American Journal of Respiratory and Critical Care Medicine

Rationale: ABCA3 mutations are known to cause fatal surfactant deficiency.

Objective: We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis.

Methods: Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting.

Results: ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro–SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro–SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro–SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively.

Conclusion: Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.

Recently, unexplained respiratory distress syndrome (URDS) of full-term newborns has been linked to mutations of the ATP-binding cassette transporter A3 (ABCA3) gene (1). ABC proteins are multispan membrane proteins that promote the efflux of specific substrates across biological membranes or function as regulatory molecules (for review, see References 2 and 3). The subfamily of ABCA transporters are full-size transporters with 12 transmembrane domains and mainly linked to phospholipid and cholesterol transport (46). The human ABCA3 gene consists of 30 coding exons. The ABCA3 protein is of special interest for surfactant homeostasis because it is predominantly expressed in type II pneumocytes of the lung and has been localized to the limiting membrane of lamellar bodies, which are the main intracellular storage organelles for pulmonary surfactant (1, 710).

Pulmonary surfactant is a complex mixture of lipids, primarily palmitoyl-phosphatidylcholine and surfactant proteins (SPs) that are synthesized by type II pneumocytes. The hydrophobic SP-B and SP-C as well as surfactant lipids are assembled and stored in lamellar bodies (11). It has been demonstrated that ABCA3 selectively facilitates the transfer of phosphatidylcholine, sphingomyelin, and cholesterol to lamellar bodies (12). ABCA3 mutations in full-term newborns with URDS are associated with a defective assembly of lamellar bodies, an abnormal staining pattern of type II pneumocytes for SP-B, and fatal surfactant deficiency (1, 13). Biochemical data indicated an abnormal expression of ABCA3 with mutations linked to URDS (12).

Because “a major obstacle involves identifying patients with ABCA3 mutations” and “a complete genetic identification of all ABCA3 mutations in all candidate patients is currently not possible” (14), we studied the protein expression of ABCA3 in type II pneumocytes in full-term newborns with URDS. Furthermore, we addressed the issue of whether an abnormal ABCA3 protein expression is associated with ABCA3 mutations. To characterize alterations of pulmonary surfactant due to impaired/defective genesis of lamellar bodies, we studied SPs A, B, and C in type II pneumocytes and in alveolar surfactant by immunohistochemistry, immunoelectron microscopy (immuno-EM), and Western blotting. Some of the results of these studies have been previously reported in form of abstracts (15, 16).

Study Group

Lung tissue from 14 full-term newborns with URDS was screened for ABCA3 protein expression in type II pneumocytes by immunohistochemistry. Ten individuals showed a greatly reduced or absent expression of ABCA3 in type II pneumocytes and were analyzed for ABCA3 mutations and expression of SP-A, SP-B, and SP-C. The patient that initially indicated a hereditary ABCA3 deficiency in a family is referred to as “index patient.” The patients' details are described in Results and summarized in Table 1. Informed consent was obtained from all parents. The German General Medical Council gave approval for immunohistochemical, ultrastructural, and mutation analysis.

TABLE 1. CHARACTERISTICS OF FAMILIES AND INFANTS


Family

Consanguinity

Race or Ethnic Group

Infants (Sex)

Duration of Mechanical Ventilation

Total Dose of Exogenous Surfactant Application

Time Span between Last Surfactant Application and Death or LTX (IP2)

