QJM: An International Journal of Medicine, 2017, 23–26

doi: 10.1093/qjmed/hcw109 Advance Access Publication Date: 2 August 2016 Original Paper

ORIGINAL PAPER Novel folliculin (FLCN) and familial Downloaded from https://academic.oup.com/qjmed/article/110/1/23/2631740 by guest on 30 September 2021 spontaneous J.-F. Zhu1,*, X.-Q. Shen2,*, F. Zhu3 and L. Tian4

From the 1Central Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, P.R. China, 2Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, People’s Republic of China, 3Department of Cardiology and 4Department of Pediatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, People’s Republic of China

Address correspondence to Dr L. Tian, Department of Pediatrics, Union Hospital, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan, Hubei 430022, People’s Republic of China. email: [email protected] *These authors contributed equally to this work.

Summary Background: Familial spontaneous pneumothorax is one of the characteristics of Birt–Hogg–Dube´ syndrome (BHDS), which is an autosomal dominant disease caused by the mutation of folliculin (FLCN). Aim: To investigate the mutation of FLCN in a familial spontaneous pneumothorax. Design: Prospective case study. Methods: Clinical and genetic data of a Chinese family with four patients who presented spontaneous pneumothorax in the absence of lesions or renal tumors were collected. CT scan of patient’s lung was applied for observation of pneumo- thorax. DNA sequencing of the coding (4–14 exons) of FLCN was performed for all 11 members of the family and 100 unrelated healthy controls. Results: CT scan of patient’s lung showed spontaneous pneumothorax. A mutation (c. 510C > G) that leads to a premature stop codon (p. Y170X) was found in the proband using DNA sequencing of coding exons (4–14 exons) of FLCN. This mutation was also observed in the other affected members of the family. Conclusions: A of FLCN was found in a spontaneous pneumothorax family. Our results expand the mu- tational spectrum of FLCN in patients with BHDS.

Introduction researchers revealed that families with a mutation in folliculin Primary spontaneous pneumothorax is a spontaneous occur- (FLCN), which results in Birt-Hogg-Dube´ syndrome (BHDS), often rence of pneumothorax in patients with no underlying lung dis- show familial spontaneous pneumothorax.2,3 BHDS is a rare, ease. Over 10% of patients with the disease report a positive autosomal dominant disorder characterised by multiple skin family history.1 Two modes of inheritance are found in families papules, hereditary renal tumours and spontaneous pneumo- with spontaneous pneumothorax: autosomal dominant inherit- thorax.4 This disease is caused by in a gene mapped ance with variable penetrance or X-linked recessive.1 Recently, in the region of 17p11.2. The FLCN gene consists of

Received: 29 January 2016; Revised (in revised form): 21 June 2016

VC The Author 2016. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: [email protected]

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14 exons and is considered to be a tumour suppressor. However, Results patients with BHDS present heterogenetic clinical characteristic Clinical features . Isolated familial spontaneous pneumothorax can occur without skin and renal manifestations. Among the three The CT examination of the proband showed several cysts on clinical features, spontaneous pneumothorax may be the first the lower lung zone on the right side and a pneumothorax 5 presentation. (Figure 2). Skin lesions and renal tumours of patient were Here, we report a case of isolated familial spontaneous excluded via a dermatologic examination and an abdominal CT pneumothorax with a novel FLCN mutation. The affected family scan, respectively. None of the family members had skin lesions members had neither the typical skin lesions nor the renal tu- or renal tumours. All of the affected members tested negative mour. A novel nonsense mutation of the FLCN gene was for alpha-1 antitrypsin deficiency and their pulmonary func- detected. tions were normal.

Gene analysis Downloaded from https://academic.oup.com/qjmed/article/110/1/23/2631740 by guest on 30 September 2021 Materials and methods A sequencing analysis revealed a novel heterozygous mutation, This study was approved by the Ethics Committee of Union c. 510C > G, which was identified in 6 of the FLCN gene in Hospital, Tongji Medical College, Huazhong University of the proband (Figure 3A). The mutation was also found in her Science and Technology. father and two other siblings with recurrent spontaneous pneumothorax (Figure 3B). This nonsense mutation changes a tyrosine at codon 170 to a nonsense codon. The mutation was History not detected in healthy family members (Table 1). Furthermore, A three-generation Chinese family with four patients suffering it was not found in any of the 100 healthy people recruited as from spontaneous pneumothorax was recruited for this study. controls, indicating that this sequence change is rare in a nor- A clustering of pneumothorax was observed in the proband’s mal population (Figure 3A). family including her father and two siblings (Figure 1). The pro- band (II-1), a 40-year-old non-smoking female, was suffering from right-sided chest pain. A chest CT-scan of the proband was taken. Spirometry and concentrations of alpha-1- anyitrpsin were tested in the patients.

