The second Newborn Sequencing in Genomic Medicine and Public Health study was a randomized, controlled trial of the effectiveness of rapid whole-genome or -exome sequencing (rWGS or rWES, respectively) in seriously ill infants with diseases of unknown etiology.Here we report comparisons of analytic and diagnostic performance. Of 1,248 ill inpatient infants, 578 (46%) had diseases of unknown etiology. 213 infants (37% of those eligible) were enrolled within 96 h of admission. 24 infants (11%) were very ill and received ultrarapid whole-genome sequencing (urWGS). The remaining infants were randomized, 95 to rWES and 94 to rWGS. The analytic performance of rWGS was superior to rWES, including variants likely to affect protein function, and ClinVar pathogenic/likely pathogenic variants (p < 0.0001). The diagnostic performance of rWGS and rWES were similar (18 diagnoses in 94 infants [19%] versus 19 diagnoses in 95 infants [20%], respectively), as was time to result (median 11.0 versus 11.2 days, respectively). However, the proportion diagnosed by urWGS (11 of 24 [46%]) was higher than rWES/rWGS (p ¼ 0.004) and time to result was less (median 4.6 days, p < 0.0001). The incremental diagnostic yield of reflexing to trio after negative proband analysis was 0.7% (1 of 147). In conclusion, rapid genomic sequencing can be performed as a first-tier diagnostic test in inpatient infants. urWGS had the shortest time to result, which was important in unstable infants, and those in whom a genetic diagnosis was likely to impact immediate management. Further comparison of urWGS and rWES is warranted because genomic technologies and knowledge of variant pathogenicity are evolving rapidly.

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero-and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na ؉ channel (hENaC), we expressed the three wild-type hENaC subunits (␣, ␤, and ␥) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three ␣, three ␤, and three ␥ subunits. Sucrose gradient sedimentation of ␣hENaC translated in vitro, as well as ␣-, ␤-, and ␥hENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.The DEG/ENaC protein family includes channels with diverse physiologic and pathophysiologic functions. Epithelial Na ϩ channels (ENaC) absorb Na ϩ in kidney, lung, and intestine (1, 2), and mutations in human ENaC (hENaC) cause disease (3-6). Several family members from Caenorhabditis elegans, including MEC-4, MEC-10, and DEG-1, play a role in mechanotransduction, and some gain-of-function mutations cause neurodegeneration (7). In Helix aspersa, the FMRFamide-gated channel (FaNaCh) functions as a neurotransmitter receptor (8). Three family members have recently been identified in the mammalian nervous system, BNC1 (MDEG, BNaC1) (9 -11), BNaC2 (ASIC) (11,12), and DRASIC (13).All members of the DEG/ENaC family appear to function as multimers. ENaC contains three homologous subunits, ␣, ␤, and ␥ (14 -18). Functional studies show that simultaneous expression of all three subunits is required to generate maximal Na ϩ current, although expression of ␣ENaC alone can produce small currents. In addition, biochemical data show that the three human ENaC (hENaC) subunits associate (19). Genetic evidence suggests that MEC-4, MEC-10, and DEG-1 also function as heteromultimers (7,20). However, the subunit stoichiometry is not known for any DEG/ENaC channel.EXPERIMENTAL PROCEDURES cDNAs and mutations were generated as described previously (9,17,18). FaNaCh was amplified by polymerase chain reaction following reverse transcription of RNA from H. aspersa. We tagged the C terminus of ␣hENaC with the sequence DYKDDDDK (␣ Flag ) for immunoprecipitation by anti-Flag M2 monoclonal antibody. This did not alter the function of the ␣ subunit in Xenopus oocytes or epithelia or its ability to associate with other subunits (19).Wild-type or mutant ␣-, ␤-, and ␥hENaC (0.2 ng each) were expressed in Xenopus oocytes by nuclear injection of cDNA (18). When a mixture of wild-type and mutant cDNAs for a subunit was coinjected, the total amount of cDNA for the subunit remained constant. 16 -24 h after injection, whole-cell Na ϩ current was measured by two-electrode voltage clamp at Ϫ60 mV (bathing solution, 116 mM NaCl, 2 mM KCl, 0.4 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, pH 7.4). To ...

