Surfactant Protein C Mutations Are the Basis of a Significant Portion of Adult Familial Pulmonary Fibrosis in a Dutch Cohort

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1 Surfactant Protein C Mutations Are the Basis of a Significant Portion of Adult Familial Pulmonary Fibrosis in a Dutch Cohort Coline H. M. van Moorsel 1,6, Matthijs F. M. van Oosterhout 2, Nicole P. Barlo 1, Pim A. de Jong 3, Joanne J. van der Vis 4, Henk J. T. Ruven 4, H. Wouter van Es 5, Jules M. M. van den Bosch 1,6, and Jan C. Grutters 1,6 1 Department of Pulmonology, 2 Department of Pathology, 4 Department of Clinical Chemistry, and 5 Department of Radiology, St Antonius Hospital, Nieuwegein, The Netherlands; and 3 Department of Radiology and 6 Division of Heart and Lung, University Medical Center Utrecht, Utrecht, The Netherlands 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 ATPbinding 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, IVS412, 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. Keywords: idiopathic pulmonary fibrosis; ABCA3; idiopathic interstitial pneumonia; lung cysts Idiopathic interstitial pneumonias (IIP) are potentially lethal lung diseases, histologically characterized by diffuse interstitial fibrosis and patchy inflammation. The most common form, idiopathic pulmonary fibrosis (IPF), is found in approximately 50% of patients with IIP and represents a category with very poor prognosis (1). Patients with IPF have a median survival of just 2 to 5 years (2). Mortality is caused by progressive respiratory insufficiency due to an unremitting fibrotic process that is not (Received in original form June 25, 2009; accepted in final form July 22, 2010) This study was supported by the Department of Pulmonology at St Antonius Hospital, Nieuwegein, The Netherlands. Correspondence and requests for reprints should be addressed to Coline H. M. van Moorsel, Ph.D., Department of Pulmonology, St. Antonius Hospital Nieuwegein, Koekoekslaan 1, 3435CM Nieuwegein, The Netherlands. c.van.moorsel@ antoniusziekenhuis.nl This article has an online supplement, which is accessible from this issue s table of contents at Am J Respir Crit Care Med Vol 182. pp , 2010 Originally Published in Press as DOI: /rccm OC on July 23, 2010 Internet address: AT A GLANCE COMMENTARY Scientific Knowledge on the Subject 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. What This Study Adds to the Field 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. responsive to therapeutic agents. Although the cause of the disease is unknown, a significant percentage of patients have a familial form of the disease, suggesting genetics might play a role (3, 4). Two to nineteen percent of patients have been reported to have at least one first-degree family member with some form of IIP (5 8), and clinical, pathological, and radiological characteristics of patients with familial pulmonary fibrosis (FPF) have been described to be generally similar to sporadic IPF (6, 9, 10). 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).

2 1420 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 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). METHODS Patient Selection 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 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. Clinical Characteristics 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). DNA Sampling 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. DNA Sequence Analysis 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 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). Mutation Analysis 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 singlenucleotide polymorphism assay (Illumina Inc., San Diego, CA). Variations SFTPC M71V, SFTPC IVS412, 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 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 IVS412 mutation (Figure 2, pedigree B IV-8) and a control and performed a polymerase chain reaction on reversetranscribed 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 (1) in Figure 2. RESULTS Characteristics of the FPF Group 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. 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). SFTPC Mutation Analysis 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 IVS412 segregated with disease in the pedigrees of FPF7 and FPF10, respectively (Figure 2). SIFT analysis predicted delete- Figure 1. Flowchart representing patient selection and availability of DNA. *Including one sib-pair: one sib was excluded from further analyses. COP 5 cryptogenic organizing pneumonia; DIP 5 desquamative interstitial pneumonia; FPF 5 familial pulmonary fibrosis; IIP 5 idiopathic interstitial pneumonia; IPF 5 idiopathic pulmonary fibrosis; NSIP 5 nonspecific interstitial pneumonia; RB-ILD 5 respiratory bronchilolitis associated interstitial lung disease.