Time Point of BAL

Clinical Outcome

Material

EM/Immuno-EM
1YesMiddle EasternAbortion
Abortion
Male3rd day of life until death200 mg/kg body weight9 dDeath at the age of 26 d
Male/ IP13rd day of life until death100 mg/kg body weight11 dDeath at the age of 14 dBALF, blood, VATS lung biopsy
MaleAlive and healthy
Male1st day of life until death100 mg/kg body weight14 d1st day of lifeDeath at the age of 17 dPostmortem lung tissue
2YesMiddle EasternFemale/IP23rd week of life until LTX200 mg/kg body weight17.5 mo21 moLTX at the age of 21 mo, death due to PTLD at the age of 4 yrBALF, blood, VATS lung biopsy, fresh-frozen lung tissue, explanted lungsYes/Yes
MaleAlive and healthyBlood
MaleAlive and healthyBlood
3YesMiddle EasternFemale/IP31st day of life until death200 mg/kg body weight20 dDeath at the age of 23 dBlood, VATS lung biopsyYes/Yes
4YesMiddle EasternFemale/IP43rd day of life until death59 mg/kg body weight10.5 wk13th day of lifeDeath at the age of 3.5 moBALF, blood, VATS lung biopsyYes/Yes
MaleAlive and healthyBlood
Abortion
5NoWhiteFemale/IP51st day of life until death200 mg/kg body weight11 d36th day of lifeDeath at the age of 47 dBALF, VATS lung biopsy
Female1st day of life until death200 mg/kg body weight2 dDeath at the age of 3 dPostmortem lung tissue
FemaleAlive and healthy
MaleAlive and healthyBlood
6NoWhiteFemaleAlive and healthy
Female/IP61st day of life until death800 mg/kg body weight23 d2nd day of lifeDeath at the age of 42 dBALF, fresh-frozen lung tissue, VATS lung biopsyYes/Yes
7YesMiddle EasternFemale/IP73rd day of life until death400 mg/kg body weight11 wk3rd day of lifeDeath at the age of 3 moBALF, blood, VATS lung biopsyYes/No
Abortion
MaleAlive and healthy
MaleAlive and healthy
8YesMiddle EasternMale /IP83rd day of life until death1000 mg/kg body weight5 d10th day of lifeDeath at the age of 22 dBALF, blood, VATS lung biopsyYes/No



Male




Alive and healthy


Definition of abbreviations: BALF = bronchoalveolar lavage fluid; EM = electron microscopy; IP = index patient; LTX = lung transplantation; NA = no information available; PTLD = post-transplant lymphoproliferative disease; VATS = video-assisted thoracoscopic surgery.

Controls for Protein Expression Analysis

Lung tissue from eight nontransplanted human donor lungs were used as controls for immunohistochemistry and immuno-EM. In addition, lung biopsies from three full-term newborns with hereditary SP-B deficiency due to homozygote 121ins2 and 122delT SFTPB mutations; two infants with heterozygote E66K and I73T SFTPC mutations (17, 18); 20 adult patients with idiopathic pulmonary alveolar proteinosis (PAP) (19), nonspecific interstitial pneumonia (NSIP), desquamative interstitial pneumonia (DIP), respiratory bronchiolitis–associated interstitial lung disease, or usual interstitial pneumonia; as well as five infants with secondary pulmonary hypertension were analyzed for ABCA3 protein expression by immunohistochemistry. For Western blot analysis of ABCA3 protein expression in membrane fractions of lung tissue, two lung specimens from tumor-free lung tissue were used. Bronchoalveolar lavage fluid (BALF) from a healthy adult and a newborn with hereditary SP-B deficiency due to 121ins2 SFTPB mutation were used as controls for Western blot analysis of SPs.

Antibody Sources

A polyclonal rabbit anti-human ABCA3 antibody was generated. Antibodies against epithelial membrane antigen (EMA), thyroid transcription factor 1 (TTF-1), SP-A, pro–SP-B, SP-B, pro–SP-C, and cathepsin D were either purchased from Dako (Glostrup, Denmark) and Chemicon (Temecula, CA) or kindly provided by S. Hawgood, Y. Suzuki, and M. F. Beers (for details, see the online supplement).

Immunohistochemistry, Electron Microscopy, and Immuno-EM of Lung Tissue

Tissue preparation, immunostaining as well as semiquantitative evaluation, and immunogold labeling of lung specimens for light and electron microscopy were performed according to standard procedures (20, 21) and are described in the online supplement.

Western Blot Analysis of ABCA3 Protein Expression in Lung Tissue

Crude membrane fractions for Western blot analysis of ABCA3 protein expression were prepared from fresh-frozen lung tissue. Detailed sample preparation and Western blot procedures are outlined in the online supplement.

