Rationale: Familial clustering of adult idiopathic interstitial pneumonias (IIP) suggests that genetic factors might play an important role in disease development. Mutations in the gene encoding surfactant protein C (SFTPC) have been found in children and families with idiopathic pneumonias, whereas cocarriage of a mutation in ATP-binding cassette subfamily A member 3 (ABCA3) was postulated to have a disease-modifying effect.
Objectives: To investigate the contribution of SFTPC mutations to adult familial pulmonary fibrosis (FPF) and the disease-modifying effect of mutations in ABCA3 within their families.
Methods: Twenty-two unrelated patients with FPF (10%) were identified within our single-center cohort of 229 patients with IIP. SFTPC was sequenced in 20 patients with FPF and 20 patients with sporadic IIP. In patients with an SFTPC mutation, sequencing of ABCA3 was performed. Discovered variants were typed in more than 100 control subjects and 121 additional patients with sporadic IIP.
Measurements and Main Results: In 5/20 unrelated patients with FPF (25%; confidence interval, 10–49) a mutation in SFTPC was detected: M71V, IVS4+2, and three times I73T. No mutations were detected in the sporadic or control cohort. Patients with SFTPC mutations presented with a histopathological pattern of usual interstitial pneumonia and nodular septa thickening and multiple lung cysts in combination with ground glass or diffuse lung involvement on chest high-resolution computed tomography. Two variants in ABCA3 were found in adult patients with FPF but not in affected children.
Conclusions: Mutations in SFTPC are a frequent cause of FPF in adult patients in our cohort. Nonclassifiable radiological patterns with cystic changes and histopathological patterns of usual interstitial pneumonia are characteristics of adult SFTPC mutation carriers.
Mutations in the gene encoding surfactant protein C (SFTPC) have been found in children and families with idiopathic pneumonias and could be a cause of sporadic and familial pulmonary fibrosis in adults.
This study demonstrates that SFTPC mutations are responsible for disease development in approximately 25% of cases with adult familial pulmonary fibrosis in a Dutch cohort. Mutation carriers are characterized by nonclassifiable high-resolution computed tomography chest scan patterns with cystic changes.
Mutations in the gene encoding surfactant protein C, SFTPC, have been frequently identified in children with severe idiopathic pneumonias (11–16). Approximately half of these children were sporadic cases with de novo mutations, and the other half had inherited the mutation from a parent (12, 13, 17). Family analysis revealed a dominant pattern of reduced penetrance and widely varying expressivity of lung disease (11, 18). In children, SFTPC mutations were associated with clinicopathological diagnoses, such as nonspecific interstitial pneumonia (NSIP), desquamative interstitial pneumonitis (DIP), and pulmonary alveolar proteinosis (PAP), whereas adults were frequently diagnosed with IPF and, to a minor degree, NSIP and DIP.
Surfactant protein C (SP-C) is a hydrophobic protein that is exclusively produced by type II pneumocytes and enhances the surface tension–reducing capacities of alveolar fluid (19). SP-C processing and secretion are dependent on ATP-binding cassette subfamily A member 3 (ABCA3), a lamellar body membrane protein (20, 21). Recently, it was shown that an unexpected high number of pediatric patients with SFTPC mutations cocarried an ABCA3 mutation, and it was therefore postulated that their combined presence modified disease severity (22).
FPF is often inherited in an autosomal dominant fashion with reduced penetrance, a model congruent with the dominant effect of carriage of an SFTPC mutation (18, 23). Although these studies have raised interest in a possible contribution of SFTPC mutations to adult FPF, no cohort analyses have been performed. Therefore, we analyzed the presence of mutations in SFTPC and possible disease-modifying effects of ABCA3 in mutation carriers in a Dutch cohort of adult patients with FPF and sporadic disease. Some of the results of these studies have been previously reported in the form of an abstract (24).
Medical records were reviewed from patients who visited the Interstitial Lung Disease outpatient clinic or had the diagnosis treatment code 1601 (Interstitial Lung Disease, not connective system disorder, not sarcoidosis) at St. Antonius Hospital Nieuwegein between 1998 and 2008. IIP diagnoses were established by a multidisciplinary team, in accordance with American Thoracic Society/European Respiratory Society criteria (25). Diagnoses made before 2002 were reviewed by an experienced clinician (J.v.d.B., J.G.) and included when current criteria were met.
Two hundred twenty-nine patients with IIP were identified (Figure 1). FPF was defined as two or more first-degree family members with IIP and was documented in 23 patients, including 1 sib-pair. In eight cases complete family history or documentation from clinical genetic centers was available. In 16 cases medical records from affected family members were reviewed; in 7 cases (i.e., FPF2, FPF8, FPF10, FPF13, FPF16, and the two patients with IPF who did not donate DNA) familial disease was based on self-reported lung disease in family members and personal communication with their respective physicians.
