Clinical and genetic characteristics of chinese patients with Birt-Hogg-Dubé syndrome

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1 Liu et l. Orphnet Journl of Rre Diseses (2017) 12:104 DOI /s RESEARCH Clinicl nd genetic chrcteristics of chinese ptients with Birt-Hogg-Dubé syndrome Open Access Yping Liu 1, Zhiyn Xu 2, Ruie Feng 3, Yongzhong Zhn 4,5, Jun Wng 4, Guozhen Li 4, Xue Li 4, Weihong Zhng 6, Xiowen Hu 7, Xinlun Tin 4*, Ki-Feng Xu 4 nd Xue Zhng 1 Abstrct Bckground: Birt-Hogg-Dubé syndrome (BHD) is n utosoml dominnt disorder, the min mnifesttions of which re fibrofolliculoms, renl tumors, pulmonry cysts nd recurrent pneumothorx. The known custive gene for BHD syndrome is the folliculin (FLCN) gene on chromosome 17p11.2. Studies of the FLCN muttion for BHD syndrome re less prevlent in Chinese popultions thn in Cucsin popultions. Our study ims to investigte the genotype spectrum in group of Chinese ptients with BHD. Methods: We enrolled 51 ptients with symptoms highly suggestive of BHD from Jnury 2014 to Februry The FLCN gene ws exmined using PCR nd Snger sequencing in every ptient, for those whose Snger sequencing showed negtive muttion results, multiplex ligtion-dependent probe mplifiction (MLPA) testing ws conducted to detect ny losses of lrge segments. Min results: Among the 51 ptients, 27 hd FLCN germline muttions. In totl, 20 muttions were identified: 14 were novel muttions, including 3 splice cceptor site muttions, 2 different deletions, 6 nonsense muttions, 1 missense muttion, 1 smll insertion, nd 1 deletion of the whole exon 8. Conclusions: We found similr genotype spectrum but different mutnt loci in Chinese ptients with BHD compred with Europen nd Americn ptients, thus providing stronger evidence for the clinicl moleculr dignosis of BHD in Chin. It suggests tht muttion nlysis of the FLCN gene should be systemticlly conducted in ptients with cystic lung diseses. Keywords: Birt-Hogg-Dubé syndrome, FLCN, Muttion spectrum Bckground Birt-Hogg-Dubé syndrome (BHD, OMIM #135150) is rre utosoml dominnt disorder, the min symptoms of which re multiple pulmonry cysts followed by recurrent pneumothorx, fibrofolliculoms, nd renl cell crcinoms. These three symptoms pper seprtely [1, 2]. Lung-relted symptoms re often the erliest phenotypicl mnifesttions to pper, but most ptients re symptomtic [3, 4]. The pulmonry mnifesttions of BHD occsionlly need to be distinguished * Correspondence: xinlun_t@sin.com Equl contributors 4 Deprtment of Respirtory Medicine, Chinese Acdemy of Medicl Sciences, Peking Union Medicl College Hospitl, Beijing , Chin Full list of uthor informtion is vilble t the end of the rticle from other conditions ssocited with diffuse cysts lung diseses (DCLD), such s lymphngioleiomyomtosis, Lngerhns cell histiocytosis, lymphocytic interstitil nd pneumonitis [5]. Lck of comprehensive understnding of BHD often leds to high misdignosis rte. The gene responsible for BHD syndrome, the folliculin (FLCN) gene on chromosome 17p11.2, is tumor suppressor gene tht ws first reported in 2002 [6] nd is known to be involved in the signling of mmmlin trget of rpmycin (mtor) [7]. The FLCN gene consists of 14 exons encoding 579-mino-cid-long protein, folliculin [8]. Disese-cusing muttions in the FLCN gene, including insertions, deletions, missense nd nonsense muttions, were found over the entire gene. Multiple in vitro studies focused on FLCN functions The Author(s) Open Access This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Public Domin Dediction wiver ( pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Liu et l. Orphnet Journl of Rre Diseses (2017) 12:104 Pge 2 of 8 suggested tht the ctivtion of the AKT-mTOR pthwy nd incresed ctivity of bsic-helix-loop-helix trnscription fctor TFE3 were relted to FLCN-deficient mouse cell lines [9 11]. To dte, 149 unique FLCN germline muttions hve been identified in BHD ptients nd ctlogued in the Leiden Open Vrition Dtbse. Since Nickerson et l. first described BHD syndrome in 2002 [6], studies of FLCN