The epithelial Na ؉ channel (ENaC) plays a critical role in Na ؉ absorption in the kidney and other epithelia. Mutations in the C terminus of the ␤ or ␥ENaC subunits increase renal Na ؉ absorption, causing Liddle's syndrome, an inherited form of hypertension. These mutations delete or disrupt a PY motif that was recently shown to interact with Nedd4, a ubiquitin-protein ligase expressed in epithelia. We found that Nedd4 inhibited ENaC when they were coexpressed in Xenopus oocytes. Liddle's syndrome-associated mutations that prevent the interaction between Nedd4 and ENaC abolished inhibition, suggesting that a direct interaction is required for inhibition by Nedd4. Inhibition also required activity of a ubiquitin ligase domain within the C terminus of Nedd4. Nedd4 had no detectable effect on the single channel properties of ENaC. Rather, Nedd4 decreased cell surface expression of both ENaC and a chimeric protein containing the C terminus of the ␤ subunit. Decreased surface expression resulted from an increase in the rate of degradation of the channel complex. Thus, interaction of Nedd4 with the C terminus of ENaC inhibits Na ؉ absorption, and loss of this interaction may play a role in the pathogenesis of Liddle's syndrome and other forms of hypertension.

The second Newborn Sequencing in Genomic Medicine and Public Health (NSIGHT2) study was a randomized, controlled trial of rapid whole-genome sequencing (rWGS) or rapid whole-exome sequencing (rWES) in infants with diseases of unknown etiology in intensive care units (ICUs). Gravely ill infants were not randomized and received ultra-rapid whole-genome sequencing (urWGS). Herein we report results of clinician surveys of the clinical utility of rapid genomic sequencing (RGS). The primary end-point-clinician perception that RGS was useful-was met for 154 (77%) of 201 infants. Both positive and negative tests were rated as having clinical utility (42 of 45 [93%] and 112 of 156 [72%], respectively). Physicians reported that RGS changed clinical management in 57 (28%) infants, particularly in those receiving urWGS (p ¼ 0.0001) and positive tests (p < 0.00001). Outcomes of 32 (15%) infants were perceived to be changed by RGS. Positive tests changed outcomes more frequently than negative tests (p < 0.00001). In logistic regression models, the likelihood that RGS was perceived as useful increased 6.7-fold when associated with changes in management (95% CI 1.8-43.3). Changes in management were 10.1-fold more likely when results were positive (95% CI 4.7-22.4) and turnaround time was shorter (odds ratio 0.92, 95% CI 0.85-0.99). RGS seldom led to clinician-perceived confusion or distress among families (6 of 207 [3%]). In summary, clinicians perceived high clinical utility and low likelihood of harm with first-tier RGS of infants in ICUs with diseases of unknown etiology. RGS was perceived as beneficial irrespective of whether results were positive or negative.

SUMMARYNecrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. During NEC pathogenesis, bacteria are able to penetrate innate immune defenses and invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Normally, Paneth cells appear in the intestinal crypts during the first trimester of human pregnancy. Paneth cells constitute a major component of the innate immune system by producing multiple antimicrobial peptides and proinflammatory mediators. To better understand the possible role of Paneth cell disruption in NEC, we quantified the number of Paneth cells present in infants with NEC and found that they were significantly decreased compared with age-matched controls. We were able to model this loss in the intestine of postnatal day (P)14-P16 (immature) mice by treating them with the zinc chelator dithizone. Intestines from dithizone-treated animals retained approximately half the number of Paneth cells compared with controls. Furthermore, by combining dithizone treatment with exposure to Klebsiella pneumoniae, we were able to induce intestinal injury and inflammatory induction that resembles human NEC. Additionally, this novel Paneth cell ablation model produces NEC-like pathology that is consistent with other currently used animal models, but this technique is simpler to use, can be used in older animals that have been dam fed, and represents a novel line of investigation to study NEC pathogenesis and treatment.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. NEC is believed to occur when intestinal bacteria invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Mucins are produced and secreted by epithelial goblet cells as a key component of the innate immune system and barrier function of the intestinal tract that help protect against bacterial invasion. To better understand the role of mucins in NEC, we quantified the number of mucus-containing small intestinal goblet cells present in infants with NEC and found they had significantly fewer goblet cells and Paneth cells compared with controls. To test whether inflammation has a developmentally dependent effect on intestinal goblet cells, TNF-α was injected into mice at various stages of intestinal development. TNF-α caused a loss of mucus-containing goblet cells only in immature mice and induced Muc2 and Muc3 mRNA upregulation only in mature ileum. Only minimal changes were seen in apoptosis and in expression of markers of goblet cell differentiation. TNF-α increased small intestinal mucus secretion and goblet cell hypersensitivity to prostaglandin E2 (PGE(2)), a known mucus secretagogue produced by macrophages. These TNF-α-induced changes in mucus mRNA levels required TNF receptor 2 (TNFR2), whereas TNF-α-induced loss of mucus-positive goblet cells required TNFR1. Our findings of developmentally dependent TNF-α-induced alterations on intestinal mucus may help explain why NEC is predominantly found in premature infants, and TNF-α-induced alterations of the intestinal innate immune system and barrier functions may play a role in the pathogenesis of NEC itself.

