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original article
ABCA3 Gene Mutations in Newborns with Fatal
Surfactant Deficiency
Sergey Shulenin, Ph.D., Lawrence M. Nogee, M.D., Tarmo Annilo, Ph.D.,
Susan E. Wert, Ph.D., Jeffrey A. Whitsett, M.D., and Michael Dean, Ph.D.
abstract
background
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 [email protected].
N Engl J Med 2004;350:1296-303.
Copyright © 2004 Massachusetts Medical Society.
Pulmonary surfactant forms a lipid-rich monolayer that coats the airways of the lung
and is essential for proper inflation and function of the lung. Surfactant is produced by
alveolar type II cells, stored intracellularly in organelles known as lamellar bodies, and
secreted by exocytosis. The gene for ATP-binding cassette transporter A3 (ABCA3) is
expressed in alveolar type II cells, and the protein is localized to lamellar bodies, suggesting that it has an important role in surfactant metabolism.
methods
We sequenced each of the coding exons of the ABCA3 gene in blood DNA from 21 racially and ethnically diverse infants with severe neonatal surfactant deficiency for
which the etiologic process was unknown. Lung tissue from four patients was examined by high-resolution light and electron microscopy.
results
Nonsense and frameshift mutations, as well as mutations in highly conserved residues
and in splice sites of the ABCA3 gene were identified in 16 of the 21 patients (76 percent). In five consanguineous families with mutations, each pair of siblings was homozygous for the same mutation and each mutation was found in only one family.
Markedly abnormal lamellar bodies were observed by ultrastructural examination of
lung tissue from four patients with different ABCA3 mutations, including nonsense,
splice-site, and missense mutations.
conclusions
Mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. ABCA3 is
critical for the proper formation of lamellar bodies and surfactant function and may
also be important for lung function in other pulmonary diseases. Since it is closely related to ABCA1 and ABCA4, proteins that transport phospholipids in macrophages
and photoreceptor cells, it may have a role in surfactant phospholipid metabolism.
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abca3 gene mutations in newborns with fatal surfactant deficiency
p
ulmonary surfactant is a complex
mixture of lipids and proteins that is essential for normal lung function. Surfactant
lowers surface tension at the air–liquid interface,
thereby preventing end-expiratory atelectasis. It is
stored within alveolar type II cells in organelles
containing multiple phospholipid layers, known
as lamellar bodies, and is secreted into the alveoli
by exocytosis. The production of pulmonary surfactant is developmentally regulated, and the respiratory distress syndrome may develop in premature
infants owing to the lack of surfactant. Homozygous loss-of-function mutations in the gene encoding the hydrophobic surfactant protein B (SFTPB)
results in fatal surfactant deficiency in full-term
newborns.1 Fatal respiratory disease has been reported in full-term infants with symptoms of surfactant deficiency in whom a deficiency of surfactant protein B was excluded, and the occurrence of
familial cases suggests that there are additional genetic mechanisms.2
The genes for ATP-binding cassette (ABC) transporters encode membrane proteins involved in the
transport of compounds across biologic membranes, and 14 ABC genes have been associated with
distinct genetic diseases in humans.3 Several ABC
transporters are involved in the transport of phospholipids and sterols. The gene encoding ABC
transporter 1 (ABCA1) is mutated in Tangier disease,
a disorder involving the accumulation of cholesterol in macrophages and peripheral tissues and a deficiency of high-density lipoproteins.4-6 The ABCA4
gene is expressed in photoreceptors and encodes a
protein that has been implicated in transporting
retinal–phosphatidylethanolamine complexes in
the photoreceptor membrane disks of rods.7,8 The
ABCA4 gene is mutated in several recessive disorders that involve retinal degeneration, including
macular dystrophy due to Stargardt’s disease, most
recessive forms of cone–rod dystrophy, and some
recessive forms of retinitis pigmentosa.9,10 The
ABCG5 and ABCG8 genes are expressed in the liver
and intestine and are mutated in patients with sitosterolemia, a disorder involving the accumulation
of cholesterol and other sterols.11,12
The ABCA3 gene encodes a 1704-amino-acid
protein highly expressed in the lung, which has
been localized to the limiting membrane of lamellar bodies,13,14 implicating ABCA3 as possibly important in the maturation of lamellar bodies and
surfactant production. Because of the probable
role of ABCA3 in lipid transport, its location within
n engl j med 350;13
alveolar type II cells, and the association of other
ABC genes with human diseases, we conducted a
study to determine whether ABCA3 is involved in
surfactant phospholipid metabolism and whether
ABCA3 is a candidate gene for unexplained surfactant deficiency in full-term infants.
methods
patients
From July 1995 until April 2003, blood samples
were collected from 337 infants with severe respiratory disease as part of a study to identify inherited
abnormalities of surfactant metabolism. The infants were of northern and southern European, African American, Asian, and Middle Eastern origin.
