Outflow Tract Defects Research Articles

Deficient GATA6-CXCR7 signaling leads to bicuspid aortic valve.

The cardiac outflow tract (OFT) transiently links the ventricles to the aortic sac and forms the arterial valves. Abnormalities in these valves, such as bicuspid aortic valve (BAV), are common congenital anomalies. GATA6-inactivating variants cause cardiac OFT defects and BAV, but their mechanisms are unclear. We generated Gata6STOP/+ mice using CRISPR-Cas9, which show highly penetrant BAV (70%) and membranous ventricular septal defects (43%). These mice exhibited decreased proliferation and increased ISL1-positive progenitor cells in the OFT, indicating abnormal cardiovascular differentiation. Gata6 deletion with the Mef2cCre driver line recapitulated Gata6STOP/+ phenotypes, indicating a cell-autonomous role for Gata6 in the second heart field. Gata6STOP/+ mice showed reduced OFT length and caliber, associated with deficient cardiac neural crest cell contribution, which may cause valvulo-septal defects. RNA-sequencing analysis showed depletion in pathways related to cell proliferation and migration, highlighting Cxcr7 (also known as Ackr3) as a candidate gene. Reduced mesenchymal cell migration and invasion were observed in Gata6STOP/+ OFT tissue. CXCR7 agonists reduced mesenchymal cell migration and increased invasion in wild-type but not in Gata6STOP/+ explants, indicating the GATA6-dependent role of CXCR7 in OFT development and its potential link to BAV.

Assisted reproductive technology and the risk of fetal congenital heart disease: insights from a tertiary-care referral center

PurposeTo investigate whether congenital heart diseases exhibit higher rates in pregnancies achieved through assisted reproductive technology (ART) compared to natural conception.MethodsIn this retrospective cohort study, multinomial logistic regression was employed to analyze the relationship between categories of congenital heart diseases and three conception groups (IVF, ICSI, and natural pregnancies). The main outcome measures are risks of congenital heart disease categories in IVF and ICSI groups using the natural group as reference. We selected fetuses referred for fetal echocardiography to IRCCS Policlinico Sant’Orsola, Bologna, between January 2005 and November 2023, diagnosed with congenital heart diseases.ResultsWe categorized the congenital heart diseases into six groups based on anatomical and embryological criteria. The estimated risk of left ventricular outflow tract, valvular, conotruncal, and atrioventricular septal defects was lower in the IVF group compared to natural conception. The estimated risk of valvular and atrioventricular septal defects was lower in the ICSI group vs natural. Conversely, the risk for right heart anomalies was higher both in the IVF and ICSI groups compared to natural conception. Heart rhythm diseases were more frequent in IVF pregnancies. When comparing ART methods, valvular defects, conotruncal defects, and right heart anomalies were more frequently observed in the ICSI group, while atrioventricular septal defects were more common in the IVF group.ConclusionSignificant differences were found in the occurrence of congenital heart diseases in pregnancies conceived through IVF and ICSI, versus those conceived naturally, underscoring the importance of further studying the underlying mechanisms of these associations.

The c.1617del variant of TMEM260 is identified as the most frequent single gene determinant for Japanese patients with a specific type of congenital heart disease

Although the molecular mechanisms underlying congenital heart disease (CHD) remain poorly understood, recent advances in genetic analysis have facilitated the exploration of causative genes for CHD. We reported that the pathogenic variant c.1617del of TMEM260, which encodes a transmembrane protein, is highly associated with CHD, specifically persistent truncus arteriosus (PTA), the most severe cardiac outflow tract (OFT) defect. Using whole-exome sequencing, the c.1617del variant was identified in two siblings with PTA in a Japanese family and in three of the 26 DNAs obtained from Japanese individuals with PTA. The c.1617del of TMEM260 has been found only in East Asians, especially Japanese and Korean populations, and the frequency of this variant in PTA is estimated to be next to that of the 22q11.2 deletion, the most well-known genetic cause of PTA. Phenotype of patients with c.1617del appears to be predominantly in the heart, although TMEM260 is responsible for structural heart defects and renal anomalies syndrome (SHDRA). The mouse TMEM260 variant (p.W535Cfs*56), synonymous with the human variant (p.W539Cfs*9), exhibited truncation and downregulation by western blotting, and aggregation by immunocytochemistry. In situ hybridization demonstrated that Tmem260 is expressed ubiquitously during embryogenesis, including in the development of cardiac OFT implicated in PTA. This expression may be regulated by a ~ 0.8 kb genomic region in intron 3 of Tmem260 that includes multiple highly conserved binding sites for essential cardiac transcription factors, thus revealing that the c.1617del variant of TMEM260 is the major single-gene variant responsible for PTA in the Japanese population.