3 van Moorsel, van Oosterhout, Barlo, et al.: SFTPC Mutations in a Dutch FPF Cohort 1421 Figure 2. Pedigrees for index patients carrying a mutation in the gene encoding surfactant protein C (SFTPC). (A) FPF7. (B) FPF10. (C) FPF9. (D) FPF18. (E ) FPF20. Arrow indicates index patient. Labels below symbols are individual identifiers and age deceased. Ltx nr 5 age at lung transplantation; plus sign indicates DNA sampled. Mutations are indicated above the symbols: left 5 SFTPC mutation; right 5 ABCA3 mutation. Filled symbol 5 pulmonary fibrosis; shaded symbol 5 lung disease: pedigree A II-1 5 lung carcinoma; pedigree B I-1 5 died from pneumonia; pedigree D II-15 died from respiratory insufficiency; pedigree E II-4 died from respiratory insufficiency. Age at diagnosis (Adx) and histopathological findings in family members: pedigree A II-2, Adx 5 35: DIP superimposed on UIP pattern; pedigree B II-1, Adx 5 35: family report pulmonary fibrosis; B IV-8, Adx 5 1: NSIP; pedigree C II-1, Adx 5 40: UIP; C III-2, Adx 5 30: UIP; pedigree D III-2, Adx 5 1: DIP/ NSIP; pedigree E II-2, Adx 5 70: UIP. rious consequences for both I73T and M71V (Figure 4C) and RNA analysis showed that the IVS412 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). ABCA3 Mutation Analysis 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 Medical History of Mutation Carriers and Family 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 TABLE 1. DEMOGRAPHICS AND CLINICAL CHARACTERISTICS OF UNRELATED PATIENTS WITH FAMILIAL PULMONARY FIBROSIS AND PATIENTS WITH SPORADIC IDIOPATHIC PULMONARY FIBROSIS FPF (n 5 22) Sporadic IPF (n 5 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) DL CO, (SD) 44% (618) 46% (617) VC (SD) 74% (622) 74% (623) Smoking, never/ former/ current 8/14/0 27/65/3 Definition of abbreviations: DL CO 5 diffusing capacity for carbon monoxide measured around diagnosis; FPF 5 familial pulmonary fibrosis; IPF 5 idiopathic pulmonary fibrosis; VC 5 vital capacity. Censoring lung transplant recipients. Expressed as percentage of predicted values. * P, 0.05.

4 1422 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL TABLE 2. CHARACTERISTICS OF UNRELATED GENOTYPED PATIENTS WITH FAMILIAL PULMONARY FIBROSIS Patient Sex Age at Diagnosis, yr Survival after Diagnosis Radiological Pattern Histological Pattern 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 UIP1mild OP FPF5 M 40 Ltx 64 mo, Reltx 65 mo, Died 84 mo NSIP UIP1DIP-like reaction FPF6 M 59 Died 13 mo UIP UIP1DIP 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 Definition of abbreviations: DIP 5 desquamative interstitial pneumonia; F 5 female; fibr 5 fibrotic; ltx 5 lung transplantation; M 5 male; np 5 not performed; NSIP 5 nonspecific interstitial pneumonia; OP 5 organizing pneumonia; reltx 5 retransplantation; UIP 5 usual interstitial pneumonia. 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). DISCUSSION 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 IVS412 mutation in FPF10 caused a splice-site alteration that resulted in deletion of exon 4 in mrna. The first ever reported SFTPC mutation, IVS411(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 smokingrelated 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 Figure 3. Differences in nucleotide composition of haplotypes of surfactant protein C with the I73T mutation in FPF9, FPF18, and FPF20. Single nucleotide polymorphisms (SNPs) are given in the following order: rs , rs , rs , rs , rs , rs , rs , rs , rs4715, rs , rs , rs , rs , rs7592, and rs Filled rectangle 5 exon; arrow 5 position of SNP; n 5 phase could not be determined.