All infants were born after a gestation of at least 36
weeks and had persistent hypoxemic respiratory
failure, with no known cause for their respiratory
disease identified at the time of enrollment. The
onset of respiratory symptoms had occurred within hours after birth, 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 15 infants: alveolar capillary dysplasia
in 4, total anomalous pulmonary venous return in
4, viral pneumonia in 3, acinar dysplasia in 2, pulmonary lymphangiectasia in 1, and mucopolysaccharidosis type II in 1. In 47 infants (14 percent),
hereditary deficiency of surfactant protein B was
identified as the basis of the lung disease, as determined by the identification of loss-of-function mutations on both alleles of the SFTPB gene. Deficiency of surfactant protein B was ruled out in the
remaining 275 infants by a combination of protein
analyses of lung fluid and tissue and genetic studies.15,16 Among these infants, 121 were analyzed
for mutations in the gene for surfactant protein C
(SFTPC); 6 of these infants were found to carry such
mutations.
Of the remaining 115 infants, a subgroup of 21
infants from 14 families who were likely to have a
genetic basis for their lung disease, on the basis of
a family history of a similarly affected sibling, consanguinity, or both, or who had fatal disease in association with low surfactant protein levels in tracheal-aspirate fluid, was selected for analysis of the
ABCA3 gene. These infants included six pairs of siblings, one of which was known to have abnormal
lamellar bodies.17 The majority of these infants
died within a month after birth (Table 1).
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Table 1. Characteristics of Full-Term Infants with Clinical Surfactant Deficiency.*
Patient
No.
Race or Ethnic
Group
Sex
Family
No.
Family History/
Consanguinity
Outcome
Histologic
Findings
ABCA3 Mutation
1
White
F
1
Yes/Yes
Death within 3 mo after birth
DIP, PAP
W1142X/W1142X
2
White
F
1
Yes/Yes
Death during neonatal period
DIP, PAP
W1142X/W1142X
3
Black
M
2
Yes/Yes
Death during neonatal period
NA
L101P/L101P
4
Black
M
2
Yes/Yes
Death during neonatal period
NA
L101P/L101P
5
White
F
3
Yes/No
Death during neonatal period
NA
4552insT/L1580P
6
White
F
3
Yes/No
Death during neonatal period
NA
4552insT/L1580P
7
White
M
4
Yes/No
Death within 3 mo after birth
PAP
G1221S/L982P
8
White
M
4
Yes/No
Death during neonatal period
PAP
G1221S/L982P
9
Middle Eastern
M
5
Yes/Yes
Death during neonatal period
DIP, PAP
L1553P/L1553P
10
Middle Eastern
M
5
Yes/Yes
Death during neonatal period
NA
L1553P/L1553P
11
White
M
6
Yes/No
Recovery from RDS
NA
None found
12
White
M
6
Yes/No
Recovery from RDS
NA
None found
13
Middle Eastern
M
7
No/Yes
Unknown
NA
1644delC/1644delC
14
Middle Eastern
M
8
Yes/No
Death during neonatal period
DIP, PAP
R106X/R106X
15
Asian
F
9†
Yes/Yes
Death during neonatal period
NA
4909+1G>A/4909+1G>A
16
White
M
10
Yes/Yes
Death during neonatal period
NA
None found
17
White
M
11
No/No
Recovery from RDS
NA
None found
18
White
F
12
No/No
Death during neonatal period
NA
None found
19
White
M
13
Yes/No
Chronic lung disease
CPI, DIP
Q1591P/—‡
20
Hispanic
M
14
No/No
Death after lung transplantation
PAP
N568D/—‡
21
Asian
F
Yes/Yes
Death during neonatal period
PAP
4909+1G>A/4909+1G>A
9†
* DIP denotes desquamative interstitial pneumonitis, PAP pulmonary alveolar proteinosis, NA not available, RDS respiratory distress syndrome,
and CPI chronic pneumonitis of infancy. Race or ethnic group was self-assigned by the parents.
† Patients 15 and 21 were cousins.
‡ No mutation was identified on one allele.
DNA was prepared from whole blood from the
infants with the use of a commercially available kit
(Gentra Systems). The protocol was approved by
the institutional review boards of the participating
institutions, and written informed consent was obtained from the parents for genetic studies.
detection of mutations
both SeqMan software (DNAStar) and Mutation
Explorer software (SoftGenetics). Variants were
identified by comparing each sequence with the reference ABCA3 sequence.18 Parental DNA was sequenced when samples were available, and nonsynonymous mutations were analyzed in at least 100
racially or ethnically matched subjects (200 chromosomes). None of the nonsynonymous mutations
were found in either the public data base of singlenucleotide polymorphisms (http://www.ncbi.nlm.
nih.gov/SNP/) or the Celera data base, which is
made up of sequences from two European Americans, one African American, one Mexican American,
and one Chinese subject.