Western Blot Analysis of SPs in BALF

Diagnostic BAL was performed according to clinical standard procedures. Until analysis, samples were stored at −80°C. Immunoblotting of BALF was performed as described previously (19) and is outlined in detail in the online supplement.

Mutation Analysis of ABCA3

Genomic DNA was extracted from ethylenediaminetetraacetic acid whole blood or fresh-frozen lung tissue (Index Patient 6). The 30 coding exons of the ABCA3 gene, including the flanking regions, were sequenced. If necessary, sequence information was verified by restriction fragment length polymorphism. A detailed method description is given in the online supplement.

Fourteen lung biopsies from full-term newborns with URDS were screened for expression of ABCA3 in type II pneumocytes by means of immunohistochemistry. Ten infants (eight index patients and two siblings) from eight different families with greatly reduced or absent expression of ABCA in type II pneumocytes were identified. Seven infants had consanguineous parents. All infants were delivered as full-term newborns after a normal pregnancy and an uneventful birth. Shortly after birth, all infants developed severe respiratory distress. Screening for metabolic, infectious, or immunologic disorders was negative. SFTPB mutations were excluded in all index patients by complete gene sequencing as described in detail previously (22).

Despite intense supportive care and mechanical ventilation, progression of respiratory insufficiency led to the death of all but one newborn within weeks or a few months. Only Index Patient 2 survived the postnatal period, and combined double lung–heart transplantation could be performed at the age of 21 mo. Subsequently, the clinical course was complicated by intermittent infections and gastrointestinal problems. Finally, the child died of post-transplant lymphoproliferative disease.

Relevant clinical information about families and patients is listed in Table 1. Pedigrees are shown in Figure 1.

Histologic examination of lung tissue of index patients and diseased siblings revealed a variable combination of NSIP-, DIP-, and PAP-like features, known as chronic pneumonitis of infancy (Figure 2a and Figures E2–E8 of the online supplement) (23). Alveolar septa were thickened due to an increased cellularity and collagen fibers (NSIP-like feature). Immunostaining for TTF-1 confirmed a marked hyperplasia of type II pneumocytes (data not shown). Furthermore, a focal intraalveolar accumulation of a fine granular, eosinophilic material (PAP-like feature) and alveolar macrophages (DIP-like feature) was observed. At the ultrastructural level, electron-dense bodies with central or peripheral core structures were found in type II pneumocytes of all index patients for whom lung tissue was available for electron microscopy (Figures 2b and 2c; Figures E3, E4, E6–E8). Only in Index Patient 2, some electron-dense bodies showed rudimentary multilamellar structures (Figures 2c and 2d) and a few regular lamellar bodies were found in type II pneumocytes (Figure 6e).

Immunohistochemical analysis of lung tissue for ABCA3 protein expression of control tissue (human nontransplanted donor lungs) as well as from adult patients with idiopathic PAP and idiopathic pneumonia (NSIP, DIP, respiratory bronchiolitis–associated interstitial lung disease, and usual interstitial pneumonia) revealed a moderate cytoplasmic ring-like staining in type II pneumocytes (immunohistochemical score 2; Figure 3a and Figure E1). The intraalveolar accumulated surfactant in adult patients with idiopathic PAP was not stained (Figure 3a). Surprisingly, in newborns with hereditary SP-B deficiency, not only a strong staining of type II pneumocytes (immunohistochemical score 3) but also a moderate staining of the intraalveolar accumulated material was observed (Figure 3b). In two infants with heterozygote SFTPC mutations, type II pneumocytes showed a moderate cytoplasmic ring-like staining pattern in type II pneumocytes (immunohistochemical score 2; Figure E1). In contrast to lung tissue from normal control subjects and adult patients with parenchymal lung disease, type II pneumocytes in the lung tissue from 10 of 14 full-term newborns with URDS showed a weak (immunohistochemical score 1) or absent (immunohistochemical score 0) ABCA3 protein expression (Figures 3c and 3d; Table E2).

