KCTD1/KCTD15 complexes control ectodermal and neural crest cell functions, and their impairment causes aplasia cutis.

Aplasia cutis congenita (ACC) is a congenital epidermal defect of the midline scalp and has been proposed to be due to a primary keratinocyte abnormality. Why it forms mainly at this anatomic site has remained a long-standing enigma. KCTD1 mutations cause ACC, ectodermal abnormalities, and kidney fibrosis, whereas KCTD15 mutations cause ACC and cardiac outflow tract abnormalities. Here, we found that KCTD1 and KCTD15 can form multimeric complexes and can compensate for each other's loss and that disease mutations are dominant negative, resulting in lack of KCTD1/KCTD15 function. We demonstrated that KCTD15 is critical for cardiac outflow tract development, whereas KCTD1 regulates distal nephron function. Combined inactivation of KCTD1/KCTD15 in keratinocytes resulted in abnormal skin appendages but not in ACC. Instead, KCTD1/KCTD15 inactivation in neural crest cells resulted in ACC linked to midline skull defects, demonstrating that ACC is not caused by a primary defect in keratinocytes but is a secondary consequence of impaired cranial neural crest cells, giving rise to midline cranial suture cells that express keratinocyte-promoting growth factors. Our findings explain the clinical observations in patients with KCTD1 versus KCTD15 mutations, establish KCTD1/KCTD15 complexes as critical regulators of ectodermal and neural crest cell functions, and define ACC as a neurocristopathy.

Double outlet right ventricle.

This review article addresses the history, morphology, anatomy, medical management, and different surgical options for patients with double outlet right ventricle.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

Spatial transcriptome profiling uncovers metabolic regulation of left-right patterning.

Left-right patterning disturbance can cause severe birth defects, but it remains least understood of the three body axes. We uncovered an unexpected role for metabolic regulation in left-right patterning. Analysis of the first spatial transcriptome profile of left-right patterning revealed global activation of glycolysis, accompanied by right-sided expression of Bmp7 and genes regulating insulin growth factor signaling. Cardiomyocyte differentiation was left-biased, which may underlie the specification of heart looping orientation. This is consistent with known Bmp7 stimulation of glycolysis and glycolysis suppression of cardiomyocyte differentiation. Liver/lung laterality may be specified via similar metabolic regulation of endoderm differentiation. Myo1d , found to be left-sided, was shown to regulate gut looping in mice, zebrafish, and human. Together these findings indicate metabolic regulation of left-right patterning. This could underlie high incidence of heterotaxy-related birth defects in maternal diabetes, and the association of PFKP, allosteric enzyme regulating glycolysis, with heterotaxy. This transcriptome dataset will be invaluable for interrogating birth defects involving laterality disturbance.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.

Profiling development of abdominal organs in the pig.

The pig is an ideal model system for studying human development and disease due to its similarities to human anatomy, physiology, size, and genome. Further, advances in CRISPR gene editing have made genetically engineered pigs viable models for the study of human pathologies and congenital anomalies. However, a detailed atlas illustrating pig development is necessary for identifying and modeling developmental defects. Here we describe normal development of the pig abdominal system and show examples of congenital defects that can arise in CRISPR gene edited SAP130 mutant pigs. Normal pigs at different gestational ages from day 20 (D20) to term were examined and the configuration of the abdominal organs was studied using 3D histological reconstructions with episcopic confocal microscopy, magnetic resonance imaging (MRI) and necropsy. This revealed prominent mesonephros, a transient embryonic organ present only during embryogenesis, at D20, while the developing metanephros that will form the permanent kidney are noted at D26. By D64 the mesonephroi are absent and only the metanephroi remain. The formation of the liver and pancreas was observed by D20 and complete by D30 and D35 respectively. The spleen and adrenal glands are first identified at D26 and completed by D42. The developing bowel and the gonads are identified at D20. The bowel appears completely rotated by D42, and testes in the male were descended at D64. This atlas and the methods used are excellent tools for identifying developmental pathologies of the abdominal organs in the pig at different stages of development.

