Aim Nasal cannulas are used to provide oxygen support for infants and have been considered as a means for delivering aerosols to the lungs. To measure mucociliary clearance in the lungs of infants with congenital heart defects, we delivered radiopharmaceutical aerosols via a nasal cannula. Here we report on the pulmonary and nasal deposition of these aerosols. Method A total of 18 infants (median age = 26 days; quartiles = 11‐74 days) performed clearance measurements soon before or after corrective cardiac surgery. The regional aerosol deposition was assessed using gamma camera imaging. Results Cannula flow rate significantly affected pulmonary dosing. Flow rates useful for oxygen support were associated with low pulmonary deposition (2 L/min; mean, 4.5% of deposited dose; range, 2%‐9%; n = 7) and high nasal deposition. Much lower cannula flow rates increased the pulmonary deposition (0.2 L/min; mean, 33.5% of deposited dose; range, 15%‐51%; n = 5; P = 0.005 vs 2 L/min). The ratio of nose/lung dosing was approximately 26:1 at 2 L/min and 2:1 at 0.2 L/min. Bench studies demonstrated cannula output rates of 10.2 ± 1.7% (2 L/min) and 3.3 ± 0.4% (0.2 L/min) of the loaded nebulizer dose during a 2‐minute delivery. Combining in vitro and in vivo results, we estimate that 0.46% of the loaded nebulizer dose reaches the lungs at 2 L/min vs 1.10% at 0.2 L/min during a 2‐minute delivery. Conclusion With the delivery system used here, pulmonary aerosol delivery via nasal cannula was very inefficient at the flow rates required to provide oxygen support. Even at low flows, nasal deposition was substantial and local toxicity must be considered.

The recent recovery of mutations in vesicular trafficking genes causing congenital heart disease (CHD) revealed an unexpected role for the endocytic pathway. We now show that mice with a C4232R missense mutation in Low density lipoprotein receptor related protein 1 (LRP1) exhibit atrioventricular septal defects with double outlet right ventricle. Lrp1m/m mice exhibit shortened outflow tracts (OFT) and dysmorphic hypocellular cushions with reduced proliferation and increased apoptosis. Lrp1m/m embryonic fibroblasts show decreased cell motility and focal adhesion turnover associated with retention of mutant LRP1 in endoplasmic reticulum and reduced LRP1 expression. Conditional deletion of Lrp1 in cardiac neural crest cells (CNC) replicates the full CHD phenotype. Cushion explants showed defective cell migration, with gene expression analysis indicating perturbation of Wnt and other signaling pathways. Thus, LRP1 function in CNCs is required for normal OFT development with other cell lineages along the CNC migratory path playing a supporting role.

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.

The etiology of congenital heart defects (CHDs), amongst the most common human birth defects, is poorly understood partly because of its complex genetic architecture. Here we show that two genes previously implicated in CHDs, Megf8 and Mgrn1, interact genetically and biochemically to regulate the strength of Hedgehog signaling in target cells. MEGF8, a singlepass transmembrane protein, and MGRN1, a RING superfamily E3 ligase, assemble to form a transmembrane 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 between components of a receptor-like ubiquitin ligase complex that tunes morphogen signaling strength can cause a birth defect syndrome inherited in an oligogenic pattern.and MGRN1 Mut2 were expressed at equivalent levels as MGRN1 ( Fig. 2F) and maintained their stable interaction with MEGF8 ( Fig. S4C), demonstrating their integrity. These results support the conclusion that both the stable interaction of MGRN1 with MEGF8 and its E3 ligase function are required to attenuate Hh signaling. The MEGF8-MGRN1 complex ubiquitinates SMOAt this point our data suggested that MGRN1 functions as a membrane-tethered ubiquitin ligase complex that attenuates Hh signaling by reducing SMO abundance at the cell surface and primary cilium. This mechanism is reminiscent of a prominent membrane-localized ubiquitination system that attenuates WNT signaling by decreasing cell-surface levels of Frizzled (FZD) proteins, receptors for WNT ligands that are the closest relatives of SMO in the GPCR superfamily (Bjarnadóttir et al., 2006). Two transmembrane RING-family E3 ubiquitin ligases, ZNRF3 and RNF43, attenuate WNT responsiveness by directly ubiquitinating FZD and promoting its clearance from the cell surface (Hao et al., 2012;Koo et al., 2012).To examine if a similar ubiquitination system regulates Hh signaling sensitivity, we measured the stability of SMO at the plasma membrane using a non-cell permeable biotinylation reagent that only labels proteins at the cell surface in wild-type, Megf8 -/-, and Mgrn1 -/-;Rnf157 -/cells (Fig. 3A). Both the steady state abundance and the stability of cellsurface SMO were markedly greater in both mutant cell lines compared to wild-type cells ( Figs. 3B and 3C). The increase in ciliary SMO abundance (Fig. 1C) is likely a secondary consequence of elevated SMO at the plasma membrane, because plasma membrane-localized SMO can enter the cilia by a lateral transport pathway (Milenkovic et al., 2009). These results are analogous to how the stability of cell-surface FZD is enhanced when the ligases ZNRF3 or RNF43 are inactivated (Hao et al., 2012;Koo et al., 2012), prompting us to consider whether SMO is a substra...

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Case Presentation: Patient 1: Female infant diagnosed with Cri du Chat Syndrome (CdCS) (Fig. 1A), hypoplastic left heart syndrome variant (Fig. 1 B), duodenal atresia (Fig. 1 E, F), IUGR. Chromosomal microarray (CMA) showed a 4.3 Mb deletion at 5p15.33 (Fig. 1A) and a 32.2 Mb duplication of 5q32. Dysmorphisms including hypertelorism, low set ears, and micrognathia were noted. Echocardiogram showed a hypoplastic left ventricle, mitral valve dysplasia (Fig. 1B), dysplastic aortic valve (Fig. 1C), interrupted aortic arch (Fig. 1D). Patient 2: Male infant prenatally diagnosed with aortic valve stenosis, aortic arch hypoplasia (Fig. 1H, I), and IUGR. Multiple dysmorphic features including microcephaly, hypertelorism, down slanting palpebral fissures, and abnormal distal extremities. CMA revealed a large deletion at 5p13.33-p13.2 (Fig 1G). He underwent aortic valvuloplasty complicated by development of a posterior left ventricular wall pseudoaneurysm (Fig. 1J). He later underwent arch reconstruction. At 2 months of age, he was diagnosed with obstructive jaundice requiring a biliary drain (Fig.1K, L). He had been gaining weight and height steadily (Fig.1M, N). We identified 5 more patients with 5p deletions and left outflow tract obstructions (LVOTO) from the Cytogenomics of Cardiovascular Malformations Consortium (Table 1). Discussion: CdCS is the most common 5p deletion syndrome and is associated with mild congenital heart defects in 15-30% of individuals. There is no reported association between LVOTO and 5p deletions. These 7 patients did not all share the same deletions and had high mortality. Further studies are needed to better understand possible genetic etiologies.

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