Because the immunohistochemical findings implicate a residual ABCA3 protein expression in some of the patients, we addressed this issue by Western blot analysis of fresh-frozen lung tissue of the explanted lung from Index Patient 2 and two healthy control subjects. The same anti-ABCA3 antibody that was used for immunohistochemistry (directed against the intracellular C-terminal region) detected a protein of approximately 150 and approximately 190 kD in lung tissue of healthy children (Figure 4, lane 1), which is similar to literature findings (10, 12). In line with the immunohistochemical findings, a greatly reduced ABCA3 protein expression compared with the healthy control subject was observed in Index Patient 2 (Figure 4, lane 2). The specificity of the results was proven by omission of the primary antibody and by preincubating the primary antibody with the corresponding blocking peptide (Figure E11).

Because we hypothesized ABCA3 mutations to be causative for URDS in patients with greatly reduced or absent ABCA3 protein expression, we continued with mutation analysis by direct sequencing of the coding exons of the ABCA3 gene, including the flanking regions. We were able to identify 13 mutations and three silent polymorphisms. With the exception of Family 5, all index patients were found to have homozygous or compound heterozygous mutations that affect the protein structure (Figure 1 and Table 2). In Family 5, material for mutation analysis was available only from both heterozygous parents and a healthy heterozygous sibling. In all families, the apparently healthy parents and siblings were shown to be heterozygous for the respective mutation. The ABCA3 mutations we found that are most likely to be causative for the observed clinical phenotype (highlighted in bold letters in Table 2) are distinct from each other and from the ones previously described (1, 13). In Families 1, 2, 4, 7, and 8, homozygous ABCA3 mutations were found that are predicted to have major impact on the protein structure (e.g., frameshift, truncation, and splice-site mutations) and to affect domains with high functional relevance (Table 2). Thus, it is conceivable that these mutations lead to impaired or even abolished function of the ABCA3 protein. The significance of missense mutations in Families 3, 5, and 6 is not yet clear. Taking into account the typical clinical phenotype as well as the consistent histologic, immunohistochemical, and ultrastructural findings, we conclude that all mutations detected are functionally relevant. The missense mutations with predicted functional relevance could not be detected in 50 control DNA samples from healthy donors.

TABLE 2. MUTATIONS OF THE ABCA3 GENE IN THE STUDY GROUP AND ABCA3 PROTEIN EXPRESSION IN TYPE II PNEUMOCYTES IN INDEX PATIENTS


Family

Localization*

Nucleotide Deviation

Structural Relevance

Affected Domain

ABCA3 Protein Expression in Type II Pneumocytes in Index Patients (immunohistochemical score)
1Exon 15c1755C > GSilent polymorphismWeak (1)
Exon 15c1814G > AR605Q (Arg > Gln)NBD 1
Exon 31c4877–8delAGFrameshift/StopC-terminus
2Exon 10c1058C > TSilent polymorphismWeak (1)
Intron 15c1897–1G > CAcceptor splice-site mutationNBD 1
3Exon 8c643C > AQ215K (Gln > Lys)First extracellular loopAbsent (0)
Exon 8c863G > AR288K (Arg > Lys)First extracellular loop
4Intron 21c3005–1G > AAcceptor splice-site mutationSecond half-size transporterWeak (1)
5Exon 5c128G > T (het)R43L (Arg > Leu)First extracellular loopAbsent (0)
Exon 8c863G > A (het)R288K (Arg > Lys)First extracellular loop
Exon 15c1755C > G (het)Silent polymorphism
Exon 31c4751delT (het)Frameshift/StopC-terminus
6Exon 14c1736T > C (het)L579P (Leu > Pro)NBD 1Weak (1)
Exon 25c3812delG (het)Frameshift/StopLast extracellular loop, C-terminus
7Exon 30c4681 C > TR1561X (Arg > Stop)C-terminusWeak (1)
8
Exon 19
c2429–30delTT
Frameshift/Stop
Second half-size transporter
Weak (1)

Definition of abbreviation: NBD = nucleotide-binding domain.

