Abstract
Familial pulmonary fibrosis is a heterogeneous group of interstitial lung diseases of unknown cause that is associated with multiple pathologic subsets. Mutations in the surfactant protein C (SP-C) gene (SFTPC) are associated with familial desquamative and nonspecific interstitial pneumonitis. Genetic studies in familial usual interstitial pneumonitis have been inconclusive. Using a candidate gene approach, we found a heterozygous exon 5 + 128 T→A transversion of SFTPC in a large familial pulmonary fibrosis kindred, including adults with usual interstitial pneumonitis and children with cellular nonspecific interstitial pneumonitis. The mutation is predicted to substitute a glutamine for a conserved leucine residue and may hinder processing of SP-C precursor protein. SP-C precursor protein displayed aberrant subcellular localization by immunostaining. Electron microscopy of affected lung revealed alveolar type II cell atypia, with numerous abnormal lamellar bodies. Mouse lung epithelial cells transfected with the SFTPC mutation were notable for similar electron microscopy findings and for exaggerated cellular toxicity. We show that an SFTPC mutation segregates with the pulmonary fibrosis phenotype in this kindred and may cause type II cellular injury. The presence of two different pathologic diagnoses in affected relatives sharing this mutation indicates that in this kindred, these diseases may represent pleiotropic manifestations of the same central pathogenesis.
The familial forms of pulmonary fibrosis (FPF) are described as the occurrence of interstitial pneumonitis in at least two members of a family. It is unknown what proportion of idiopathic pulmonary fibrosis (IPF) is familial, but it is estimated that 0.5 to 2.2% of cases have a genetic basis (1). To date, over 68 kindreds presumed to have IPF have been reported (1–3). Diagnostic heterogeneity cannot be excluded in a number of these reports, however, as many of these families lack surgical lung biopsy specimens. Marshall reviewed the clinical and epidemiologic findings of 67 patients from 25 families with IPF, from which 32% of cases had surgical lung biopsies performed. Clinical signs/symptoms, treatment outcomes, and histologic findings (usual interstitial pneumonitis [UIP], the pathologic correlate of IPF) were indistinguishable from sporadic IPF, except that cases were younger at diagnosis (1). Familial forms of desquamative interstitial pneumonitis (4) and lymphocytic interstitial pneumonitis have also been described (5). Several FPF kindreds have been reported that include affected adults and children (6–9). The familial form of IPF/UIP is likely transmitted as an autosomal dominant trait with reduced penetrance (1–3, 10, 11). Genetic studies analyzing candidate loci near the human leukocyte antigen region of chromosome 6 in familial IPF/UIP (9) and in sporadic pulmonary fibrosis have been largely inconclusive (12, 13). Polymorphisms in the genes encoding interleukin-1 receptor antagonist (14), tumor necrosis factor-α (14), and angiotensin-converting enzyme (15) have been suggested to play a role in sporadic forms of pulmonary fibrosis.
Recently, a mutation in the gene (SFTPC) encoding the hydrophobic, lung-specific surfactant protein C (SP-C) was discovered in association with an infant and mother with cellular nonspecific interstitial pneumonitis (NSIP) and desquamative interstitial pneumonitis, respectively (16). A heterozygous G to A transition was identified at the first base of intron 4 (IVS4+1 G→A) of both patients' DNA that abolished the normal IVS4 5′ splice site, resulting in a deletion of 37 amino acids from the carboxy-terminal (C-terminal) region of SFTPC.
We report here our study of SFTPC in a large FPF kindred, including 14 affected members spanning 6 decades. The clinical findings of three members of this family have been previously described (6, 17).
The Vanderbilt University Institutional Review Board approved this investigation. This kindred (Figure 1)
Figure 1. Condensed pedigree of kindred. Generation (I–V): identifier number is listed below each family member (circle = female, square = male, open symbol = unaffected, closed circle = affected, slash through symbol = deceased).