Primers were designed to amplify each of the 30
coding exons of the ABCA3 gene (see Supplementary Appendix 1, available with the full text of this
article at www.nejm.org), and the purified polymerase-chain-reaction products spanning the exons and their respective splice junctions were sequenced on both strands with the use of ABI BigDye
Terminator sequencing reagents (Applied Biosys- phylogenetic analysis
tems) and an ABI 3730 sequencer (Applied Biosys- To analyze the evolutionary (phylogenetic) relation
tems). The results were analyzed with the use of between ABCA3 and related proteins from differ-
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abca3 gene mutations in newborns with fatal surfactant deficiency
ent organisms, we used the deduced amino acid sequence of ABCA3 (GenBank accession number
NP_001080) to search the sequence data base using the BLAST program (http://www.ncbi.nlm.nih.
gov/blast/). Similar proteins were also identified in
other vertebrate as well as invertebrate species. The
amino acid sequences were aligned with the use of
the Clustal X program.19 The alignment was used
for phylogenetic analyses involving the Mega2
program (http://www.megasoftware.net/).20 This
method generates a dendrogram (or tree) indicating the extent to which the sequences are evolutionarily related. To test the reliability of the tree, a
bootstrap test with 1000 replications was implemented in the Mega2 program. In this test, the same
number of amino acids as in the original data set
are randomly sampled from the set of sequences
and analyzed. The percentage of the times each
branch of the tree has the same topology as the
original set of sequences is reported. A bootstrap
value of 95 percent or higher provides strong supporting evidence of an evolutionary relation between the particular branch of the tree and the original set of sequences.
used to assess the concordance of the six pairs of
siblings for ABCA3 haplotypes. One pair of siblings
could be excluded from the analysis of recessive
mutations in ABCA3, since the siblings were discordant for ABCA3 haplotypes (Fig. 1). The remaining
five pairs of siblings were concordant for ABCA3
haplotypes, and the affected infants from consanguineous families were all homozygous for ABCA3
haplotypes.
Mutations were identified in the ABCA3 gene in
16 of the 21 infants (76 percent) (Fig. 2 and Table 2).
These included homozygous nonsense mutations
in codons 106 and 1142, a homozygous frameshift
mutation, and heterozygous insertion mutations
and splice-site mutations. Seven missense mutations were identified in conserved amino acids (Fig.
2), including homozygous substitutions of proline
for leucine in codons 101 and 1553 (L101P and
L1553P, respectively) and heterozygous substitutions of aspartic acid for asparagine at position
568 (N568D), proline for leucine at position 982
(L982P), serine for glycine at position 1221
(G1221S), proline for leucine at position 1580
(L1580P), and proline for glutamine at position
1591 (Q1591P). These missense alleles were not
ultrastructural analysis
found in control subjects. Several polymorphisms
Tissue for electron microscopy was fixed in modi- in the introns and exons were also identified (Table
fied Karnovsky’s fixative (2 percent paraformalde- 2). The N568 residue is within the N-terminal ATPhyde plus 2 percent glutaraldehyde in 0.1 M sodi- binding domain and is conserved in the mammalium cacodylate buffer), post-fixed with 1 percent
osmium tetroxide, stained en bloc with cold 4 perFamily 1
Family 2
Family 3
cent uranyl acetate to preserve the lamellar-body
phospholipids, and embedded in EMbed 812 (Electron Microscopy Services) as described previously.21 Plastic sections that were 1 µm thick were
22 22
11 11
12 12
stained with 1 percent toluidine blue (in 1 percent
sodium borate in water) and assessed with the use
Family 4
Family 5
Family 6
of a wide-field microscope (Nikon FXA-Microphot).
Plastic sections that were 0.1 µm thick were cut from
the same blocks as the semithin sections, stained
23 23
44 44
14 24
with uranyl acetate and lead citrate, and photographed with a transmission electron microscope
Figure 1. Pedigrees of Patients with Surfactant Deficiency.
(Jeol 1230, Jeol).
Solid symbols indicate patients. The haplotype of ABCA3 polymorphisms is
results
dna sequencing of abca3
To test the hypothesis that the ABCA3 gene is mutated in some infants with surfactant deficiency, we
sequenced each of the coding exons of the gene and
the flanking splice sites in samples from 21 infants.
Polymorphisms identified in this study were first
n engl j med 350;13
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.
