A clinical scoring system for early onset (neonatal) Marfan syndrome.

PURPOSE: This study aimed to develop objective diagnostic criteria for early onset Marfan syndrome (eoMFS) to facilitate early diagnosis and timely interventions. METHODS: On the basis of an extensive literature review and the responses from a survey distributed among providers with expertise in the diagnosis and management of eoMFS, we developed an age-based, diagnostic scoring system encompassing 10 features common to eoMFS (9 clinical + 1 laboratory) and divided them into cardiac, systemic, and FBN1 (on the basis of the location of the pathogenic FBN1 variant) scores. RESULTS: In total, 77 individuals with eoMFS (13 newly reported) and 49 individuals diagnosed with classical Marfan syndrome during early childhood were used to validate the criteria. Median cardiac (8 vs 0, P < .001), systemic (11 vs 3, P < .001), FBN1 (5 vs 0, P < .001), and total (23 vs 4, P < .001) scores were significantly higher in individuals with eoMFS than in those without. A proposed clinical score (cardiac + systemic) cutoff of ≥14 points showed excellent sensitivity (100%), specificity (92%), and reliability (correctly classified = 94%). CONCLUSION: Distinct from classical Marfan syndrome in phenotype and morbidity, eoMFS can be diagnosed clinically using an objective scoring system encompassing the typical physical features and cardiac disease manifestations. Although genetic testing can be suggestive of eoMFS, genetic testing alone is insufficient for diagnosis.

Deficiency of Circulating Monocytes Ameliorates the Progression of Myxomatous Valve Degeneration in Marfan Syndrome.

BACKGROUND: Myxomatous valve degeneration (MVD) involves the progressive thickening and degeneration of the heart valves, leading to valve prolapse, regurgitant blood flow, and impaired cardiac function. Leukocytes composed primarily of macrophages have recently been detected in myxomatous valves, but the timing of the presence and the contributions of these cells in MVD progression are not known. METHODS: We examined MVD progression, macrophages, and the valve microenvironment in the context of Marfan syndrome (MFS) using mitral valves from MFS mice (Fbn1C1039G/+), gene-edited MFS pigs (FBN1Glu433AsnfsX98/+), and patients with MFS. Additional histological and transcriptomic evaluation was performed by using nonsyndromic human and canine myxomatous valves, respectively. Macrophage ontogeny was determined using MFS mice transplanted with mTomato+ bone marrow or MFS mice harboring RFP (red fluorescent protein)-tagged C-C chemokine receptor type 2 (CCR2) monocytes. Mice deficient in recruited macrophages (Fbn1C1039G/+;Ccr2RFP/RFP) were generated to determine the requirements of recruited macrophages to MVD progression. RESULTS: MFS mice recapitulated histopathological features of myxomatous valve disease by 2 months of age, including mitral valve thickening, increased leaflet cellularity, and extracellular matrix abnormalities characterized by proteoglycan accumulation and collagen fragmentation. Diseased mitral valves of MFS mice concurrently exhibited a marked increase of infiltrating (MHCII+, CCR2+) and resident macrophages (CD206+, CCR2-), along with increased chemokine activity and inflammatory extracellular matrix modification. Likewise, mitral valve specimens obtained from gene-edited MFS pigs and human patients with MFS exhibited increased monocytes and macrophages (CD14+, CD64+, CD68+, CD163+) detected by immunofluorescence. In addition, comparative transcriptomic evaluation of both genetic (MFS mice) and acquired forms of MVD (humans and dogs) unveiled a shared upregulated inflammatory response in diseased valves. Remarkably, the deficiency of monocytes was protective against MVD progression, resulting in a significant reduction of MHCII macrophages, minimal leaflet thickening, and preserved mitral valve integrity. CONCLUSIONS: All together, our results suggest sterile inflammation as a novel paradigm to disease progression, and we identify, for the first time, monocytes as a viable candidate for targeted therapy in MVD.

Global genetic analysis in mice unveils central role for cilia in congenital heart disease.

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.

Cannabinoid receptor signaling regulates liver development and metabolism.

Endocannabinoid (EC) signaling mediates psychotropic effects and regulates appetite. By contrast, potential roles in organ development and embryonic energy consumption remain unknown. Here, we demonstrate that genetic or chemical inhibition of cannabinoid receptor (Cnr) activity disrupts liver development and metabolic function in zebrafish (Danio rerio), impacting hepatic differentiation, but not endodermal specification: loss of cannabinoid receptor 1 (cnr1) and cnr2 activity leads to smaller livers with fewer hepatocytes, reduced liver-specific gene expression and proliferation. Functional assays reveal abnormal biliary anatomy and lipid handling. Adult cnr2 mutants are susceptible to hepatic steatosis. Metabolomic analysis reveals reduced methionine content in Cnr mutants. Methionine supplementation rescues developmental and metabolic defects in Cnr mutant livers, suggesting a causal relationship between EC signaling, methionine deficiency and impaired liver development. The effect of Cnr on methionine metabolism is regulated by sterol regulatory element-binding transcription factors (Srebfs), as their overexpression rescues Cnr mutant liver phenotypes in a methionine-dependent manner. Our work describes a novel developmental role for EC signaling, whereby Cnr-mediated regulation of Srebfs and methionine metabolism impacts liver development and function.