From 22 unrelated patients with FPF and 95 patients with sporadic IPF, we collected demographics, age at diagnosis, survival, lung function, and smoking history. In total, 72 patients had died and 12 patients were transplanted.
The Mann-Whitney U test was used to determine differences between the FPF and sporadic IPF group. The Kaplan-Meier method was used to describe survival time in each group, which was statistically evaluated for differences between the two groups with the log-rank test (SPSS 15; Chicago, IL).
Blood for DNA extraction was donated by 141 (136 white/5 nonwhite) patients with IIP (Figure 1). Among the 20 unrelated patients with FPF, 18 were Dutch; FPF4 is Indonesian, and FPF16 is from India. Family members of patients with FPF volunteered to donate DNA. DNA from the mother (II-2) of FPF7 was obtained from biopsied material. The study protocol was approved by the medical ethical committee from our hospital and all subjects gave formal written informed consent.
We sequenced SFTPC coding regions in 20 unrelated patients with FPF and 20 patients with sporadic disease. The 20 sporadic cases were relatively young at diagnosis (mean 43, range 26–55 yr) and had a disease that had in literature been described to occur in patients with SFTPC mutations (i.e., IIP or PAP). This sporadic group consisted of five patients with IPF, two patients with NSIP, nine patients with DIP, and four patients with PAP.
In patients with an SFTPC mutation we subsequently sequenced ABCA3 coding regions and available family members (see Tables E1 and E2 in the online supplement for primers).
The frequency of sequence variations was determined in 121 unrelated patients with IIP and in our Dutch control group of 100 self-reported healthy hospital employees. Mutations SFTPC I73T and ABCA3 S1262G were genotyped with an Illumina GoldenGate bead single-nucleotide polymorphism assay (Illumina Inc., San Diego, CA). Variations SFTPC M71V, SFTPC IVS4+2, and ABCA3 R288K were analyzed with high-resolution melting analysis (ABI Fast 7500RT; Applied Biosystems, Foster City, CA). Prediction of deleterious amino acid changes was performed online at http://sift.jcvi.org using default settings in Sorting Intolerant From Tolerant (SIFT) (26). To detect a splice site alteration we isolated RNA from explant lung tissue from a patient with IVS4+2 mutation (Figure 2, pedigree B IV-8) and a control and performed a polymerase chain reaction on reverse-transcribed cDNA. The delta exon 4 polymerase chain reaction product was isolated from gel and subsequently sequenced.
I73T-carrying haplotypes were deduced from familial segregation patterns of single-nucleotide polymorphisms in sequenced SFTPC regions. Sampled family members are indicated (+) in Figure 2.
Twenty-three patients with FPF were identified within a cohort of 229 patients with IIP, including one sib-pair. Demographics and disease characteristics in the FPF group were similar to those in the sporadic IPF group, except for a significantly lower age at diagnosis (Table 1). There was no indication of a relatively recent common ancestor for patients with FPF based on their places of birth or residence.
FPF (n = 22)
Sporadic IPF (n = 95)
|Patients, male/ female||15/7||79/16|
|Mean age at diagnosis, yr (range)||54 (29–75)*||63 (36–85)|
|Median survival,† mo, (range)||49 (3–88)||42 (1–90)|
|DlCO,‡ (SD)||44% (±18)||46% (±17)|
|VC‡ (SD)||74% (±22)||74% (±23)|
|Smoking, never/ former/ current||8/14/0||27/65/3|
Characteristics of the 20 patients with FPF who had donated DNA are summarized in Table 2, and pedigrees are given in Figure 2 and Figure E1. Biopsies were all characterized by a usual interstitial pneumonia (UIP) pattern, in two cases differentially diagnosed as fibrotic NSIP. In three patients with FPF, co-occurrence of UIP with DIP or organizing pneumonia patterns was found. Radiological patterns varied between UIP/possible UIP, NSIP/possible NSIP, and nonclassifiable (Table 2).
