’ Eur Respir J 2008; 32: 1316–1320 DOI: 10.1183/09031936.00132707 CopyrightßERS Journals Ltd 2008 Novel mutations in the folliculin gene associated with spontaneous pneumothorax B.A. Fro¨hlich*, C. Zeitz#,",G.Ma´tya´s#, H. Alkadhi+, C. Tuor*, W. Berger# and E.W. Russi* ABSTRACT: Spontaneous pneumothorax is mostly sporadic but may also occur in families with AFFILIATIONS genetic disorders, such as Birt–Hogg–Dube´ syndrome, which is caused by mutations in the *Pulmonary Division, +Institute of Diagnostic Radiology, folliculin (FLCN) gene. University Hospital of Zurich, The aim of the present study was to investigate the presence and type of mutation in a Swiss #Division of Medical Molecular pedigree and in a sporadic case. Clinical examination, lung function tests and high-resolution Genetics and Gene Diagnostics, computed tomography were performed. All coding exons and flanking intronic regions of FLCN Institute of Medical Genetics, University of Zurich, Zurich, were amplified by PCR and directly sequenced. The amount of FLCN transcripts was determined Switzerland, and by quantitative real-time RT-PCR. "Institut de la Vision, INSERM U592, Two novel mutations in FLCN were identified. Three investigated family members with a history Universite´ Pierre et Marie Curie6, of at least one spontaneous pneumothorax were heterozygous for a single nucleotide substitution Paris, France. (c.779G A) that leads to a premature stop codon (p.W260X). Quantitative real-time RT-PCR CORRESPONDENCE revealed a reduction of FLCN transcripts from the patient compared with an unaffected family E.W. Russi member. DNA from the sporadic case carried a heterozygous missense mutation (c.394G.A). Pulmonary Division Lung function of this patient was normal and computed tomography showed similar bilateral University Hospital Raemistrasse 100 cysts, as observed in the two members of the unrelated Swiss family. CH-8091 Zurich Mutations in the folliculin gene are associated with cystic lung lesions in an otherwise Switzerland morphological normal lung and predispose to spontaneous pneumothorax. Fax: 41 442554451 E-mail: [email protected] KEYWORDS: Familial pneumothorax, folliculin gene, mutation, pneumothorax Received: October 09 2007 Accepted after revision: spontaneous pneumothorax is a collec- and renal cancer. It is also associated with an June 12 2008 tion of air in the pleural space of the lung, increased incidence of spontaneous pneumothorax A causing the lung to collapse. A majority of [8, 9]. In a study of 198 patients with BHDS, SUPPORT STATEMENT individuals with a spontaneous pneumothorax pneumothorax occurred in 24% of the cases [10]. It This study was funded by Forschungskredit University of Zurich have no obvious lung disease (‘‘primary’’ sponta- has been shown that mutations in the folliculin and by the Foundation Voir et neous pneumothorax), but intraoperative inspec- (FLCN) gene on chromosome 17p11.2 can cause this Entendre. tion or preoperative computed tomography (CT) syndrome [11]. Other mutations in FLCN have been scans generally reveal the presence of small associated with spontaneous pneumothorax and STATEMENT OF INTEREST subpleural blebs of the lung [1, 2]. The pathogen- bullous lung disease in the absence of the oncolo- Statements of interest for C. Zeitz and esis of these subpleural blebs is probably related to gical manifestations of BHDS [12, 13]. the study itself can be found at airway inflammation secondary, to some extent, to www.erj.ersjournals.com/ cigarette smoking in many cases. Pneumothorax The current authors have recently studied a Swiss misc.statements.shtml also occurs with increased rate in patients suffer- pedigree, with several members who had experi- ing from hereditary connective tissue disorders, enced a pneumothorax, and a 27-yr-old female such as Marfan syndrome and Ehlers–Danlos sporadic case after a first pneumothorax event syndrome [3, 4]. The clustering of pneumothorax and striking parenchymal lung changes on a events in families not affected by a connective high-resolution CT (HRCT). Based on the asso- tissue disease is well known, and autosomal ciation of lung cysts and pneumothoraces with dominant, autosomal recessive, X-linked reces- BHDS, affected family members from two gen- sive, as well as polygenic, inheritances have been erations and one sporadic case with similar cystic suggested [5–7]. The autosomal dominant Birt– lung structures have been screened for mutations European Respiratory Journal Hogg–Dube´ syndrome (BHDS) is a genoderma- in the FLCN gene. Two novel disease-associated Print ISSN 0903-1936 tosis predisposing patients to benign skin tumours DNA sequence alterations were detected. Online ISSN 1399-3003 1316 VOLUME 32 NUMBER 5 EUROPEAN RESPIRATORY JOURNAL B.A. FRO¨HLICH ET AL. FLCN