Influence of Congenital Heart Defect Types over Cardiopulmonary Bypass, Aortic Cross-Clamping, and Intensive Care Unit Length of Stay and Their Association with Genetic Factors

Aim: Surgical correction of congenital heart defects (CHD) often requires interruption of blood flow through cardiopulmonary bypass (CPB) and aortic cross-clamping (ACC), for which duration(s) are considered to be prognostic factors, along with intensive care unit (ICU) length of stay (ICULOS). The aim of this study was to evaluate these surgical prognostic factors in pediatric patients with different types of CHD regarding their type of lesion and associated genetic factors.
 Study Design: Cross-sectional cohort study with 307 pediatric patients.
 Place and Duration of Study: Pediatric Intensive Care Unit (ICU) of Hospital da Criança Santo Antônio, in Porto Alegre/RS, Brazil, from 2006-2009 (3 years)
 Methodology: After inclusion criteria, we studied 266 pediatric patients admitted for the first time in a reference cardiac pediatric ICU from Southern Brazil following cardiac surgery. Intraoperative prognostic factors such as duration of CPB, ACC and ICULOS, in addition to dysmorphological and cytogenetic examinations were compiled and analyzed. P-values of <0.05 were considered significant.
 Results: CPB time was associated to four outflow tract defects (Tetralogy of Fallot [ToF], transposition of the great arteries [TGA], double outlet right ventricle, and truncus arteriosus [TA]), atrioventricular septal defect, and hypoplastic left heart syndrome (HLHS) (P < 0.001). ACC duration was associated with three outflow tract defects (ToF, TGA, and TA) and HLHS (P < 0.001). Moreover, CPB and ACC times showed an association with cyanotic and complex heart defects, as well as prolonged ICULOS (P < 0.001). There was no relationship between these prognostic factors and syndromic aspects or cytogenetic findings.
 Conclusions: CHD type has an impact over CPB and ACC duration and ICULOS, whereas genetic factors are not associated with those prognostic factors.

Morphologic classification of tracheobronchial arborization in children with congenital tracheobronchial stenosis and the associated cardiovascular defects.

We sought to classify patients with congenital tracheal stenosis (CTS) according to tracheobronchial morphology and determine anatomic features associated with tracheobronchial anomalies (TBAs) and concurrent cardiovascular defects (CVDs). We enrolled 254 patients who underwent tracheoplasty between November 1, 2009 and December 30, 2018. The anatomic features of the tracheobronchial tree and cardiovascular system were abstracted from bronchoscopy, echocardiography, computerized tomography, and operative reports. Four types of tracheobronchial morphology were identified: Type-1, which included normal tracheobronchial arborization (Type-1A, n = 29) and tracheal bronchus (Type-1B, n = 22); Type-2 (tracheal trifurcation; n = 49), and Type-3 (typical bridging bronchus; n = 47). Type-4 (bronchus with an untypical bridging pattern) was divided into Type-4A (involving bronchial diverticulum; n = 52) and Type-4B (absent bronchus; n = 55). Carinal compression and tracheomalacia were significantly more frequent in Type-4 patients than in the other patients (P < 0.01). CVDs were common in patients with CTS, especially in patients with Type-3 and Type-4 (P < 0.01). Persistent left superior vena cava was most common among patients with Type-3 (P < 0.01), and pulmonary artery sling was most frequent among those with Type-4 (P < 0.01). Outflow tract defects were most likely to occur in Type-1B. Early mortality was detected in 12.2% of all patients, and young age (P = 0.02), operation in the early era (P < 0.01), and bronchial stenosis (P = 0.03) were proven to be risk factors. We demonstrated a useful morphological classification for CTS. Bridging bronchus was most closely linked with vascular anomalies, while tracheal bronchus was frequently associated with outflow tract defects. These results may provide a clue to CTS pathogenesis.