Inhibition of the Translation Initiation Factor eIF4A Enhances Tumor Cell Radiosensitivity.

A fundamental component of cellular radioresponse is the translational control of gene expression. Because a critical regulator of translational control is the eukaryotic translation initiation factor 4F (eIF4F) cap binding complex, we investigated whether eIF4A, the RNA helicase component of eIF4F, can serve as a target for radiosensitization. Knockdown of eIF4A using siRNA reduced translational efficiency, as determined from polysome profiles, and enhanced tumor cell radiosensitivity as determined by clonogenic survival. The increased radiosensitivity was accompanied by a delayed dispersion of radiation-induced γH2AX foci, suggestive of an inhibition of DNA double-strand break repair. Studies were then extended to (-)-SDS-1-021, a pharmacologic inhibitor of eIF4A. Treatment of cells with the rocaglate (-)-SDS-1-021 resulted in a decrease in translational efficiency as well as protein synthesis. (-)-SDS-1-021 treatment also enhanced the radiosensitivity of tumor cell lines. This (-)-SDS-1-021-induced radiosensitization was accompanied by a delay in radiation-induced γH2AX foci dispersal, consistent with a causative role for the inhibition of double-strand break repair. In contrast, although (-)-SDS-1-021 inhibited translation and protein synthesis in a normal fibroblast cell line, it had no effect on radiosensitivity of normal cells. Subcutaneous xenografts were then used to evaluate the in vivo response to (-)-SDS-1-021 and radiation. Treatment of mice bearing subcutaneous xenografts with (-)-SDS-1-021 decreased tumor translational efficiency as determined by polysome profiles. Although (-)-SDS-1-021 treatment alone had no effect on tumor growth, it significantly enhanced the radiation-induced growth delay. These results suggest that eIF4A is a tumor-selective target for radiosensitization.

Genetic resiliency associated with dominant lethal TPM1 mutation causing atrial septal defect with high heritability.

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 eight individuals with large atrial septal defect (ASD) in a five-generation pedigree. Mice with Tpm1 mutation exhibit early embryonic lethality with disrupted myofibril assembly and no heartbeat. However, patient-induced pluripotent-stem-cell-derived cardiomyocytes show normal beating with mild myofilament defect, indicating disease suppression. A variant in TLN2, another myofilament actin-binding protein, is identified as a candidate suppressor. Mouse CRISPR knock-in (KI) of both the TLN2 and TPM1 variants rescues heart beating, with near-term fetuses exhibiting large ASD. Thus, the role of TPM1 in ASD pathogenesis unfolds with suppression of its embryonic lethality by protective TLN2 variant. These findings provide evidence that genetic resiliency can arise with genetic suppression of a deleterious mutation.

Common deletion variants causing protocadherin-α deficiency contribute to the complex genetics of BAV and left-sided congenital heart disease.

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.

Cardiovascular Development and Congenital Heart Disease Modeling in the Pig.

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.

Inhibition of translation initiation factor eIF4a inactivates heat shock factor 1 (HSF1) and exerts anti-leukemia activity in AML.

Eukaryotic initiation factor 4A (eIF4A), the enzymatic core of the eIF4F complex essential for translation initiation, plays a key role in the oncogenic reprogramming of protein synthesis, and thus is a putative therapeutic target in cancer. As important component of its anticancer activity, inhibition of translation initiation can alleviate oncogenic activation of HSF1, a stress-inducible transcription factor that enables cancer cell growth and survival. Here, we show that primary acute myeloid leukemia (AML) cells exhibit the highest transcript levels of eIF4A1 compared to other cancer types. eIF4A inhibition by the potent and specific compound rohinitib (RHT) inactivated HSF1 in these cells, and exerted pronounced in vitro and in vivo anti-leukemia effects against progenitor and leukemia-initiating cells, especially those with FLT3-internal tandem duplication (ITD). In addition to its own anti-leukemic activity, genetic knockdown of HSF1 also sensitized FLT3-mutant AML cells to clinical FLT3 inhibitors, and this synergy was conserved in FLT3 double-mutant cells carrying both ITD and tyrosine kinase domain mutations. Consistently, the combination of RHT and FLT3 inhibitors was highly synergistic in primary FLT3-mutated AML cells. Our results provide a novel therapeutic rationale for co-targeting eIF4A and FLT3 to address the clinical challenge of treating FLT3-mutant AML.

