Background Pulmonary surfactant forms a lipid-rich monolayerthat coats the airways of the lung and is essential for properinflation and function of the lung. Surfactant is produced byalveolar type II cells, stored intracellularly in organellesknown as lamellar bodies, and secreted by exocytosis. The genefor ATP-binding cassette transporter A3 (ABCA3) is expressedin alveolar type II cells, and the protein is localized to lamellarbodies, suggesting that it has an important role in surfactantmetabolism.
Methods We sequenced each of the coding exons of the ABCA3 genein blood DNA from 21 racially and ethnically diverse infantswith severe neonatal surfactant deficiency for which the etiologicprocess was unknown. Lung tissue from four patients was examinedby high-resolution light and electron microscopy.
Results Nonsense and frameshift mutations, as well as mutationsin highly conserved residues and in splice sites of the ABCA3gene were identified in 16 of the 21 patients (76 percent).In five consanguineous families with mutations, each pair ofsiblings was homozygous for the same mutation and each mutationwas found in only one family. Markedly abnormal lamellar bodieswere observed by ultrastructural examination of lung tissuefrom four patients with different ABCA3 mutations, includingnonsense, splice-site, and missense mutations.
Conclusions Mutation of the ABCA3 gene causes fatal surfactantdeficiency in newborns. ABCA3 is critical for the proper formationof lamellar bodies and surfactant function and may also be importantfor lung function in other pulmonary diseases. Since it is closelyrelated to ABCA1 and ABCA4, proteins that transport phospholipidsin macrophages and photoreceptor cells, it may have a role insurfactant phospholipid metabolism.
Pulmonary surfactant is a complex mixture of lipids and proteinsthat is essential for normal lung function. Surfactant lowerssurface tension at the air–liquid interface, thereby preventingend-expiratory atelectasis. It is stored within alveolar typeII cells in organelles containing multiple phospholipid layers,known as lamellar bodies, and is secreted into the alveoli byexocytosis. The production of pulmonary surfactant is developmentallyregulated, and the respiratory distress syndrome may developin premature infants owing to the lack of surfactant. Homozygousloss-of-function mutations in the gene encoding the hydrophobicsurfactant protein B (SFTPB) results in fatal surfactant deficiencyin full-term newborns.1 Fatal respiratory disease has been reportedin full-term infants with symptoms of surfactant deficiencyin whom a deficiency of surfactant protein B was excluded, andthe occurrence of familial cases suggests that there are additionalgenetic mechanisms.2
The genes for ATP-binding cassette (ABC) transporters encodemembrane proteins involved in the transport of compounds acrossbiologic membranes, and 14 ABC genes have been associated withdistinct genetic diseases in humans.3 Several ABC transportersare involved in the transport of phospholipids and sterols.The gene encoding ABC transporter 1 (ABCA1) is mutated in Tangierdisease, a disorder involving the accumulation of cholesterolin macrophages and peripheral tissues and a deficiency of high-densitylipoproteins.4,5,6 The ABCA4 gene is expressed in photoreceptorsand encodes a protein that has been implicated in transportingretinal–phosphatidylethanolamine complexes in the photoreceptormembrane disks of rods.7,8 The ABCA4 gene is mutated in severalrecessive disorders that involve retinal degeneration, includingmacular dystrophy due to Stargardt's disease, most recessiveforms of cone–rod dystrophy, and some recessive formsof retinitis pigmentosa.9,10 The ABCG5 and ABCG8 genes are expressedin the liver and intestine and are mutated in patients withsitosterolemia, a disorder involving the accumulation of cholesteroland other sterols.11,12
The ABCA3 gene encodes a 1704-amino-acid protein highly expressedin the lung, which has been localized to the limiting membraneof lamellar bodies,13,14 implicating ABCA3 as possibly importantin the maturation of lamellar bodies and surfactant production.Because of the probable role of ABCA3 in lipid transport, itslocation within alveolar type II cells, and the associationof other ABC genes with human diseases, we conducted a studyto determine whether ABCA3 is involved in surfactant phospholipidmetabolism and whether ABCA3 is a candidate gene for unexplainedsurfactant deficiency in full-term infants.
