Lamellar Bodies In Alveolar Type Research Articles

HPS6 Deficiency Leads to Reduced V-ATPase and Impaired Biogenesis of Lamellar Bodies in Alveolar Type II Cells.

Lamellar body (LB) is a tissue-specific lysosome-related organelle in type II alveolar cells, which is the main site for the synthesis, storage and secretion of pulmonary surfactants. Defects in pulmonary surfactants lead to a variety of respiratory and immune-related disorders. LB biogenesis is closely related to its function, but the underlying regulatory mechanism is largely unclear. Here, we found that deficiency of HPS6, a subunit of BLOC-2 (biogenesis of lysosome-related organelles complex-2), led to the reduction of the steady-state level of V-ATPase and the increase of luminal pH of LB. Furthermore, we observed increased LB size, accumulated surfactant proteins, and altered lipid profiling of lung tissue and bronchoalveolar lavage fluid due to HPS6 deficiency. These findings suggest that HPS6 regulates the distribution of V-ATPase on LBs to maintain its luminal acidity and LB homeostasis. This may provide new insights into the LB pathology.

Life cell imaging of amiodarone sequestration into lamellar bodies of alveolar type II cells

Amiodarone is widely used to treat cardiac arrhythmias and is very effective in preventing these disorders. However, its use is limited by a wide range of adverse effects, mainly affecting the lungs, and ranging from mild shortness of breath to pulmonary fibrosis. Amiodarone has been shown to accumulate strongly in lung tissue, exceeding its plasma concentration by a hundredfold. However, the site of accumulation and the mechanisms of transport are not fully understood. In this study, we used live cell imaging of primary rat alveolar type II cells to show that amiodarone specifically accumulates in large amounts in lamellar bodies, the surfactant storage organelles. Fluorescence imaging and correlation, and colocalization studies combined with confocal Raman microscopy identified these organelles as a major target for sequestration. Accumulation was rapid, on the order of a few hours, while storage was much more persistent. Partial uptake was observed in chemically fixed, dead cells, or cells treated with bafilomycin A1. Not only was uptake pH dependent, but intraluminal pH, measured with lysosomotropic pH sensitive dyes, was also affected. From these observations and from the physicochemical properties of amiodarone, we propose that passive diffusion, ion-trapping and lipophilic interactions are the main mechanisms for intracellular bioaccumulation. Furthermore, we demonstrate that measurement of amiodarone autofluorescence is highly useful for tracking cellular uptake and sequestration.

Biologic characterization of ABCA3 variants in lung tissue from infants and children with ABCA3 deficiency.

ABCA3 is a phospholipid transporter protein required for surfactant assembly in lamellar bodies of alveolar type II cells. Biallelic pathogenic ABCA3 variants cause severe neonatal respiratory distress syndrome or childhood interstitial lung disease. However, ABCA3 genotype alone does not explain the diversity in disease presentation, severity, and progression. Additionally, monoallelic ABCA3 variants have been reported in infants and children with ABCA3-deficient phenotypes. The effects of most ABCA3 variants identified in patients have not been characterized at the RNA level. ABCA3 allele-specific expression occurs in some cell types due to epigenetic regulation. We obtained lung tissue at transplant or autopsy from 16 infants and children with ABCA3 deficiency due to compound heterozygous ABCA3 variants for biologic characterization of the predicted effects of ABCA3 variants at the RNA level and determination of ABCA3 allele expression. We extracted DNA and RNA from frozen lung tissue and reverse-transcribed cDNA from mRNA. We performed Sanger sequencing to assess allele-specific expression by comparing the heights of variant nucleotide peaks in amplicons from genomic DNA and cDNA. We found similar genomic and cDNA variant nucleotide peak heights and no evidence of allele-specific expression among explant or autopsy samples with biallelic missense ABCA3 variants (n = 6). We observed allele-specific expression of missense alleles in trans with frameshift (n = 4) or nonsense (n = 1) variants, attributable to nonsense-mediated decay. The missense variant c.53 A > G;p.Gln18Arg, located near an exon-intron junction, encoded abnormal splicing with skipping of exon 4. Biologic characterization of ABCA3 variants can inform discovery of variant-specific disease mechanisms.