Neurogenic Defects Occur in LRIG2-Associated Urinary Bladder Disease

Urofacial, or Ochoa, syndrome (UFS) is an autosomal recessive disease featuring a dyssynergic bladder with detrusor smooth muscle contracting against an undilated outflow tract. It also features an abnormal grimace. Half of individuals with UFS carry biallelic variants in HPSE2, whereas other rare families carry variants in LRIG2.LRIG2 is immunodetected in pelvic ganglia sending autonomic axons into the bladder. Moreover, Lrig2 mutant mice have abnormal urination and abnormally patterned bladder nerves. We hypothesized that peripheral neurogenic defects underlie LRIG2-associated bladder dysfunction. We describe a new family with LRIG2-associated UFS and studied Lrig2 homozygous mutant mice with exvivo physiological analyses. The index case presented antenatally with urinary tract (UT) dilatation, and postnatally had urosepsis and functional bladder outlet obstruction. He had the grimace that, together with UT disease, characterizes UFS. Although HPSE2 sequencing was normal, he carried a homozygous, predicted pathogenic, LRIG2 stop variant (c.1939C>T; p.Arg647∗). Lrig2 mutant mice had enlarged bladders. Exvivo physiology experiments showed neurogenic smooth muscle relaxation defects in the outflow tract, containing the urethra adjoining the bladder, and in detrusor contractility. Moreover, there were nuanced differences in physiological outflow tract defects between the sexes. Putting this family in the context of all reported UT disease-associated LRIG2 variants, the full UFS phenotype occurs with biallelic stop or frameshift variants, but missense variants lead to bladder-limited disease. Our murine observations support the hypothesis that UFS is a genetic autonomic neuropathy of the bladder affecting outflow tract and bladder body function.

Surgical correction of a ventricular septal defect in a child with spinal muscular atrophy type 2 treated with nusinersen sodium: a case report

IntroductionSpinal muscular atrophy (SMA) is a severe, inherited neuromuscular disorder characterized by progressive muscle weakness and atrophy. Cardiac pathology co-existence is reported more frequently in the severely affected patient groups. Structural heart anomalies, mainly septal, and outflow tract defects are commonly observed pathologies.Case presentationWe herein report the case of a 23 days-old female patient with the diagnosis of spinal muscular atrophy type 2 complicated with structural heart defects. Successful pulmonary banding, and at the age of 17 months, subsequent surgical atrial and ventricular septal defect closure were performed on our patient who was under treatment of Nusinersen Sodium. Post-operative recovery was uncomplicated. Cardiac assessments were normal, and the patient was neurologically improving in her recent follow-up.ConclusionIn the literature, there are no reported cases of successful surgical repair of heart defects in spinal muscular atrophy patients. These patients can be perceived as risky surgical candidates with suboptimal postoperative recovery given the unfavorable disease prognosis of SMA in untreated patients. We report our promising experience with a SMA type 2 patient undergoing a disease-modifying medical treatment. The SMA patients under treatment may be potential candidates for successful surgical cardiac correction given their overall improved prognosis.

STRA6 is essential for induction of vascular smooth muscle lineages in human embryonic cardiac outflow tract development.