Macrophage Transitions in Heart Valve Development and Myxomatous Valve Disease.

OBJECTIVE: Hematopoietic-derived cells have been reported in heart valves but remain poorly characterized. Interestingly, recent studies reveal infiltration of leukocytes and increased macrophages in human myxomatous mitral valves. Nevertheless, timing and contribution of macrophages in normal valves and myxomatous valve disease are still unknown. The objective is to characterize leukocytes during postnatal heart valve maturation and identify macrophage subsets in myxomatous valve disease. APPROACH AND RESULTS: Leukocytes are detected in heart valves after birth, and their numbers increase during postnatal valve development. Flow cytometry and immunostaining analysis indicate that almost all valve leukocytes are myeloid cells, consisting of at least 2 differentially localized macrophage subsets and dendritic cells. Beginning a week after birth, increased numbers of CCR2+ (C-C chemokine receptor type 2) macrophages are present, consistent with infiltrating populations of monocytes, and macrophages are localized in regions of biomechanical stress in the valve leaflets. Valve leukocytes maintain expression of CD (cluster of differentiation) 45 and do not contribute to significant numbers of endothelial or interstitial cells. Macrophage lineages were examined in aortic and mitral valves of Axin2 KO (knockout) mice that exhibit myxomatous features. Infiltrating CCR2+ monocytes and expansion of CD206-expressing macrophages are localized in regions where modified heavy chain hyaluronan is observed in myxomatous valve leaflets. Similar colocalization of modified hyaluronan and increased numbers of macrophages were observed in human myxomatous valve disease. CONCLUSIONS: Our study demonstrates the heterogeneity of myeloid cells in heart valves and highlights an alteration of macrophage subpopulations, notably an increased presence of infiltrating CCR2+ monocytes and CD206+ macrophages, in myxomatous valve disease.

Selenoprotein H is an essential regulator of redox homeostasis that cooperates with p53 in development and tumorigenesis.

Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels.

Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate.

During heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCgamma and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.

Macrophage lineages in heart valve development and disease.

Heterogeneous macrophage lineages are present in the aortic and mitral valves of the heart during development and disease. These populations include resident macrophages of embryonic origins and recruited monocyte-derived macrophages prevalent in disease. Soon after birth, macrophages from haematopoietic lineages are recruited to the heart valves, and bone marrow transplantation studies in mice demonstrate that haematopoietic-derived macrophages continue to invest adult valves. During myxomatous heart valve disease, monocyte-derived macrophages are recruited to the heart valves and they contribute to valve degeneration in a mouse model of Marfan syndrome. Here, we review recent studies of macrophage lineages in heart valve development and disease with discussion of clinical significance and therapeutic applications.

Endothelial Cell Lineage Analysis Does Not Provide Evidence for EMT in Adult Valve Homeostasis and Disease.

Epithelial-to-mesenchymal transition (EMT) enables stationary epithelial cells to exhibit migratory behavior and is the key step that initiates heart valve development. Recent studies suggest that EMT is reactivated in the pathogenesis of myxomatous valve disease (MVD), a condition that involves the progressive degeneration and thickening of valve leaflets. These studies have been limited to in vitro experimentation and reliance on histologic costaining of epithelial and mesenchymal markers as evidence of EMT in mouse and sheep models of valve disease. However, longitudinal analysis of cell lineage origins and potential pathogenic or reparative contributions of newly generated mesenchymal cells have not been reported previously. In this study, a genetic lineage tracing strategy was pursued by irreversibly labeling valve endothelial cells in the Osteogenesis imperfecta and Marfan syndrome mouse models to determine whether they undergo EMT during valve disease. Tie2-CreER T2 and Cdh5(PAC)-CreER T2 mouse lines were used in combination with colorimetric and fluorescent reporters for longitudinal assessment of endothelial cells. These lineage tracing experiments showed no evidence of EMT during adult valve homeostasis or valve pathogenesis. Additionally, CD31 and smooth muscle α-actin (αSMA) double-positive cells, used as an indicator of EMT, were not detected, and levels of EMT transcription factors were not altered. Interestingly, contrary to the endothelial cell-specific Cdh5(PAC)-CreER T2 driver line, Tie2-CreER T2 lineage-derived cells in diseased heart valves also included CD45+ leukocytes. Altogether, our data indicate that EMT is not a feature of valve homeostasis and disease but that increased immune cells may contribute to MVD. Anat Rec, 302:125-135, 2019. © 2018 Wiley Periodicals, Inc.

Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer.

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options1. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families2. We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases3. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

MRI supersedes ictal EEG when other presurgical data are concordant.

PURPOSE: When ictal EEG is discordant with MRI and other presurgical data, our group has sometimes discounted the ictal findings and proceeded with epilepsy surgical resection based on MRI. We aimed to evaluate the prudence of such practice by comparing the outcome of MRI-lesional epilepsy surgery patients with discordant ictal EEG with those with concordant ictal EEG. METHOD: We retrospectively studied 115 children with epilepsy who underwent surgical resection of an MRI lesion that was corroborated as the epileptogenic focus by other presurgical findings. Ictal findings on video-EEG were categorized as: "positive ictal EEG" if the ictal onset localization was concordant with MRI and other presurgical data; "negative ictal EEG" if the ictus was discordant with them. Seizure-free outcome at 2 years was compared between the "positive" and the "negative" ictal EEG groups. RESULTS: Seizure-free outcome did not differ between children with positive ictal EEG (73%) and those with negative ictal EEG (80%). Positive ictal EEG did not result in better outcome regardless of the location of the surgery or the pathology of the lesion. Ictal EEG with 73% positive predictive value provided no added benefit in this cohort whose seizure-free outcome was of 77% irrespective of ictal EEG findings. CONCLUSIONS: In our selected cohort of pediatric epilepsy surgery patients with an epileptogenic lesion on MRI and concordant other data, ictal EEG had limited predictive value. This calls into question the additive role of ictal recordings in patients with an MRI lesion and concordant other presurgical data.

Phenotyping cardiac and structural birth defects in fetal and newborn mice.

Mouse models are invaluable for investigating the developmental etiology and molecular pathogenesis of structural birth defects. While this has been deployed for studying a wide spectrum of birth defects, mice are particularly valuable for modeling congenital heart disease, given they have the same four-chamber cardiac anatomy as in humans. We have developed the use of noninvasive fetal ultrasound together with micro-computed tomography (micro-CT) imaging for high throughput phenotyping of mice for congenital heart defects (CHD) and other developmental anomalies. We showed the efficacy of fetal ultrasound and micro-CT imaging for diagnosis of a wide spectrum of CHD. With fetal ultrasound, longitudinal scans can be conducted to track the developmental profile of disease pathogenesis, providing both structural information with two-dimensional (2D) imaging, as well as functional data from hemodynamic assessments with color flow and spectral Doppler imaging. In contrast, with micro-CT imaging, virtual necropsies can be conducted rapidly postmortem for diagnosis of not only CHD, but also other structural birth defects. To validate the CHD diagnosis, we further showed the use of a novel histological technique with episcopic confocal microscopy to obtain rapid 3D reconstructions for accurate diagnosis of even the most complex anatomical defect. The latter histological imaging technique when combined with the use of ultrasound and micro-CT imaging provides a powerful combination of imaging modalities that will be invaluable in meeting the accelerating demands for high throughput mouse phenotyping of genetically engineered mice in the coming age of functional genomics. Birth Defects Research 109:778-790, 2017. © 2017 Wiley Periodicals, Inc.

Seizure Outcomes in Children Following Electrocorticography-Guided Single-Stage Surgical Resection.

BACKGROUND: In children with abnormal imaging, single-stage epilepsy surgery is an attractive alternative to the two-stage approach that relies on invasive recording of seizures. Implanted electrodes carry risks of their own and extend hospitalization, but the efficacy of one-stage resections in a variety of pathologies and cerebral locations is not well established. We report our center's experience with single-stage epilepsy surgery guided by intraoperative electrocorticography (ECoG). METHODS: We retrospectively analyzed 130 consecutive patients who underwent single-stage epilepsy surgery before age 19 years and had at least a two-year follow-up. Intraoperative ECoG was available for review in 113. Patients were considered seizure-free if they were continuously Engel Class I up to the two-year postoperative mark. ECoG findings were classified according to the presence of interictal attenuation, spikes, both, or neither. Complications and hospital length of stay were evaluated. RESULTS: Eighty percent of 130 patients were seizure-free at two years. All but one had an abnormal MRI. Patients with tumor had a better seizure outcome than patients with cortical malformation. Frontal resections had worse outcome, especially among tumors. Intraoperative ECoG revealed both attenuation and spikes in 48%, attenuation only in 23%, spikes only in 20%, and neither in 9%. The complication rate was 6.9%, with no major neurological complications. The average length of stay was 5.7 nights. CONCLUSIONS: With ECoG-guided single-stage surgery, we achieved results comparable with other pediatric surgical series and with a low complication rate. An extensive two-stage approach may not be required when there is a lesion on imaging and other information is concordant, even when the MRI abnormality is subtle and unclearly delineated. Frontal foci may present a challenge because of their proximity to "eloquent" nonresectable cortex or critical structures.