Methods
Patients
From July 1995 until April 2003, blood samples were collectedfrom 337 infants with severe respiratory disease as part ofa study to identify inherited abnormalities of surfactant metabolism.The infants were of northern and southern European, AfricanAmerican, Asian, and Middle Eastern origin. All infants wereborn after a gestation of at least 36 weeks and had persistenthypoxemic respiratory failure, with no known cause for theirrespiratory disease identified at the time of enrollment. Theonset of respiratory symptoms had occurred within hours afterbirth, and all infants had clinical or radiographic findings(or both) that were consistent with surfactant deficiency.
A cause of the lung disease was subsequently identified in 15infants: alveolar capillary dysplasia in 4, total anomalouspulmonary venous return in 4, viral pneumonia in 3, acinar dysplasiain 2, pulmonary lymphangiectasia in 1, and mucopolysaccharidosistype II in 1. In 47 infants (14 percent), hereditary deficiencyof surfactant protein B was identified as the basis of the lungdisease, as determined by the identification of loss-of-functionmutations on both alleles of the SFTPB gene. Deficiency of surfactantprotein B was ruled out in the remaining 275 infants by a combinationof protein analyses of lung fluid and tissue and genetic studies.15,16Among these infants, 121 were analyzed for mutations in thegene for surfactant protein C (SFTPC); 6 of these infants werefound to carry such mutations.
Of the remaining 115 infants, a subgroup of 21 infants from14 families who were likely to have a genetic basis for theirlung disease, on the basis of a family history of a similarlyaffected sibling, consanguinity, or both, or who had fatal diseasein association with low surfactant protein levels in tracheal-aspiratefluid, was selected for analysis of the ABCA3 gene. These infantsincluded six pairs of siblings, one of which was known to haveabnormal lamellar bodies.17 The majority of these infants diedwithin a month after birth (Table 1).
Table 1. Characteristics of Full-Term Infants with Clinical Surfactant Deficiency.
DNA was prepared from whole blood from the infants with theuse of a commercially available kit (Gentra Systems). The protocolwas approved by the institutional review boards of the participatinginstitutions, and written informed consent was obtained fromthe parents for genetic studies.
Detection of Mutations
Primers were designed to amplify each of the 30 coding exonsof the ABCA3 gene (see Supplementary Appendix 1, available withthe full text of this article at www.nejm.org), and the purifiedpolymerase-chain-reaction products spanning the exons and theirrespective splice junctions were sequenced on both strands withthe use of ABI BigDye Terminator sequencing reagents (AppliedBiosystems) and an ABI 3730 sequencer (Applied Biosystems).The results were analyzed with the use of both SeqMan software(DNAStar) and Mutation Explorer software (SoftGenetics). Variantswere identified by comparing each sequence with the referenceABCA3 sequence.18 Parental DNA was sequenced when samples wereavailable, and nonsynonymous mutations were analyzed in at least100 racially or ethnically matched subjects (200 chromosomes).None of the nonsynonymous mutations were found in either thepublic data base of single-nucleotide polymorphisms (http://www.ncbi.nlm.nih.gov/SNP/)or the Celera data base, which is made up of sequences fromtwo European Americans, one African American, one Mexican American,and one Chinese subject.
Phylogenetic Analysis
To analyze the evolutionary (phylogenetic) relation betweenABCA3 and related proteins from different organisms, we usedthe deduced amino acid sequence of ABCA3 (GenBank accessionnumber NP_001080
[GenBank]
) to search the sequence data base using theBLAST program (http://www.ncbi.nlm.nih.gov/blast/). Similarproteins were also identified in other vertebrate as well asinvertebrate species. The amino acid sequences were alignedwith the use of the Clustal X program.19 The alignment was usedfor phylogenetic analyses involving the Mega2 program (http://www.megasoftware.net/).20This method generates a dendrogram (or tree) indicating theextent to which the sequences are evolutionarily related. Totest the reliability of the tree, a bootstrap test with 1000replications was implemented in the Mega2 program. In this test,the same number of amino acids as in the original data set arerandomly sampled from the set of sequences and analyzed. Thepercentage of the times each branch of the tree has the sametopology as the original set of sequences is reported. A bootstrapvalue of 95 percent or higher provides strong supporting evidenceof an evolutionary relation between the particular branch ofthe tree and the original set of sequences.