Rab38 Mutation and the Lung Phenotype.

Rab38 is highly expressed in alveolar type II cells, melanocytes, and platelets. These cells are specifically-differentiated cells and contain characteristic intracellular organelles called lysosome-related organelles, i.e., lamellar bodies in alveolar type II cells, melanosomes in melanocytes, and dense granules in platelets. There are Rab38-mutant rodents, i.e., chocolate mice and Ruby rats. While chocolate mice only show oculocutaneous albinism, Ruby rats show oculocutaneous albinism and prolonged bleeding time and, hence, are a rat model of Hermansky-Pudlak syndrome (HPS). Most patients with HPS suffer from fatal interstitial pneumonia by middle age. The lungs of both chocolate mice and Ruby rats show remarkably increased amounts of lung surfactant and conspicuously enlarged lysosome-related organelles, i.e., lamellar bodies, which are also characteristic of the lungs in human HPS. There are 16 mutant HPS-mouse strains, of which ten mutant genes have been identified to be causative in patients with HPS thus far. The gene products of eight of the ten genes constitute one of the three protein complexes, i.e., biogenesis of lysosome-related organelle complex-1, -2, -3 (BLOC-1, -2, -3). Patients with HPS of the mutant BLOC-3 genotype develop interstitial pneumonia. Recently, BLOC-3 has been elucidated to be a guanine nucleotide exchange factor for Rab38. Growing evidence suggests that Rab38 is an additional candidate gene of human HPS that displays the lung phenotype.

Pulmonary surfactant protein SP‐B promotes exocytosis of lamellar bodies in alveolar type II cells

The release of pulmonary surfactant by alveolar type II (ATII) cells is essential for lowering surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to alveolar collapse. Hydrophobic surfactant protein (SP)-B ensures the proper packing of newly synthesized surfactant particles, promotes the formation of the surface active film at the alveolar air-liquid interface and maintains its proper structure along the respiratory dynamics. We report that membrane-associated SP-B efficiently induces secretion of pulmonary surfactant by ATII cells, at the same level as potent secretagogues such as ATP. The presence in the extracellular medium of lipid-protein complexes containing SP-B activates the P2Y2 purinergic signaling pathway that ultimately triggers exocytosis of lamellar bodies by ATII cells. Our data suggest that SP-B prompts Ca2+-dependent surfactant secretion via ATP release from ATII cells. This result implies that SP-B is not only an essential component for the biophysical function of surfactant but is also a central element in the alveolar homeostasis by eliciting autocrine and paracrine cell stimulation.-Martínez-Calle, M., Olmeda, B., Dietl, P., Frick, M., Pérez-Gil, J. Pulmonary surfactant protein SP-B promotes exocytosis of lamellar bodies in alveolar type II cells.

Exogenous gene transfer of Rab38 small GTPase ameliorates aberrant lung surfactant homeostasis in Ruby rats

BackgroundRab38 small GTPase regulates intracellular transport in melanocytes and alveolar type II epithelial cells. Ruby rats carrying Rab38 and other gene mutations exhibit oculocutaneous albinism, bleeding diathesis, and hence, are a rat model of human Hermansky-Pudlak syndrome (HPS). We previously showed that Long Evans Cinnamon (LEC) rats, one strain of the Ruby rats, developed aberrant lung surfactant homeostasis with remarkably enlarged lamellar bodies in alveolar type II cells.MethodsA replication-deficient recombinant adenovirus expressing rat Rab38 (Ad-Rab38) was constructed. Alveolar type II cells were isolated from the LEC rats and tested for lung surfactant phosphatidylcholine secretion. The rats were also examined whether exogenous expression of Ad- Rab38 could rescue the altered lung surfactant homeostasis in the lungs.ResultsIsolated type II cells infected with Ad-Rab38 exhibited improved secretion patterns of [3H]phosphatidylcholine, i.e. increased basal hyposecretion and decreased agonist-induced hypersecretion. Endobronchial administration of Ad-Rab38 improved the morphology of type II cells and lamellar bodies, reducing their sizes close to those of wild-type rats. The increased amounts of phosphatidylcholine and surfactant protein B in the lamellar body fractions were decreased in the Ad-Rab38 infected lungs.ConclusionsThese results provide strong evidence that the aberrant lung surfactant homeostasis in the LEC rats is caused by Rab38 deficit, and suggest that endobronchial delivery of the responsive transgene could be an effective method to ameliorate the abnormal lung phenotype in the animal model of HPS.

