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Depts of 1 Internal Medicine, 2 Biochemistry, and, 3 Anaesthesiology, Intensive Care Medicine and Pain Therapy, Faculty of Medicine, University of Giessen Lung Center, Giessen, and Depts of 4 Anaesthesiology and Intensive Care Medicine, and 5 Gastroenterology, Charité, Campus Virchow-Klinikum, Universitatsmedizin Berlin, Berlin, Germany. 6 Authors contributed equally to the study.
CORRESPONDENCE: P. Markart, Dept of Internal Medicine, Faculty of Medicine, University of Giessen Lung Center, Klinikstr. 36, 35392 Giessen, Germany. Fax: 49 6419942509. E-mail: philipp.markart{at}innere.med.uni-giessen.de
Keywords: Genetics, interstitial lung disease, interstitial pneumonia, pulmonary fibrosis, surfactant protein C
Received: March 10, 2006
Accepted September 1, 2006
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ABSTRACT |
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In total, 35 adult patients with sporadic idiopathic interstitial pneumonia and 50 healthy subjects were investigated for SFTPC mutations by direct DNA sequencing. Of the patients with sporadic idiopathic interstitial pneumonia, 25 suffered from idiopathic pulmonary fibrosis and 10 patients from nonspecific interstitial pneumonia.
Only two frequent nonsynonymous variants, T138N and S186N, were detected. Allele frequencies of both variations as well as of other identified noncoding alterations did not differ significantly between the diverse patient groups and control subjects.
In conclusion, mutations in the gene encoding surfactant protein C are not common in sporadic cases of idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia, suggesting that the mutated gene does not play an important role in the pathogenesis of these forms of idiopathic interstitial pneumonia.
Idiopathic interstitial pneumonias comprise seven different entities including idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP) 1. IPF is characterised by the histological appearance of usual interstitial pneumonia (UIP) and by a poor prognosis with a median survival of 2–3 yrs 2. Approximately 3% of IPF cases are estimated to be familial 3.
Recent studies have suggested that some cases of familial interstitial pneumonias are associated with mutations in the gene encoding surfactant protein C (SFTPC). Most of these mutations occur in the C-terminal BRICHOS domain of the proprotein 4. This region is important for proper processing and folding of pro-SFTPC 5–7.
In 2001, Nogee et al. 8 reported a SFTPC splice site mutation affecting the first base of intron 4 (c.460+1G>A) in a mother and her infant, which resulted in skipping of exon 4 and a truncated protein with a deletion of 37 amino acids of the carboxyterminal region. The infant suffered from cellular NSIP, whereas the mother displayed desquamative interstitial pneumonitis. In vitro studies showed that the mutant causes apoptotic cell death by two mechanisms: on the one hand, this mutant accumulated in the endoplasmic reticulum (ER) leading to the induction of ER stress characterised by an exaggerated unfolded protein response; on the other hand, enhanced deposition of cellular aggregates of the mutated protein with an inhibition of proteasome activity indicating disruption of the ubiquitin/proteasome system was demonstrated 4.
For the first time, a SFTPC mutation associated with UIP, the histological correlate of IPF, was demonstrated in 2002. In this study, Thomas et al. 9 reported a heterozygous T to A transversion in exon 5, leading to the substitution of a highly conserved leucine residue by a glutamine residue at codon 188 (L188Q) in a large familial pulmonary fibrosis kindred. In this kindred of 97 members, 11 had pulmonary fibrosis, six adults showed a UIP pattern and three children a cellular NSIP pattern. An abnormal lamellar body formation and aberrant subcellular localisation of pro-SFTPC was demonstrated in type II cells of affected family members. Furthermore, in vitro studies demonstrated exaggerated toxicity of the L188Q mutant 9.
In 2002, Nogee et al. 10 analysed SFTPC in infants with chronic lung diseases of unknown aetiology and found heterozygous mutations in 11 out of 34 patients. In another study investigating 34 sporadic or familial cases with unexplained respiratory distress, two heterozygous SFTPC mutations, I73T and R167Q, were identified and found to be associated with pulmonary alveolar proteinosis with or without fibrosing lung disease 11.
In a recent study investigating SFTPC mutations in sporadic cases of adult IPF and NSIP, 13 out of 135 patients showed SFTPC variations that were not found in controls, but only one patient possessed an amino acid changing variant, I73T 12.
These data support the hypothesis that SFTPC mutations contribute to the pathophysiology of some types of interstitial lung diseases. The present study therefore investigated 35 adult patients with sporadic idiopathic interstitial pneumonia for SFTPC mutations.
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METHODS |
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shows the demographic characteristics, 6-min walk distance and results of pulmonary function testing of the patient cohort. Control subjects were 50 healthy German individuals without pulmonary diseases (medical staff and students; 23 female, 27 male; mean (range) age 34.4 (24–61) yrs).
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PCR was performed using 0.75 U AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA, USA), 400 µM deoxynucleoside triphosphates, 1.5 mM MgCl2 and 0.1 µM of each primer in a total volume of 25 µL. Cycle conditions were as follows: initial denaturation for 12 min at 95°C; 48 cycles of 20-s denaturation at 95°C, 40 s of annealing at 64°C and 90 s of primer extension at 72°C; and a final extension for 2 min at 72°C.
