588 LETTER TO JMG Mutations of the Birt–Hogg–Dube´ gene in patients with multiple lung cysts and recurrent pneumothorax Yoko Gunji, Taeko Akiyoshi, Teruhiko Sato, Masatoshi Kurihara, Shigeru Tominaga, Kazuhisa Takahashi, Kuniaki Seyama ......
J Med Genet 2007;44:588–593. doi: 10.1136/jmg.2007.049874
been reported that FLCN interacts with FLCN-interacting Rationale: Birt–Hogg–Dube´ (BHD) syndrome, a rare inherited protein 1 (FNIP1) and may be involved in energy and/or autosomal genodermatosis first recognised in 1977, is nutrient sensing through the 59 AMP-activated protein kinase characterised by fibrofolliculomas of the skin, an increased and mammalian target of rapamycin signalling pathway.16 risk of renal tumours and multiple lung cysts with spontaneous Once the BHD gene was identified, enabling genetic testing, pneumothorax. The BHD gene, a tumour suppressor gene the clinical features of BHD syndrome were gradually specified, located at chromosome 17p11.2, has recently been shown to giving a better understanding of the disease. Analysis of a large be defective. Recent genetic studies revealed that clinical cohort of families with BHD syndrome revealed that the pictures of the disease may be variable and may not always characteristic triad of the phenotype (involvement of skin, present the full expression of the phenotypes. lung and kidney) may not always be apparent in people with Objectives: We hypothesised that mutations of the BHD gene BHD germline mutation and that BHD patients show diverse 17 are responsible for patients who have multiple lung cysts of clinical heterogeneity. For example, it was reported that which the underlying causes have not yet been elucidated. isolated familial spontaneous pneumothorax with no fibrofol- Methods: We studied eight patients with lung cysts, without liculomas and no renal tumour was caused by mutations of the BHD gene.18 19 Accordingly, we hypothesised that patients with skin and renal disease; seven of these patients have a history of multiple lung cysts with undetermined causes might be part of spontaneous pneumothorax and five have a family history of the clinical spectrum of BHD syndrome even if no skin and pneumothorax. The BHD gene was examined using PCR, renal lesions exist . denaturing high-performance liquid chromatography and In this study, we performed BHD mutation analysis in eight direct sequencing. patients with multiple lung cysts identified by a chest CT scan Main results: We found that five of the eight patients had a that was prompted by the occurrence or history of pneumo- BHD germline mutation. All mutations were unique and four of thorax or other predisposing reasons. Although all patients had them were novel, including three different deletions or neither skin nor renal lesions, five of the eight patients were insertions detected in exons 6, 12 and 13, respectively and found to carry BHD germline mutations. one splice acceptor site mutation in intron 5 resulting in an in- frame deletion of exon 6. MATERIALS AND METHODS Conclusions: We found that germline mutations of the BHD Study population gene are involved in some patients with multiple lung cysts and This study was approved by the ethics committees of Juntendo pneumothorax. Pulmonologists should be aware that BHD University. Written informed consent was obtained from all syndrome can occur as an isolated phenotype with pulmonary subjects as required. We enrolled a total of eight patients (two involvement. male, six female), who have been receiving medical care at our hospital since 1998 and had multiple lung cysts on high- resolution CT (HRCT) of the chest (table 1). High-resolution 2 mm sections were obtained through the chest at 10 mm irt–Hogg–Dube´ (BHD) syndrome is a rare inherited intervals. We counted the number of cysts identified in all autosomal genodermatosis first recognised in 1977 by sections of the chest HRCT but regarded a cyst to be the same three Canadian doctors.1 They studied a large family whose B one when it was located in the same area in consecutive HRCT members were affected with multiple, small, white or skin- sections. All patients but one (patient B5) had a medical history coloured papules on the face, neck and upper trunk that of pneumothorax, and most of the patients had repeated developed after the age of 25 years. These lesions, called episodes of pneumothorax in both lungs except for patients B4 fibrofolliculomas, are proliferating benign hamartomas of the and B8. Patient B4 had a pneumothorax but could not hair follicle. Subsequent reports have described patients whose remember which side was affected, and patient B8 had cutaneous lesions are associated with renal tumours,2–4 sponta- experienced episodes of right-sided pneumothorax. Mean neous pneumothorax or lung cysts,45 colon polyps and colon (SD) age at time of pneumothorax (except for patient B5) carcinomas.6–11 was 30.4 (10.9) years and age at time of enrolment was 41.8 The genetic defect responsible for BHD syndrome was (17.1) years. All patients but one (patient B4) had undergone mapped to chromosome 17p11.2212 and thereafter the BHD 13 video-assisted thoracic surgery for resection of the bullous part gene, consisting of 14 exons, was cloned. Disease-causing of the lungs, but this did not yield any pathological results that mutations were found over the entire region of the BHD gene, including insertions, deletions and nonsense mutations that were predicted to truncate the BHD protein.13 The BHD gene Abbreviations: BHD, Birt–Hogg–Dube´; DHPLC, denaturing high- performance liquid chromatography; EBV-LCL, Epstein–Barr virus- codes a protein called folliculin (FLCN)which is expressed transformed lymphoblastoid cell line; FLCN, folliculin; FNIP1, folliculin- 13 14 widely in skin, kidney, lung and other organs. It is supposed interacting protein 1; HRCT, high-resolution