Gene analysis For the genetic analysis, genomic DNA was extracted from each of the 11 members of the family and 100 unrelated healthy con- trols using the DNA Isolation Kit for Cells and Tissues (Roche Diagnostic Co., Indianapolis, IN) from venous blood. The con- centration and quality of the genomic DNA were evaluated by Nanodrop (ND-1000, Thermo Scientific, Wilmington, DE, USA). Primers designed to amplify exons 4–14 and the flanking introns of FLCN were used. PCR was performed on 50 ng of genomic DNA under the following amplification conditions: 94 C for 5 min followed by 10 cycles at 94 C for 30 s, 61.5 C for 30 s and 72 C for 45 s, then another 30 cycles at 94 C for 30 s, 55 C for 30 s and 72 C for 45 s. The PCR products were then purified and both strands were sequenced on an ABI 3730 automated se- quencer using BigDye terminator cycle sequencing reagents Figure 2. HRCT scan of the proband, who having episodes of spontaneous (Applied Biosystems). pneumothorax.

Figure 1. Pedigree of the patient family. Generations is identified by Roman numerals and individuals with Arabic numbers. The proband: II-1. J.-F. Zhu et al. | 25 Downloaded from https://academic.oup.com/qjmed/article/110/1/23/2631740 by guest on 30 September 2021

Figure 3. A direct sequencing of 11 coding exons of FLCN (exon 4–14) revealed the rare nonsense mutation: c. 510C > G (p.Tyr170X) on exon 6 in patient and not in healthy people. B All chromatograms of other patients in the family were also showed.

Table 1. Genotypes in FLCN in patient family members found in patients with spontaneous pneumothorax, but not in the family members with no incidence of pneumothorax, sug- Patient family member Exon 6 c.510 gesting that there is a strong correlation between the gene mu- I-1 CG tation and pneumothorax. I-2 GG More than 100 mutation patterns have been reported for the II-1 CG FLCN gene. Here, we defined a novel nonsense mutation that re- II-2 CG sults in a premature stop in exon 6. This mutation induces the II-3 CG truncation of the FLCN , leading to the loss of the protein II-4 GG function. The function of the FLCN protein is known to be asso- II-5 GG ciated with autophagy as a tumour suppressor. The lack of FLCN II-6 GG leads to an enhanced MTORC1 activity, which results in a sup- III-1 GG pressive effect of autophagy10 and increases downstream mol- III-2 GG ecules of the mTOR pathway, such as the vascular endothelial III-3 GG growth factor, which may lead to cyst formation.11,12 Recently, Khabibullin et al.13 found significantly increased cell-cell adhe- sion forces in FLCN-deficient HBE cells, suggesting that FLCN de- Discussion ficiency may result in an increased vulnerability to respiration- induced physical forces due to altered cell–cell adhesion in the This study presented the clinical and genetic data of a Chinese lung. family in which four patients have spontaneous pneumothorax. Renal tumours and skin lesions are the other two major A CT scan of the proband’s lung revealed lung cysts and characteristics of BHDS, and these manifestations can develop pneumothorax. An exon 6 c.510C > A mutation has already with age.14 In our study, patients with the mutation did not pre- been reported (https://grenada.lumc.nl/LOVD2/shared1/vari sent any other features of BHDS. A previous study of nonsense ants.php?select_db¼FLCN&action¼view&view¼0000259%2C000 mutations in FLCN in familial spontaneous pneumothorax 0122%2C0); here we reported a heterozygous mutation c.510C > showed that neither of the two independently affected family A in exon 6 with a different allele. Following the diagnostic cri- members had the typical skin lesions or renal tumours.2 Ren teria for BHDS proposed by Menko et al.,4 the patient could be et al.15 reported that an in-frame mutation in exon 6 was de- diagnosed as BHDS in the absence of skin lesions or renal tected in a Chinese family who presented with spontaneous tumours. pneumothorax only. A further follow-up study is needed. Numerous clinical manifestations, including fibrofollicu- In summary, we report a Chinese family with spontaneous loma, renal cancer, multiple lung cysts and pneumothorax, can pneumothorax caused by a nonsense mutation in exon 6 of the be found in patients with BHDS. Among them, spontaneous FLCN gene. pneumothorax is the most common presentation of pulmonary involvement with BHDS. A CT examination of the thorax detects multiple bilateral pulmonary cysts in >80% of BHDS-affected in- dividuals,6 which develop at a median age of 30–40 years.7 According to Zbar, patients with BHDS have a 50-fold increase Funding in the risk of developing pneumothorax, and this may relate to This study was supported by grants from the National lung cysts.8 The location of cysts in cases of BHDS is different Natural Science Foundation of China (No.31301024); from the typical apical location seen in cases of primary spon- Doctoral Fund of Ministry of Education of the People’s taneous pneumothorax or emphysema; in BHDS patients, the Republic of China (20120142120080). cyst is most often located in the basal lung regions.9 After sequencing, a heterozygous mutation c. 510C > G in exon 6 was Conflict of interest: None declared. 26 | QJM: An International Journal of Medicine, 2017, Vol. 110, No. 1

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