Sequence information is based on reference sequences NM_001089 and NP_001080, with the numbering beginning at the ATG start codon. The ABCA3 mutations found that are most likely to be causative for the observed clinical phenotype are highlighted in bold letters.

*Structural information regarding the ABCA3 gene refers to Shulenin and colleagues (1).

Semiquantitative immunohistochemical score of ABCA3 protein expression: 0 = absent, 1 = weak, 2 = moderate, 3 = strong.

The information concerning base positions as well as exon/intron structure of the ABCA3 gene is based on reference sequence NM_001089 and refers to reference 1. According to this nomenclature, the ABCA3 gene consists of 30 coding and 3 noncoding exons (total of 33 exons). Latest mapping approaches may favor a different structure (with only two noncoding exons instead of three). The details are described in the online Methods.

The mutations of the ABCA3 gene that have been identified to date are distinct from each other and affect different protein domains (Table E2). The localization within the putative model of the ABCA3 protein is depicted in Figure E10. Clusters of mutations seem to be confined mainly to the N-terminal nucleotide-binding domain and the C-terminus.

We then addressed the issue whether hereditary ABCA3 deficiency affects trafficking and processing of SPs within type II pneumocytes. Therefore, we continued with immunohistochemical, immuno-EM, and Western blot analysis of SPs in the index patients. By immunohistochemistry, type II pneumocytes and the granular material that focally filled the alveoli showed a strong staining for SP-A (Figure 5a) and precursors of SP-B (Figure 5b), whereas the staining for SP-B was only weak (Figure 5c). Higher magnification revealed a dotlike staining pattern of type II pneumocytes for SP-B (Figure 5d). In all patients, anti–pro-SP-C moderately stained type II pneumocytes (Figure 5e), whereas a weak staining of the granular material in the alveoli was observed only in Index Patient 4.

At the ultrastructural level, SP-A (10-nm gold particles) was localized in small vesicles in type II pneumocytes and over “proteinosis-like” multilamellated surfactant figures in the alveolar space (Figures 6a and 6b). In line with the immunohistochemical dotlike staining pattern, SP-B (10-nm gold particles) was found in multivesicular bodies and over core structures of electron-dense bodies in type II pneumocytes (Figures 6c and 6d; Figures E3, E4, and E6). Only in Index Patient 2, we detected a few regular lamellar bodies with SP-B that was localized over the projection core (Figure 6e) as previously described in normal human lungs (24). Furthermore, we detected SP-B over dense-core particles within “proteinosis-like” multilamellated surfactant figures only in Index Patient 2 (Figure 6f). These dense-core particles were similar to those found in tubular myelin figures in normal human lungs (24).

Because electron-dense bodies resembled lysosomal organelles, we performed an immuno-double-labeling for the lysosomal aspartic protease cathepsin D (5-nm gold particles) and SP-B (15-nm gold particles). Although SP-B (15-nm gold particles) was found over core structures, cathepsin D (5-nm gold particles) was localized within the matrix of electron-dense bodies (Figure 6g, arrows). Furthermore, precursors of SP-C (10-nm gold particles) were found not only in multivesicular bodies but also within the matrix of electron-dense bodies (Figure 6h, arrows).

To study secretion of hydrophobic surfactant-associated proteins B and C, Western blot analysis of BALF from a healthy adult, a full-term newborn with hereditary SP-B deficiency due to a 121ins2 SFTPB mutation, and seven index patients was performed (Table 3). In the healthy control subject, we clearly identified mature dimeric SP-B (band at ∼ 18 kD) and mature monomeric (band at ∼ 4 kD) as well as dimeric SP-C (band at ∼ 8 kD), but no precursors of SP-B or SP-C. In the BALF of the full-term newborn with hereditary SP-B deficiency, neither precursors of SP-B nor mature SP-B were detectable. Furthermore, many precursors of SP-C (bands at ∼ 6, ∼ 10, ∼ 12, ∼ 14, ∼ 16, ∼ 21, and ∼ 23 kD), but no mature SP-C, were found. In line with immunohistochemical analysis, precursors of SP-B (bands at ∼ 23 and ∼ 17 kD), but only very low levels of mature SP-B (band at ∼ 18 kD) were found in BALF of index patients. Only in Index Patient 4 were we able to detect precursors of SP-C (bands at ∼ 5 and ∼ 10 kD). Trace amounts of mature SP-C were detectable only in Index Patient 6. Results of the Western blot analysis are summarized in Table 3, and a representative Western blot is shown in the online supplement (Figure E9).