Age at Diagnosis, yr
Survival after Diagnosis
|FPF1||M||59||Died 3 mo||No classification||No classification|
|FPF2||M||64||Alive 52 mo||Possible UIP||UIP|
|FPF3||F||42||Ltx 45 mo, alive 67 mo||NSIP||Np|
|FPF4||M||57||Died 87 mo||Possible UIP||UIP+mild OP|
|FPF5||M||40||Ltx 64 mo, Reltx 65 mo, Died 84 mo||NSIP||UIP+DIP-like reaction|
|FPF6||M||59||Died 13 mo||UIP||UIP+DIP|
|FPF7||M||30||Alive 39 mo||No classification||UIP|
|FPF8||M||75||Died 58 mo||UIP||UIP|
|FPF9||F||29||Ltx 46 mo, alive 75 mo||No classification||UIP|
|FPF10||M||32||Died 10 mo||No classification||UIP|
|FPF11||M||63||Died 5 mo||Possible UIP||UIP|
|FPF12||F||53||Ltx 8 mo, alive 50 mo||No classification||UIP/fibr. NSIP|
|FPF13||F||74||Alive 40 mo||No classification||UIP/fibr. NSIP|
|FPF14||F||47||Ltx 20 mo, alive 38 mo||Possible UIP||UIP|
|FPF15||M||43||Died 18 mo||NSIP||UIP|
|FPF16||F||60||Alive 30 mo||NSIP||UIP|
|FPF17||M||58||Alive 9 mo||Possible NSIP||Np|
|FPF18||M||55||Alive 66 mo||No classification||UIP|
|FPF19||M||53||Alive 12 mo||Possible NSIP||UIP|
|FPF20||M||72||Alive 10 mo||No classification||Np|
SFTPC sequence analysis identified mutations in 5 out of 20 patients with FPF (25%; confidence interval, 10–49%); in the other 15 patients with FPF, wild-type SFTPC alleles were present. Mutation I73T was detected in three patients: FPF9, FPF18, and FPF20; however, haplotypes of I73T-carrying alleles were different in each family (Figure 3). Mutations M71V and IVS4+2 segregated with disease in the pedigrees of FPF7 and FPF10, respectively (Figure 2). SIFT analysis predicted deleterious consequences for both I73T and M71V (Figure 4C) and RNA analysis showed that the IVS4+2 mutation caused a splice site alteration with subsequent deletion of exon 4 (Figures 4A and 4B). None of mutations were present in the rest of patients with sporadic IIP or healthy control subjects (Table 3).
|Gene||cDNA Position||Variant Name||Consequence||FPF||sp.IIP||Control Subjects|
|c.218 T>C||I73T||Nonsynonymous||FPF9, FPF18, FPF20||0||0|
|c.435+2 T>C||IVS4+2||Splice site||FPF10||0||0|
Sequencing of exonic ABCA3 gene regions in patients with FPF with SFTPC mutations revealed two amino acid substitutions: S1262G and R288K (Table 3, Figure 2). In the kindred of FPF9, S1262G did not segregate with disease but was inherited from her healthy father. ABCA3-R288K was found in FPF20, whereas a third ABCA3 variant, R280H, was found in one patient with sporadic disease. No consequences on protein function were predicted by the SIFT analysis for either variant. All ABCA3 variants were also present in control subjects and had an allele frequency less than 0.02.
In patients with FPF and family members with SFTPC mutations, the age at disease onset ranged from 1 to 72 years. SFTPC mutation carriers FPF7, FPF9, FPF10, and FPF18 presented with a pattern of UIP on biopsy specimens. Although a biopsy was not performed on FPF20, a specimen from his sister (Figure 2, pedigree E II-2) was classified as UIP. Surprisingly, all mutation carriers presented with a chest high-resolution computed tomography (HRCT) pattern that was nonclassifiable and uncharacteristic for IPF, but revealed remarkable similarities that can be summarized as the presence of nodular septa thickening and multiple lung cysts between 5 and 30 mm (at least three cysts per patient) in combination with ground glass and/or diffuse lung involvement (Figure 5).
Within our center we identified 22 unrelated adult patients with FPF within a cohort of 229 patients with IIP. With that, our number of patients with FPF is one of the highest ever found. In the past, 8% of published cases with Hamman-Rich were found to have familial disease, whereas 19% of patients in an IPF lung transplant program reported a positive family history (5, 8). However, in more detailed studies, the frequency was estimated to be significantly lower: between 0.5 and 3.7% (6, 7). The high number of patients with FPF in our cohort might be a consequence of referral bias of relatively young and increasingly aware patients to our tertiary referral center. Even so, comparison between our FPF and sporadic IPF cohort showed that all variables correspond to those expected internationally (Table 1). We found disease development to be similar between the two groups and the age at diagnosis to be significantly lower in FPF, a result repeatedly found before, with FPF cohorts presenting on average 3.5 to 12 years earlier then patients with sporadic disease (6, 7, 9).
Our selection of patients with FPF consisted completely of adult patients with first-degree family members with IIP. It is remarkable that pediatric disease was only reported in three families that also carry an SFTPC mutation. The presence of pediatric lung disease in families with adult FPF might therefore be a characteristic of families carrying SFTPC mutations.
