Familial Cancer (2013) 12:373–379 DOI 10.1007/s10689-012-9593-8
ORIGINAL ARTICLE
A de novo FLCN mutation in a patient with spontaneous pneumothorax and renal cancer; a clinical and molecular evaluation Fred H. Menko • Paul C. Johannesma • R. Jeroen A. van Moorselaar • Rinze Reinhard • Jan Hein van Waesberghe • Erik Thunnissen • Arjan C. Houweling Edward M. Leter • Quinten Waisfisz • Martijn B. van Doorn • Theo M. Starink • Pieter E. Postmus • Barry J. Coull • Maurice A. M. van Steensel • Johan J. P. Gille
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Published online: 22 December 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract Birt–Hogg–Dube´ syndrome (BHD) is an autosomal dominant condition due to germline FLCN (folliculin) mutations, characterized by skin fibrofolliculomas, lung cysts, pneumothorax and renal cancer. We identified a de novo FLCN mutation, c.499C[T (p.Gln167X), in a patient who presented with spontaneous pneumothorax. Subsequently, typical skin features and asymptomatic renal cancer were diagnosed. Probably, de novo FLCN mutations are rare. However, they may be under-diagnosed if BHD is not considered in sporadic patients who present with one or more of the syndromic features. Genetic and immunohistochemical analysis of the renal tumour indicated features compatible with a tumour suppressor role of FLCN. The finding that mutant FLCN was expressed in the tumour might indicate residual functionality of mutant FLCN, a notion which will be explored in future studies. Keywords Birt–Hogg–Dube´ syndrome Folliculin de novo mutation Pneumothorax Renal cancer
F. H. Menko (&) A. C. Houweling E. M. Leter Q. Waisfisz J. J. P. Gille Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands e-mail:
[email protected] P. C. Johannesma P. E. Postmus Department of Pulmonology, VU University Medical Center, Amsterdam, The Netherlands R. J. A. van Moorselaar Department of Urology, VU University Medical Center, Amsterdam, The Netherlands R. Reinhard J. H. van Waesberghe Department of Radiology, VU University Medical Center, Amsterdam, The Netherlands
Introduction In 1977 Birt, Hogg and Dube´ described a three-generation pedigree affected with multiple fibrofolliculomas [1]. Subsequently, it was shown that patients with Birt–Hogg–Dube´ syndrome (BHD) can also develop renal cancer and pneumothorax [2, 3]. The causative gene was mapped to chromosome 17p11.2 and germline mutations in the FLCN (folliculin) gene were identified in BHD families [4]. FLCN mutations have now been detected not only in classical BHD families but also in pneumothorax and renal cancer patients and families [5–7]. The spectrum of FLCN mutations has been outlined in detailed reports [8–10] and summarized in two databases [11, 12]. The function of folliculin is complex and involves several molecular pathways including mTOR and vesicular transport [13–15]. The European BHD Consortium recently summarized diagnosis and management of this syndrome [16]. In autosomal dominant tumour syndromes a varying proportion of index patients have a de novo mutation,
E. Thunnissen Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands M. B. van Doorn T. M. Starink Department of Dermatology, VU University Medical Center, Amsterdam, The Netherlands B. J. Coull M. A. M. van Steensel Department of Dermatology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands M. A. M. van Steensel Department of Clinical Genetics, GROW School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands
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which is an alteration in a gene that is present for the first time in a family member as a result of a mutation in a germ cell of one of the parents, or a mutation that arises in the fertilized egg itself during early embryogenesis. For example, in Lynch syndrome (hereditary nonpolyposis colorectal cancer) about 1–5 % of patients have de novo mutations in DNA mismatch repair genes [17], whereas in familial adenomatous polyposis de novo APC mutations occur in about 10–25 % of cases [18]. The phenomenon of de novo mutations is important from a clinical point of view, since it implies that a negative family history for syndromic features does not exclude an autosomal dominant condition. To our knowledge, in BHD de novo mutations have thus far not been reported. Here we describe a patient who presented with spontaneous pneumothorax due to a de novo FLCN mutation. Subsequently, skin fibrofolliculomas were diagnosed and Magnetic Resonance Imaging (MRI) of the kidneys revealed a small left-sided renal cancer, treated by partial left nephrectomy. Histologically, the tumour was a chromophobe renal cancer. Since the molecular pathogenesis of renal cancer in BHD is incompletely understood, we analysed the tumour for loss of heterozygosity and expression of FLCN. Patients and methods Patient and family data The proband, a 30-year-old man, had recently been admitted to our hospital due to pneumothorax. He was referred to the department of clinical genetics by the dermatologist, who had identified multiple skin-coloured centro-facial papules histologically compatible with fibrofolliculomas typical for Birt–Hogg–Dube´ syndrome (Fig. 1). We collected patient and family data and performed FLCN mutation analysis. In addition, renal imaging was performed by MRI and renal ultrasound. Mutation analysis For FLCN mutation analysis genomic DNA was extracted from blood samples after the patient gave informed consent. Primers for the amplification and sequencing of the 14 exons were designed as detailed by Nickerson et al. [4]. PCR amplification was performed using a PE 9700 thermocycler (Applied Biosystems, Forster City, CA, USA). Sequencing reactions were performed using the Big Dye Terminator system (Applied Biosystems) and run on an ABI 3100XL or ABI 3730 genetic analyzer (Applied Biosystems). For the detection of deletions and duplications of one or more exons MLPA analysis was performed using MLPA kit P256 (MRC Holland, www.mrc-holland.com).