Retinoic acid (RA) signalling is essential for heart development, and dysregulation of the RA signalling can cause several types of cardiac outflow tract (OFT) defects, the most frequent congenital heart disease (CHD) in humans. Matthew-Wood syndrome is caused by inactivating mutations of a transmembrane protein gene STRA6 that transports vitamin A (retinol) from extracellular into intracellular spaces. This syndrome shows a broad spectrum of malformations including CHD, although murine Stra6-null neonates did not exhibit overt heart defects. Thus, the detailed mechanisms by which STRA6 mutations could lead to cardiac malformations in humans remain unclear. Here, we investigated the role of STRA6 in the context of human cardiogenesis and CHD. To gain molecular signatures in species-specific cardiac development, we first compared single-cell RNA sequencing (RNA-seq) datasets, uniquely obtained from human and murine embryonic hearts. We found that while STRA6 mRNA was much less frequently expressed in murine embryonic heart cells derived from the Mesp1+ lineage tracing mice (Mesp1Cre/+; Rosa26tdTomato), it was expressed predominantly in the OFT region-specific heart progenitors in human developing hearts. Next, we revealed that STRA6-knockout human embryonic stem cells (hESCs) could differentiate into cardiomyocytes similarly to wild-type hESCs, but could not differentiate properly into mesodermal nor neural crest cell-derived smooth muscle cells (SMCs) in vitro. This is supported by the population RNA-seq data showing down-regulation of the SMC-related genes in the STRA6-knockout hESC-derived cells. Further, through machinery assays, we identified the previously unrecognized interaction between RA nuclear receptors RARα/RXRα and TBX1, an OFT-specific cardiogenic transcription factor, which would likely act downstream to STRA6-mediated RA signalling in human cardiogenesis. Our study highlights the critical role of human-specific STRA6 progenitors for proper induction of vascular SMCs that is essential for normal OFT formation. Thus, these results shed light on novel and human-specific CHD mechanisms, driven by STRA6 mutations.

Pax3 Hypomorphs Reveal Hidden Pax7 Functional Genetic Compensation in Utero

Pax3 and Pax7 transcription factors are paralogs within the Pax gene family that that are expressed in early embryos in partially overlapping expression domains and have distinct functions. Significantly, mammalian development is largely unaffected by Pax7 systemic deletion but systemic Pax3 deletion results in defects in neural tube closure, neural crest emigration, cardiac outflow tract septation, muscle hypoplasia and in utero lethality by E14. However, we previously demonstrated that Pax3 hypomorphs expressing only 20% functional Pax3 protein levels exhibit normal neural tube and heart development, but myogenesis is selectively impaired. To determine why only some Pax3-expressing cell lineages are affected and to further titrate Pax3 threshold levels required for neural tube and heart development, we generated hypomorphs containing both a hypomorphic and a null Pax3 allele. This resulted in mutants only expressing 10% functional Pax3 protein with exacerbated neural tube, neural crest and muscle defects, but still a normal heart. To examine why the cardiac neural crest appears resistant to very low Pax3 levels, we examined its paralog Pax7. Significantly, Pax7 expression is both ectopically expressed in Pax3-expressing dorsal neural tube cells and is also upregulated in the Pax3-expressing lineages. To test whether this compensatory Pax7 expression is functional, we deleted Pax7 both systemically and lineage-specifically in hypomorphs expressing only 10% Pax3. Removal of one Pax7 allele resulted in partial outflow tract defects, and complete loss of Pax7 resulted in full penetrance outflow tract defects and in utero lethality. Moreover, combinatorial loss of Pax3 and Pax7 resulted in severe craniofacial defects and a total block of neural crest cell emigration from the neural tube. Pax7Cre lineage mapping revealed ectopic labeling of Pax3-derived neural crest tissues and within the outflow tract of the heart, experimentally confirming the observation of ectopic activation of Pax7 in 10% Pax3 hypomorphs. Finally, genetic cell ablation of Pax7Cre-marked cells is sufficient to cause outflow tract defects in hypomorphs expressing only 10% Pax3, confirming that ectopic and induced Pax7 can play an overlapping functional genetic compensational role in both cardiac neural crest lineage and during craniofacial development, which is normally masked by the dominant role of Pax3.