Homooligomerization of ABCA3 and its functional significance.

ABCA3 is a surfactant lipid transporter in the limiting membrane of lamellar bodies in alveolar typeII cells. Mutations in the ATP-binding cassette, sub-familyA(ABC1), member3(ABCA3) gene cause respiratory distress syndrome in newborns, and chronic interstitial lung disease in children and adults. ABCA3 belongs to the class of full ABCtransporters, which are supposed to be functional in their monomeric forms. Although other family members e.g.,ABCA1 and ABCC7 have been shown to function as oligomers, the oligomerization state of ABCA3 is unknown. In the present study, the oligomerization of ABCA3 was investigated in cell lysates and crude membrane preparations from transiently and stably transfected 293cells using blue native PAGE(BN-PAGE), gel filtration and co-immunoprecipitation. Additionally, homooligomerization was examined invivo in cells using bioluminescence resonance energy transfer(BRET). Using BN-PAGE and gel filtration, we demonstrate that non-denatured ABCA3 exists in different oligomeric forms, with monomers(45%) and tetramers(30%) being the most abundant forms. Furthermore, we also show the existence of 20%dimers and 5%trimers. BRET analyses verified intermolecular interactions invivo. Our results also demonstrated that the arrest of ABCA3 in the endoplasmic reticulum(ER), either through drug treatment or induced by mutations in ABCA3, inhibited the propensity of the protein to form dimers. Based on our results, we suggest that transporter oligomerization is crucial for ABCA3 function and that a disruption of oligomerization due to mutations represents a novel pathomechanism in ABCA3-associated lung disease.

Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases.

RationaleABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.MethodsThe exact site where ABCA3 is cleaved was localized using mass spectrometry (MS). Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3’s cleavage region, followed by MS analysis.ResultsWe found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins’ first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.ConclusionWe show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

Synaptotagmin-7 links fusion-activated Ca²⁺ entry and fusion pore dilation.

ABSTRACTCa2+-dependent regulation of fusion pore dilation and closure is a key mechanism determining the output of cellular secretion. We have recently described ‘fusion-activated’ Ca2+ entry (FACE) following exocytosis of lamellar bodies in alveolar type II cells. FACE regulates fusion pore expansion and facilitates secretion. However, the mechanisms linking this locally restricted Ca2+ signal and fusion pore expansion were still elusive. Here, we demonstrate that synaptotagmin-7 (Syt7) is expressed on lamellar bodies and links FACE and fusion pore dilation. We directly assessed dynamic changes in fusion pore diameters by analysing diffusion of fluorophores across fusion pores. Expressing wild-type Syt7 or a mutant Syt7 with impaired Ca2+-binding to the C2 domains revealed that binding of Ca2+ to the C2A domain facilitates FACE-induced pore dilation, probably by inhibiting translocation of complexin-2 to fused vesicles. However, the C2A domain hampered Ca2+-dependent exocytosis of lamellar bodies. These findings support the hypothesis that Syt7 modulates fusion pore expansion in large secretory organelles and extend our picture that lamellar bodies contain the necessary molecular inventory to facilitate secretion during the exocytic post-fusion phase. Moreover, regulating Syt7 levels on lamellar bodies appears to be essential in order that exocytosis is not impeded during the pre-fusion phase.

LysoTracker is a marker of differentiated alveolar type II cells

BackgroundLysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.MethodsMouse lung cells were sorted on the basis of CD45negCD31negEpCAMposLysoTrackerpos expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay. To track AT2 cell differentiation, lung epithelial stem and progenitor cells were cultured in a CFU-Epi assay with LysoTracker-supplemented media.ResultsThe purity of sorted AT2 cells as determined by SP-C staining was 97.4% and viability was 85.3%. LysoTrackerpos AT2 cells generated SP-Cpos alveolar epithelial cell colonies in culture, and when added to the CFU-Epi culture medium, LysoTracker marked the differentiation of stem/progenitor-derived AT2 cells.ConclusionsThis study describes a novel method for isolating AT2 cells from mouse lungs. The high purity and viability of cells attained by this method, makes them suitable for functional analysis in vitro. The application of LysoTracker to live cell cultures will allow better assessment of the cellular and molecular mechanisms that regulate AT2 cell differentiation.