TABLE 3. RESULTS OF WESTERN BLOT ANALYSIS OF HYDROPHOBIC SURFACTANT PROTEINS B AND C IN BRONCHOALVEOLAR LAVAGE FLUID OF A HEALTHY CONTROL SUBJECT, A NEWBORN WITH HEREDITARY SP-B DEFICIENCY DUE TO 121 INS2 SFTPB MUTATION, AND INDEX PATIENTS




Healthy Control

Hereditary SP-B Deficiency

IP1

IP2

IP4

IP5

IP6

IP7

IP8
Precursors of SP-BNoNoYesYesYesn.d.YesYesYes
Mature SP-BYesNoReducedReducedReducedReducedReducedReducedReduced
Precursors of SP-CNoYesNoNoYesn.d.NoNon.d.
Mature SP-C
Yes
No
No
No
No
n.d.
Reduced
No
No

Definition of abbreviations: IP = index patient; n.d. = not done; SP = surfactant protein.

The human ABCA3 transporter appears to play an important role in lung surfactant homeostasis because (1) it was found to be mutated in newborns with fatal surfactant deficiency and (2) it seems to be involved in the formation of lamellar bodies (1, 7, 10, 12). So far, the pattern of ABCA3 protein expression in full-term newborns with hereditary ABCA3 deficiency as well as consequences of ABCA3 mutations for processing and trafficking of SPs are unknown. We provide data that underscore the relevance of an immunhistochemical analysis of the ABCA3 protein expression in type II pneumocytes to detect ABCA3 mutations and elucidate important aspects regarding the impact of ABCA3 mutations on surfactant homeostasis.

Immunohistochemical analysis of the ABCA3 protein revealed a cytoplasmic ring-like staining pattern in type II pneumocytes in normal human lungs, lung biopsies from pediatric patients with secondary pulmonary hypertension, and SFTPC mutations, as well as in adult patients with parenchymal lung disease. This staining pattern fits very well with the assumed localization of the ABCA3 protein in the limiting membrane of lamellar bodies (7, 10). Because no nonspecific background staining could be observed, the immunohistochemical analysis underscores the specificity of the ABCA3 antibody.

Immunohistochemical screening of lung biopsies from 14 full-term infants with URDS showed a greatly reduced or even absent ABCA3 protein expression in type II pneumocytes of 10 infants from eight different consanguineous and nonconsanguineous families. Sequencing of the ABCA3 gene revealed splice-site, frameshift, and missense mutations in homozygote as well as compound heterozygote allelic combinations in these patients. All mutations that are predicted to have functional relevance are distinct from the ones previously described by Shulenin and colleagues and Bullard and colleagues (1, 13). For several reasons, the reported genetic deviations are likely to have functional relevance and do not represent silent polymorphisms or rare variants: First, immunohistochemical and Western blot analysis revealed a greatly reduced or absent ABCA3 protein expression in type II pneumocytes that was clearly distinguishable from control subjects and patients with other parenchymal lung diseases. Second, in six index patients whose lung tissue was available for ultrastructural analysis, we detected unique electron-dense bodies in type II pneumocytes. These organelles have been described in full-term babies with ABCA3 mutations and appear to be highly specific for ABCA3 mutations, as they have not been reported in association with other diseases (1). Third, immunohistochemical analysis revealed a staining pattern for mature SP-B that was previously described in pediatric patients with ABCA3 mutations (1, 13). Fourth, based on pedigree analysis, the causative ABCA3 mutations seem to be inherited in an autosomal-recessive fashion. Fifth, the mutations that were found are distinct from each other and are predicted to have a strong structural impact in the majority of cases (like frameshift, truncation, and splice-site mutations). The splice-site mutations c1897–1G > C and c3005–1G > A are likely to affect the structure of large portions of the ABCA3 protein, including the nucleotide-binding domains that are prerequisites for the function of ABC transporters. The nonsense frameshift mutations c2429-30delTT, c3812delG, c4751delT, and c4877–8delAG severely alter either the second half-size of the ABCA3 transporter or the C-terminal region. Similar to other membrane proteins, the C-terminal region is believed to mediate interactions with intracellular molecules that are relevant to protein function (25, 26). Also, the missense mutations with single amino acid substitutions at least partially affect domains with known or assumed structural relevance like the first extracellular loop (believed to mediate extracellular interactions) and the nucleotide-binding domains (Figure E10) (27). Sixth, the missense mutations were not detected in 50 DNA control samples from healthy control subjects (representing 100 alleles).