[More] [Minimize]Patient | Sex | Age at Dx | Year of Dx | Year of Death | Clinical and Pathologic Descriptors |
|---|---|---|---|---|---|
| II:1 | M | 29 | 1942 | 1952 | Dyspnea, cough, clubbing; CXR with “diffuse granular infiltration”; path report “fibrocystic pulmonary dysplasia.” |
| II:2 | F | 57 | 1976 | 1986 | Dyspnea, cough; cause of death “pulmonary fibrosis.” |
| II:3 | M | 20 | 1945 | 1990 | Dyspnea, cough, clubbing; CXR with “bilateral interstitial fibrosis”; TLC 52%, DlCO 51%. |
| II:4 | F | 41 | 1967 | 1986 | Dyspnea, clubbing; cause of death “pulmonary fibrosis.” |
| III:1 | F | 17 | 1959 | 1964 | Dyspnea, cough, clubbing; CXR “extensive, scattered nodular infiltrations throughout both lungs”; FVC 21%; Asian influenza pneumonia before onset; path report “interstitial pulmonary fibrosis (Hamman-Rich Disease).” |
| III:2 | M | 20 | 1965 | 1991 | Dyspnea, cough; path report UIP. |
| III:3 | M | 32 | 1986 | Alive | Dyspnea; CXR “diffuse coarse reticulation with multiple lucencies”; DLT 2000; path UIP by slide review. |
| III:4 | M | 34 | 1989 | 2000 | Dyspnea, cough, clubbing; TLC 60%, DlCO 41%; CXR “diffuse interstitial infiltrates, worse at bases”; DLT 2000; path UIP by slide review. |
| III:5 | F | 6 mo | 1952 | 1953 | Failure to thrive, cough, cyanosis; CXR “ground glass appearance with fine fibrillary infiltration through both lungs”; path NSIP by slide review. |
| III:6 | M | 40 | 2001 | Alive | Pulmonary fibrosis diagnosed as a child; mild dyspnea in adulthood; age 40 CXR “worsening reticulonodular interstitial infiltrates.” |
| III:7 | M | 44 | 1990 | Alive | Diagnosed as “environmental lung scarring”; CXR “bilateral scarring.” |
| IV:1 | F | 37 | 1999 | Alive | Dyspnea, cough; TLC 70%, DlCO 60%; chest CT “bilateral patchy interstitial opacities and honeycombing”; path report UIP. |
| V:1 | F | 17 mo | 1997 | Alive | Respiratory failure; RSV pneumonia before disease onset; path report NSIP. |
| V:2 | M | 4 mo | 1998 | Alive | Respiratory failure; influenza B pneumonia before disease onset; path report NSIP. |
Figure 2. Photomicrographs of representative lung tissue. (A) UIP. This low power view of patient III:3's lung biopsy shows coarse scarring with airspace dilatation and metaplastic cuboidal to columnar epithelium (left), normal alveolar tissue (right), and two fibroblast foci at the interface (arrowheads; hematoxylin and eosin ×12.5). (B) Cellular NSIP. Autopsy lung tissue from patient III:5 shows homogenous alveolar septal thickening by a cellular infiltrate and intra-alveolar cells and granular material (hematoxylin and eosin ×12.5).
[More] [Minimize]Blood and paraffin-embedded lung tissue was used for isolation of DNA using a PureGene Kit (Gentra Systems, Minneapolis, MN). Control DNA was the Ethnic Diversity Set (DNA Polymorphism Discovery Resource; Coriell Cell Repositories, Camden, NJ). Control lung tissue was resected for malignancy.
A microsatellite marker within 9 kb of SFTPC was used for linkage analysis. Primers (fluorescently labeled) used for polymerase chain reaction of the marker have been described (18). Alleles were analyzed on an automated gene sequencer (Applied Biosystems, Foster City, CA). A logarithm of the odds score was calculated assuming autosomal dominant inheritance of a gene with a disease allele frequency of 0.0001. The marker variant was assumed to have an equally rare allele frequency. Using the program VITESSE (19), analysis was done assuming that apparently unaffected individuals were not disease gene carriers, using phenotype information on only affected individuals.
For mutational analysis, polymerase chain reaction primers (Integrated DNA Technologies, Coralville, IA) used to amplify SFTPC have been described (20) (Figure 3)
Figure 3. Diagram of SFTPC gene. (A) Introns (open bars), exons I–V (closed bars), and untranslated exons V–VI (cross-hatched bars) are shown. Scale below gene is in kilobases. Approximate location of exon 5 + 128 T→A mutation is shown above exon 5. Location of polymerase chain reaction primers (A–I, 1–15) is shown below gene. The right arrow represents forward primer, and the left arrow represents reverse primer (A and B = first segment, C and D = second segment, C and E = nested second segment, F and G = restriction analysis segment, H and I = mutation site direct primers). (B) Sequence of SFTPC primers A–G. (C) Sequence of SFTPC dideoxy-fingerprinting primers 1–15.