muttions nd relted mnifesttions hve been populr in Europe nd the United Sttes. Anlysis of Cucsin dt demonstrted tht the frequency of 1-bp deletion or insertion within hypermutble C8 trct in exon 11 of FLCN ws high; further moleculr reserch lso confirmed tht the poly(c) trct in exon 11 of FLCN is muttion hot spot [12]. Studies of FLCN muttions for BHD syndrome re less prevlent in Chin thn they re in Europe nd Americ. Jpnese reserchers reported gene muttions of FLCN in five ptients with BHD in 2007; ll of the muttions were unique, nd four were novel [13]. A recent genetic study of Jpnese ptients with BHD syndrome published in 2016 included 312 ptients from 120 different fmilies nd identified 31 FLCN sequence vrints; two different muttion hot spots, c.1533_1536delgatg in exon 13 nd c.1347_1353dupccaccct in exon 12, were found [14]. The difference between rces my result in different muttion spectrum in Asin compred with Cucsin popultions. Nevertheless, studies of BHD syndrome in Asi re rre. BHD syndrome muttion nlysis in Jpnese popultions concluded tht isolted type with pulmonry involvement nd recurrent episodes of pneumothorces were more informtive s dignostic criteri for BHD in the Asin Jpnese popultion [13, 14]. In Chin, Ren et l. reported spordic nd fmilil isolted primry spontneous pneumothorx (PSP) nd found tht 10 PSP ptients hd FLCN gene muttions, none of whom hd other fetures of BHD [15]. However, further studies of BHD syndrome in Chinese popultions re wrrnted. This study ims to discover the muttion spectrum of the FLCN gene in Chinese ptients with BHD nd ttempts to relte the muttion spectrum to the known phenotypes through literture review. Methods Study popultion A totl of 51 ptients who hd been receiving medicl cre t Peking Union Medicl College Hospitl (PUMCH) nd who hd pulmonry cysts in clinicl dignosis from Jnury 2014 to Februry 2017 were included in the study. The inclusion criteri were BHD symptoms, s described in the review of BHD syndrome dignosis nd mngement published in Lncet Oncol, 2009 [16]. Ptients with the following conditions were highly suspected of hving BHD: multiple bilterl bslly locted lung cysts (Fig. 1) with no other pprent cuse, history of episodes of pneumothorces, first-degree reltive with BHD, multiple fibrofolliculoms or trichodiscoms confirmed by dermtologists, probble nephropthy. High-resolution computed tomogrphy (HRCT) results of ech ptient were ssessed by two pulmonry physicins independently. Those with rdiology imges highly suggestive of other diffuse cystic lung diseses were excluded; these diseses included lymphgioleiomyomtosis, light chin deposition disese, myloidosis, infectious pneumocystis, tuberous sclerosis, lymphoid interstitil pneumoni nd pulmonry Lngerhns cell histiocytosis. Ptients ccompnied by specific symptoms of hereditry syndromes, such s cystic fibrosis, Ehlers-Dnlos syndrome, homocystinuri, Mrfn syndrome nd α1- ntitrypsin deficiency, were lso excluded from the study [17, 18]. The protocol of this study ws pproved by the Institutionl Review Bord committee t PUMCH. Muttion nlysis of the FLCN gene Genomic DNA from ll 51 ptients ws extrcted from peripherl blood leukocytes using Qigen minibox (ccording to the mnufcturer s instructions. A totl of 14 coding exons with the flnking sequences of the FLCN gene were mplified. The softwre Primier-Primer5 ws used to design exon primers. The primer sequences re listed in Additionl file 1: Tble S1. The mplifiction rection mixture (25 μl) ws subjected to denturtion t 95 C for 5 min, followed by 35 cycles t 95 C for 1 min, nneling t C for 1 min, 72 C for 3 min, nd finl extension t 72 C for 15 min. Snger sequencing ws crried out on the smples to identify the muttions in ech mplicon. Muttions were described ccording to the recommended nomenclture t [19]. Nucleotide numbers re derived from GenBnk ccession number NM_144997, ssuming tht nucleotide 456 is the A of the first ATG trnsltion initition codon. All muttions were checked in the Humn Gene Muttion Dtbse, which is the gold stndrd resource for comprehensive dt on published humn inherited disese muttions. Those tht hd not been previously reported re mrked s novel muttions in this rticle. Furthermore, for those whose Snger sequencing showed negtive muttion results, multiplex ligtiondependent probe mplifiction (MLPA) test ws pplied. MLPA cn be used to detect whole-exon deletions nd duplictions tht re not detectble by trditionl Snger sequencing [20, 21]. Results Germline muttion of the FLCN gene FLCN muttions were identified in 27 independent individuls from different fmilies. A totl of 20 muttions,