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Fig. 1 Multiple smooth facial papules in the proband
Paternity confirmation To confirm paternity and exclude sample mix-up DNA samples of the patient and both parents were analysed using the Powerplex 16 system (Promega Madison, WI, USA). Molecular analysis of the renal tumour (a) Mutation analysis Genomic DNA was isolated from paraffin-embedded tumour material with the Macherey– Nagel kit for FFPE material (Macherey–Nagel, Du¨ren, Germany) according to the manufacturer’s instructions. Tumour tissue was isolated by needle scraping of macroscopically visible cancerous areas. Loss of heterozygosity was assessed by amplifying exon 6 using a Corbett Rotorgene 6000 (Qiagen, Venlo, the Netherlands) real-time system (primer sequences and conditions available on request). Sequencing reactions were performed with the PCR primers using the ABI BigDye terminator (v 1.1) kit according to the manufacturer’s instructions and analyzed on an ABI 3100 capillary system (Applied Biosystems, Carlsbad, CA, USA). Sequence traces were assembled and examined using the PhredPhrap-Consed software package. The amplified tumour DNA was cloned into the pCR2.1TOPO vector (Invitrogen, Groningen, The Netherlands) to determine whether the second hit had occurred in cis or in trans to the germline mutation. (b) Immunohistochemistry Procedures used for immunohistochemistry have been described in detail elsewhere [19]. Polyclonal FLCN antibody (rabbit) was a kind gift of professor Arnim Pause (McGill University, Montreal, Canada). Four lm formalin-fixed, paraffin-embedded (FFPE) sections of tumour sample, obtained during surgery, were deparaffinized in xylene and dehydrated through graded ethanol concentrations. Endogenous peroxidase activity was blocked by incubation in 3 % (w/v) hydrogen peroxide (H2O2) in methanol for 30 min, followed by microwave treatment using 10 mmol/L citrate buffer (pH 6) for 10 min (90 W) to facilitate antigen retrieval. Non-specific protein
A de novo FLCN mutation in a patient with spontaneous pneumothorax
binding was blocked using 3 % bovine-serum-albumin in tris-buffered saline tween-20. Primary antibody was diluted in Dako Antibody diluent and incubated for 1 h at room temperature. Secondary detection was done by use of the Envision detection system (Dako Netherlands BV, Heverlee, Belgium) for 30 min and bound antibody was visualized by using 3,3-diaminobenzidine (DAB) for 10 min. Tissue was counterstained with Gill II haematoxylin, dehydrated and coverslipped. Dako Washbuffer was used throughout for washing.