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4909+1G>A
A4771C(Q1591P)
T4739C(L1580P)
T4657C(L1553P)
4552insT
G3661A(G1221S)
G3426A(W1142X)
1644delC
Missense
T2945C(L982P)
Splice
A1702G(N568D)
Frameshift
C316T(R106X)
T301C(L101P)
Nonsense
new england journal
ATP-binding domains
Human
Mouse
Rat
Puffer fish
Zebra fish
L101P N568D
ETVRRALVIN NGAGKTT
ETVKREFMIK NGAGKTT
EAVRREFMIK NGAGKTT
EDVRGKLELS NGAGKTT
QDVQQNLVRG NGAGKTT
L982P
QQLSEHL
QQLSENL
QQLSEHL
G1221S
LSGIAT
LSGIAT
LSGIAT
L1553P L1580P
VARRLL ECEALC
VARRLL ECEALC
VARRLL ECEALC
VARRLL
Q1591P
LAIMVQGQFKC
LAIMVQGQFKC
LAIMVQGQFKC
LAVMVNGQFKC
LAVMVNGQFKC
Figure 2. Location of ABCA3 Mutations.
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.
an and fish ABCA3 genes as well as almost all other
members of the ABC type A subfamily (Fig. 2). The
corresponding residue is mutated in ABCA1 in patients with Tangier disease and in ABCA4 in patients
with Stargardt’s disease.
ultrastructural analysis
Histologic findings in the nine patients with ABCA3
mutations from whom lung tissue was obtained
(Table 1) included hyperplasia of alveolar type II
cells, accumulations of alveolar macrophages in distal air spaces with various amounts of proteinaceous
material and interstitial thickening, findings consistent with the presence of infantile desquamative
interstitial pneumonitis, and neonatal alveolar proteinosis. Plastic sections that were 1 µm thick and
stained with toluidine blue were obtained from four
patients (Patients 1, 8, 9, and 21), and light-microscopical examination demonstrated alveolar type II
cells with homogeneous cytoplasm, without the typical inclusions of lamellar bodies. Electron micrographs of lung tissue from Patient 21 (who was ho-
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mozygous for the 4909+1G>A mutation) revealed
lamellar bodies (Fig. 3) that were smaller than those
from control lung tissue, with more densely packed
membranes and eccentrically placed, dense inclusion bodies, similar to those previously described
in Patients 1 and 2, who were homozygous for the
W1142X mutation.17 Similarly abnormal lamellar
bodies were observed in lung tissue from Patient 8
(who was heterozygous for the G1221S and L982P
mutations) and Patient 9 (who was homozygous
for the L1553P mutation).
evolutionary analysis of abca3
A missense variant may be a benign polymorphism
instead of a deleterious mutation. The variant is
more likely to be a deleterious mutation if it is absent in controls and if it affects an amino acid residue that is conserved across species. We therefore
aligned the human, mouse, and rat amino acid sequences of ABCA3 with partial abca3 sequences of
the puffer fish (Takifugu rubripes) and zebra fish (Danio rerio). Nearly all the missense mutations we iden-
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abca3 gene mutations in newborns with fatal surfactant deficiency
tified occur in residues that are highly conserved
(Fig. 2). The amino acid alignment was used to produce a phylogenetic tree of the ABCA3-related proteins showing the relation of the proteins 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 proteins and are distinct from
other, more distant ABCA-family proteins, such as
the mouse Abca14, Abca15, and Abca16 proteins
and the sea-urchin ABCA proteins (see Supplementary Appendix 2. These latter genes are all expressed
exclusively in testes and are therefore distinct from
ABCA3 genes in both structure and expression.
discussion
We have demonstrated that the ABCA3 gene is frequently mutated in patients with severe neonatal
lung disease and symptoms of surfactant deficiency. Our patients were from several major racial or
ethnic groups and our findings therefore indicate
that such mutations are not confined to a single
group. Most of our patients had mutations predicted to inactivate the gene or protein and died shortly
after birth. Electron micrographs of patients’ lung
tissue demonstrated abnormal lamellar bodies, a
finding that is consistent with a role of ABCA3 in
the formation of lamellar bodies. All the infants
presented with clinical and radiographic findings
of surfactant deficiency. Since ABCA3 is related to
other transporters of phospholipids and cholesterol, our findings suggest that ABCA3 transports
phospholipids that are critical for surfactant function into lamellar bodies. Defective transport of one
or more components would be expected to lead to
ineffective assembly of the structure and abnormal
surfactant. Alternatively, ABCA3 could transport lipids that are deleterious to the function of surfactant
out of lamellar bodies.
Missense variants in conserved amino acids were
identified in some patients. We were unable to find
these same variants in the public polymorphism
data bases or in 100 racially or ethnically matched
controls, but in the absence of a functional test, we
cannot rule out the possibility that these are neutral
variants. In the case of the L101P and L1553P mutations, each of which affected one pair of siblings
from two different families, the two pairs of siblings were both homozygous for the variant and
homozygous for all other polymorphisms that we
found in the gene — findings that are consistent
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Table 2. Mutations and Polymorphisms Identified in the ABCA3 Gene.