MUTATIONS AND SPONTANEOUS PNEUMOTHORAX METHODS FLCN and POLR2A. After normalisation to POLR2A,theFLCN Patient recruitment and examination transcript expression was calculated relative to that of a All participants gave written informed consent for molecular and calibrator sample of normal, healthy family member III.1. clinical testing. Skin changes were excluded by physical examina- Repeated observations of relative expressions were analysed tion and kidney manifestations by abdominal ultrasound. by descriptive statistics. For the arithmetic mean, upper and lower confidence limits were calculated using critical values of Lung function paired t-test distribution [16]. Spirometry, whole body plethysmography and measurement of The ratio of allele-specific transcripts was quantified basically carbon monoxide diffusing capacity (DL,CO) were performed according to a procedure outlined by QIU et al. [17] by sequencing (6200 Autobox SensorMedics, Yorba Linda, CA, USA) according of mutation-harboring RT-PCR products on an ABI PRISM 3100 to standard criteria [14]. Reference values were in accordance to Genetic Analyzer (Applied Biosystems) using primers specific to the European Community for Steel and Coal [15]. exons 6 and 8 (details on reaction conditions are available upon request). CT Thin-section CT of the chest was performed with a 64-slice CT RESULTS scanner (Somatom Sensation 64; Siemens Medical Solutions, History and clinical features Forchheim, Germany). Patients were examined in the supine None of the individuals analysed in the present study had position. Inspiratory scans were obtained during suspended a-antitrypsin deficiency, a connective tissue disorder such as deep inspiration from the apices of the lung to the costophrenic Marfan or Ehlers-Danlos syndrome, nor skin or kidney angles. Examination parameters were 120 kV and 150 mAs, manifestations of BHDS. using a 5126512 matrix. Images with a slice thickness of 1 mm and an increment of 0.8 mm were reconstructed with a high The index patient of the Swiss family (III.3) was a 56-yr-old spatial frequency algorithm and analysed at window settings female (fig. 1) who consulted her family doctor after an appropriate for viewing lung parenchyma (window centre episode of shortness of breath during her preceding holidays. -600 HU; window width 1,500 HU). No intravenous contrast Her history is remarkable for a first pneumothorax after giving material was administered. birth to her oldest child (IV.3), and a second event three weeks before the birth of her second child (IV.4). A HRCT scan of the Mutation analysis thorax showed bilateral cystic lung lesions preferentially in Genomic DNA was isolated from circulating leukocytes using both lower lobes within otherwise radiologically normal lung the chemagic Magnetic Separation Module I (Chemagen parenchyma (fig. 2a). Her lung function was normal. Biopolymer-Technology AG, Baesweiler, Germany). The patient’s family was of Swiss descent. Deceased family All 11 coding exons and flanking intronic sequences of the members, i.e. her father (II.1), an uncle (II.3) and her grand- FLCN gene were amplified and sequenced. PCR amplifica- father (I.1), were also known to have experienced at least one tions, PCR product purification and cycle sequencing were pneumothorax episode. The patient’s brother (III.5) and her performed under routine conditions (details are available upon request). The detected sequence variants were verified by I repeated sequencing on newly amplified PCR products. The 1 2 control panel included .210 alleles from unrelated unaffected individuals of the Swiss population. FLCN transcript analyses II Total RNA was isolated using the PAXgene Blood RNA kit 1 23 (Qiagen, Hombrechtikon, Switzerland) from venous blood collected into PAXgene Blood RNA Tubes (Becton Dickinson, Basel, Switzerland) according to the manufacturers’ instruc- tions. The quality of the purified RNA (as determined by RNA III integrity number (RIN)) was assessed using an Agilent 2100 # 1234¶ # ¶ 56 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). RNA samples with RIN .5 were used as template for cDNA synthesis, which was carried out with a commercially available kit for reverse transcription using 4 mg total RNA and random IV primers (Superscript III kit; Invitrogen, Basel, Switzerland). 1 2 3 4 5 6 ¶# The amount of FLCN transcripts was determined by quantita- tive real-time RT-PCR performed on an ABI PRISM 7900 HT FIGURE 1. Pedigree of a Swiss family with spontaneous pneumothorax. Sequence Detection System (Applied Biosystems, Rotkreuz, Circles indicate females, squares indicate males, open symbols indicate unaffected Switzerland) using primers specific for FLCN and the endogen- individuals and filled symbols indicate affected individuals;