Application of Biosheets as Right Ventricular Outflow Tract Repair Materials in a Rat Model

PurposesWe report the experimental use of completely autologous biomaterials (Biosheets) made by “in-body tissue architecture” that could resolve problems in artificial materials and autologous pericardium. Here, Biosheets were implanted into full-thickness right ventricular outflow tract defects in a rat model. Their feasibility as a reparative material for cardiac defects was evaluated.MethodsAs the evaluation of mechanical properties of the biosheets, the elastic moduli of the biosheets and RVOT-free walls of rats were examined using a tensile tester. Biosheets and expanded polytetrafluoroethylene sheet were used to repair transmural defects surgically created in the right ventricular outflow tracts of adult rat hearts (n = 9, each patch group). At 4 and 12 weeks after the operation, the hearts were resected and histologically examined.ResultsThe strength and elastic moduli of the biosheets were 421.3 ± 140.7 g and 2919 ± 728.9 kPa, respectively, which were significantly higher than those of the native RVOT-free walls (93.5 ± 26.2 g and 778.6 ± 137.7 kPa, respectively; P < 0.005 and P < 0.001, respectively). All patches were successfully implanted into the right ventricular outflow tract-free wall of rats. Dense fibrous adhesions to the sternum on the epicardial surface were also observed in 7 of 9 rats with ePTFE grafts, whereas 2 of 9 rats with biosheets. Histologically, the vascular-constructing cells were infiltrated into Biosheets. The luminal surfaces were completely endothelialized in all groups at each time point. There was also no accumulation of inflammatory cells.ConclusionsBiosheets can be formed easily and have sufficient strength and good biocompatibility as a patch for right ventricular outflow tract repair in rats. Therefore, Biosheet may be a suitable material for reconstructive surgery of the right ventricular outflow tract.

Maternal effect genes as risk factors for congenital heart defects.

SummaryMaternal effect genes (MEGs) encode factors (e.g., RNA) in the oocyte that control embryonic development prior to activation of the embryonic genome. Over 80 mammalian MEGs have been identified, including several that have been associated with phenotypes in humans. Maternal variation in MEGs is associated with a range of adverse outcomes, which, in humans, include hydatidiform moles, zygotic cleavage failure, and offspring with multi-locus imprinting disorders. In addition, data from both animal models and humans suggest that the MEGs may be associated with structural birth defects such as congenital heart defects (CHDs). To further investigate the association between MEGs and CHDs, we conducted gene-level and gene-set analyses of known mammalian MEGs (n = 82) and two common groups of CHDs: conotruncal heart defects and left ventricular outflow tract defects. We identified 14 candidate CHD-related MEGs. These 14 MEGs include three (CDC20, KHDC3L, and TRIP13) of the 11 known human MEGs, as well as one (DNMT3A) of the eight MEGs that have been associated with structural birth defects in animal models. Our analyses add to the growing evidence that MEGs are associated with structural birth defects, in particular CHDs. Given the large proportion of individuals with structural birth defects for whom etiology of their condition is unknown, further investigations of MEGs as potential risk factors for structural birth defects are strongly warranted.

Right Ventricular Outflow Tract Surgical Resection in Young, Large Animal Model for the Study of Alternative Cardiovascular Patches.

Tetralogy of Fallot (ToF) is a severe congenital heart defect (CHD) that requires surgical reconstruction soon after birth. Reconstructive surgery involves the implantation of synthetic cardiovascular patches to widen the right ventricular outflow tract (RVOT) and repair defects in the septal wall. However, synthetic patches can cause complications for these patients later in life as they do not integrate or adapt in the tissue of a growing patient; a limitation that could be solved with the development of a patch fabricated from a degradable biomaterial. Unfortunately, the lack of appropriate pre-clinical models has hindered the development of novel patch materials. Currently, most studies use rodent models to study the efficacy of new patch materials; however, large animal models are necessary to develop realistically sized patches in a clinically relevant growing heart where gradients in diffusion and length scales for cell migration are more similar to the human. Here, we describe a novel method by which a Satinsky vascular clamp is used to isolate RVOT muscle for resection followed by implantation of a cardiovascular patch in an appropriately young, rapidly growing porcine model.