Site Affected
Patient No. Nucleotide Affected* or Outcome SNP No.†
Variant
Mutation
Exon 5
3, 4
T301C
L101P‡
Exon 5
14
C316T
R106X‡
Exon 14
20
A1702G
N568D
Exon 14
13
1644delC
Frameshift‡
Exon 21
7, 8
T2945C
L982P
Exon 23
1, 2
G3426A
W1142X‡
Exon 24
7, 8
G3661A
G1221S
Exon 30
9, 10
T4657C
L1553P
Exon 30
5, 6
4552insT
Frameshift
Exon 31
15, 21
4909+1G>A
Splice site‡
Exon 31
5, 6
T4739C
L1580P
Exon 31
19
A4772C
Q1591P
Exon 5
Multiple
Exon 5+50A/G
Intron
Exon 6
20
393C/T
A131A
Exon 6
18
Exon 6+119G/A
Intron
Exon 7
19
Exon 7–14C/G
Intron
Exon 8
14
681C/T
A227A
Exon 10
Multiple
Exon 10–105C/A
Intron
Exon 10
Multiple
Exon 10–20C/T
Intron
Exon 10
Multiple
1058C/T
F353F
Exon 14
Multiple
Exon 14+33G/A
Intron
rs170447
Exon 15
Multiple
1755C/G
P585P
rs323043
Exon 18
13
Exon 18–17G/A
Intron
Exon 18
1
2340C/T
H780H
Exon 21
Multiple
Exon 21–20C/G
Intron
rs313908
Exon 21
Multiple
Exon 21+34C/T
Intron
rs313909
Exon 27
Multiple
4116C/T
S1372S
rs149532
Exon 32
11
4944C/T
V1648V
Polymorphism
rs46725
rs323059
rs323066
* Nucleotides are numbered from the ATG start codon.
† Polymorphisms with entries in the public data base of single-nucleotide polymorphisms (SNPs) (http://www.ncbi.nlm.nih.gov/SNP/) are indicated.
‡ This sample was homozygous for the mutation.
with the occurrence of a recessive mutation in these
consanguineous families. The N568D mutation is
in a highly conserved residue in the ATP-binding
domain, and it almost certainly disrupts the function of the protein. The clinical phenotype and histopathological findings in patients with missense
mutations were similar to those in patients with apparent loss-of-function mutations, supporting the
notion that the mutations were deleterious rather
than neutral variants.
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A
B
C
Figure 3. Ultrastructure of Alveolar Type II Cells.
A representative electron micrograph of normal lung tissue shows normal lamellar bodies (Panel A, ¬15,000).
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, ¬15,000), and many have dense peripheral inclusions (Panel C, ¬80,000).
In two patients, a mutation was identified on
only one allele. Although the ABCA3 sequence variants in these children may have been unrelated to
their lung disease, the similarity of their clinical presentation and the severe nature of their lung disease
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suggest that these children probably had a second
mutation on the other allele, which may have been
mutations within introns or regulatory regions or
large rearrangements or deletions. These variations
would not have been detected by our sequencing
strategy. Similar rates of detection of mutations have
been reported among patients with mutations in the
genes for other ABC transporters such as the cystic
fibrosis transmembrane conductance regulator and
ABCA4.22,23
These findings indicate that families in which
mutations are identified may benefit from genetic
counseling and prenatal or preimplantation diagnoses. We found different mutations in the different families, suggesting that there are no common
alleles that confer this condition. One patient, who
had a missense mutation (Q1591P) on one allele
and an unknown mutation on the other allele, is
still alive at six years of age and has chronic lung
disease, suggesting that some ABCA3 mutations
are not fatal. ABCA3 is thus a candidate gene for
other pulmonary disorders involving surfactant dysfunction, including the neonatal respiratory distress
syndrome and disorders with a later onset, such as
asthma and the acute respiratory distress syndrome. There is considerable heterogeneity in the
presentation and severity of the respiratory distress
syndrome in premature infants, and different ABCA3
variants could influence the severity of that condition. We did not find many nonsynonymous variants
in ABCA3 that represent candidate single-nucleotide polymorphisms for the 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 might also be important.
ABCA3 mutations were identified in 16 of our 21
patients (76 percent), and 3 of the 5 patients without ABCA3 mutations recovered completely from
their initial lung disease and thus did not have the
identical phenotype. The high percentage of patients with ABCA3 mutations in the group of 21 infants suggests that ABCA3 deficiency may account
for a substantial number of cases of fatal lung disease among full-term infants for which no specific
cause can be identified, but further study is needed
to address the relative contributions of mutations
in SFTPB, SFTPC, and ABCA3 to neonatal lung disease. The absence of mutations in some full-term
infants with fatal surfactant deficiency indicates that
other, as yet unidentified genes are essential for surfactant production. This possibility is not surpris-
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abca3 gene mutations in newborns with fatal surfactant deficiency
ing, given the complex nature of surfactant and the
many different types of proteins in lamellar bodies.