Ultrastructural Analysis
Tissue for electron microscopy was fixed in modified Karnovsky'sfixative (2 percent paraformaldehyde plus 2 percent glutaraldehydein 0.1 M sodium cacodylate buffer), post-fixed with 1 percentosmium tetroxide, stained en bloc with cold 4 percent uranylacetate to preserve the lamellar-body phospholipids, and embeddedin EMbed 812 (Electron Microscopy Services) as described previously.21Plastic sections that were 1 µm thick were stained with1 percent toluidine blue (in 1 percent sodium borate in water)and assessed with the use of a wide-field microscope (NikonFXA-Microphot). Plastic sections that were 0.1 µm thickwere cut from the same blocks as the semithin sections, stainedwith uranyl acetate and lead citrate, and photographed witha transmission electron microscope (Jeol 1230, Jeol).
Results
DNA Sequencing of ABCA3
To test the hypothesis that the ABCA3 gene is mutated in someinfants with surfactant deficiency, we sequenced each of thecoding exons of the gene and the flanking splice sites in samplesfrom 21 infants. Polymorphisms identified in this study werefirst used to assess the concordance of the six pairs of siblingsfor ABCA3 haplotypes. One pair of siblings could be excludedfrom the analysis of recessive mutations in ABCA3, since thesiblings were discordant for ABCA3 haplotypes (Figure 1). Theremaining five pairs of siblings were concordant for ABCA3 haplotypes,and the affected infants from consanguineous families were allhomozygous for ABCA3 haplotypes.
Figure 1. Pedigrees of Patients with Surfactant Deficiency.
Solid symbols indicate patients. The haplotype of ABCA3 polymorphisms is shown below each child (triangular symbols) in the pedigree. In Family 6, the two siblings are discordant for ABCA3 haplotypes, ruling out this gene as the cause of the disorder. All other families have at least one mutation identified in the gene. The haplotype is composed of the following polymorphisms: exon 10–20C/T, F353F(1058C/T), exon 14+33G/A and P585P(1755C/G). Haplotype 1 is C-C-A-C for these polymorphisms in the order given. Haplotype 2 is C-C-G-C, haplotype 3 is T-T-G-G, and haplotype 4 is C-C-G-G. Double lines indicate consanguinity. Circles denote female family members, and squares male family members.
Mutations were identified in the ABCA3 gene in 16 of the 21infants (76 percent) (Figure 2 and Table 2). These includedhomozygous nonsense mutations in codons 106 and 1142, a homozygousframeshift mutation, and heterozygous insertion mutations andsplice-site mutations. Seven missense mutations were identifiedin conserved amino acids (Figure 2), including homozygous substitutionsof proline for leucine in codons 101 and 1553 (L101P and L1553P,respectively) and heterozygous substitutions of aspartic acidfor asparagine at position 568 (N568D), proline for leucineat position 982 (L982P), serine for glycine at position 1221(G1221S), proline for leucine at position 1580 (L1580P), andproline for glutamine at position 1591 (Q1591P). These missensealleles were not found in control subjects. Several polymorphismsin the introns and exons were also identified (Table 2). TheN568 residue is within the N-terminal ATP-binding domain andis conserved in the mammalian and fish ABCA3 genes as well asalmost all other members of the ABC type A subfamily (Figure 2).The corresponding residue is mutated in ABCA1 in patientswith Tangier disease and in ABCA4 in patients with Stargardt'sdisease.
Exons encoding the ATP-binding domains are shown in green. The chart below the diagram shows the degree of conservation of residues involved in missense mutations in the ABCA3 protein, predicted on the basis of the sequences in various murine and vertebrate species. The sequences of the puffer fish and the zebra fish are not complete, resulting in some gaps in this information in the case of L982P, G1221S, L1552P, L1553P, and L1580P. The first three noncoding exons are not shown.
Table 2. Mutations and Polymorphisms Identified in the ABCA3 Gene.