It is not yet completely clear whether patients with hereditary ABCA3 deficiency have a characteristic histologic pattern. Previously, histologic features of some newborns with ABCA3 mutations were described as PAP (1). Congenital, idiopathic, and secondary PAP is characterized by an intraalveolar accumulation of SP-A (19, 28). In index patients, SP-A was localized over intraalveolar multilamellated surfactant forms that were characterized in adult patients with PAP (29). However, the intraalveolar accumulation of SP-A was only patchy in contrast to PAP (19). Furthermore, histologic examination also revealed a variable combination of patterns of NSIP and DIP in index patients. A similar morphologic picture has been described in infants with a severe and potentially lethal form of surfactant dysfunction, known as chronic pneumonitis of infancy (23).

The hydrophobic mature SP-B is the most critical component of the pulmonary surfactant, and hereditary SP-B deficiency due to mutations in the SFTPB gene is a well-established cause of fatal surfactant deficiency in full-term newborns (30). Because clinical features of index patients were suggestive of fatal surfactant deficiency, SFTPB mutations had been excluded and the impact of hereditary ABCA3 deficiency on the pulmonary surfactant system was studied. Similar to newborns with hereditary SP-B deficiency (31), the pulmonary surfactant was completely disorganized and tubular myelin figures as well as regular lamellar bodies were completely absent in index patients except for Index Patient 2. Despite a strong expression of precursors of SP-B, only a very weak and dotlike cytoplasmic staining pattern of type II pneumocytes for mature SP-B was found. Immuno-EM analysis indicated that the abnormal staining pattern was due to the formation of aggregates of mature SP-B within electron-dense bodies in type II pneumocytes. It has been speculated that the abnormal staining may reflect decreased availability of the SP-B epitopes for the antibodies (1). In a brother of Index Patient 1, who had been a subject of a previous case report and from whom lung tissue was available for expression analysis of SPs, a low level of mature SP-B was found (32). The authors hypothesized that the reduced amounts of mature SP-B were related to a “12-nucleotide deletion at the beginning of exon 8” in the SP-B messenger RNA, which “results in the loss of four amino acids in the SP-B precursor protein.” However, different antibodies against precursors of SP-B and mature SP-B showed the same staining pattern and Western blot analysis of BALF from seven index patients confirmed an intraalveolar accumulation of precursors of SP-B, but greatly reduced levels of mature SP-B. Therefore, it is unlikely that reduced amounts of mature SP-B in index patients were due to an artificial immunohistochemical staining or alterations of SP-B messenger RNA.