We observed distinct ultrastructural changes in
lamellar bodies in association with ABCA3 mutations, and abnormal lamellar bodies were also seen
in association with a deficiency of surfactant protein B.24 These findings illustrate the potential importance of electron microscopy in the examination of lung tissue from infants 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 predates the development of the lung. This possibility is consistent with
morphologic studies showing that the surfactants
in the swim bladders of teleost (bony) fish contain
phospholipids and proteins similar to those found
in the mammalian lung.25 Surfactants are also
found in the airways of reptiles, salamanders, and
lungfish. The conservation of the ABCA3 gene across
diverse vertebrate species supports a role of the
ABCA3 protein in surfactant lipid metabolism and
cellular homeostasis, although the protein may have
other functions as well.
Supported by grants from the National Institutes of Health (HL54703, 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 for
technical assistance, to the families who participated in these studies, to the physicians and nurses who cared for the children, and to
Henry Shuman for helpful suggestions.
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3. Dean M, Rzhetsky A, Allikmets R. The
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4. Bodzioch M, Orso E, Klucken J, et al.
The gene encoding ATP-binding cassette
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al. Mutations in ABC1 in Tangier disease
and familial high-density lipoprotein deficiency. Nat Genet 1999;22:336-45.
6. Rust S, Rosier M, Funke H, et al. Tangier
disease is caused by mutations in the gene
encoding ATP-binding cassette transporter
1. Nat Genet 1999;22:352-5.
7. Mata NL, Tzekov RT, Liu X, Weng J,
Birch DG, Travis GH. Delayed dark-adaptation and lipofuscin accumulation in abcr+/mice: implications for involvement of ABCR
in age-related macular degeneration. Invest
Ophthalmol Vis Sci 2001;42:1685-90.
8. Sun H, Molday RS, Nathans J. Retinal
stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease. J Biol Chem
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9. Allikmets R, Singh N, Sun H, et al.
A photoreceptor cell-specific ATP-binding
transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet
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et al. Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice
site mutations in the Stargardt’s disease gene
ABCR. Hum Mol Genet 1998;7:355-62.
11. Berge KE, Tian H, Graf GA, et al. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC
transporters. Science 2000;290:1771-5.
12. Lu K, Lee MH, Hazard S, et al. Two
genes that map to the STSL locus cause sitosterolemia: genomic structure and spectrum
of mutations involving sterolin-1 and sterolin-2, encoded by ABCG5 and ABCG8, respectively. Am J Hum Genet 2001;69:278-90.
13. Mulugeta S, Gray JM, Notarfrancesco
KL, et al. Identification of LBM180, a lamellar body limiting membrane protein of alveolar type II cells, as the ABC transporter
protein ABCA3. J Biol Chem 2002;277:
22147-55.
14. Yamano G, Funahashi H, Kawanami O,
et al. ABCA3 is a lamellar body membrane
protein in human lung alveolar type II cells.
FEBS Lett 2001;508:221-5.
15. Nogee LM, Wert SE, Proffit SA, Hull
WM, Whitsett JA. Allelic heterogeneity in
hereditary surfactant protein B (SP-B) deficiency. Am J Respir Crit Care Med 2000;161:
973-81.
16. Nogee LM, Dunbar AE III, Wert SE,
Askin F, Hamvas A, Whitsett JA. A mutation
in the surfactant protein C gene associated
with familial interstitial lung disease. N Engl
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17. Tryka AF, Wert SE, Mazursky JE, Arrington RW, Nogee LM. Absence of lamellar
bodies with accumulation of dense bodies
characterizes a novel form of congenital
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18. Connors TD, Van Raay TJ, Petry LR,
Klinger KW, Landes GM, Burn TC. The
cloning of a human ABC gene (ABC3) mapping to chromosome 16p13.3. Genomics
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19. Thompson JD, Gibson TJ, Plewniak F,
Jeanmougin F, Higgins DG. The CLUSTAL_X
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Weaver TE. An en bloc staining protocol improves the preservation of lamellar bodies in
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23. Lewis RA, Shroyer NF, Singh N, et al.
Genotype/phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease. Am
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24. deMello DE, Heyman S, Phelps DS, et al.
Ultrastructure of lung in surfactant protein
B deficiency. Am J Respir Cell Mol Biol 1994;
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25. Wood PG, Lopatko OV, Orgeig S, Joss
JM, Smits AW, Daniels CB. Control of pulmonary surfactant secretion: an evolutionary perspective. Am J Physiol Regul Integr
Comp Physiol 2000;278:R611-R619.
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march 25, 2004
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1303
Who Should Receive Myeloablative Therapy for Lymphoma?