DNA analysis of 20 unrelated patients with FPF revealed that 5 carried a mutation in SFTPC that segregated with disease within their respective families and, in case of I73T, had originated independently on different haplotypes (Figures 2 and 3). The I73T mutation is well known to cause childhood and adult ILD (12, 13, 17, 27). The newly discovered IVS4+2 mutation in FPF10 caused a splice-site alteration that resulted in deletion of exon 4 in mRNA. The first ever reported SFTPC mutation, IVS4+1(11), had similar consequences and forms toxic intracellular aggresomes of mutant SP-C protein (28, 29).
Another new mutation, M71V, was found in FPF7. His mother, a never smoker, had been diagnosed with DIP, but review of the biopsy specimen showed the DIP pattern to be superimposed on a background of UIP, and macrophages did not contain the dark pigment that is characteristic for smoking-related DIP. Her sister had died of lung cancer, but medical documents describe the presence of extensive fibrosis in resectioned lung tissue. The mutation segregated with disease within this family (Figure 2) was absent in healthy control subjects and was in silico predicted to have deleterious functional consequences (Figure 4C). Furthermore, Dr. Kammenscheidt (Ambry Genetics) has recently found M71V in an infant with pulmonary disease who tested negative for mutations in surfactant protein B and ABCA3 (personal communication).
All five families with SFTPC mutations exhibited variable disease expressivity. Although children present with NSIP- and DIP-like features, most adults have histopathological findings of UIP. The largest FPF collection has been described by Steele and coworkers and contains 118 families consisting of approximately 80% patients with IPF (3). They and others discovered multiple IIP types per family (3, 30). Interestingly, our mutation carriers showed nonclassifiable chest HRCTs that could be characterized by nodular septa thickening and multiple lung cysts in combination with ground glass or diffuse lung involvement (Figure 5). Numerous articles describe pathohistological consequences of SFTPC mutations in children and sometimes in adults (11, 14, 16, 27, 31). However, descriptions of HRCT patterns in adult mutation carriers are rare. Thomas and colleagues summarized pathological findings in a kindred with the L188Q mutation and cited from old chest radiograph reports. Diffuse patterns, lucencies, nodular infiltrations, and ground glass were reported, whereas typical UIP features such as bibasilar predominantly peripheral changes were not described (31).
Genetically, heterozygosity for ABCA3 mutations was previously postulated to have a modifying effect on disease presentation (22). We found genetic changes in ABCA3 in two families, but neither change was predicted to have deleterious consequences. In the pedigree with FPF9 it can be seen that the sisters cocarrying the ABCA3 variation presented approximately 15 years earlier than those without the variation, allowing a possible influence on timing of disease onset (Figure 2). However, such an influence is not supported by the absence of variations in affected children and the presence in one of the oldest patients with FPF (FPF20). Thus, the effect of carriership of ABCA3 variations is not simply understood and a well-designed case-control study should be undertaken to investigate a possible influence on development of idiopathic lung fibrosis.
We are the first to report on the possible contribution of SFTPC mutations to disease development in a cohort of adult patients with FPF and found that 5/20 (25%; confidence interval, 10–40) carried a mutation. In recent studies, mutations in the genes encoding telomerase reverse transcriptase (TERT), telomerase RNA component (TERC), or surfactant protein A2 (SFTPA2) were detected in up to 15% of patients with FPF and in some sporadic cases (32–34). Future studies should be directed toward replication of these findings to estimate the true contribution of SFTPC, TERT, TERC, and SFTPA2 to FPF.
Limitations of this study are that from deceased relatives mutation carriership could not always be confirmed due to missing DNA, and that phenotypic data in the pedigrees was incomplete. Furthermore, we did not find SFTPC mutations in patients with sporadic disease, but only 20 patients with sporadic disease were sequenced. Two other studies evaluated a larger number of patients with sporadic disease, including 35 and 135 patients with IIP, respectively, and only 1 individual with an SFTPC mutation (I73T) was identified within a subgroup of 89 patients with UIP (35, 36).
In conclusion, in this study, SFTPC mutations were found in 5 out of 20 adult patients with FPF, but not in sporadic disease. Although a clear role for additional ABCA3 variations cannot be deduced from our samples, their absence in affected children shows that it is obviously not the only modifier of disease expression. Adult SFTPC mutation carriers were characterized by a histopathologically proven UIP pattern, and nonclassifiable, but characteristic HRCT patterns with cystic changes throughout the lung. This could help identify this particular group of patients for SFTPC mutation analysis.
The authors thank N. Pot and J. Broess for technical assistance in the laboratory. They also thank Dr Nossent and the University Medical Centers of Groningen and Utrecht for preparation of lung tissue for DNA and RNA analysis.
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