Ultrastructural Analysis
Histologic findings in the nine patients with ABCA3 mutationsfrom whom lung tissue was obtained (Table 1) included hyperplasiaof alveolar type II cells, accumulations of alveolar macrophagesin distal air spaces with various amounts of proteinaceous materialand interstitial thickening, findings consistent with the presenceof infantile desquamative interstitial pneumonitis, and neonatalalveolar proteinosis. Plastic sections that were 1 µmthick and stained with toluidine blue were obtained from fourpatients (Patients 1, 8, 9, and 21), and light-microscopicalexamination demonstrated alveolar type II cells with homogeneouscytoplasm, without the typical inclusions of lamellar bodies.Electron micrographs of lung tissue from Patient 21 (who washomozygous for the 4909+1G>A mutation) revealed lamellarbodies (Figure 3) that were smaller than those from controllung tissue, with more densely packed membranes and eccentricallyplaced, dense inclusion bodies, similar to those previouslydescribed in Patients 1 and 2, who were homozygous for the W1142Xmutation.17 Similarly abnormal lamellar bodies were observedin lung tissue from Patient 8 (who was heterozygous for theG1221S and L982P mutations) and Patient 9 (who was homozygousfor the L1553P mutation).
Figure 3. Ultrastructure of Alveolar Type II Cells.
A representative electron micrograph of normal lung tissue shows normal lamellar bodies (Panel A, x15,000). In lung tissue from Patient 21, who was homozygous for an ABCA3 splicing mutation (4909+1G>A), cytoplasmic lamellar bodies are smaller and denser (arrowheads in Panel B, x15,000), and many have dense peripheral inclusions (Panel C, x80,000).
Evolutionary Analysis of ABCA3
A missense variant may be a benign polymorphism instead of adeleterious mutation. The variant is more likely to be a deleteriousmutation if it is absent in controls and if it affects an aminoacid residue that is conserved across species. We thereforealigned the human, mouse, and rat amino acid sequences of ABCA3with partial abca3 sequences of the puffer fish (Takifugu rubripes)and zebra fish (Danio rerio). Nearly all the missense mutationswe identified occur in residues that are highly conserved (Figure 2).The amino acid alignment was used to produce a phylogenetictree of the ABCA3-related proteins showing the relation of theproteins from different organisms (see Supplementary Appendix 2,available with the full text of this article at www.nejm.org).The fish ABCA3 proteins cluster with the mammalian ABCA3 proteinsand are distinct from other, more distant ABCA-family proteins,such as the mouse Abca14, Abca15, and Abca16 proteins and thesea-urchin ABCA proteins (see Supplementary Appendix 2). Theselatter genes are all expressed exclusively in testes and aretherefore distinct from ABCA3 genes in both structure and expression.
Discussion
We have demonstrated that the ABCA3 gene is frequently mutatedin patients with severe neonatal lung disease and symptoms ofsurfactant deficiency. Our patients were from several majorracial or ethnic groups and our findings therefore indicatethat such mutations are not confined to a single group. Mostof our patients had mutations predicted to inactivate the geneor protein and died shortly after birth. Electron micrographsof patients' lung tissue demonstrated abnormal lamellar bodies,a finding that is consistent with a role of ABCA3 in the formationof lamellar bodies. All the infants presented with clinicaland radiographic findings of surfactant deficiency. Since ABCA3is related to other transporters of phospholipids and cholesterol,our findings suggest that ABCA3 transports phospholipids thatare critical for surfactant function into lamellar bodies. Defectivetransport of one or more components would be expected to leadto ineffective assembly of the structure and abnormal surfactant.Alternatively, ABCA3 could transport lipids that are deleteriousto the function of surfactant out of lamellar bodies.
Missense variants in conserved amino acids were identified insome patients. We were unable to find these same variants inthe public polymorphism data bases or in 100 racially or ethnicallymatched controls, but in the absence of a functional test, wecannot rule out the possibility that these are neutral variants.In the case of the L101P and L1553P mutations, each of whichaffected one pair of siblings from two different families, thetwo pairs of siblings were both homozygous for the variant andhomozygous for all other polymorphisms that we found in thegene — findings that are consistent with the occurrenceof a recessive mutation in these consanguineous families. TheN568D mutation is in a highly conserved residue in the ATP-bindingdomain, and it almost certainly disrupts the function of theprotein. The clinical phenotype and histopathological findingsin patients with missense mutations were similar to those inpatients with apparent loss-of-function mutations, supportingthe notion that the mutations were deleterious rather than neutralvariants.