[More] [Minimize]Using a Bsr1 restriction site created by the mutation, the presence or absence of the exon 5 + 128 T→A mutation was screened from DNA of available family members and 88 control chromosomes. A 549 base pair (bp) exon 5 polymerase chain reaction amplicon was assessed for the presence of a Bsr1 site with restriction products (New England Biolabs, Beverly, MA) resolved on a 2% metaphor agarose gel.
Lung tissue sections (3–5 μm) from patient III:3 were deparafinized in xylene and hydrated through graded ethanols. Using the Ventana ES (Ventana Medical Systems, Tucson, AZ), immunostaining was performed using diluted rabbit polyclonal antibody to SP-C precursor protein (proSP-C). Avidin-biotin staining with diaminobenzidine as the chromogen and counterstaining with hematoxylin were performed.
Normal full-length SFTPC was amplified from unaffected family member DNA. pGEMTeasy was transformed with this amplicon. Inserts were excised and ligated into pCINeo using Not1, Nhe1, and Spe1 sites. This plasmid/insert was used to create the mutation by site-directed mutagenesis using the Quickchange kit (Stratagene, La Jolla, CA) and mutation-overlapping primers (Figure 3). Mouse lung epithelial (MLE12) cells were transfected with plasmids containing normal and mutant SFTPC, using FuGENE6 (Roche, Indianapolis, IN). Pooled stable lines were grown in Hite's media under G418 selection (American Type Culture Collection, Manassas, VA). Supernatants and lysates of 105 viable nontransfected, wild-type, or mutant SFTPC-transfected MLE cells incubated for 24 hours were assayed for cellular toxicity using the lactate dehydrogenase (LDH)-based CytoTox96 Assay (Promega, Madison, WI). Statistical significance was tested with the unpaired Student's t test.
Electron microscopy (EM) methods have been described (21). Briefly, lung tissue was fixed in Karnovsky's solution and then postfixed and dehydrated. Embedded sections were examined by light microscopy. Thin sections were cut and mounted on grids that were observed on a Phillips 300 electron microscope.
Under the assumed autosomal dominant inheritance, a logarithm of the odds score of 4.33 at a recombination fraction of 0.00 was generated between the FPF phenotype and the marker. An abnormal dideoxy-fingerprinting pattern was obtained from polymerase chain reaction fragments containing exon 5 from DNAs of three affected family members (data not shown). Sequencing of these three affected member's DNA revealed a heterozygous exon 5 + 128 T→A transversion (Figure 4)
Figure 4. Sequencing gel of three affected patients and one control subject. Numbers below gel represent patient identifiers shown in Figure 1. (A) Heterozygous T→A mutation is evident at genomic position exon 5 + 128 in three patients (arrows). Sequence of segment is shown to right of gel. (B) Amino acid sequence homology of carboxy terminal region of SFTPC containing leucine at position 188 (bold text), which is highly conserved across species (underlined).
[More] [Minimize]
Figure 5. Restriction analysis. (A) Condensed pedigree of family. Numbers below symbols correspond to patient identifiers shown in Figure 1 (circle = female, square = male, open symbol = unaffected, close symbol = affected, slash through symbol = deceased). (B) Bsr1 restriction digest of 546 base pair (bp) fragment containing exon 5 + 128 T→A mutation. Each lane corresponds to pedigree symbol directly above. Affected individuals have three bands with sizes in bp noted on the right. Unaffected individuals have single band. (C) Exon 5 amplicon showing sequence of mutant and normal DNA. Bsr1 restriction site is underlined.