Bronchopulmonary dysplasia is a common pulmonary complication of extreme prematurity. Arrested lung development leads to bronchopulmonary dysplasia, but the molecular pathways that cause this arrest are unclear. Lung injury and inflammation increase disease risk, but the cellular site of the inflammatory response and the potential role of localized inflammatory signaling in inhibiting lung morphogenesis are not known. Here we show that tissue macrophages present in the fetal mouse lung mediate the inflammatory response to lipopolysaccharide and that macrophage activation inhibits airway morphogenesis. Macrophage depletion or targeted inactivation of the NF-κB signaling pathway protected airway branching in cultured lung explants from the effects of lipopolysaccharide. Macrophages also appear to be the primary cellular site of IL-1β production following lipopolysaccharide exposure. Conversely, targeted NF-κB activation in transgenic macrophages was sufficient to inhibit airway morphogenesis. Macrophage activation in vivo inhibited expression of multiple genes critical for normal lung development, leading to thickened lung interstitium, reduced airway branching, and perinatal death. We propose that fetal lung macrophage activation contributes to bronchopulmonary dysplasia by generating a localized inflammatory response that disrupts developmental signals critical for lung formation.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is found at the apical region of exocrine epithelial cells, both at the cell surface and in an apically localized Intracellular compartment. To determine if this internal pool was due to endocytosis, a technique was developed that allows the rate of CFTR internalization from the cell surface to be monitored. A two-step periodate/hydrazide biotinylation procedure was used to derivatize cell surface glycocoijugates. Because both of these steps are required for derivitization and are conducted at 4°C, the iclusion of a 3rC incubation between the treatments resulted in an assay for the internalization of cell surface glycocoajugates. CFTR was found to be targeted to a rapidly recycling endocytic pathway, as .'.O% of cell surface CFTR was internalized within minutes and unavailable for biotinylation. In contrast, the major glycoproteins ofthe apical surface were not significantly endocytosed during even longer incubations at 3TC. Elevating cAMP levels either by forskolin or cAMP analogs, which has been shown to activate CFTR chloride channel activity, inhibited CFTR internalization. However, cAMP did not affect the internalization of G551D CFTR, a naturally occurring Gly-551 -S Asp mutant that is expressed at the cell surface but lacks normal ion-channel function. In addition, the inhibition by cAMP of CFTR was not observed when cells were depleted of cellular chloride. The presence of CFTR in epithelial cells had previously been shown to confer a cAMP-mediated inhibition on the rate of fluid-phase endocytosis. This effect was not seen in chloride-depleted cells, suggesting that CFTR's ion-channel function and localization to incipient endosomes may be responsible for the observed inhibition. The finding that CFTR is targeted to the endocytic pathway may provide insight into the role of CFTR in normal exocrine function. In addition, these fndings suggest that the expression of a regulated ion channel in a membranous subcellular compartment provides a mechanim by which a cell can regulate vesicular trafficking through that compartment.