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Novel mutations in the folliculin (FLCN) gene associated with
Fröhlich, B A; Zeitz, C; Mátyás, G; Alkadhi, H; Tuor, C; Berger, W; Russi, E W
Fröhlich, B A; Zeitz, C; Mátyás, G; Alkadhi, H; Tuor, C; Berger, W; Russi, E W (2008). Novel mutations in the
folliculin (FLCN) gene associated with spontaneous pneumothorax. European Respiratory Journal : official journal
of the European Society for Clinical Respiratory Physiology:Epub ahead of print.
Postprint available at:
Posted at the Zurich Open Repository and Archive, University of Zurich.
Originally published at:
European Respiratory Journal : official journal of the European Society for Clinical Respiratory Physiology 2008,
:Epub ahead of print.
Novel mutations in the folliculin (FLCN) gene associated with
Spontaneous pneumothorax (SP) is mostly sporadic but may also occur in families with genetic
disorders such as Birt-Hogg-Dubé syndrome (BHDS), which is caused by mutations in the folliculin
gene FLCN.To investigate the presence and type of mutation in a Swiss pedigree and in a sporadic
case.Clinical examination, lung function tests and HRCT. All coding exons and flanking intronic
regions of FLCN were amplified by PCR and directly sequenced. The amount of FLCN transcripts was
determined by quantitative real-time RT-PCR.We identified two novel mutations in FLCN. Three
investigated family members with a history of at least one SP were heterozygous for a single nucleotide
substitution (c.779G>A) that leads to a premature stop codon (p.W260X). Quantitative real-time
RT-PCR revealed a reduction of FLCN transcripts from the patient compared to an unaffected family
member. DNA from the sporadic case carried a heterozygous missense mutation (c.394G>A). Lung
function of this patient was normal and the CT showed similar bilateral cysts as observed in the two
members of the unrelated Swiss family.Mutations in FLCN are associated with cystic lung lesions in an
otherwise morphological normal lung and predispose to SP.
Novel mutations in the folliculin (FLCN) gene associated with
1Benjamin A. Fröhlich, 2, 4Christina Zeitz, 2Gábor Mátyás, 3Hatem Alkadhi, 1Christoph Tuor,
2Wolfgang Berger, and 1Erich W. Russi
1Pulmonary Division, University Hospital of Zurich, Switzerland, 2Division of Medical
Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of
Zurich, Switzerland, 3Institute of Diagnostic Radiology, University Hospital of Zurich,
Switzerland. 4Institut de la Vision, INSERM, U592, Université Pierre et Marie Curie6, Paris,
Key Words familial pneumothorax, folliculin gene, mutation, pneumothorax
Address for correspondence:
Erich W. Russi, MD, FCCP
CH-8091 Zurich, Switzerland
Tel.: +41 44 255 38 28
FAX: +41 44 255 44 51
. Published on June 25, 2008 as doi: 10.1183/09031936.00132707
Copyright 2008 by the European Respiratory Society.
Background: Spontaneous pneumothorax (SP) is mostly sporadic but may also occur in
families with genetic disorders such as Birt-Hogg-Dubé syndrome (BHDS), which is caused
by mutations in the folliculin gene FLCN.
Aim: To investigate the presence and type of mutation in a Swiss pedigree and in a sporadic
Methods: Clinical examination, lung function tests and HRCT. All coding exons and flanking
intronic regions of FLCN were amplified by PCR and directly sequenced. The amount of
FLCN transcripts was determined by quantitative real-time RT-PCR.
Results: We identified two novel mutations in FLCN. Three investigated family members
with a history of at least one SP were heterozygous for a single nucleotide substitution
(c.779G>A) that leads to a premature stop codon (p.W260X). Quantitative real-time RT-PCR
revealed a reduction of FLCN transcripts from the patient compared to an unaffected family
member. DNA from the sporadic case carried a heterozygous missense mutation (c.394G>A).