5 van Moorsel, van Oosterhout, Barlo, et al.: SFTPC Mutations in a Dutch FPF Cohort 1423 Figure 4. Consequences of mutations in the gene encoding surfactant protein C (SFTPC). (A) Polymerase chain reaction (PCR) on reverse transcribed RNA from lung tissue. Lanes 1 and 5: ladder with band sizes indicated at the left. Lane 2: wild type. Lane 3: IVS412 carrier. Lane 4: water. Expected length of PCR product is 311 bp; deletion of exon 4 causes a product of 200 bp (arrow). (B) Sequence of small IVS412 PCR product that was isolated from gel, demonstrating the absence of exon 4. (C ) Scaled probability matrix for SFTPC amino acid positions 67 to 77 produced by the program Sorting Intolerant From Tolerant (SIFT). Left column: reference amino acid position and composition; header row in silico evaluated mutations. Substitutions predicted to be intolerant are highlighted in red (P, 0.05). Values for mutations M71V and I73T are underlined. 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 welldesigned 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 TABLE 3. NEWLY IDENTIFIED VARIANTS IN SURFACTANT PROTEIN C AND ATP-BINDING CASSETTE SUBFAMILY A MEMBER 3 Allele Frequency Gene cdna Position Variant Name Consequence FPF sp.iip Control Subjects SFTPC c.211 A.G M71V Nonsynonymous FPF7 0 0 c.218 T.C I73T Nonsynonymous FPF9, FPF18, FPF c T.C IVS412 Splice site FPF ABCA3 c.839g.a R280H Nonsynonymous c.863g.a R288K Nonsynonymous FPF c.3784 A.G S1262G Nonsynonymous FPF Definition of abbreviations: ABCA3 5 gene encoding ATP-binding cassette subfamily A member 3; SFTPC 5 gene encoding surfactant protein C; sp.iip 5 sporadic idiopathic interstitial pneumonia. For frequency determination 121 patients with sp.iip and 100 healthy control subjects were used. SFTPC I73T and ABCA3 S1262G were genotyped in 511 healthy control subjects.

6 1424 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL ; international project number 76, European Pulmonary Alveolar Proteinosis Network: Molecular Determinants of Causes, Variability and Outcome. Acknowledgment: 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. Figure 5. High-resolution computed tomography chest scans of carriers of mutations in the gene encoding surfactant protein C. FPF7: Diffuse lung disease without anterior-posterior, apical-basal, or central-peripheral gradient. Mild abnormalities consisting of mild nodular septa thickening and seven round cysts of 5 to 15 mm in diameter. FPF9: Diffuse lung disease without anterior-posterior, apicalbasal, or central-peripheral gradient. Severe ground glass, mild nodular septa thickening. Approximately 15% of the lung volume is occupied with mostly intraparenchymal, round clustered cysts sized 5 to 15 mm. FPF10: Diffuse lung disease without anterior-posterior, apical-basal, or central-peripheral gradient. Severe ground glass, severe nodular septa thickening. Approximately 20% of the lung volume is occupied with mostly intraparenchymal, round clustered cysts sized 5 to 20 mm. FPF18: Diffuse lung disease without anterior-posterior, apical-basal, or centralperipheral gradient. Approximately 30% of the lung volume is occupied by largely clustered, intraparenchymal, round cysts with diameters ranging from 5 to 30 mm. Severe septal thickening, with moderate nodular component. Diffuse ground glass. FPF20: Mild, predominantly peripherally located lung abnormalities consisting of ground glass and moderate nodular septa thickening. Three lung cysts sized 10 mm. 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. Author Disclosure: C.H.M.V.M. received $10,001 $50,000 from ZonMW as an EU research grant for alveolar proteinoses. M.F.M.V.O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.P.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.A.D.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.J.V.D.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.J.T.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.W.V.E. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.M.M.V.D.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.C.G. received $10,001 $50,000 from Erare PAP as a ZonMw grant, project number References 1. Garantziotis S, Steele MP, Schwartz DA. Pulmonary fibrosis: thinking outside of the lung. J Clin Invest 2004;114: Meltzer EB, Noble PW. Idiopathic pulmonary fibrosis. Orphanet J Rare Dis 2008;3:8. 3. Steele MP, Speer MC, Loyd JE, Brown KK, Herron A, Slifer SH, Burch LH, Wahidi MM, Phillips JA, Sporn TA, et al. Clinical and pathologic features of familial interstitial pneumonia. Am J Respir Crit Care Med 2005;172: Grutters JC, du Bois RM. Genetics of fibrosing lung diseases. Eur Respir J 2005;25: Loyd JE. 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