A Membrane-Tethered Ubiquitination Pathway Regulates Hedgehog Signaling and Heart Development.

The etiology of congenital heart defects (CHDs), which are among the most common human birth defects, is poorly understood because of its complex genetic architecture. Here, we show that two genes implicated in CHDs, Megf8 and Mgrn1, interact genetically and biochemically to regulate the strength of Hedgehog signaling in target cells. MEGF8, a transmembrane protein, and MGRN1, a RING superfamily E3 ligase, assemble to form a receptor-like ubiquitin ligase complex that catalyzes the ubiquitination and degradation of the Hedgehog pathway transducer Smoothened. Homozygous Megf8 and Mgrn1 mutations increased Smoothened abundance and elevated sensitivity to Hedgehog ligands. While mice heterozygous for loss-of-function Megf8 or Mgrn1 mutations were normal, double heterozygous embryos exhibited an incompletely penetrant syndrome of CHDs with heterotaxy. Thus, genetic interactions can arise from biochemical mechanisms that calibrate morphogen signaling strength, a conclusion broadly relevant for the many human diseases in which oligogenic inheritance is emerging as a mechanism for heritability.

Rocaglates Induce Gain-of-Function Alterations to eIF4A and eIF4F.

Rocaglates are a diverse family of biologically active molecules that have gained tremendous interest in recent years due to their promising activities in pre-clinical cancer studies. As a result, this family of compounds has been significantly expanded through the development of efficient synthetic schemes. However, it is unknown whether all of the members of the rocaglate family act through similar mechanisms of action. Here, we present a comprehensive study comparing the biological activities of >200 rocaglates to better understand how the presence of different chemical entities influences their biological activities. Through this, we find that most rocaglates preferentially repress the translation of mRNAs containing purine-rich 5' leaders, but certain rocaglates lack this bias in translation repression. We also uncover an aspect of rocaglate mechanism of action in which the pool of translationally active eIF4F is diminished due to the sequestration of the complex onto RNA.

Structure-property studies of an imidazoquinoline chemotype with antitrypanosomal activity.

Human African trypanosomiasis is a neglected tropical disease (NTD) that is fatal if left untreated. Although approximately 13 million people live in moderate- to high-risk areas for infection, current treatments are plagued by problems with safety, efficacy, and emerging resistance. In an effort to fill the drug development pipeline for HAT, we have expanded previous work exploring the chemotype represented by the compound NEU-1090, with a particular focus on improvement of absorption, distribution, metabolism and elimination (ADME) properties. These efforts resulted in several compounds with substantially improved aqueous solubility, although these modifications typically resulted in a loss of trypanosomal activity. We herein report the results of our investigation into the antiparasitic activity, toxicity, and ADME properties of this class of compounds in the interest of informing the NTD drug discovery community and avoiding duplication of effort.

Targeting translation initiation by synthetic rocaglates for treating MYC-driven lymphomas.