[More] [Minimize]In lung from patient III:3, immunostaining for proSP-C was evident without antigen retrieval within rows of abnormal type II cells lining thickened alveoli and terminal airways. Large, dysplastic, cuboidal type II cells were seen proliferating throughout affected areas of lung. Staining for proSP-C revealed diffuse cytoplasmic distribution in type II cells. In contrast, in control type II cells, proSP-C was localized adjacent to lamellar bodies (Figure 6)
Figure 6. Photomicrographs of immunohistochemistry. (A) Lung of a normal adult subject immunostained for proSP-C. Type II cell shows predominately focal brown staining of the cytoplasm adjacent to lamellar bodies, which are evident as clear vesicles unstained (arrows) (×175). (B) Lung immunostained for proSP-C, taken from an explanted lung from FPF patient III:3. Two cuboidal type II cells show diffuse brown cytoplasmic staining. No obvious lamellar bodies are seen (×175).
[More] [Minimize]Lung tissue from two affected family members was notable for dense fibrosis and distorted cellular architecture. Dysplastic type II cells contained many abnormal-appearing lamellar bodies. Some lamellar bodies were seen associated with normal-appearing multivesicular bodies. Many type II cells appeared to be sloughing from their underlying basement membranes and were notable for questionable cell viability. Frequently, type II cells displayed unidentifiable electron-dense cytoplasmic dark bodies, which may represent multivesicular bodies emerging from the Golgi apparatus unable to be completely processed. MLE cells transfected with mutant SFTPC revealed dysplastic, atypical morphology compared with wild-type SFTPC-transfected MLE cells. Mutant MLE cells contained excessive, atypical inclusions resembling lamellar bodies that were not seen in wild-type MLE cells (Figure 7)
Figure 7. Electron microscopy images. (A) Lung from FPF patient IV:1. Type II cell (center) shows abundant, atypical lamellar bodies (arrow) accumulating in the cytoplasm. A loss of basement membrane and cell–cell contact (bottom) is present as type II cell sloughs into airway filled with amorphous debris (top, right). Dense fibrosis is evident (left) (×8,000). (B) Type II cell from FPF patient IV:1 shows cytoplasmic electron-dense dark bodies (arrows) (×40,000). (C) MLE cells transfected with normal SFTPC (×5,600). (D) MLE cell transfected with SFTPC containing the exon 5 + 128 T→A transversion. Excessive inclusions resembling lamellar bodies are evident accumulating in the cytoplasm (arrows) (×4,400).
Mutant SFTPC-transfected cells displayed sluggish growth rates compared with nontransfected and wild-type SFTPC-transfected cells, taking several days longer to grow to confluence on culture plates. The percentage of nontransfected, wild-type SFTPC-transfected, and mutant SFTPC-transfected MLE cells displaying cytotoxicity (percentage of cytotoxicity = LDH activity supernatant/LDH activity cell lysate) was 5.8 ± 1.4%, 4.6 ± 2.3%, and 11.9 ± 1.9%, respectively (p < 0.05 for comparison between both nontransfected and mutant MLE cells and between wild-type and mutant MLE cells) (Figure 8)
Figure 8. MLE cytotoxicity assay. Cell lines are shown on x axis (NT = nontransfected MLE cells, WT = normal SFTPC-transfected MLE cells, MUT = exon 5 + 128 T→A SFTPC-transfected MLE cells). The percentage of cytotoxicity = LDH activity of cell supernatant/LDH activity of cell lysate. Data shown represents average of eight experiments, with standard deviation represented by error bars. *p < 0.05 for comparison between MUT-NT and between MUT-WT.
[More] [Minimize]Evidence that the SFTPC exon 5 + 128 T→A transversion in our family causes disease is supported by the following findings: (1) The SFTPC marker variant segregated with disease in all available affected individuals and two unaffected obligate heterozygotes. Logarithm of the odds score calculations confirmed complete coinheritance and gave a highly significant score of 4.33 at no recombination. This logarithm of the odds score represents nominal odds of over 20,000:1 that this gene is associated with FPF in this family. (2) The mutation was not seen in 88 control chromosomes and thus is not likely to be a polymorphism. (3) The mutation encodes substitution of a highly conserved nonpolar leucine residue by a polar glutamine residue (L188Q) and occurs in the C-terminal domain of proSP-C, a region proven to be crucial to proper folding/processing of proSP-C (22–24). (4) The mutation was present on only one allele, consistent with the previously reported autosomal dominant mode of transmission in FPF (1–3, 10, 11). (5) In vitro, cells stablely transfected with SFTPC containing the point mutation displayed inclusions resembling aberrant lamellar bodies and were associated with slow growth rates and excess cellular toxicity, indicating deranged cell function.
