Genetic defects in surfactant protein A2 are associated with pulmonary fibrosis and lung cancer

Abstract

Idiopathic pulmonary fibrosis (IPF) is a lethal scarring lung disease that affects older adults. Heterozygous rare mutations in the genes encoding telomerase are found in approximately 15% of familial cases. We have used linkage to map another disease-causing gene in a large family with IPF and adenocarcinoma of the lung to a 15.7 Mb region on chromosome 10. We identified a rare missense mutation in a candidate gene, SFTPA2, within the interval encoding surfactant protein A2 (SP-A2). Another rare mutation in SFTPA2 was identified in another family with IPF and lung cancer. Both mutations involve invariant residues in the highly conserved carbohydrate-recognition domain of the protein and are predicted to disrupt protein structure. Recombinant proteins carrying these mutations are retained in the endoplasmic reticulum and are not secreted. These data are consistent with SFTPA2 germline mutations that interfere with protein trafficking and cause familial IPF and lung cancer.

Figure 1

Mutations in SFTPA2 Segregate with Familial Lung Cancer and Pulmonary Fibrosis (A) Abridged pedigrees are shown. The arrow indicates the index case. Circles represent females; squares represent males. Symbols with a slash through them indicate deceased subjects. Individuals with pulmonary fibrosis, lung cancer, or undefined lung disease are indicated by blue, red, and green symbols, respectively. Numbers below each symbol correspond to the individuals described in Table S1; numbers in parentheses indicate individuals for whom no DNA sample was available. The presence or absence of a mutation in SFTPA2 is indicated by plus or minus signs, respectively. When the mutation was inferred based upon the pattern of inheritance, the plus sign is placed in parentheses. The current age or the age at death is indicated to the upper right of each symbol. The SFTPA2 mutations and predicted amino acid changes are listed above each family. (B) A region of chromosome 10 cosegregates with pulmonary fibrosis and lung cancer. The genes that encode paralogs of both surfactant protein A and surfactant protein D lie within the linked region. The DNA sequence of a segment of SFTPA2 exon 6 is shown for the heterozygous CKG810 (left) and the proband of family F27 (right). A single base substitution (arrow) changes the wild-type thymidine to cytosine and leads to the predicted substitution of phenylalanine to serine at codon 198 (left); substitution of the wild-type guanine to thymidine leads to the predicted substitution of glycine for valine at codon 231 (right). Both mutations affect the coding region of the carbohydrate-recognition domain of surfactant protein A2. (C) The terminal portion of the amino acid sequence of the carbohydrate-recognition domain of surfactant A proteins is shown from diverse vertebrate species. This domain is highly conserved. Both the phenylalanine and glycine at positions 198 and 231, respectively, are conserved in all species shown. All sequences were obtained from NCBI; the comparison of the different proteins was based on a ClustalW-generated alignment. (D) The ribbon diagram of the crystal structure of the monomeric carbohydrate recognition domain (CRD) and neck domains of rat surfactant protein A is shown; the positions of amino acids corresponding to codons 198 and 231 of the human sequence are indicated by arrows.

Figure 2

Histology and Immunohistochemical Staining of SP-A in Lung Specimens from Affected Individuals in Family F27 The hemotoxylin and eosin-stained slides (A–J) show different pathologic subtypes of pulmonary fibrosis with and without adenocarcinoma with features of bronchioloalveolar cell carcinoma (BAC). Specimens from the proband IV:8 (A, B) and III:11 (C, D) demonstrate the pathologic findings of usual interstitial pneumonia, consistent with IPF. The specimen shown from IV:6 (E and F), a 29-year-old female diagnosed with hypersensitivity pneumonitis resulting from bird exposures, shows organizing pneumonitis with minimal fibrosis; her disease remitted with prednisone treatment and avoidance of birds. The specimen from III:12 (G and H) shows pulmonary fibrosis and adenocarcinoma with features of BAC. An autopsy sample from IV:7 (I and J) shows extensive severe interstitial fibrosis with cystic remodeling and marked epithelial proliferation. In some areas the epithelial cells show severe cytologic atypical and papillary and acinar growth patterns, consistent with adenocarcinoma with features of BAC confined to the lungs. Photographs were obtained as previously described. Slides from a normal subject (K), a sporadic patient with IPF without any mutations in SFTPA2 (L), a patient with adenocarcinoma with BAC features and no SFTPA2 genomic mutations (N) and F27 family members IV:8 (M), IV:7 (O), and III:12 (P) were stained for immunochemical localization of SP-A. Type II cells lining the alveoli and neoplastic adenocarcinoma cells demonstrate SP-A staining (indicated by the brown color). Slides were deparaffinized with xylene for 10 min two times and rehydrated by immersion in serial ethanol baths: 100% twice, 95% once, 75% once, 50% once, and then three washes in PBS for 5 min each. Antigen retrieval was performed with 1× Citra buffer (Biogenex, San Ramon, CA) for 10 min at 100°C. Endogenous peroxidase activity was quenched by incubation with 0.6% H₂O₂ in methanol for 30 min at room temperature. Immunohistochemical staining was performed with the Vectastain Elite ABC kit (Vector Laboratories) according to the manufacturer's protocol with anti-SP-A antibody (1:2000 dilution with overnight incubation at 4°C), biotinylated GAR (1:500 with a 1 hr room-temperature incubation) and the chromagen DAB (Dako). Slides were counterstained with Mayer's Hematoxylin (Dako, Carpinteria, CA) and mounted with VectaMount AQ (Vector Laboratories). Scale bars represent 1 mm and 100 μm for the low-power (A, C, E, G, and I) and high-power (B, D, F, H, and J–P) magnifications, respectively.

Figure 3

The Effects of the G231V and F198S SFTPA2 Mutations on Protein Expression, Secretion, Interactions, and Endoglycosidase Sensitivity in Transiently Transfected A549 Cells (A) A549 cells were transiently transfected with vector plasmid (Mock), the wild-type SP-A2 expression construct and various untagged SP-A2 constructs (upper panels) or V5-tagged constructs (lower panels). Rare variants are those that were found at a frequency of <5%. Aliquots of cellular lysates (C, 40 μg) and medium (M, 80 μg) were analyzed via immunoblotting with either a rabbit polyclonal that was raised against purified human SP-A (upper panel) or a mouse monoclonal antibody that recognizes the V5 epitope (lower panel). Blots were exposed for 1–20 min or 1–20 s for the anti-SP-A and anti-V5 antibodies, respectively. (B) A549 cells transfected with empty vector (Mock), plasmid expressing the Myc-tagged SP-A1 or SP-A2, and the V5-tagged wild-type or variant SP-A2. Forty-eight hours after transfection the cell lysates (C) and medium (M) were split into two aliquots. The Myc-tagged SP-A proteins were immunoprecipitated from the cellular lysates and media, subjected to SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with antibodies directed against the Myc or V5 epitopes (upper panels). The arrow and arrowhead indicate the position of the Myc-tagged SP-A and V5-tagged SP-A2, respectively. Aliquots of cell lysates (C) and medium (M) (40 μg) were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and immunobloted with antibodies directed against the Myc or V5 epitopes (lower panels). (C) A549 cells were transiently transfected with V5-tagged SP-A2 wild-type, G231V, or F198S constructs. Aliquots of lysates (C) or media (M) were treated with the indicated endoglycosidases and subjected to immunoblot analysis. Untreated samples were treated in parallel tubes without the addition of enzyme.