Múltiplas denominações para um fenótipo complexo: contribuição de dois casos clínicos para o reconhecimento da síndrome de deleção 22q11.2

22q11.2 microdeletion is the most common microdeletion syndrome in humans and occurs with a frequency of 1 in 2,000-6,000 live births. Its main clinical features are associated with a failure to form derivatives of the third and fourth branchial arches during embryonic development. The phenotype includes heart outflow tract defects, craniofacial dysmorphisms (prominent and bulbous tip, external ears malformations, and posterior cleft palate) and endocrinological and immune system dysfunctions, as well as neurological and psychiatric disorders. Patients with this syndrome received different names, such as DiGeorge syndrome and velocardiofacial syndrome, since the recognition was based on the phenotype. Recently, molecular and genomic techniques have shown that this syndrome has great phenotypic heterogeneity. Two children with quite distinct phenotypes from this microdeletion syndrome are described in order to review the most common clinical manifestations and highlight that the great clinical heterogeneity can complicate its recognition by pediatricians.

Concomitant genetic defects potentiate the adverse impact of prenatal alcohol exposure on cardiac outflow tract maturation.

Prenatal alcohol exposure (PAE) is associated with an increased incidence of congenital heart defects (CHD), in particular outflow tract (OFT) defects. However, the variability in the incidence of CHD following PAE has not been fully explored. We hypothesize that a concomitant, relevant genetic defect would potentiate the adverse effect of PAE and partially explain the variability of PAE-induced CHD incidence. The OFT is formed by the second heart field (SHF). Our PAE model consisted of two intraperitoneal injections (3 g/kg, separated by 6 hr) of 30% ethanol on E6.5 during SHF specification. The impact of genetic defects was studied by SHF-specific loss of Delta-like ligand 4 (Dll4), fibroblast growth factor 8 (Fgf8) and Islet1. Acute PAE alone significantly increased CHD incidence (4% vs. 26%, p= .015) with a particular increase in OFT alignment defects, viz., double outlet right ventricle (0 vs. 9%, p= .02). In embryos with a SHF genetic defect, acute PAE significantly increased CHD incidence (14 vs. 63%, p< .001), including double outlet right ventricle (6 vs. 50%, p< .001) compared to controls. PAE (p= .01) and heterozygous loss of Dll4 (p= .04) were found to independently contribute to CHD incidence, while neither Islet1 nor Fgf8 defects were found to be significant. Our model recapitulates the increased incidence of OFT alignment defects seen in the clinic due to PAE. The presence of a concomitant SHF genetic mutation increases the incidence of PAE-related OFT defects. An apparent synergistic interaction between PAE and the loss of DLL4-mediated Notch signaling in OFT alignment requires further analysis.

Abstract 10425: Epigenetic Regulation of Notch Signaling by Prenatal Alcohol Exposure Impacts Cardiac Outflow Tract Defect Development