Results Patient and family data Both parents and the two siblings of the proband were healthy. The multiple fibrofolliculomas in the proband which started to develop from the age of about 25 years are depicted in Fig. 1. A few weeks prior to referral the patient had been treated for left-sided spontaneous pneumothorax. He had experienced two previous episodes of left-sided spontaneous pneumothorax, at the ages of 19 and 22 years, treated by drainage and drainage plus tetracycline pleurodesis, respectively. A recent thoracic Computer Tomography (CT) showed a left-sided pneumothorax (Fig. 2) but no intrapulmonary cystic lesions apart from a small left-sided subpleural apical bleb. Since pneumothorax had recurred despite former drainage and pleurodesis, video-assisted thoracoscopic surgery was performed, which revealed a small rim of fibrosis in the left upper lobe probably due to collapsed bullae. Excision of the apex of the left upper lobe and pleurectomy were performed. Histologically, collapsed bullae associated with mild pleuritis, minor local emphysematous changes and a
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small rim of fibrosis with focal excentric intima fibrosis of the pulmonary artery were observed (Fig. 3). Abdominal MRI revealed a 18 mm left renal lesion which—notably—was not detected on renal ultrasound. The differential diagnosis was renal cancer or oncocytoma. A follow-up MRI with contrast made 3 months later showed that the lesion had not grown (Fig. 4). After weighing the various management options (follow-up, nephron sparing treatment) a left partial nephrectomy was performed, which revealed a pT1aN0 chromophobe renal cancer (Fig. 5). Molecular data In the proband a pathogenic FLCN mutation c.499C[T, p.Gln167X, located within exon 6 was identified (Fig. 6), which was absent in his parents and both siblings who had no signs of BHD. VNTR markers were tested in the patient and his parents. For all markers tested the patient showed one maternal and one paternal allele thus confirming paternity and excluding sample mix-up. Mutation analysis of the tumour indicated the presence of a second somatic hit, c.397-7_404del15, which deletes the exon 6 splice acceptor site and which is expected to result in exon skipping and production of a truncated protein. There was no material available for RNA isolation and therefore we were not able to analyze the consequences of the mutation. Subsequent cloning of amplified tumour DNA showed that the somatic mutation had occurred in trans to the germline mutation (Fig. 7). Immunohistochemical staining of the tumour showed robust FLCN expression; normal kidney tubules and skin also expressed FLCN (Fig. 8).
Discussion
Fig. 2 Left-sided pneumothorax; of note, pulmonary cysts were absent at all levels
Among the more than 35 BHD families with pathogenic germline FLCN mutations identified in our centre, most of which have been published [20, 21], this is the only de novo FLCN mutation identified. This type of FLCN mutation, c.499C[T, p.Gln167X, can be classified as pathogenic and causative of BHD since it leads to a premature stop. The FLCN mutation identified in this case has not been listed in the FLCN mutation databases [11, 12]. Since the proband had all three major manifestations of BHD, i.e. fibrofolliculomas, pneumothorax and renal cancer, a somatic mosaic of the FLCN mutation is unlikely. The absence of the mutation in both parents is consistent with a de novo mutation, but somatic mosaicism in one of the parents cannot be excluded. We cannot correlate the clinical expression in this patient to characteristics of the
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Fig. 3 a 2,5 9 (H & E staining) overview of a lung bulla (asterisks) in the collapsed resection specimen. b and c (elastin and H & E stain, respectively) show thick elastotic alveolar walls and fibrosis in subpleural alveolar spaces and local marginal fibrotic thickening of the pleura
Fig. 4 The asymptomatic left renal tumour in the proband as revealed by MRI
specific mutation since thus far, in BHD, no clear genotype-phenotype correlations have been found. To our knowledge, de novo FLCN mutations have not been reported previously. The frequency of de novo FLCN mutations is probably low, but may be under diagnosed if BHD is not considered in sporadic patients who show one or more of the syndromic clinical features.
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Fig. 5 Histopathology of the chromophobe kidney cancer (overview, 2.5 9); the insert (40 9) shows the monotonous cytonuclear appearance
For clinical diagnosis the time scale for the various manifestations of BHD is also relevant. For example, pneumothorax may be the first manifestation in a patient who will develop fibrofolliculomas and/or renal cancer at a later age. If pneumothorax is the only manifestation the diagnosis BHD may easily be missed. In de novo cases, the
A de novo FLCN mutation in a patient with spontaneous pneumothorax
Fig. 6 Electropherogram of part of exon 6 of FLCN. a sequence of the DNA of the patient showing the FLCN mutation c.499C [ T; due to the mutation codon 167, which is underlined, is changed from CAG encoding Gln into TAG encoding a stop; b wild type sequence
absence of a positive family history for syndromic features increases the difficulty of recognizing the condition. Skin lesions may be absent in up to 20 % of FLCN mutation carriers and if typical skin lesions are present they may not be recognized as marker lesions for the syndrome. Apart from the unusual cause of BHD in this patient—a de novo FLCN mutation—his clinical features are also
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remarkable and are therefore considered below in some detail. The recurrent spontaneous pneumothorax in this patient was initially interpreted as primary, which is the common form of this disease. The pathogenesis of primary spontaneous pneumothorax (PSP) is incompletely understood. Probably, PSP is associated with a focal inflammatory process related to the pleura and underlying lung. Small subpleural blebs or larger subpleural bullae, which are found in the majority of these patients, are only one feature of a complex and not fully understood pathogenetic mechanism. It remains unclear whether these emphysema-like changes are the cause of the air leak or merely a coincidental phenomenon. Subpleural blebs or bullae are also observed in about 15 % of the normal population [22, 23]. In BHD, spontaneous pneumothorax has been linked directly to multiple bilateral and mostly basally located lung cysts, which are found in the majority of patients. In the patient described here thoracic CT did not show lung cysts but only a left apical subpleural bleb. The absence of lung cysts in a BHD patient who exhibits pneumothorax is remarkable: all 48 BHD patients with pneumothorax described by Toro et al. [24] had multiple lung cysts. However, in BHD, the occurrence of pneumothorax without lung cysts on thoracic CT has been described previously [25, 26]. Thus, the relationship between lung cysts and pneumothorax in BHD has not yet been fully clarified.