Abruptly attenuated terminal ictal pattern in pediatrics.

PURPOSE: We examined the ictal discharges at the end of pediatric seizures and categorized the various patterns. One particular pattern, termed "abruptly attenuated termination" was studied in detail. METHODS: Ictal segments captured on video-EEG monitoring during a 26-month interval were analyzed for a variety of ictal termination patterns, including one that we rigorously defined as abruptly attenuated termination pattern (AAT). We studied the associations between AAT and the other ictal EEG and clinical features. RESULTS: AAT was noted in 16 of 200 (8%) pediatric seizures. All 16 were immediately preceded by repetitive spikes or spike-waves. The presence of AAT also correlated with ictal spread pattern, initial ictal pattern, laterality of onset, seizure duration, age, and epilepsy etiology. AAT is more often noted in children older than 6 months and in children with idiopathic or cryptogenic forms of epilepsy. CONCLUSIONS: The minority of pediatric seizures recorded in a tertiary epilepsy monitoring unit end with diffuse, synchronized abrupt attenuation. AAT probably is the result of an active process that is developmentally related. It appears to require some degree of mature and intact neurophysiology and may involve the thalamocortical circuit.

[Not Quite] The Ketogenic Diet in a Pill.

Researchers at Okayama University, Japan showed lactate dehydrogenase (LDH) inhibition suppresses neuronal excitation in vitro, reduces EEG discharges and seizures in rodent models, and may provide a novel mechanism for anticonvulsant medications in human patients.

A practical, simple, and useful method of categorizing interictal EEG features in children.

We introduce a simple scheme of categorizing interictal EEG in patients with pediatric epilepsy. Five patterns of EEG can be determined by using 2 interictal EEG domains: organization of the background activity and a morphology/topography of epileptiform discharges. These patterns relate to commonly recognized categories of pediatric epilepsy: familial epilepsies, genetic generalized epilepsies, self-limited epilepsies, epilepsies with encephalopathy, and focal structural epilepsies. Each group has distinguishable clinical presentations, inheritance patterns, and outcomes. This categorization may be a useful educational tool; it may also guide decisions about further testing and management.

Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis.

NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting.

Interictal attenuation in pediatric electrocorticography can be reliably detected by EEG readers.

Intraoperative electrocorticography (ECoG) helps to demarcate epileptogenic cortex, but a commonly observed feature, interictal attenuation, has received little attention. This may limit its use in the determination of the resection margin. In order to test how reliably EEGers can discern attenuation, we assessed how well EEGers agree with each other and with an objective, quantified measure of attenuation. ECoG segments (n=34) were evaluated for attenuation by two EEGers independently and in consensus, and by an amplitude spectral analysis-based quantitative method. A third EEGer divided the 34 ECoG segments into 3 subgroups-physiologic field present, physiologic field uncertain, and physiologic field absent-based on the clustering patterns of the attenuated electrodes. Inter-rater agreement between two independent EEGers (kappa=0.56) was moderate, and between consensus EEGers and the quantitative method (kappa=0.71) was substantial. These agreements were especially good among the physiologic field present subgroup where the attenuation clearly involved contiguous electrodes, and thus, more likely pathologic (kappa=0.64 for two independent EEGers and kappa=0.78 for consensus EEGers and quantitative method). Our results suggest that interictal attenuation, especially when involving contiguous electrodes, is an ECoG marker that can be consistently and reliably discerned by trained EEGers.

Prostaglandin E2 regulates liver versus pancreas cell-fate decisions and endodermal outgrowth.

The liver and pancreas arise from common endodermal progenitors. How these distinct cell fates are specified is poorly understood. Here we describe prostaglandin E2 (PGE2) as a regulator of endodermal fate specification during development. Modulating PGE2 activity has opposing effects on liver versus pancreas specification in zebrafish embryos as well as mouse endodermal progenitors. The PGE2 synthetic enzyme cox2a and receptor ep2a are patterned such that cells closest to PGE2 synthesis acquire a liver fate, whereas more distant cells acquire a pancreas fate. PGE2 interacts with the bmp2b pathway to regulate fate specification. At later stages of development, PGE2 acting via the ep4a receptor promotes outgrowth of both the liver and pancreas. PGE2 remains important for adult organ growth, as it modulates liver regeneration. This work provides in vivo evidence that PGE2 may act as a morphogen to regulate cell-fate decisions and outgrowth of the embryonic endodermal anlagen.

Interrogating congenital heart defects with noninvasive fetal echocardiography in a mouse forward genetic screen.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.