MYC-driven lymphomas, especially those with concurrent MYC and BCL2 dysregulation, are currently a challenge in clinical practice due to rapid disease progression, resistance to standard chemotherapy, and high risk of refractory disease. MYC plays a central role by coordinating hyperactive protein synthesis with upregulated transcription in order to support rapid proliferation of tumor cells. Translation initiation inhibitor rocaglates have been identified as the most potent drugs in MYC-driven lymphomas as they efficiently inhibit MYC expression and tumor cell viability. We found that this class of compounds can overcome eIF4A abundance by stabilizing target mRNA-eIF4A interaction that directly prevents translation. Proteome-wide quantification demonstrated selective repression of multiple critical oncoproteins in addition to MYC in B-cell lymphoma including NEK2, MCL1, AURKA, PLK1, and several transcription factors that are generally considered undruggable. Finally, (-)-SDS-1-021, the most promising synthetic rocaglate, was confirmed to be highly potent as a single agent, and displayed significant synergy with the BCL2 inhibitor ABT199 in inhibiting tumor growth and survival in primary lymphoma cells in vitro and in patient-derived xenograft mouse models. Overall, our findings support the strategy of using rocaglates to target oncoprotein synthesis in MYC-driven lymphomas.

Amidino-Rocaglates: A Potent Class of eIF4A Inhibitors.

Rocaglates share a common cyclopenta[b]benzofuran core that inhibits eukaryotic translation initiation by modifying the behavior of the RNA helicase, eIF4A. Working as interfacial inhibitors, rocaglates stabilize the association between eIF4A and RNA, which can lead to the formation of steric barriers that block initiating ribosomes. There is significant interest in the development and expansion of rocaglate derivatives, as several members of this family have been shown to possess potent anti-neoplastic activity in vitro and in vivo. To further our understanding of rocaglate diversity and drug design, herein we explore the RNA clamping activity of >200 unique rocaglate derivatives. Through this, we report on the identification and characterization of a potent class of synthetic rocaglates called amidino-rocaglates. These compounds are among the most potent rocaglates documented to date and, taken together, this work offers important information that will guide the future design of rocaglates with improved biological properties.

Oxo-aglaiastatin-Mediated Inhibition of Translation Initiation.

Translation is a highly regulated process that is perturbed in human cancers, often through activation of the PI3K/mTOR pathway which impacts directly on the ribosome recruitment phase of translation initiation. While significant research has focused on "drugging" components of the PI3K/mTOR network, efforts have also been directed towards inhibiting eukaryotic initiation factor (eIF) 4F-dependent translation. Small molecule inhibitors of this complex have been identified, characterized, and used to validate the rationale of targeting this step to curtail tumor cell growth and modulate chemotherapy response. One such class of compounds are the rocaglates, secondary metabolites from the plant genus Aglaia, which target the RNA helicase subunit of eIF4F, eIF4A. Here we explore the ability of synthetic derivatives of aglaiastatins and an aglaroxin derivative to target the translation process in vitro and in vivo and find the synthetic derivative oxo-aglaiastatin to possess such activity. Oxo-aglaiastatin inhibited translation in vitro and in vivo and synergized with doxorubicin, ABT-199 (a Bcl-2 antagonist), and dexamethasone when tested on hematological cancer cells. The biological activity of oxo-aglaiastatin was shown to be a consequence of inhibiting eIF4A1 activity.

Simplified Luminal Water Imaging for the Detection of Prostate Cancer From Multiecho T2 MR Images.