Cell‐specific expression of alveolar TNFR1

Ligation of the cytokine, tumor necrosis factor alpha (TNFα) to TNFα receptor 1 (TNFR1) causes lung inflammation. Therefore, the organization and extent of TNFα‐induced lung inflammation is likely to depend on the distribution and density of TNFR1 in the alveolar epithelium (AE). We addressed this issue by real‐time confocal microscopy of intact alveoli in the isolated, blood‐perfused mouse lung (n=3). To visualize the AE, we microinjected alveoli with the cytosolic marker, calcein green, and with LysoTracker Red (LTR) which localizes to lamellar bodies in alveolar type 2 (AT2) cells. To determine TNFR1 expression, we injected an Alexa‐633‐conjugated anti‐TNFR1 mAb that recognizes an extracellular domain of mouse TNFR1. TNFR1 fluorescence was accepted at ≥50 grey levels. In the imaged region, each alveolus contained 1‐2 AT2 cells, consistent with the understanding that the majority of the alveolar surface area is provided by alveolar type 1 (AT1) cells. TNFR1 expression was spatially heterogeneous, since the receptor fluorescence co‐localized with AT1, but not AT2 cells (P<0.05). Our findings suggest that the primary alveolar inflammatory response to TNFα is driven by TNFR1‐induced signaling in the AT1 cell (HL57556, HL64896).

Molecular and cellular characteristics of ABCA3 mutations associated with diffuse parenchymal lung diseases in children

ABCA3 (ATP-binding cassette subfamily A, member 3) is expressed in the lamellar bodies of alveolar type II cells and is crucial to pulmonary surfactant storage and homeostasis. ABCA3 gene mutations have been associated with neonatal respiratory distress (NRD) and pediatric interstitial lung disease (ILD). The objective of this study was to look for ABCA3 gene mutations in patients with severe NRD and/or ILD. The 30 ABCA3 coding exons were screened in 47 patients with severe NRD and/or ILD. ABCA3 mutations were identified in 10 out of 47 patients, including 2 homozygous, 5 compound heterozygous and 3 heterozygous patients. SP-B and SP-C expression patterns varied across patients. Among patients with ABCA3 mutations, five died shortly after birth and five developed ILD (including one without NRD). Functional studies of p.D253H and p.T1173R mutations revealed that p.D253H and p.T1173R induced abnormal lamellar bodies. Additionally, p.T1173R increased IL-8 secretion in vitro. In conclusion, we identified new ABCA3 mutations in patients with life-threatening NRD and/or ILD. Two mutations associated with ILD acted via different pathophysiological mechanisms despite similar clinical phenotypes.

Vesicular calcium channels as regulators of the exocytotic post-fusion phase

Regulated secretion is a fundamental cellular process in many different types of eukaryotic cells with Ca2+-triggered exocytosis taking centre stage. Elevations of the cytoplasmic Ca2+ concentration ([Ca2+]c) regulate multiple steps from vesicle fusion with the plasma membrane to fusion pore dilation and subsequent retrieval of spent vesicles. The general view is that the rise in [Ca2+]c initiates during the pre-fusion stage and either results from Ca2+-influx via Ca2+ channels in the plasma membrane or from release from intracellular Ca2+-stores. However, there is increasing evidence that exocytosis of secretory vesicles triggers additional, localised Ca2+ signals via insertion of vesicle-associated Ca2+ channels into the cell surface. These restricted Ca2+ signals following fusion are ideally suited for regulating the post-fusion fate of individual secretory vesicles. In invertebrates they have been shown to trigger compensatory endocytosis. Recently we have reported that exocytosis of lamellar bodies in alveolar type II epithelial cells results in a localized Ca2+-influx via vesicular P2X4 receptors which regulates fusion pore expansion and vesicle content release. This finding expands the emerging picture that post-fusion Ca2+ entry via vesicle-associated Ca2+ channels plays a central role for regulated exocytosis.

