William A. Devine

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.

Cardiac swelling associated with linear radiofrequency ablation in the atrium.

AbstractObjectives: To characterize myocardial swelling in response to application of endocardial radio- frequency ablation lesions. Background: In individual patients, we have observed that ablation in the posterior right atrium was associated with echocardiographic evidence of atrial and contiguous right pulmonary vein wall swelling. Methods: 1. Human Subjects: “linear” ablation was performed in the posterior right atrium in 10 subjects; a portion of the ablation lesion was contiguous to the right pulmonary vein; this area was defined as the “contiguity zone”. In the contiguity zone, right atrial wall thickness and pulmonary vein lumen diameter were measured utilizing intracardiac echocardiography. Measurements were made just prior to (baseline) and immediately after ablation.2. Porcine Subjects: linear ablation was performed in the posterior right atrium of 14 pigs. In the contiguity zone, atrial wall thickness, interstitial space thickness, right pulmonary vein wall thickness and lumen diameter were measured using intracardiac echocardiography. Measurements were made at baseline, immediately after ablation, and at 1, 4, 8 or 12 weeks after ablation (followup). Post-mortem pathologic evaluation of the contiguity zone was performed. Results: 1. Human Subjects: Immediately after ablation, relative to baseline right atrial wall thickness was significantly increased (9.4±3.1[emsp4 ]mm versus 5.4±1.5[emsp4 ]mm) and right pulmonary vein lumen diameter was significantly decreased (6.2±2.9[emsp4 ]mm versus 8.1±2.9[emsp4 ]mm).2. Porcine Subjects: Immediately after ablation, right atrial wall thickness (4.1±1.2[emsp4 ]mm), interstitial space thickness (1.9±1.1[emsp4 ]mm), and right pulmonary vein wall thickness (1.2±0.4[emsp4 ]mm) were each significantly increased relative to baseline (1.0±0.3[emsp4 ]mm, 0±0[emsp4 ]mm, and 0.7±0.2[emsp4 ]mm, respectively) and pulmonary vein lumen diameter was significantly decreased (5.0±1.4[emsp4 ]mm versus 6.9±2.2[emsp4 ]mm). Similar findings were made at the 1 week followup interval. At 4, 8 and 12 week followup intervals, thicknesses and lumen diameter were not significantly different from baseline. At post-mortem examination, direct measurements of wall thickness were significantly correlated with echocardiographic measurements. Histologic analysis demonstrated edema to be the cause of the early wall thickness and lumen diameter changes. Ablation lesions were transmural in the right atria of all animals; in some animals, lesion formation was also observed in the pulmonary vein wall. Conclusions: Cardiac edema resulting from right atrial linear ablation results in swelling of atrial and contiguous right pulmonary vein walls, as well as the interposed extracardiac interstitial space. These changes are associated with a decrease in pulmonary vein lumen diameter. Swelling evolves rapidly and resolves within 4 weeks.

Congenital heart disease and the specification of left-right asymmetry

Complex congenital heart disease (CHD) is often seen in conjunction with heterotaxy, the randomization of left-right visceral organ situs. However, the link between cardiovascular morphogenesis and left-right patterning is not well understood. To elucidate the role of left-right patterning in cardiovascular development, we examined situs anomalies and CHD in mice with a loss of function allele of Dnaic1, a dynein protein required for motile cilia function and left-right patterning. Dnaic1 mutants were found to have nodal cilia required for left-right patterning, but they were immotile. Half the mutants had concordant organ situs comprising situs solitus or mirror symmetric situs inversus. The remaining half had randomized organ situs or heterotaxy. Looping of the heart tube, the first anatomical lateralization, showed abnormal L-loop bias rather than the expected D-loop orientation in heterotaxy and nonheterotaxy mutants. Situs solitus/inversus mutants were viable with mild or no defects consisting of azygos continuation and/or ventricular septal defects, whereas all heterotaxy mutants had complex CHD. In heterotaxy mutants, but not situs solitus/inversus mutants, the morphological left ventricle was thin and often associated with a hypoplastic transverse aortic arch. Thus, in conclusion, Dnaic1 mutants can achieve situs solitus or inversus even with immotile nodal cilia. However, the finding of abnormal L-loop bias in heterotaxy and nonheterotaxy mutants would suggest motile cilia are required for normal heart looping. Based on these findings, we propose motile nodal cilia patterns heart looping but heart and visceral organ lateralization is driven by signaling not requiring nodal cilia motility.

Airway ciliary dysfunction and sinopulmonary symptoms in patients with congenital heart disease

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

The morphology of aortopulmonary windows with regard to their classification and morphogenesis

We examined 25 heart specimens with aortopulmonary windows in order to review the variety of the lesion and its associated malformations. Unlike common arterial trunk, the aortopulmonary window involves a deficiency of the wall of the ascending aorta and that of the pulmonary trunk. The window was in proximal position in three specimens, intermediate position in three specimens, distal position in 16 specimens and was confluent in three specimens. The size of the window varied from 27 to 100% of the total length of the pulmonary trunk but size did not bear any apparent relationship to the position of the defect. The shape of the window was tunnel-like in one case. Of the 16 specimens with distal windows, the orifice of the right pulmonary artery arose from the aorta in seven specimens and was overriding the plane of the window in one specimen. The window occurred as an isolated lesion in four specimens. It was associated with interruption of the aortic arch at the isthmus in eight specimens and between the left carotid and left subclavian arteries in three specimens. A further three specimens had isthmal stenosis. Among the other associated defects were complete transposition (two specimens), tetralogy of Fallot (one specimen), and double outlet right ventricle (one specimen). A ventricular septal defect was present in six of the 25 specimens. The associated lesions found with aortopulmonary window are seldom encountered with common arterial trunk, suggesting these two arterial lesions have different pathogenesis and are not variants of the same entity.