Lung function of this patient was normal and the CT showed similar bilateral cysts as
observed in the two members of the unrelated Swiss family.
Conclusions: Mutations in FLCN are associated with cystic lung lesions in an otherwise
morphological normal lung and predispose to SP.
SP: Spontaneous pneumothorax; BHDS: Birt-Hogg-Dubé syndrome; FLCN: folliculin; PCR:
polymerase chain reaction; HRCT: high-resolution computed tomography; UTR: untranslated
region; NMD: nonsense mediated mRNA decay.
A spontaneous pneumothorax (SP) is a collection of air in the pleural space of the lung,
causing the lung to collapse. A majority of individuals with a spontaneous pneumothorax
have no obvious lung disease (“primary” spontaneous pneumothorax), but intraoperative
inspection or preoperative CT scans generally reveal the presence of small subpleural blebs of
the lung [1, 2]. The pathogenesis of these subpleural blebs is probably related to airway
inflammation secondary to some extent to cigarette smoking in many cases. Pneumothorax
also occurs with increased rate in patients suffering from hereditary connective tissue
disorders, such as Marfan syndrome and Ehlers-Danlos syndrome [3, 4]. The clustering of
pneumothorax events in families not affected by a connective tissue disease is well known,
and autosomal dominant, autosomal recessive, X-linked recessive as well as polygenic
inheritance have been suggested [5, 6, 7]. The autosomal dominant Birt-Hogg-Dubé
syndrome (BHDS) is a genodermatosis predisposing patients to benign skin tumours and renal
cancer. It is also associated with an increased incidence of spontaneous pneumothorax [8, 9].
In a study of 198 patients with BHDS, pneumothorax occurred in 24% of the cases . It has
been shown that mutations in the folliculin gene (FLCN) on chromosome 17p11.2 can cause
this syndrome . Other mutations in FLCN have been associated with spontaneous
pneumothorax and bullous lung disease in the absence of the oncologic manifestations of
BHDS [12, 13].
We have recently studied a Swiss pedigree, with several members who had experienced a
pneumothorax and a 27-year-old female sporadic case after a first pneumothorax event and
striking parenchymal lung changes on a high-resolution computed tomography (HRCT).
Based on the association of lung cysts and pneumothoraces with BHDS we now have
screened affected family members from two generations and one sporadic case with similar
cystic lung structures for mutations in the FLCN gene and detected two novel disease-
associated DNA sequence alterations.
Patient Recruitment & Examination
All participants gave written informed consent for molecular und clinical testing. Skin
changes were excluded by physical examination and kidney manifestations by abdominal
Spirometry, whole body plethysmography, and measurement of carbon monoxide diffusing
capacity (DLco) were performed (6200 Autobox SensorMedics, Yorba Linda, CA, USA)
according to standard criteria . Reference values were in accordance to the European
Community for Steel and Coal .
Thin-section computed tomography (CT) of the chest was performed with a 64-slice CT
scanner (Somatom Sensation 64, Siemens Medical Solutions, Forchheim, Germany). Patients
were examined in the supine position. Inspiratory scans were obtained during suspended deep
inspiration from the apices of the lung to the costophrenic angles. Examination parameters
were 120 kV and 150 mAs, using a 512 × 512 matrix. Images with a slice thickness of 1 mm
and an increment of 0.8 mm were reconstructed with a high-spatial-frequency algorithm and
analyzed at window settings appropriate for viewing lung parenchyma (window center –600
HU; window width, 1500 HU). No intravenous contrast material was administered.
Genomic DNA was isolated from circulating leukocytes using the chemagic Magnetic
Separation Module I (Chemagen Biopolymer-Technology AG, Baesweiler, Germany).
All eleven coding exons and flanking intronic sequences of the FLCN gene were amplified
and sequenced. PCR amplifications, PCR product purification, and cycle sequencing were
performed under routine conditions (details are available upon request). The detected
sequence variants were verified by repeated sequencing on newly amplified PCR products.