Secretagogues of lung surfactant increase annexin A7 localization with ABCA3 in alveolar type II cells

Membrane fusion between the lamellar bodies and plasma membrane is an obligatory event in the secretion of lung surfactant. Previous studies have postulated a role for annexin A7 (A7) in membrane fusion during exocytosis in some cells including alveolar type II cells. However, the intracellular trafficking of A7 during such fusion is not described. In this study, we investigated association of endogenous A7 with lamellar bodies in alveolar type II cells following treatment with several secretagogues of lung surfactant. Biochemical studies with specific antibodies showed increased membrane-association of cell A7 in type II cells stimulated with agents that increase secretion through different signaling mechanisms. Immuno-fluorescence studies showed increased co-localization of A7 with ABCA3, the lamellar body marker protein. Because these agents increase surfactant secretion through activation of PKC and PKA, we also investigated the effects of PKC and PKA inhibitors, bisindolylmaleimideI (BisI) and H89, respectively, on A7 partitioning. Western blot analysis showed that these inhibitors prevented secretagogue-mediated A7 increase in the membrane fractions. These inhibitors also blocked increased co-localization of A7 with ABCA3 in secretagogue-treated cells, as revealed by immuno-fluorescence studies. In vitro studies with recombinant A7 showed phosphorylation with PKC and PKA. The cell A7 was also phosphorylated in cells treated with surfactant secretagogues. Thus, our studies demonstrate that annexin A7 relocates to lamellar bodies in a phosphorylation-dependent manner. We suggest that activation of protein kinase promotes phosphorylation and membrane-association of A7 presumably to facilitate membrane fusion during lung surfactant secretion.

Endoplasmic Reticulum Proteins BiP and PDI are Localized to Lamellar Bodies of Alveolar Type 2 Epithelial Cells

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Harlequin ichthyosis model mouse reveals alveolar collapse and severe fetal skin barrier defects

Harlequin ichthyosis (HI), which is the most severe genodermatosis, is caused by loss-of-function mutations in ABCA12, a member of the ATP-binding cassette transporter family. To investigate the pathomechanism of HI and the function of the ABCA12 protein, we generated ABCA12-deficient mice (Abca12(-/-)) by targeting Abca12. Abca12(-/-) mice closely reproduce the human HI phenotype, showing marked hyperkeratosis with eclabium and skin fissure. Lamellar granule abnormalities and defective ceramide distribution were remarkable in the epidermis. Skin permeability assay of Abca12(-/-) fetuses revealed severe skin barrier dysfunction after the initiation of keratinization. Surprisingly, the Abca12(-/-) mice also demonstrated lung alveolar collapse immediately after birth. Lamellar bodies in alveolar type II cells of the Abca12(-/-) mice lacked normal lamellar structures. The level of surfactant protein B, an essential component of alveolar surfactant, was reduced in the Abca12(-/-) mice. Fetal therapeutic trials with systemic administration of retinoid or dexamethasone, which are effective for HI and respiratory distress, respectively, to the pregnant mother mice neither improved the skin phenotype nor extended the survival period. Our HI model mice reproduce the human HI skin phenotype soon after the initiation of fetal skin keratinization and provide evidence that ABCA12 plays pivotal roles in lung and skin barrier functions.

Annexin A2 Interactions with Rab14 in Alveolar Type II Cells

Annexin A2, a calcium-dependent phospholipid-binding protein, is abundantly expressed in alveolar type II cells where it plays a role in lung surfactant secretion. Nevertheless, little is known about the details of its cellular pathways. To identify annexin A2-regulated or associated proteins, we silenced endogenous annexin A2 expression in rat alveolar type II cells by RNA interference and assessed the change of the cellular transcriptome by DNA microarray analysis. The loss of annexin A2 resulted in the change of 61 genes. Thirteen of the selected genes (11 down-regulated and 2 up-regulated genes) were validated by real time quantitative PCR. When the loss of rat annexin A2 was rescued by overexpressing EGFP-tagged human annexin A2, six of seven selected targets returned to their normal expression level, indicating that these genes are indeed annexin A2-associated targets. One of the targets, Rab14, co-immunoprecipitated with annexin A2. Rab14 also co-localized in part with annexin A2 and lamellar bodies in alveolar type II cells. The silencing of Rab14 resulted in a decrease in surfactant secretion, suggesting that Rab14 may play a role in surfactant secretion.