BACKGROUND: Luminal water imaging (LWI) suffers less from imaging artifacts than the diffusion-weighted imaging used in multiparametric MRI of the prostate. LWI obtains multicompartment tissue information from a multiecho T2 dataset. PURPOSE: To compare a simplified LWI technique with apparent diffusion coefficient (ADC) in classifying lesions based on groupings of PI-RADS v2 scores. Secondary aims were to investigate whether LWI differentiates between histologically confirmed tumor and normal tissue as effectively as ADC, and whether LWI is correlated with the multicompartment parameters of the vascular, extracellular, and restricted diffusion for cytometry in tumors (VERDICT) diffusion model. STUDY TYPE: A subset of a larger prospective study. POPULATION: In all, 65 male patients aged 49-79 were scanned. FIELD STRENGTH/SEQUENCE: A 32-echo T2 and a six b-value diffusion sequence (0, 90, 500, 1500, 2000, 3000 s/mm2 ) at 3T. ASSESSMENT: Regions of interest were placed by a board-certified radiologist in areas of lesion and benign tissue and given PI-RADS v2 scores. STATISTICAL TESTS: Receiver operating characteristic and logistic regression analyses were performed. RESULTS: LWI classifies tissue as PI-RADS 1,2 or PI-RADS 3,4,5 with an area under curve (AUC) value of 0.779, compared with 0.764 for ADC. LWI differentiated histologically confirmed malignant from nonmalignant tissue with AUC, sensitivity, and specificity values of 0.81, 75%, and 87%, compared with 0.75, 83%, and 67% for ADC. The microstructural basis of the LWI technique is further suggested by the correspondence with the VERDICT diffusion-based microstructural imaging technique, with α, A1 , A2 , and LWF showing significant correlations. DATA CONCLUSION: LWI alone can predict PI-RADS v2 score groupings and detect histologically confirmed tumors with an ability similar to ADC alone without the limitations of diffusion-weighted MRI. This is important, given that ADC has an advantage in these tests as it already informs PI-RADS v2 scoring. LWI also provides multicompartment information that has an explicit biophysical interpretation, unlike ADC. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:910-917.

Airway ciliary dysfunction and respiratory symptoms in patients with transposition of the great arteries.

BACKGROUND: Our prior work on congenital heart disease (CHD) with heterotaxy, a birth defect involving randomized left-right patterning, has shown an association of a high prevalence of airway ciliary dysfunction (CD; 18/43 or 42%) with increased respiratory symptoms. Furthermore, heterotaxy patients with ciliary dysfunction were shown to have more postsurgical pulmonary morbidities. These findings are likely a reflection of the common role of motile cilia in both airway clearance and left-right patterning. As CHD comprising transposition of the great arteries (TGA) is commonly thought to involve disturbance of left-right patterning, especially L-TGA with left-right ventricular inversion, we hypothesize CHD patients with transposition of great arteries (TGA) may have high prevalence of airway CD with increased respiratory symptoms. METHODS AND RESULTS: We recruited 75 CHD patients with isolated TGA, 28% L and 72% D-TGA. Patients were assessed using two tests typically used for evaluating airway ciliary dysfunction in patients with primary ciliary dyskinesia (PCD), a recessive sinopulmonary disease caused by respiratory ciliary dysfunction. This entailed the measurement of nasal nitric oxide (nNO), which is typically low with PCD. We also obtained nasal scrapes and conducted videomicroscopy to assess respiratory ciliary motion (CM). We observed low nNO in 29% of the patients, and abnormal CM in 57%, with 22% showing both low nNO and abnormal CM. No difference was observed for the prevalence of either low nNO or abnormal ciliary motion between patients with D vs. L-TGA. Respiratory symptoms were increased with abnormal CM, but not low nNO. Sequencing analysis showed no compound heterozygous or homozygous mutations in 39 genes known to cause PCD, nor in CFTR, gene causing cystic fibrosis. As both are recessive disorders, these results indicate TGA patients with ciliary dysfunction do not have PCD or cystic fibrosis (which can cause low nNO or abnormal ciliary motion). CONCLUSIONS: TGA patients have high prevalence of abnormal CM and low nNO, but ciliary dysfunction was not correlated with TGA type. Differing from PCD, respiratory symptoms were increased with abnormal CM, but not low nNO. Together with the negative findings from exome sequencing analysis, this would suggest TGA patients with ciliary dysfunction do not have PCD but nevertheless may suffer from milder airway clearance deficiency. Further studies are needed to investigate whether such ciliary dysfunction is associated with increased postsurgical complications as previously observed in CHD patients with heterotaxy.

The complex genetics of hypoplastic left heart syndrome.

Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. 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 HLHS. 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.