Fig. 7 Sequence traces of cloned FLCN exon 6 fragments amplified from tumour DNA showing the second hit mutation in the bottom panel, with a wild type sequence in the top for comparison. Since the sequence trace only shows one mutation (the second hit) and not the germ-line change (not shown due to space constraints), it must have occurred in trans to the germ line mutation. The reverse strand is shown as it had the best read quality. The 15 bp deletion is shown in the shaded area and is emphasized with a continuous line. The dashed lines indicate how the sequence has changed. The intron–exon boundary is indicated by a vertical arrow
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Fig. 8 Immunohistochemical staining of the renal tumour with a polyclonal FLCN antibody. a Unaffected kidney tissue of the proband. Strong FLCN expression throughout the kidney tubules, but not in a glomerulus. b The kidney tumour shows pronounced staining, consistent with expression of one or both the mutant alleles.
c Positive control: normal skin from an unrelated healthy individual. Uniform staining in the epidermis and hair follicle as previously reported. Note the presence of FLCN in sebaceous glands. d Negative control, secondary antibody only on normal skin
Few case studies have reported lung histopathology in BHD [5, 27–30]. Recently, distinct microscopic features were documented [29, 30]. In the case described here the surgical specimen showed subpleural fibrosis in pre-existing alveolar walls associated with focal excentric intima fibrosis in a small pulmonary artery. Since these are morphologic changes of a small infarction, due to local hypoxia, a local infarction may have led to a local bulla. The molecular pathogenesis of renal cancer in BHD has been evaluated in a series of studies. Vocke et al. [31] demonstrated a second somatic FLCN mutation or loss of heterozygosity in the majority (70 %) of 77 BHD-associated renal tumours. These findings were interpreted as supportive for a tumour suppressor role for FLCN. By immunohistochemical staining we detected strong FLCN expression. In theory, this could be due to expression in cells in which a wild type allele is still present. However, when sequencing cloned tumour DNA in order to determine whether the second hit we found was indeed in trans, we detected no wild type sequences. We therefore suggest that at least one of the mutant alleles is expressed in the tumour. The second hit mutation is predicted to result in skipping of exon 6 in which case the proper reading frame for FLCN is maintained. This mRNA encodes for a mutant protein
lacking the amino acids encoded by exon 6 and detected by the FLCN antibody, thus explaining the IHC result. As FLCN truncations are considered to result in a non-functional protein, our observations are consistent with a tumour suppressor role for FLCN. An important goal of diagnosing BHD is prevention of disease burden and death due to renal cancer. In the present case, early diagnosis of a chomophobe renal cancer allowed curative surgical treatment. The renal tumour identified with MRI was not detected by renal ultrasound. Undoubtedly, MRI is more sensitive than ultrasound for the detection of small renal lesions and therefore MRI is generally advised for surveillance of individuals at high renal cancer risk. In summary, the case described here with a de novo FLCN mutation shows that BHD should be considered in patients with a negative family history who present with one or more of the syndromic features. We also showed that mutant FLCN can be expressed in BHD-associated renal cancer, raising the possibility that BHD-associated mutations do not result in complete loss of functionality.
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Acknowledgments MvS and BJC are supported by grants from the Dutch Cancer Society KWF (UM2009-4352), the Association for International Cancer Research AICR (11-0687) and the Annadal Foundation.
A de novo FLCN mutation in a patient with spontaneous pneumothorax
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