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.

The Rh antigen is a multi-subunit complex composed of Rh polypeptides and associated glycoproteins (Rh50, CD47, LW and glycophorin B); these interact in the red cell membrane and are lacking or severely reduced in Rhnull cells. As a result, individuals with Rhnull suffer chronic haemolytic anaemia known as the Rh-deficiency syndrome. Most frequently, Rhnull phenotypes are caused by homozygosity of an autosomal suppressor gene unlinked to the RH locus (Rhnull regulator or Rhmod types). We have analysed the genes and transcripts encoding Rh, CD47 and Rh50 proteins in five such unrelated Rhnull cases. In all patients, we identified alteration of Rh50--frameshift, nucleotide mutations, or failure of amplification--which correlated with Rhnull phenotype. We propose that mutant alleles of Rh50, which map to chromosome 6p11-21.1, are likely candidates for suppressors of the RH locus accounting for most cases of Rh-deficiency.

Planar cell polarity (PCP) is controlled by a conserved pathway that regulates directional cell behavior. Here, we show that mutant mice harboring a newly described mutation termed Beetlejuice (Bj) in Prickle1 (Pk1), a PCP component, exhibit developmental phenotypes involving cell polarity defects, including skeletal, cochlear and congenital cardiac anomalies. Bj mutants die neonatally with cardiac outflow tract (OFT) malalignment. This is associated with OFT shortening due to loss of polarized cell orientation and failure of second heart field cell intercalation mediating OFT lengthening. OFT myocardialization was disrupted with cardiomyocytes failing to align with the direction of cell invasion into the outflow cushions. The expression of genes mediating Wnt signaling was altered. Also noted were shortened but widened bile ducts and disruption in canonical Wnt signaling. Using an in vitro wound closure assay, we showed Bj mutant fibroblasts cannot establish polarized cell morphology or engage in directional cell migration, and their actin cytoskeleton failed to align with the direction of wound closure. Unexpectedly, Pk1 mutants exhibited primary and motile cilia defects. Given Bj mutant phenotypes are reminiscent of ciliopathies, these findings suggest Pk1 may also regulate ciliogenesis. Together these findings show Pk1 plays an essential role in regulating cell polarity and directional cell migration during development.

A central role for cilia in congenital heart disease (CHD) was recently identified in a largescale mouse mutagenesis screen. Although the screen was phenotype-driven, the majority of genes recovered were cilia-related, suggesting that cilia play a central role in CHD pathogenesis. This partly reflects the role of cilia as a hub for cell signaling pathways regulating cardiovascular development. Consistent with this, many cilia-transduced cell signaling genes were also recovered, and genes regulating vesicular trafficking, a pathway essential for ciliogenesis and cell signaling. Interestingly, among CHD-cilia genes recovered, some regulate left-right patterning, indicating cardiac left-right asymmetry disturbance may play significant roles in CHD pathogenesis. Clinically, CHD patients show a high prevalence of ciliary dysfunction and show enrichment for de novo mutations in cilia-related pathways. Combined with the mouse findings, this would suggest CHD may be a new class of ciliopathy.

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.

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<0.05), but not to specific heart lesions or white matter injury. Neurodevelopmental assessment of 20 patients at 18 months showed higher BDI score correlated with reduction in Total Developmental Quotient (p <0.05) and Adaptive Developmental Quotient (p<0.009). 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.

Myelomeningocele and gastroschisis, on their own, are both relatively common entities encountered in pediatric surgical care. Coexistence of these pathologies, however, is exceedingly rare. The authors report on 2 patients who presented with myelomeningocele and gastroschisis at birth. They obtained blood for whole-exome analysis for one of the patients and identified 3 mutations that could be related to the underlying anomalies: homozygous mutations in FAM171B and ABCA1 and a hemizygous (X-linked) mutation in COL4A5. Of these, FAM171B and ABCA1 both have function that may be related to the underlying disease.

Proper control of multipotent/stem cell number and fate is essential for ensuing organ formation during development. β1-integrin, a subfamily of cell surface receptors, has a conserved role in maintenance of multipotent/stem cells, including renal progenitor cells, follicle stem cells, epidermal stem cells and neural stem cells. However, it remains unclear whether β1-integrin has a role in cardiac progenitor cell (CPC) development. Here we show that a mesodermal deletion of β1-integrin decreases Isl1 cell number in the second pharyngeal arch (PA2), where CPCs undergo renewal and expansion. Mesp1 lineage-specific mosaicism revealed that β1-integrin-deleted Isl1 cells do not proliferate in the PA2. Consistently, β1-integrin-deleted Isl1 CPCs failed to expand in vitro, independent of PA2 cells. β1-integrin co-localized and physically associated with Numb, a crucial regulator of CPC renewal and expansion. Importantly, Numb/Numbl-deleted CPCs showed dramatic reduction in β1-integrin levels. These findings suggest that β1-integrin is a key mediator of the Numb pathway in CPC maintenance.