The control panel included >210 alleles from unrelated unaffected individuals of the Swiss
FLCN Transcript Analyses
Total RNA was isolated using the PAXgene Blood RNA kit (Qiagen, Hombrechtikon,
Switzerland) from venous blood collected into PAXgene Blood RNA Tubes (Becton
Dickinson, Basel, Switzerland) according to the manufacturers’ instructions. The quality of
the purified RNA (RNA integrity number, RIN) was assessed using an Agilent 2100
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 (RT) using 4µg total RNA and random primers (Superscript III kit,
Invitrogen, Basel, Switzerland).
The amount of FLCN transcripts was determined by quantitative real-time RT-PCR
performed on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems,
Rotkreuz, Switzerland) using primers specific for FLCN and the endogenous reference
POLR2A as well as TaqMan probe (FLCN) or SybrGreen I dye (POLR2A) as a reporter.
Amplicons were run as triplicates and standard curves were prepared for both FLCN and
POLR2A. After normalization to POLR2A, the FLCN transcript expression was calculated
relative to that of a calibrator sample of normal, healthy family member III.1. Repeated
observations of relative expressions were analyzed by descriptive statistics. For the arithmetic
mean, upper and lower confidence limits were calculated using critical values of Student`s t-
The ratio of allele-specific transcripts was quantified basically according to a procedure
outlined by Qiu et al. by sequencing of mutation-harboring RT-PCR products on an ABI
PRISM 3100 Genetic Analyzer (Applied Biosystems) using primers specific to exons 6 and 8
(details on reaction conditions are available upon request) .
History and clinical features
None of the individuals analyzed in this study had α-antitrypsin-deficiency, a connective
tissue disorder such as Marfan or Ehlers-Danlos syndrome, nor skin or kidney manifestations
The index patient of the Swiss family (III.3) is a 56-year-old female (fig 1), who consulted her
family doctor after an episode of shortness of breath during her preceding holidays. Her
history is remarkable for a first pneumothorax after giving 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
thorax shows bilateral cystic lung lesions preferentially in both lower lobes within otherwise
radiological normal lung parenchyma (fig 2a). Her lung function is normal.
The patient’s family is of Swiss descent. Deceased family members as her father (II.1), an
uncle (II.3), and her grandfather (I.1) are also known to have experienced at least one
pneumothorax episode. The patient’s brother (III.5) und her son (IV.3), both with a typical
history for at least one SP event, as well as her healthy sister (III.1) and her daughter (IV.4),
who denied previous episodes of chest pain and/or shortness of breath, were available for
testing. Except the non consanguineous husband (III.4) of the index patient all participant are
non-smokers. The lung function i.e. dynamic and static lung volumes as well as diffusing
capacity for carbon monoxide was normal in the index patients sister and her children and
revealed mild obstruction to airflow in her brother, who is known to suffer from bronchial
asthma. HRCT revealed the same but less striking alterations consisting of small cysts within
otherwise unremarkable lung parenchyma, but was completely normal in her son, who had a
pneumothorax event at the age of 22 years.
The family of the sporadic female case is unknown, since the 27-year-old nonsmoking woman
has been adopted as a child from Brazil and does not know any consanguineous family
members. One week before admission she experienced pain in her right chest followed by
shortness of breath and a right sided pneumothorax was diagnosed. Since an air leak persisted
for three days after the insertion of a chest tube video-assisted thoracoscopy was indicated.
Blebs were seen at the pleural surface and a wedge resection of the right upper lobe, a pleural
abrasion and talk poudrage for pleurodesis was performed. Histology revealed small blebs
within normal lung tissue but without features of lymphangioleiomyomatosis and Langerhans
cell histiocytosis and negative staining for HBM-45 and S-100, respectively. The HRCT of
this patient shows small cysts besides normal lung structure (fig 2b), strikingly similar to the
index patient of the large Swiss family.
