguineous Bedouin-Arabic family has recently revealed a homozygous protein truncating variant in CCDC151 (c.925G>T; p.Glu308*). Parallel exome sequencing combined with autozygosity mapping in a consanguineous UK-Pakistani-origin family highlighted a large autozygous region on chr 19p13 harbouring a homozygous CCDC151 protein-truncating variant (c.1256C>T; pSer419*). Sanger sequencing of CCDC151 in 150 more PCD cases identified another individual carrying c.925G>T. Transmission electron microscopy of respiratory cilia from individuals carrying CCDC151 mutations showed loss of ODA. Consistent with laterality defects in these individuals, we find Ccdc151 expressed in vertebrate left-right organizers. Both homozygous zebrafish and mouse Ccdc151-deficient mutants display situs defects associated with complex heart defects. Immunofluorescence analysis in patients shows that CCDC151 mutations abolish assembly of CCDC151 into respiratory cilia, and furthermore cause a failure in assembly of the ODA component DNAH5 and ODA docking proteins CCDC114 and ARMC4. We conclude that CCDC151 mutations appear to cause PCD by disruption of the axonemal ODA docking complex machinery.

Recent studies identified a previously uncharacterized gene C5ORF42 (JBTS17) as a major cause of Joubert syndrome (JBTS), a ciliopathy associated with cerebellar abnormalities and other birth defects. Here we report the first Jbts17 mutant mouse model, Heart Under Glass (Hug), recovered from a forward genetic screen. Exome sequencing identified Hug as a S235P missense mutation in the mouse homolog of JBTS17 (2410089e03rik). Hug mutants exhibit multiple birth defects typical of ciliopathies, including skeletal dysplasia, polydactyly, craniofacial anomalies, kidney cysts and eye defects. Some Hug mutants exhibit congenital heart defects ranging from mild pulmonary stenosis to severe pulmonary atresia. Immunostaining showed JBTS17 is localized in the cilia transition zone. Fibroblasts from Hug mutant mice and a JBTS patient with a JBTS17 mutation showed ciliogenesis defects. Significantly, Hug mutant fibroblasts showed loss of not only JBTS17, but also NPHP1 and CEP290 from the cilia transition zone. Hug mutants exhibited reduced ciliation in the cerebellum. This was associated with reduction in cerebellar foliation. Using a fibroblast wound-healing assay, we showed Hug mutant cells cannot establish cell polarity required for directional cell migration. However, stereocilia patterning was grossly normal in the cochlea, indicating planar cell polarity is not markedly affected. Overall, we showed the JBTS pathophysiology is replicated in the Hug mutant mice harboring a Jbts17 mutation. Our findings demonstrate JBTS17 is a cilia transition zone component that acts upstream of other Joubert syndrome associated transition zone proteins NPHP1 and CEP290, indicating its importance in the pathogenesis of Joubert syndrome.

Ciliopathies such as cranioectodermal dysplasia, Sensenbrenner syndrome, short-rib polydactyly, and Jeune syndrome are associated with respiratory complications arising from rib cage dysplasia. While such ciliopathies have been demonstrated to involve primary cilia defects, we show motile cilia dysfunction in the airway of a patient diagnosed with cranioectodermal dysplasia. While this patient had mild thoracic dystrophy not requiring surgical treatment, there was nevertheless newborn respiratory distress, restrictive airway disease with possible obstructive airway involvement, repeated respiratory infections, and atelectasis. High-resolution videomicroscopy of nasal epithelial biopsy showed immotile/dyskinetic cilia and nasal nitric oxide was reduced, both of which are characteristics of primary ciliary dyskinesia, a sinopulmonary disease associated with mucociliary clearance defects due to motile cilia dysfunction in the airway. Exome sequencing analysis of this patient identified compound heterozygous mutations in WDR35, but no mutations in any of the 30 known primary ciliary dyskinesia genes or other cilia-related genes. Given that WDR35 is only known to be required for primary cilia function, we carried out WDR35 siRNA knockdown in human respiratory epithelia to assess the role of WDR35 in motile cilia function. This showed WDR35 deficiency disrupted ciliogenesis in the airway, indicating WDR35 is also required for formation of motile cilia. Together, these findings suggest patients with WDR35 mutations have an airway mucociliary clearance defect masked by their restrictive airway disease.

Patients with congenital heart disease with heterotaxy exhibit a high prevalence of abnormal airway ciliary motion and low nasal nitric oxide, characteristics associated with primary ciliary dyskinesia, a reflection of the role of motile cilia in airway clearance and left-right patterning.To assess the potential broader clinical significance of airway ciliary dysfunction in congenital heart disease, we assessed the prevalence of ciliary dysfunction versus respiratory symptoms in patients with congenital heart disease with or without heterotaxy.Patients with a broad spectrum of congenital heart disease were recruited (n = 218), 39 with heterotaxy. Nasal nitric oxide measurements and nasal biopsies for ciliary motion video microscopy were conducted. Sinopulmonary symptoms were reviewed by questionnaire.A high prevalence of ciliary motion defects (51.8%) and low or borderline low nasal nitric oxide levels (35.5%) were observed in patients with congenital heart disease with or without heterotaxy. Patients with ciliary motion defects or low nasal nitric oxide showed increased sinopulmonary symptoms, with most respiratory symptoms seen in those with both abnormal ciliary motion and low nitric oxide. Multivariate analysis showed that abnormal ciliary motion and low nasal nitric oxide were more important in determining risk of sinopulmonary symptoms than heterotaxy status.Patients with congenital heart disease without heterotaxy exhibit a high prevalence of abnormal ciliary motion and low nasal nitric oxide. This was associated with more sinopulmonary symptoms. These findings suggest that patients with a broad spectrum of congenital heart disease and respiratory symptoms may benefit from screening for ciliary dysfunction and implementation of medical interventions to reduce sinopulmonary morbidities.

A forward genetic screen in fetal mice to identify genes involved in congenital heart disease (CHD) reveals that a large proportion of genes associated with CHD are related to cilia and cilia-transduced cell signalling, with potential implications for the human disease. The identification of genes causing congenital heart disease (CHD) has been challenging, in part because of the difficulty of distinguishing pathogenic mutations from random sequence genetic variability. Cecilia Lo and colleagues have therefore used a large-scale mouse forward genetic screen with chemical mutagenesis to recover mutations causing congenital heart disease. They identify 218 mouse models of the condition and, using whole-exome sequencing, 91 recessive mutations in 61 genes. A larger than expected proportion of these genes was found to be related to cilia and cilia-transduced cell signalling. 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.

The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6(Streaker) and Nek8(Roc). Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6(Strkr) mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8(Roc) mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.

Background— 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. Methods and Results— 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. Conclusions— 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.