PERSPECTIVE
conventional-dose therapy and can be pushed toward cure by very intensive therapy. The results
constitute a modest advance, and it is questionable whether they justify recommending such
therapy for all patients with diffuse large-B-cell
lymphoma of low-to-intermediate risk. Clearly, at
least two thirds of the patients who have been cured
in this way would have been cured with CHOP alone
and were therefore overtreated, with an approach
that must be associated with greater short-term
and long-term morbidity and mortality. Such overtreatment cannot be good for the individual patient
or for the health care system. Better prognostic indexes are needed — and may soon be at hand, with
the aid of molecular technology.
As Milpied et al. themselves point out, in determining how to act on their results, we must remember that CHOP is no longer the gold standard for the
treatment of the most common of the aggressive
lymphomas, diffuse large-B-cell lymphoma. The
combination of anti-CD20 (rituximab) and CHOP
has been shown to be superior to CHOP alone in
both older and younger patients with non-Hodgkin’s lymphoma; chemoimmunotherapy is becom-
ing the order of the day. Shortening the interval between cycles of CHOP by a week (from 21 days to 14
days) is also finding favor. What result would have
been obtained had Milpied et al. compared CHOP
plus rituximab administered every 14 days with myeloablative therapy? Is myeloablative therapy appropriate for the substantial proportion of patients who
present in later life with coexisting conditions?
The answers to these questions notwithstanding, this critique does not devalue the relevance of
the data presented by Milpied et al. These investigators have shown that considerable intensification of treatment is feasible for most patients with
aggressive lymphoma of low-to-intermediate risk
and that the fraction of patients who are cured may
well be increased. It is incumbent on us to build on
this information, in the context of the new treatments available today and our new tools for understanding the nature and prognoses of this heterogeneous group of diseases, so that we can continue
to make incremental improvements in care.
From the Department of Medical Oncology, St. Bartholomew’s
Hospital, London.
Lung Surfactant, Respiratory Failure, and Genes
Mikko Hallman, M.D., Ph.D.
After the first observation of surfactant deficiency in
infants who were dying of the respiratory distress
syndrome (also known as hyaline membrane disease), a series of investigations led to effective therapies, including surfactant therapy given at birth.
These therapies, in turn, have led to a dramatic decrease in mortality associated with the respiratory
distress syndrome, from nearly 100 percent to less
than 10 percent. Most infants who die from the syndrome are preterm. Surfactant therapy is usually
not required beyond the first day of life, because
the turnover of surfactant is slow in newborns, and
the rapid differentiation of alveolar tissue leads to
sufficient endogenous secretion within days after
birth in most cases. However, there are still some
cases of the respiratory distress syndrome that are
resistant to treatment, and an understanding of
1278
what causes them may lead to strategies for prevention and new therapies.
The synthesis, storage, secretion, recycling, and
catabolism of alveolar surfactant take place in alveolar type II cells (see Figure), which are characterized by lamellar bodies — organelles containing
concentric, onion-like layers of surfactant. Alveolar
macrophages also mediate the catabolism of surfactant. In the alveolar lining, surfactant released by
lamellar bodies (when the limiting membrane fuses with the cell membrane) is transformed into aggregates made up of tubular myelin that can both
enter the air–water interface exceptionally quickly by
means of adsorption and nearly eliminate the surface tension of that interface. Low surface tension
in the lining of the tiny spherical air spaces prevents
generalized atelectasis (collapse) and its devastat-
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march 25, 2004
Lung Surfactant, Respiratory Failure, and Genes
PERSPECTIVE
(SP-C). In this issue of the Journal, Shulenin et al.
(pages 1296–1303) describe a third genetic cause
in 16 cases: recessive mutations in the gene encoding the ATP-binding cassette (ABC) transporter A3
(ABCA3).1 Close examination of alveolar tissue
from four patients uncovered small, abnormal
lamellar bodies, leading the authors to propose
that ABCA3 is critical to the formation of lamellar
bodies.
Although the molecular action of ABCA3 is un-
ing consequences. Ninety percent of the surfactant
complex consists of specific lipids enriched with dipalmitoyl phosphatidylcholines that form the film at
the air–water interface. Of the four surfactant proteins A, B, C, and D, three (hydrophobic transmembrane surfactant proteins B and C and collectin surfactant protein A) bind to surfactant lipids.
Hereditary respiratory failure in term infants has
been attributed to mutations in the genes encoding
surfactant protein B (SP-B) and surfactant protein C
Alveolar
space
Monolayer–multilayer film
Adsorption
Surfactant
Tubular
myelin
ABCA3
protein
Lipid vesicle
Secretion
Air
Turnover
Lamellar
body
Liquid
Recycling
Golgi
complex
Multivesicular
bodies
Clearance
Alveolar
macrophage
Alveolar
type II cell
Alveolar
type I cell
Figure. Lung Surfactant and the Lamellar Body.