In two patients, a mutation was identified on only one allele.Although the ABCA3 sequence variants in these children may havebeen unrelated to their lung disease, the similarity of theirclinical presentation and the severe nature of their lung diseasesuggest that these children probably had a second mutation onthe other allele, which may have been mutations within intronsor regulatory regions or large rearrangements or deletions.These variations would not have been detected by our sequencingstrategy. Similar rates of detection of mutations have beenreported among patients with mutations in the genes for otherABC transporters such as the cystic fibrosis transmembrane conductanceregulator and ABCA4.22,23
These findings indicate that families in which mutations areidentified may benefit from genetic counseling and prenatalor preimplantation diagnoses. We found different mutations inthe different families, suggesting that there are no commonalleles that confer this condition. One patient, who had a missensemutation (Q1591P) on one allele and an unknown mutation on theother allele, is still alive at six years of age and has chroniclung disease, suggesting that some ABCA3 mutations are not fatal.ABCA3 is thus a candidate gene for other pulmonary disordersinvolving surfactant dysfunction, including the neonatal respiratorydistress syndrome and disorders with a later onset, such asasthma and the acute respiratory distress syndrome. There isconsiderable heterogeneity in the presentation and severityof the respiratory distress syndrome in premature infants, anddifferent ABCA3 variants could influence the severity of thatcondition. We did not find many nonsynonymous variants in ABCA3that represent candidate single-nucleotide polymorphisms forthe respiratory distress syndrome and other disorders. However,the ABCA3 gene is hormonally regulated,13,14 and genetic variants(in other genes) that indirectly affect its regulation mightalso be important.
ABCA3 mutations were identified in 16 of our 21 patients (76percent), and 3 of the 5 patients without ABCA3 mutations recoveredcompletely from their initial lung disease and thus did nothave the identical phenotype. The high percentage of patientswith ABCA3 mutations in the group of 21 infants suggests thatABCA3 deficiency may account for a substantial number of casesof fatal lung disease among full-term infants for which no specificcause can be identified, but further study is needed to addressthe relative contributions of mutations in SFTPB, SFTPC, andABCA3 to neonatal lung disease. The absence of mutations insome full-term infants with fatal surfactant deficiency indicatesthat other, as yet unidentified genes are essential for surfactantproduction. This possibility is not surprising, given the complexnature of surfactant and the many different types of proteinsin lamellar bodies.
We observed distinct ultrastructural changes in lamellar bodiesin association with ABCA3 mutations, and abnormal lamellar bodieswere also seen in association with a deficiency of surfactantprotein B.24 These findings illustrate the potential importanceof electron microscopy in the examination of lung tissue frominfants who are dying from a lung disease of unclear causation.
The ABCA3 gene is highly conserved in both mammals and fish,suggesting that its role in the production of surfactants predatesthe development of the lung. This possibility is consistentwith morphologic studies showing that the surfactants in theswim bladders of teleost (bony) fish contain phospholipids andproteins similar to those found in the mammalian lung.25 Surfactantsare also found in the airways of reptiles, salamanders, andlungfish. The conservation of the ABCA3 gene across diversevertebrate species supports a role of the ABCA3 protein in surfactantlipid metabolism and cellular homeostasis, although the proteinmay have other functions as well.
Supported by grants from the National Institutes of Health (HL-54703,to Dr. Nogee, and HL-56387, to Drs. Wert, Nogee, and Whitsett)and from the Eudowood Foundation (to Dr. Nogee).
We are indebted to Bernard Gerrard and Georgianne Cirado fortechnical assistance, to the families who participated in thesestudies, to the physicians and nurses who cared for the children,and to Henry Shuman for helpful suggestions.
Source Information
From the Human Genetics Section, Laboratory of Genomic Diversity, National Cancer Institute — Frederick, Frederick, Md. (S.S., T.A., M.D.); the Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore (L.M.N.); and Cincinnati Children's Hospital Medical Center and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati (S.E.W., J.A.W.).
Address reprint requests to Dr. Dean at Bldg. 560, Rm. 21-18, NCI — Frederick, Frederick, MD 21702, or at dean{at}ncifcrf.gov.
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