Lysosomal acid lipase over‐expression disrupts lamellar body genesis and alveolar structure in the lung

The functional role of neutral lipids in the lung is poorly understood. Lysosomal acid lipase (LAL) is a critical enzyme in hydrolysis of cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. Human LAL was over-expressed in a doxycycline-controlled system in mouse respiratory epithelial cells to accelerate intracellular neutral lipid degradation and perturb the surfactant homeostasis in the lung. In this animal system, neutral lipid concentrations of pulmonary surfactant were reduced in bronchoalveolar lavage fluid (BALF) in association with decrease of surfactant protein C (SP-C) gene expression. The size and the number of lamellar bodies in alveolar type II epithelial cells (AT II cells) were significantly reduced accordingly. The number of macrophages required for surfactant recycling in BALF was also significantly reduced. As a result of these combinatory effects, emphysema of the alveolar structure was observed. Taken together, neutral lipid homeostasis is essential for maintenance of lamellar body genesis and the alveolar structure in the lung.

ABCA3 as a Lipid Transporter in Pulmonary Surfactant Biogenesis

ABCA3 protein is expressed predominantly at the limiting membrane of the lamellar bodies in alveolar type II cells, and mutations in the ABCA3 gene cause lethal respiratory distress in newborn infants. To investigate the function of ABCA3 protein, we generated Abca3-deficient mice by targeting Abca3. Full-term Abca3(-/-) newborn pups died within an hour after birth because of acute respiratory failure. Ultrastructural analysis revealed abnormally dense lamellar body-like organelles and no normal lamellar bodies in Abca3(-/-) alveolar type II cells. TLC and electrospray ionization mass spectrometry analyses of lipids in the pulmonary interstitium showed that phosphatidylcholine and phosphatidylglycerol, which contain palmitic acid and are abundant in normal surfactant lipids, were dramatically decreased in Abca3(-/-) lung. These findings indicate that ABCA3 plays an essential role in pulmonary surfactant lipid metabolism and lamellar body biogenesis, probably by transporting these lipids as substrates.

166 The E292V Variant in ABCA3 is Over-Represented in Newborns with Respiratory Distress Syndrome (RDS)

Background: ABCA3 is an ATP Binding Cassette protein localized to the limiting membrane of lamellar bodies in alveolar Type II cells. Recessive mutations in ABCA3 disrupt surfactant composition and function and cause lethal respiratory distress in newborns. A valine for glutamic acid substitution at codon 292 (E292V) has been found in unrelated individuals with chronic respiratory insufficiency.Objective: To determine the prevalence of the E292V variant in infants with and without RDS. Design/Methods: We obtained genomic DNA and clinical data from an unselected cohort of newborns with RDS (RDS, n=125, gestational age [GA] 31±5 wks), newborns with RDS referred for evaluation of familial or atypical RDS (REF, n=56, GA 34±5 wks), and normal newborns (noRDS, n=167, GA 39±2 wks). We amplified a 637 base pair product that included codon 292 and subjected amplicons to BsrG1 restriction analysis and agarose gel electrophoresis.Results: Excluding 1 each of concordant twins in the REF and noRDS groups, 3 RDS (2.4%), 5 REF (9.1%), and 2 noRDS (1.2%) infants were heterozygous for E292V (P=0.009). Males and females were equally represented. One infant with E292V in the noRDS group was Black, all others with E292V were White. Symptomatic infants (REF+RDS) with E292V were more mature than those without E292V (35±3 vs 32±5 wks, P=0.007). Duration of mechanical ventilation, and need for supplemental oxygen were not significantly different. Infants with RDS and E292V developed pneumothoraces more frequently (78%) than infants with RDS but without E292V (9%) (P<0.001). Six infants had resolution of symptoms within 1 month of birth; the noRDS individuals with E292V remained asymptomatic.Conclusions: The E292V variant in ABCA3 is significantly more prevalent in infants with RDS than in asymptomatic controls. Patients heterozygous for E292V come to clinical attention by presenting with RDS that is more severe than anticipated for gestational age. E292V may be a genetic influence for the risk and severity of RDS.