Mutation Analysis of the FLCN Gene
Direct sequencing of all eleven coding exons of FLCN (exons 4-14) amplified from genomic
DNA of three affected family members revealed a novel heterozygous nonsense mutation in
exon 7 (c.779G>A, p.W260X; fig 3B). It leads to a premature termination codon at position
260 instead of 581 in the open reading frame of FLCN isoform 1. The mutation c.779G>A
was present in all three affected family members while 346 control alleles did not carry this
sequence variant. The three affected patients (III.3, III.5, IV.3) had all at least one SP during
their lifetime. In addition, III.3 and III.5 but not IV.3 had an abnormal HRCT (see above).
In the sporadic female case we found a novel heterozygous missense mutation (fig 3B). The
transition c.394G>A in exon 5 leads to a single glutamic acid to a lysine substitution at codon
position 132 (p.E132K). This substitution affects an evolutionary highly conserved amino
acid, indicating an important role of the glutamic acid at position 132 for the correct function
of the FLCN protein . In addition it did not appear in 356 control alleles. According to
Collins and Schwartz a sequence alteration shows a high probability to be pathogenic if it is
not found in more than 210 control alleles .
The high conservation and the absence of the p.E132K amino acid substitution in the
unaffected population suggest it as a disease-causing mutation.
Transcript Analyses of FLCN
Premature termination codons are known to cause nonsense mediated decay (NMD), a
mechanism of mRNA surveillance to prevent the expression of truncated proteins. This is
achieved by a selective degradation of the respective mRNA molecules. In order to quantify
FLCN mRNA levels in affected family members carrying the premature termination codon
we performed real-time RT-PCR analyses. RNA extracted from peripheral blood of patient
III.5 carrying the heterozygous c.779G>A (p.W260X) mutation revealed a significantly
reduced amount of FLCN transcripts (43±11%, P=0.05) compared to a healthy family
member (III.1) (100±12%; P=0.05) (fig 3C). We also performed semi-quantitative sequencing
of RT-PCR products from patient III.5 and detected a highly reduced amount of mutated
transcripts (A allele) in comparison to wild-type transcripts (G allele at position c.779, data
After the first published observation of an increased frequency of SP in patients with BHDS
other individuals with a family history of SP but without clinical features of BHDS have been
described. Some of them showed a mutation in the folliculin gene, FLCN, with lung cysts as
the morphological basis for pneumothorax events . Our findings confirm that in
individuals with a family SP history or in persons with a SP and multiple lung cysts,
mutations of FLCN may be found even in the absence of the typical dermatologic findings for
BHDS, i.e. fibrofolliculomas of the skin.
There are several considerations about the function of FLCN protein in the cell, e.g. tumour
suppressor activity by involvement in mTOR signalling has been suggested . Expression
studies revealed, that the folliculin mRNA is widely, but not universally expressed in human
organs and tissues including skin, lung and kidney . In the lung FLCN is transcribed in
type 1 pneumocytes and stroma cells including fibroblasts and macrophages. An imbalance
may either induce an inflammatory response or alter matrix degradation and remodelling.
A variety of mutations in all eleven coding exons of the folliculin gene have been detected
since the first description of the clinical manifestations of mutations in this gene. The most
frequent comprise frameshift or nonsense mutations that are predicted to introduce a
premature termination codon. However, also missense and splice site mutations were
associated with SP . No clear cut correlation between the type of folliculin mutation and
the disease phenotype, i.e. BHDS or SP without BHDS features has been found so far.
Recently, an extensive investigation on lung cysts and pneumothorax comprising 198 patients
in 89 families with BHDS was published. In this study, FLCN mutations in exons 9 and 12
were associated with a higher number of cysts, larger cyst diameters and a higher incidence of
pneumothorax events .