Introduction: Prenatal alcohol exposure (PAE) leads to congenital heart defects (CHD) with incomplete penetrance and phenotypic heterogeneity. We have demonstrated a gene-environment interaction in this context wherein acute PAE synergizes with second heart field (SHF) haploinsufficiency of Notch signaling, resulting in highly penetrant outflow tract (OFT) defects. Hypothesis: We hypothesize that acute PAE-induced hyperacetylation at histone marker 3 lysine residue 9 (aH3K9) epigenetically inhibits Notch signaling. This potentiates the impact of a pre-existing genetic defect that results in partial loss of Notch signaling leading to loss of viability of SHF progenitors necessary for OFT elongation and alignment. Methods: We studied the effects of acute PAE in vivo in our murine model (two injections of 3g/kg 30% ethanol on E6.5) on acetylation, SHF viability, OFT length, and Notch intracellular domain (NICD) localization. Complimentarily, we evaluated alcohol exposure in vitro using our established thoracic organ culture model. Acetylation was reversed using pan-histone acetyltransferase inhibitor anacardic acid. Results: At E9.5, acute PAE resulted in a significant increase in H3K9 acetylation in SHF associated with restriction of NICD to the cell membrane. This resulted in a 0.28-fold reduction in Notch signaling, as evidenced by reduced Notch reporter expression, and 0.52-fold reduction in SHF cell proliferation (both p&lt;0.001). Loss of SHF progenitors needed for incorporation into the developing OFT reduced OFT length 26% (p&lt;0.001). In vitro thoracic organ culture analyses recapitulated increased acetylation, NICD restriction to cell membrane and reduced SHF proliferation by 60% (p&lt;0.001). Anacardic acid reversed the increase of aH3K9 and allowed NICD nuclear translocation. Notch signaling and SHF proliferation returned to baseline. Conclusions: Acute PAE induces hyperacetylation in the SHF and inhibits Notch signaling by restricting NICD translocation. Lack of Notch signaling reduces SHF viability and prevents elongation of the developing OFT. This hereto unexplored impact of alcohol on the Notch pathway provides a novel area of investigation regarding gene-environment interaction in alcohol teratogenesis.

Proprotein convertase furina is required for heart development in zebrafish.

Cardiac looping and trabeculation are key processes during cardiac chamber maturation. However, the underlying mechanisms remain incompletely understood. Here, we report the isolation, cloning and characterization of the proprotein convertase furina from the cardiovascular mutant loft in zebrafish. loft is an ethylnitrosourea-induced mutant and has evident defects in the cardiac outflow tract, heart looping and trabeculation, the craniofacial region and pharyngeal arch arteries. Positional cloning revealed that furina mRNA was barely detectable in loft mutants, and loft failed to complement the TALEN-induced furina mutant pku338, confirming that furina is responsible for the loft mutant phenotypes. Mechanistic studies demonstrated that Notch reporter Tg(tp1:mCherry) signals were largely eliminated in mutant hearts, and overexpression of the Notch intracellular domain partially rescued the mutant phenotypes, probably due to the lack of Furina-mediated cleavage processing of Notch1b proteins, the only Notch receptor expressed in the heart. Together, our data suggest a potential post-translational modification of Notch1b proteins via the proprotein convertase Furina in the heart, and unveil the function of the Furina-Notch1b axis in cardiac looping and trabeculation in zebrafish, and possibly in other organisms.

Msx1 haploinsufficiency modifies the Pax9-deficient cardiovascular phenotype

BackgroundSuccessful embryogenesis relies on the coordinated interaction between genes and tissues. The transcription factors Pax9 and Msx1 genetically interact during mouse craniofacial morphogenesis, and mice deficient for either gene display abnormal tooth and palate development. Pax9 is expressed specifically in the pharyngeal endoderm at mid-embryogenesis, and mice deficient for Pax9 on a C57Bl/6 genetic background also have cardiovascular defects affecting the outflow tract and aortic arch arteries giving double-outlet right ventricle, absent common carotid arteries and interruption of the aortic arch.ResultsIn this study we have investigated both the effect of a different genetic background and Msx1 haploinsufficiency on the presentation of the Pax9-deficient cardiovascular phenotype. Compared to mice on a C57Bl/6 background, congenic CD1-Pax9–/– mice displayed a significantly reduced incidence of outflow tract defects but aortic arch defects were unchanged. Pax9–/– mice with Msx1 haploinsufficiency, however, have a reduced incidence of interrupted aortic arch, but more cases with cervical origins of the right subclavian artery and aortic arch, than seen in Pax9–/– mice. This alteration in arch artery defects was accompanied by a rescue in third pharyngeal arch neural crest cell migration and smooth muscle cell coverage of the third pharyngeal arch arteries. Although this change in phenotype could theoretically be compatible with post-natal survival, using tissue-specific inactivation of Pax9 to maintain correct palate development whilst inducing the cardiovascular defects was unable to prevent postnatal death in the mutant mice. Hyoid bone and thyroid cartilage formation were abnormal in Pax9–/– mice.ConclusionsMsx1 haploinsufficiency mitigates the arch artery defects in Pax9–/– mice, potentially by maintaining the survival of the 3rd arch artery through unimpaired migration of neural crest cells to the third pharyngeal arches. With the neural crest cell derived hyoid bone and thyroid cartilage also being defective in Pax9–/– mice, we speculate that the pharyngeal endoderm is a key signalling centre that impacts on neural crest cell behaviour highlighting the ability of cells in different tissues to act synergistically or antagonistically during embryo development.