The composition of lung surfactant is critical to lung function, and the subcellular lamellar body of the type II alveolar cell has a major role in
maintaining the composition of surfactant. The distribution of ATP-binding cassette (ABC) transporter A3 (ABCA3) is shown in red. It is possible that ABCA3 targets surfactant-containing vesicles to the lamellar bodies. The lamellar body is formed through the fusion of several multivesicular bodies, and its lipid bilayers are converted to tubular myelin as they are turned out into the alveolar space. Surfactant protein A,
secreted primarily by a constitutive pathway that does not involve the lamellar body, is required for the formation of tubular myelin. Surfactant
aggregate, such as tubular myelin, is the precursor of surfactant at the air–water interface (inset). Cationic transmembrane proteins (surfactant protein B or surfactant protein C or both [not depicted in the monolayer–multilayer film]) together with anionic phospholipids (phosphatidylglycerol or phosphatidylinositol or both [green]) facilitate the entry of dipalmitoyl phosphatidylcholines (blue) into the monolayer at
the interface, maintaining a low surface tension. During tidal breathing, tubular myelin surfactant is converted to smaller aggregates that are
taken up by type II cells and alveolar macrophages. Arrows depict the direction of surfactant flux.
n engl j med 350;13
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1279
Lung Surfactant, Respiratory Failure, and Genes
PERSPECTIVE
clear, we know about the biologic behavior of other
ABCA proteins, which may be relevant to that of
ABCA3. The ABCA subfamily consists of 12 transporter genes, some of which are known to mediate
ATP-driven lipid transport across a lipid-bilayer
membrane. Tangier disease, a multiorgan hyperlipidemia syndrome, is caused by a loss-of-function
mutation in the ABCA1 gene; ABCA1 controls the
extrusion of the membrane lipid toward extracellular acceptors, the apolipoproteins. Stargardt’s disease, which affects the retina, is caused by loss-offunction mutations in the ABCA4 gene. This protein
serves as a “flippase”; it catalyzes a 180° rotation of
a specific phospholipid derivative and simultaneously moves it from the inner leaflet of the lipid bilayer to the outer leaflet within the disk membrane
of the rod cell. ABCA3 is found in the outer limiting
membrane of lamellar bodies, multivesicular bodies, and segments of luminal plasma membrane of
alveolar type II cells. This pattern of expression, together with the known functions of ABCA1 and
ABCA4, is consistent with the possibility that ABCA3
mediates the targeting of surfactant-containing vesicles to the lamellar bodies.
The phenotype of the patients characterized by
Shulenin et al. resembles that of patients with mutations of the SP-B gene: both groups of patients
have fatal respiratory failure and highly abnormal
lamellar bodies, and in both, histologic analysis reveals neonatal alveolar proteinosis.2 In contrast, the
phenotypes of patients with a mutation of the SP-C
gene are varied: some have mild respiratory symptoms, whereas others have severe respiratory failure.
Histologic analysis reveals interstitial lung disease.2
In fatal respiratory failure due to the absence of surfactant protein B, surfactant protein C precursor
protein is not cleaved to form mature surfactant protein C, so the precursor protein accumulates in the
alveolar space. A dominant negative mutation of
1280
SP-C, on the other hand, may result in the aggregation of misfolded surfactant protein C precursor
protein and subsequent injury and inflammation.
The lungs of the patients studied by Shulenin et
al. showed hyperplasia of alveolar macrophages
and alveolar type II cells, as well as indications of
desquamative interstitial pneumonitis or alveolar
proteinosis. The abnormally small, densely packed
lamellar bodies suggest the presence of abnormal
surfactant that probably does not have the near-zero
surface tension required for alveolar stability. Further study to determine the distribution of surfactant and other membrane components in bronchoalveolar lavage fluid and in lung compartments
— together with experimental studies using mouse
models — will contribute to a more accurate picture
of ABCA3 deficiency.
Although heritable cases of the respiratory distress syndrome can be prevented through genetic
counseling and prenatal diagnosis, we may not be
aware of all the genes that can fatally affect the surfactant system. Nor do we yet know the proportion
of cases of the respiratory distress syndrome that are
caused by a mutation of ABCA3, which may be similar to the proportion caused by a mutation of the
SP-B gene (in the range of 1 case in 1 million newborns). It is also possible that the common allelic
variants of the genes encoding the surfactant proteins, together with environmental factors, influence susceptibility to serious respiratory disease.3
From the Biocenter Oulu and the Department of Pediatrics, University of Oulu — both in Oulu, Finland.
1. Shulenin S, Nogee LM, Annilo T, Wert SE, Whitsett JA, Dean
M. ABCA3 gene mutations in newborns with fatal surfactant deficiency. N Engl J Med 2004;350:1296-303.
2. Whitsett JA, Weaver TE. Hydrophobic surfactant proteins in
lung function and disease. N Engl J Med 2002;347:2141-8.
3. Haataja R, Hallman M. Surfactant proteins as genetic determinants of multifactorial pulmonary diseases. Ann Med 2002;34:
324-33.
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