Recently, it has been shown that ABCA1 is involved in the basolateral efflux of lipids in type II pneumocytes, whereas ABCA3 is believed to be involved in the apical secretion pathway (33). Both mature SP-B and precursors of SP-C are delivered together by multivesicular bodies to lamellar bodies and will be secreted into the alveolar space after final proteolytic remodeling of the N-terminal propeptide of SP-C (20, 24). Despite a colocalization of mature SP-B and precursors of SP-C in multivesicular and electron-dense bodies in type II pneumocytes, only in two index patients were either trace amounts of mature SP-C or precursors of SP-C detected in the alveolar surfactant. A lack of mature SP-C, but intraalveolar accumulation of precursors of SP-C due to an aberrant processing of SP-C, was described in newborns with hereditary SP-B deficiency (34). Surprisingly, immunohistochemical analysis of lung tissue sections of newborns with hereditary SP-B deficiency showed an intraalveolar accumulation of precursors of not only SP-C but also of ABCA3. A nonspecific staining appears unlikely, because the abnormal intraalveolar surfactant in idiopathic PAP was negative. Therefore, our data indicate an important role and interaction of ABCA3 and SP-B not only in the formation of lamellar bodies and processing of SP-C but also in the apical secretion pathway. Furthermore, immuno-EM and Western blot results provide evidence of an impaired processing and routing of SP-B as well as SP-C in hereditary ABCA3 deficiency.

In a wide range of tissues, secretory granules that appear lysosome-like and lysosomes that look and behave like secretory granules were described (35). Regular lamellar bodies contain many lysosomal proteins, such as hydrolases, CD63, LAMP-1, and cathepsin H as well as napsin A, but not the ubiquitous lysosomal protease cathepsin D (20, 3639). Previously, it has been shown that ABCA3 is localized to membranes of both secretory lysosomes and lamellar bodies and is required for the conversion of secretory lysosomes to lamellar bodies (12). Because we found in patients with hereditary ABCA3 deficiency many electron-dense bodies in type II pneumocytes that appeared lysosome-like, an immuno-double-labeling for SP-B and cathepsin D was performed. In contrast to regular lamellar bodies, the colocalization of cathepsin D and mature SP-B in electron-dense bodies is indicative not only for an essential function of ABCA3 for lamellar body formation but also supports the concept of electron-dense bodies representing abnormal lysosome-like granules (40).

In conclusion, our data emphasize the importance of ABCA3 in surfactant homeostasis. Even though genetic deviations within the ABCA3 gene are highly heterogeneous regarding their localization, they cluster within vulnerable domains of the ABCA3 protein. They lead not only to an aberrant expression of ABCA3, SP-B, and SP-C but also to the formation of electron-dense bodies in type II pneumocytes. Thus, the combination of immunohistochemistry and electron microscopy seems to be an appropriate initial tool to screen lung biopsies of patients with suspected hereditary ABCA3 deficiency. Because the frequency of ABCA3 mutations that may allow for an adequate expression of a nonfunctional ABCA3 protein is unknown and the functional relevance of some mutations is difficult to estimate, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.

The authors thank the families of index patients for their willingness to participate in the study and the physician and nurses who cared for them. Furthermore, the authors thank V. Rigourd and L. Chevret from Service de Réanimation Néonatale, Institut de Puériculture et de Périnatalogie and Service de Réanimation Pédiatrique, CHU de Bicêtre, Le Kremlin-Bicêtre, Paris, France, for the referral of lung biopsies, blood, and BALF as well as clinical data from infants with URDS and their families. They thank Dr. Richter, Children's Hospital auf der Bult, Hannover, Germany, for contributing one of his patients to the study. The authors acknowledge the confirmation of the ABCA3 mutation found in Index Patient 1 by L. M. Nogee, Division of Neonatology, The Johns Hopkins Hospital, Baltimore, MD. The excellent technical assistance of H. Hühn (Göttingen), M. Kochem, S. Geiger, and U. Thomek (Bochum) as well as of F. Walther, B. Wilhelm, M. Schlangstedt, D. Richter, and E. Kowalewski (Regensburg) is highly appreciated.

Information for enrollment of patients into ongoing studies designed to detect defects in surfactant metabolism is available through contact with Dr. F. Brasch (E-mail: ).

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Correspondence and requests for reprints should be addressed to Prof. Dr. G. Schmitz, M.D., Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. E-mail:

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