SP-C is a hydrophobic integral membrane protein expressed exclusively by type II epithelial cells in the lung. Human SFTPC is located on chromosome 8p21 and is translated as a large precursor (197 amino acids, 21 kD). Posttranslational processing of proSP-C involves folding and modification in the endoplasmic reticulum and Golgi body, followed by proteolysis of the N- and C-terminal domains in the multivesicular body, completed with storage of mature SP-C (35 amino acids, 3.7 kD) in the lamellar body associated with phospholipids (25). From this compartment, it is secreted into the alveolar space. Together with SP-B, mature SP-C maintains the biophysical surface activity of lipids through adsorption at the air–fluid interface of the alveolar lining and may also protect the surfactant film layer from protein and fluids (26).
In vitro studies have shown that the C-terminal domain plays a critical role in targeting and processing of proSP-C. For instance, in Chinese hamster ovary cells transfected with a mutant form of SFTPC whose truncated proSP-C product lacks the C-terminal 22 amino acids, proSP-C is restricted to the endoplasmic reticulum (23). Likewise, in A549 cells transfected with SFTPC deletional mutants whose products lack the C-terminal 10 amino acids of proSP-C, the proprotein remains localized in the endoplasmic reticulum without proper proteolysis (22). The conserved L188 residue that is altered by the mutation found in this kindred lies within this critical region. In addition, in studies using similar in vitro constructs containing a rat proSP-C C-terminal point mutation (C186G) of a conserved cysteine residue corresponding to the human proSP-C residue (C189) adjacent to L188, proSP-C is not processed correctly and is retained in early secretory compartments associated with misfolded protein aggregates (24).
There is increasing evidence that implicates SP-C in the pathophysiology of some types of chronic lung disease. Intracellular accumulation of incompletely processed proSP-C has been demonstrated in SP-B–deficient infants with congenital alveolar proteinosis (27). Multiple heterozygous mutations in SFTPC have been reported in association with children suffering from interstitial lung disease (16, 28), including familial and sporadic occurrences. Most of these cases likely represent desquamative interstitial pneumonitis or NSIP. Similar to this report, many of these mutations occur within the C terminus of proSP-C, including a patient with a mutation encoding substitution (L188R) of the same conserved leucine residue that is altered in our kindred (L188Q). In addition, a deficiency of SP-C has been described in a small kindred suffering from a poorly defined form of interstitial pneumonitis, despite no sequence variation in SFTPC (29). To our knowledge, the SFTPC exon 5 + 128 T→A transversion that we describe in this kindred is the first description of an SP mutation associated with UIP.
Therefore, we propose the L188Q mutation may cause misfolding and trapping of proSP-C in or near the endoplasmic reticulum without delivery to distal secretory compartments. This is supported by immunohistochemistry, as unlike control lung tissue, proSP-C did not localize adjacent to type II cell lamellar bodies in diseased lung tissue but was found diffusely throughout the cytoplasm (Figure 6). The many abnormal inclusions resembling lamellar bodies seen by EM of affected lung tissue and mutant SFTPC-transfected MLE cells could possibly be secondary to this abnormal processing of proSP-C. Although the identity of the dark bodies seen by EM accumulating in the cytoplasm of affected lung type II cells is unclear, electron-dense inclusions possibly representing early multivesicular bodies have been described in EM studies of infants with respiratory distress and SP-B deficiency (30), SP-B −/− deficient mice with unprocessed proSP-C (21) and in in vitro transfection studies with C-terminal proSP-C point mutants associated with misfolded protein aggregates (24).