Identification of disease-relevant modulators of the SHH pathway in the developing brain.

Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes. To identify such modifiers, we established novel congenic mouse models. LRP2-deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on a FVB/N background, indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders.

The particular anatomy of ventricular septal defect in patients with Down syndrome and complete atrioventricular septal defect: An echocardiographic study

Complete atrioventricular septal defect (CAVSD) is associated with Down syndrome (DS) in almost half of cases, and is due in this setting to an abnormal development of the vestibular spine, derived from the posterior second heart field (SHF). Clinical studies have shown that Tetralogy of Fallot (TF), an outflow tract defect involving the anterior SHF, is more frequent in CAVSD patients with DS. We therefore decided to look for an outlet extension of the VSD in DS and non-DS. We reviewed retrospectively all 208 consecutive patients hospitalized in our unit with the diagnosis of CAVSD between 01/01/2017 and 09/12/2019. All echocardiographic examinations were screened by 2 pediatric cardiologists (ZB/LH). We excluded 46 patients whose VSD anatomical type could not be determined. We used the classification as agreed upon by ICD-11 to determine the VSD anatomical type in the 162 patients included. Among the cohort, 101/162 had DS (62.7%). An outlet extension of the VSD, with anterior malalignment of the outlet septum, was found in 88/101 (87.1%) DS vs. 4/61 (6.5%) non-DS ( P < 0.0001). Associated TF was found in 12 DS vs. 1 in non-DS ( P = ns). Ventricles were balanced in 126 patients, 23 had a hypoplastic left ventricle (LV), 13 had a hypoplastic right ventricle (RV). There was no significant difference regarding balanced or not ventricles between DS and non-DS. However, the hypoplastic ventricle was always the LV in non-DS, and was the RV in 62% of DS ( P = 0.006). This echocardiographic study describes for the first time that outlet extension of the VSD is the rule in CAVSD with DS and that hypoplastic RV occurs only in DS. This seems to confirm the involvement of the two parts of the second heart field in the morphogenesis of CAVSD in DS patients. The presence of a malaligned outlet septum in a fetus with CAVSD should raise awareness of the high risk of associated DS.

Common Variation in Cytoskeletal Genes is Associated with Conotruncal Heart Defects.

There is strong evidence for a genetic contribution to non-syndromic congenital heart defects (CHDs). However, exome- and genome-wide studies conducted at the variant and gene-level have identified few genome-wide significant CHD-related genes. Gene-set analyses are a useful complement to such studies and candidate gene-set analyses of rare variants have provided insight into the genetics of CHDs. However, similar analyses have not been conducted using data on common genetic variants. Consequently, we conducted common variant analyses of 15 CHD candidate gene-sets, using data from two common types of CHDs: conotruncal heart defects (1431 cases) and left ventricular outflow tract defects (509 cases). After Bonferroni correction for evaluation of multiple gene-sets, the cytoskeletal gene-set was significantly associated with conotruncal heart defects (βS = 0.09; 95% confidence interval (CI) 0.03–0.15). This association was stronger when analyses were restricted to the sub-set of cytoskeletal genes that have been observed to harbor rare damaging genotypes in at least two CHD cases (βS = 0.32, 95% CI 0.08–0.56). These findings add to the evidence linking cytoskeletal genes to CHDs and suggest that, for cytoskeletal genes, common variation may contribute to the risk of CHDs.