computed tomography; LAM, to function as a tumour suppressor gene,15 and it has recently lymphangioleiomyomatosis; LCH, Langerhans cell histiocytosis www.jmedgenet.com Birt-Hogg-Dube´ mutations in multiple lung cysts and recurrent pneumothorax 589 could identify causes for cystic formation in the lungs. Patient Reverse transcription PCR of BHD mRNA B8 underwent transbronchial lung biopsy, but did not result in An Epstein–Barr virus-transformed lymphoblastoid cell line definite diagnosis. There was thus no positive evidence, either (EBV-LCL) was established from peripheral blood mononuclear clinical or pathological, to support the diagnosis of any cystic cells using standard methods and maintained in RPMI 1640 lung disease including lymphangioleiomyomatosis, pulmonary medium supplemented with 10% fetal calf serum. Total RNA Langerhans cell histiocytosis (LCH), Sjo¨gren syndrome, lym- was isolated from EBV-LCL (RNeasy Plus Mini Kit; Invitrogen) phoproliferative disorders, amyloidosis, alpha-1-antitrypsin and cDNA synthesised by reverse transcriptase (RT) PCR deficiency, Ehlers–Danlos syndrome or Marfan syndrome. All (ThermoScript RT-PCR system; Invitrogen), under the same patients were evaluated with abdominal CT scan and consulted conditions as described above. dermatologists. RESULTS Mutation analysis of the BHD gene Germline mutation of the BHD gene Genomic DNA was isolated from peripheral blood leukocytes. BHD mutations were identified in five patients (table 2). All Exons with flanking intronic sequences of the BHD gene were mutations detected were unique and four of them were novel. amplified by PCR using genomic DNA. Each PCR was All were insertions or deletions; no missense or nonsense performed in a 25 ml reaction mixture containing 100 ng of mutations were found. Four different insertions or deletions genomic DNA, 1 mmol/l of each primer, PCR buffer II (Applied (patients B1, B2, B3 and B7) cause frameshifts leading to Biosystems, Foster City, California, USA), 1.0,2.0 mmol/l premature truncation of the protein. The mutation identified in MgCl2, 0.2 mmol/l dNTPs and 0.625 U of AmpliTaq Gold patient B2 was a cytosine insertion in the mononucleotide tract (Applied Biosystems). PCR primers were prepared according (hot spot) of eight cytosines (nucleotides 1733–1740, C8) in to the method of Nickerson et al.13 PCR conditions were 94˚C for exon 11.13 Figure 1 shows a representative mutation analysis 4 min; 35 cycles of 94˚C for 30 sec, 55˚C for 30 sec, 72˚C for result and family pedigree of patient B1. The fifth mutation was 1 min; and 72˚C for 5 min. Each PCR product was first screened a deletion including a splice acceptor site of intron 5 (patient for mutations by denaturing high-performance liquid chroma- B6; figure 2). As the mutation is expected to cause problems in tography (DHPLC) (WAVE; Transgenomic, Omaha, Nebraska, BHD mRNA splicing, we examined the effect of the mutation USA) and followed by sequence analysis if heteroduplex on the mRNA transcript using EBV-LCL established from formation was detected. Although genomic sequencing may patient B6. RT-PCR demonstrated two different PCR products be more sensitive to detect mutations, we used DHPLC because on agarose gel (figure 2C), one was the size expected from it is sufficiently sensitive to screen for genetic alterations and normally spliced transcript (453 bp) and the other expected commonly used for mutation analysis of hereditary diseases.20 21 from an exon 6-skipped transcript (231 bp): direct sequencing Sequencing was performed using commercial reagents and of the each PCR product confirmed that this assumption was an automated sequencer (ABI Prism BigDye Terminator v1.1 correct (data not shown). However, a tiny amount of exon 6- Cycle Sequencing Kit and ABI 3130 Genetic Analyzer; both skipped transcript appears to be generated in controls too, Applied Biosystems). Both strands were sequenced to confirm suggesting that the intron 5 splice acceptor site may be ‘‘leaky’’, nucleotide alterations. If direct sequencing revealed a super- at least in EBV-LCL. imposed nucleotide chromatogram suggesting nucleotide In this study, we identified four nucleotide alterations that alteration of either insertion or deletion, the PCR products are considered to be polymorphisms in the BHD gene (table 3). were cloned (TA Cloning Kit; Invitrogen, Carlsbad, California, One is located in the non-coding region of exon 1, two exist in USA) and then sequenced. an intronic sequence and there is one silent mutation in exon
Table 1 Clinical data of the eight subjects
No. and No of location of Family history Family history of carcinoma/ pulmonary Patient Sex Age* PTX episodes1 Smoking history Medical history of PTX neoplasm` cysts
B1 F 23 (30) L (1) R (1) Never Endometriosis, PCOS, ovarian cyst, Brother, aunt Grandfather, uncle (no details), 23 lipoma grandmother (colon) B2 F 16 (38) L (3) R (3) 20–34 years, Myoma of the uterus, ureterolithiasis Cousin Grandmother (liver) 134 2.2 pack-years B3 F 25 (40) L (3) R (2) Never None Father, brother Father (kidney) 71 B4 F 49 (83) Unknown (1) Never Pyothorax, myoma of the uterus, None Father (tonsil), mother (liver) 387 chronic pancreatitis, gallstone disease, sinusitis B5 M 26 (30) None 20–22 years, Colon polyp None Grandmother (lung), aunt 552 0.45 pack-years (oesophageal), mother (uterus), sister (colon polyp) B6 F 35 (37) L (1) R (1) Never Myoma of the uterus, gastric polyp Father, uncle, Grandmother (lung), aunt 14 aunts, cousins (kidney) B7 F 28 (38) L (2) R (1) 20–25 years, Vocal cord nodules, myoma of the Grandmother, Great-grandmother (stomach), 17 0.6 pack-years uterus, carcinoma of thyroid gland, mother grandmother (uterus), great- recurrent tonsillitis uncles and aunts (stomach, breast, thyroid gland, lung), mother (kidney, thyroid gland) B8 M 37 (38) L (0) R (2) 20–38 years, Lung cancer