Identification and analysis of left atrial isomerism

The veno-atrial connections, atrial morphology, atrioventricular (AV) junction, ventricular mass, ventriculoarterial (VA) connection and great arteries in 22 autopsied hearts, diagnosed as having bilateral left-sidedness because of the morphology of the atrial appendages, were studied. The findings were correlated with the arrangement of the thoracic-abdominal organs. A solitary spleen was found in 3 and double spleens in 2 hearts (the remaining 17 hearts had multiple spleens) but left bronchial isomerism existed in all hearts in which bronchial arrangement could be determined. The heart was in the left chest in 14 cases, in the right chest in 5 and midline in 3. The apex pointed to the left in 18 hearts while in 4 hearts it pointed to the right. Fifteen hearts had a biventricular and ambiguous AV connection, 3 hearts had an absent left AV connection and 4 had double-inlet connection via a common valve (to the left ventricle in 3 and the right ventricle in 1). The VA connection was concordant in 14 hearts, discordant in 1, double outlet from the right ventricle in 4, double outlet from a solitary indeterminate ventricle in 1 and single outlet from the right ventricle through a pulmonary trunk with aortic atresia in 2 hearts. Superior caval veins were present bilaterally in 13 hearts. There was interruption of the infrahepatic inferior caval vein with azygos or hemiazygos continuation in 19 hearts while in 3 hearts the inferior caval vein continued upwards to drain into the right-sided morphologically left atrium.(ABSTRACT TRUNCATED AT 250 WORDS)

The surgical anatomy of tetralogy of Fallot with pulmonary atresia rather than pulmonary stenosis.

We examined the pertinent surgical features of the anatomy of 56 hearts having tetralogy of Fallot with pulmonary atresia instead of stenosis, or malformations with pulmonary atresia closely related to tetralogy. We took particular cognizance of the pulmonary arterial supply in 15 hearts in which this was derived through systemic‐to‐pulmonary collateral arteries, dissecting, as far as possible, the bronchopulmonary segmental distribution of the collateral arteries compared to the intrapericardial pulmonary arteries in 11 of these hearts. Two of the hearts had absence of intrapericardial pulmonary arteries, so that a solitary arterial trunk left the base of the heart. Evidence of an atretic subpulmonary infundibulum was found in 40 of the hearts, while such an infundibulum was lacking in the remainder. The pulmonary atresia was muscular in 43 hearts, valvar in 11, while the pulmonary trunk was absent in the other two hearts. In the hearts with collateral arteries, on average 2.6 collaterals were found in each case, varying from two to five per case. Only one of these arose from a brachiocephalic artery, the others all arising from the descending aorta. The distribution of collateral arteries in two cases was remarkably reminiscent of the arrangement of bronchial arteries. As far as could be judged, 16.5 bronchopulmonary segments on average were supplied in each heart, 5.1 exclusively by collateral arteries, 11.8 by intrapericardial pulmonary arteries and an average of 0.64 segments per case having a shared supply.

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.

Morphometric analysis of atrioventricular septal defect with common valve orifice

OBJECTIVES We sought to analyze morphometric features of atrioventricular septal defect (AVSD) in autopsy specimens and to consider the developmental implications of obstruction in either ventricular outflow tract. BACKGROUND Left ventricular outlet obstruction (LVO) is more prevalent in patients with Rastelli type A morphology. When tetralogy of Fallot (ToF) complicates this malformation, there is usually a free-floating superior bridging leaflet. The reasons for these associations are uncertain. METHODS In 133 hearts with AVSD and common atrioventricular (AV) valve orifice, we measured the degrees of horizontal and anterior deviation of the great arteries from the AV valve, the diameters of the ventricular outlets and the great arteries and the degree of deficiency of the ventricular septum. RESULTS In Rastelli type A morphology, the great arteries were deviated more leftward than in type C morphology (p < 0.01). Type A hearts also had a relatively small aorta, with a long and narrow subaortic tract. The presence of obstruction in either ventricular outlet was associated with a more oblique arrangement of the great arteries, with the pulmonary trunk being more leftward than in hearts without LVO (p < 0.01). In combination with ToF, the aorta was dextroposed and the pulmonary trunk was located more posteriorly (p < 0.01). No heart with type A morphology showed ToF (p < 0.01). CONCLUSIONS The geometric arrangement of the great arteries correlated significantly with obstruction in either ventricular outflow tract and with the Rastelli subtypes. Malrotation of the developing outlet septum may be an embryologic factor producing obstruction, with horizontal deviation of the outlets also influencing the morphology of the superior bridging leaflet.

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.