The mutation we report has the potential to cause alveolar type II cellular injury, as transfection of MLE cells with SFTPC differing in only one single bp (exon 5 + 128 T→A) results in a threefold increase in cytotoxicity compared with MLE cells transfected with the normal gene. On the EM of affected lung tissue, type II cellular injury is suggested, as many cells lack basement membranes and lose structural integrity or cell–cell contact. Such morphologic findings have been described in early type II cellular death or apoptosis (31, 32). Alveolar type II cell injury and altered re-epithelialization associated with an excessive fibrotic phenotype has been implicated in pulmonary fibrosis (33, 34). Although the exact mechanism of alveolar epithelial injury associated with this SFTPC mutation will likely require animal models to be elucidated, accumulation of misfolded proSP-C could eventually lead to type II cellular damage. Similar pathophysiology is seen in forms of α1-antitrypsin deficiency, where the Z mutant protein is misfolded, leading to hepatocyte injury and cirrhosis (35). The list of genetic disorders associated with protein misfolding is growing, including autosomal dominant diseases such as congenital nephrogenic diabetes insipidus, protein C deficiency, and hereditary blindness (36).
The presence of the exon 5 + 128 T→A mutation on one allele suggests that it may have a dominant negative effect on the function or processing of SP-C. SP-C can form oligomers and interacts with surfactant protein B (37). Oligomeric sorting in the trafficking of proSP-C was recently shown to occur in vitro, as proSP-C was retained in early secretory compartments in deletional mutants lacking endogenous targeting signals, but the same cells cotransfected with full length proSP-C were capable of secretion to late compartments (38). Thus, the abnormal protein theoretically could hinder trafficking of normal proSP-C or possibly surfactant protein B. Also, a deficiency in mature secreted SP-C may be contributing to the pathophysiology of this disease as well. This is supported by the observation that SP-C −/− mice have normal lung function at birth but survive into adulthood with abnormal surfactant that is unstable at low lung volumes (39). Deficiency of SP-C could theoretically promote alveolar instability, leading to lung inflammation and injury.
Interestingly, two different subsets of pulmonary fibrosis were found by pathologic examination to exist in members of our family who share a mutation in SFTPC. We cannot exclude the possibility of cellular NSIP occurring as a precursor lesion to UIP in this family. Although some investigators believe that predominately inflammatory subsets of pulmonary fibrosis such as desquamative interstitial pneumonitis are early stages of UIP (40, 41), current evidence suggests they are separate pathologic and clinical entities (42). More likely, cellular NSIP and UIP are pleiotropic effects of the genetic defect that are occurring in our kindred. It is possible that the forms of FPF differ on the molecular level from sporadic pulmonary fibrosis, resulting in greater diversity in clinical and pathologic features. This is evident in this study, where some patients with a pathologic diagnosis of UIP had disease onset in childhood or early adulthood with long durations of illness, in contrast to the more aggressive course of sporadic IPF with later disease onset. The genetic defect in this family does not display complete penetrance, as two unaffected (to date) family members heterozygous for the mutation were found in this kindred. Reduced penetrance in familial IPF has similarly been reported by multiple authors (1–3, 10, 11). Thus, secondary modifiers likely affect the penetrance of this SFTPC mutation. Although the role of environmental influence on SFTPC mutations has not been studied, triggers such as infection or toxins may influence the wide diversity in clinical presentation of SFTPC-associated pulmonary fibrosis. Respiratory viral infections were temporally related to onset of disease in at least three members of this family. Multiple viral agents have been suggested to play a role in pulmonary fibrosis, including Epstein-Barr virus, influenza, and hepatitis C (43). Unlike surfactant proteins A and D, no antiviral or immune properties have been associated with SP-C (25).
In conclusion, we report a mutation in SFTPC associated with a large FPF kindred, including adults with the most common pathologic subset of pulmonary fibrosis, UIP, and children with cellular NSIP. The mutation in vitro is associated with cellular toxicity; thus, type II cell damage may underlie the pathogenesis of pulmonary fibrosis associated with this mutation. Our results suggest that within this kindred, different pathologic forms of FPF may represent pleiotropic manifestations of the same SFTPC mutation.
The authors thank the subject family for participation in this study and multiple physicians and colleagues, including Sandy Olsen, Brent Weedman, and Terry Johnson, for technical assistance and Dr. Jeff Whitsett, University of Cincinnati Hospital (MLE cells, proSP-C antibody). For the loan of tissue specimens, the authors thank Drs. J. Douglass Rolf, Dean Chamberlain, Rajni Chibbar, Ernest Cutz, Ted Jones, Lakshmi Puttagunta, and D. L. Webber. For the loan of medical records, the authors thank Dr. Edward Poon.
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