The nonsense mutation in exon 7 (c.779G>A, p.W260X) may lead to nonsense mediated
mRNA decay (NMD) . To investigate the effect of the nonsense mutation on RNA
expression, we compared the amounts of FLCN transcript in patient III.5 with an unaffected
family member III.1 of comparable age. Indeed, this analysis revealed a 60%-reduction of the
transcript level in the patients´ RNA, probably due to NMD. The missense mutation
c.394G>A (p.E132K) in exon 5 of FLCN affects an amino acid residue, which is highly
conserved across species. Future studies will show whether or not this mutations leads to a
reduced amount of FLCN transcripts as reported for missense mutations in other genes .
Our findings lead to the conclusion that the pathogenic defect of SP is at least for the
nonsense mutation due to the lower amount of normal FLCN transcript compared to
unaffected persons. Discovery whether our observation also apply to lung tissues require the
investigation of affected human lung tissues. It would be also of interest if other FLCN
mutations associated with SP in general leads to an FLCN transcript reduction and if this is
different in patients with BHDS and FLCN mutations.
In patients with SP computed tomography of the lung shows either a normal lung structure or
few blebs in the apical region of the lung. The lung cysts, which may be found in patients
with mutations in FLCN with or without features of BHDS, are unique and distinct from the
cystic lesions found in pulmonary emphysema or in Langerhans cell histiocytosis, lung
diseases where the cystic lesions are accompanied by other radiomorphological alterations
typical for the underlying disease. The cysts resemble punched holes in an otherwise
normally structured lung and are not preferentially located in the upper lobes of the lung. As
recently described in 48 patients with BHDS with a history of pneumothorax the cysts may
vary in size from a few millimetres to several centimetres and from a few up to over 100 .
Lymphangioleiomyomatosis, a disease almost exclusively affecting women in the third and
fourth decade of her life, may be more difficult to distinguish radiologically from the BHD
syndrome, since also in this disease the cysts may vary in size and are randomly distributed
throughout the lungs with no noticeable changes in the intervening lung parenchyma.
In a retrospective study of the Mayo Clinic, including five patients during an 8-year period
(1998-2005), pulmonary function results were available in four patients and reported to be
normal in the one single patient, who never has smoked . Our study is the first, where
lung function measurements were performed systematically in non-smoking individuals with
a folliculin gene defect. The functional data are consistent with the radiomorphologic findings
and indicate that apart from lung cysts the pulmonary parenchyma in the investigated patients
seems not defective, which is in contrast to other cystic lung diseases such as lymphangioleio-
myomatosis, Langerhans cell histiocytosis, and pulmonary emphysema.
We thank Dr. Eva Achermann (Spital Limmattal, CH-8952 Schlieren) for referring the index
Supported by an unrestricted grant from AstraZeneca. This study was funded by
“Forschungskredit University of Zurich” and by the Foundation “Voir et Entendre”.
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LEGENDS TO FIGURES
Figure 1 Pedigree of a Swiss family with SP. Circles indicate females, squares indicate males,
open symbols indicate unaffected individuals, filled symbols indicate affected individuals,
and lines across symbols indicate deceased individuals. Generations and individuals are
identified by roman and arabic numerals, respectively. The determined genotype is given
below each symbol representing investigated individuals.
Figure 2a Chest high-resolution computed tomography of patient III.3.
Figure 2b Chest high-resolution computed tomography of the sporadic case.
Figure 3 A, Exon structure of FLCN. Boxed symbols indicate exons, lines indicate introns,
filled boxes indicate coding exons, unfilled boxes the 5’ and 3’ UTR, waved caret indicates
the position of TaqMan probe used for transcript measurement. B, Electropherograms
showing the two novel FLCN mutations compared with a control. Arrows indicate the site of
mutation, lowercase letters indicate intronic sequences, and uppercase letters indicate exonic
sequences. C, Relative Expression of FLCN transcript in patient III.5 compared to unaffected
family member III.1. Vertical lines indicate confident intervals (P=0.05).
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