DNA sequencing
PCR products were digested with shrimp alkaline phosphatase (USB, Staufen, Germany) and exonuclease I (USB) and performed cycle sequencing using BigDye terminator mix (Applied Biosystems). For sequencing, the oligonucleotides used were: 5'-CCCAGGTTTGCTCTTGCTGG-3' and 5'-GAG-GAGGCAGGGCCCATCAC-3' (fragment 1); 5'-TCCAGCCCTAGGACGCCGTG-3', 5'-CTGTCTGGCATGTCCTGTGC-3', 5'-GATGGGTACCACTGGCTGAG-3', 5'-TGGGTC-AGGGAGAGAGCAGG-3' and 5'-CACTCCTCCCAGCAGCCCTG-3' (fragment 2); and 5'-GTCCCACAATAAGGGCTGCAC-3', 5'-GGGAGTGGGAAGTACCGGTC-3' and 5'-CTGGGACAGAGGGCGAATGG-3' (fragment 3). In doing so, the entire coding region, including the 5'- and 3'-UTR and the promoter sequence from -400, was analysed. Cycle conditions were as follows: initial denaturation for 3 min followed by 30 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 20 s and elongation at 60°C for 30 s. The reaction products were purified with Sephadex G-50 (Amersham, Freiburg, Germany) and loaded onto an ABI 3100 fluorescence sequencer (Applied Biosystems).
Statistical analysis
Statistical analysis was carried out using Fisher's exact test and p-values <0.05 were considered statistically significant.
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RESULTS |
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Strong linkage disequilibrium was found between the two coding SNPs, T138N and S186N, as previously described by a Finnish group: 138N was almost exclusively in cis with 186N 13. As for the allele frequencies of both SNPs, the frequencies of the estimated haplotypes did not differ significantly between the different patient groups and control subjects.
Additional variations located in the promotor, UTR or intronic regions were identified. Again, no significant differences in allele frequencies between patients and controls were observed. Table 2 summarises the allele frequencies of the detected variations in patients and control subjects.
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DISCUSSION |
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In general, data regarding a possible genetic basis of sporadic forms of interstitial pneumonia are rare. Polymorphisms in the genes coding for tumour necrosis factor-
, interleukin-1 receptor antagonist, angiotensin-converting enzyme and complement receptor 1 have been linked to sporadic cases of IPF 14–16. Transforming growth factor-ß1 polymorphisms have also been associated with the progression of IPF 17. To date, only one study has investigated SFTPC mutations in adult patients with sporadic forms of IPF or NSIP. In this study, 89 patients with IPF and 46 with NSIP were analysed, but only one IPF patient possessed a genetic sequence variation that predicted a change in the amino acid sequence (I73T) 12. In a 13-month-old infant with severe respiratory insufficiency, the same mutation was associated with combined histological patterns of NSIP and pulmonary alveolar proteinosis. Functional analyses showed that expression of mutant surfactant protein C propeptide (proSP-C) results in abnormal proprotein trafficking, leading to an accumulation of aberrantly processed proSP-C in the alveoli 18.
In contrast to sporadic forms of pulmonary fibrosis, SFTPC mutations might be more important in familial forms. Over 68 kindreds with familial idiopathic pulmonary fibrosis have been described so far 3, 19, 20. These familial forms are most probably transmitted in an autosomal manner with reduced penetrance. Some cases of familial pulmonary fibrosis were found to be associated with heterozygous SFTPC mutations 8, 9. Interestingly, different histopathological types of pulmonary fibrosis were found in members of the same kindred who shared the identical SFTPC mutation. These different forms may represent pleiotropic manifestations of the same genetic defect 9. Reduced penetrance is another feature of SFTPC-associated familial forms of pulmonary fibrosis, suggesting that additional endogenous or exogenous factors contribute to the marked diversity of pulmonary fibrosis predisposed by SFTPC mutations 9.
There are several mechanisms by which SFTPC mutations may contribute to the pathophysiology of pulmonary fibrosis. Recent in vitro studies indicate that an intracellular accumulation of incompletely processed and misfolded SFTPC may cause alveolar type II cell injury and apoptosis, thereby initiating fibrosis 4. This is in line with the currently accepted hypothesis that chronic injury of the alveolar epithelium is the underlying pathogenic event for initiating the fibrotic response in IPF 21, 22. While protein misfolding is one possible explanation for the development of fibrosis in patients with SFTPC mutations, it is also imaginable that fibrosis is caused by the lack of mature SFTPC; SFTPC-deficient mice develop severe progressive pulmonary disorder displaying histological features consistent with interstitial pneumonitis 23. Interestingly, mature SFTPC was absent in the lung tissue or bronchoalveolar lavage fluid of the infant with the recently described splice site mutation (c.460+1 G>A) 8. Furthermore, SFTPC deficiency has been described in a kindred with interstitial pneumonitis without a detectable SFTPC mutation 24.
In addition to mutations in SFTPC, mutations in another molecule associated with the pulmonary surfactant system were recently shown to occur in paediatric interstitial pneumonias; mutations in the ATP-binding cassette protein (ABC)A3 gene have been associated with fatal respiratory failure in neonates without deficiency in the surfactant proteins B and C and with non-fatal chronic interstitial pneumonitis in one older child 25. Further studies are necessary to elucidate the role of ABCA3 mutations in adult forms of interstitial pneumonias.
In summary, no pathogenic mutations of the surfactant protein C gene were identified in 35 adult patients with sporadic idiopathic pulmonary fibrosis or nonspecific interstitial pneumonia, indicating that, in contrast to familial pulmonary fibrosis, mutation of the surfactant protein C gene represents a rare cause of sporadic idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia.
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ACKNOWLEDGEMENTS |
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