3 Liu et l. Orphnet Journl of Rre Diseses (2017) 12:104 Pge 3 of 8 Fig. 1 Chest CT showing multiple thin-wlled cysts in Ptient B24 Tble 1 Results of muttion nlysis of the FLCN gene Ptient Position (hg19) (NM_144997) Nucleotide chnge Amino cid chnge B1 B2 Vrint clssifiction (ACMG guideline) MAF (ExAC, est Asin) Exon 9 ( _449) c.946_947delag p.ser316tyrfs*73 Exon 9 ( ) c.933delt p.pro311profs*11 B3 Intron 7 ( ) c.780-1g > T Splicing B4 Exon 7 ( ) c.658c > T p.gln220* B5 Intron 5 ( ) c.397-1g > C Splicing B6 Intron 4 ( ) c G > T Splicing B7 Exon 4 ( ) c.214dela p.ser72alfs*104 B8 Exon 4 ( ) c.157c > T p.gln53* B9 Exon 13 ( ) c.1533g > A p.trp511* B10 Exon 12 ( ) c.1429c > T p.arg477* B11, B12, B13,B19, B20 Exon 11 ( ) c.1285dupc p.his429profs* B14, B15, B24, B27 Exon 11 ( ) c.1285delc p.his429thrfs* B16 Exon 10 ( ) c.1067 T > C p.leu356pro B17 Exon 8 (brek points were not determined) ΔE8 p.trp260cysfs*12 B18 Exon 9 ( ) c.1015c > T p.gln339* B21 Intron 10 ( _827) c _1179-8deltcc Splicing B22 Exon 10 ( ) c.1165g > T p.glu389* B23 Exon 14 ( ) c.1658g > A p.trp553* B25 Exon 7 ( _836) c.747_756insgtgatgacaa p.asn249lysfs*1 B26 Exon 4 ( ) novel muttions identified in this study *designtes stop codon c.145g > T p.glu49*

4 Liu et l. Orphnet Journl of Rre Diseses (2017) 12:104 Pge 4 of 8 with 14 novel nd 6 previously known heterozygous FLCN muttions (Tble 1), were identified in this study. The muttion spectrum of FLCN mong our ptients is illustrted in Fig. 2. Clinicl chrcteristics of these ptients re listed in Tble 2. The clinicl dignoses of FLCN negtive ptients re vilble in Additionl file 2: Tble S2. Two novel muttions, c G > T, c.780-1g > T, involving cnonicl splicing sites, were expected to cuse problems in FLCN mrna splicing. One novel muttion, c _1179-8deltcc, ws predicted to crete berrnt splicing of FLCN mrna by Humn Splicing Finder. Smll deletions involve the following 2 loci: c.933delt, single bp deletion in exon 9 of the FLCN gene leds to premture termintion codon 11 mino cids wy from the deletion site, nd c.214dela, single bp deletion in exon 4 of FLCN leds to frmeshift muttion tht genertes stop codon 104 mino cids downstrem. One smll insertion in exon 7 ws noted: c.747_756insgtgat- GACAA, p.asn249lysfs*1. Six nonsense muttions, c.157c > T, p.gln53*; c.658c > T, p.gln220*; c.1165g > T, p.glu389*; c.145g > T, p.glu49*; c.1015c > T, p.gln339*; nd c.1658g > A, p.trp553* were detected in exons 4, 7, 10, 4, 9 nd 14, respectively. One missense muttion, c.1067 T > C, p.leu356pro, in exon 10 ws discovered. Among the previously known heterozygous muttions, single deletion or insertion of cytosine in codon 1285 ws identified in nine ptients, suggesting tht codon 1285 of exon 11 ws lso muttion hot spot in Chinese Asins compred with Western Cucsins [22]. In ddition to the point muttions we hve found using Snger Sequencing, whole-exon 8 deletion ws detected in one ptient using MLPA (Fig. 3). Clinicl chrcteristics of these ptients There were 27 independent individuls from different fmilies dignosed with BHD in our group. Only three ptients were mle. The medin dignostic ge is 48 (29-65 yers old). Only seven ptients did not hve history of pneumothorx, nd two ptients hd history of smoking. A totl of 22 ptients hd fmily history of pulmonry bull or pneumothorx. Only three of our ptients hd skin lesions t the time of dignosis, nd two of them hd fibrofolliculom nd one hd srcom cutis. Renl involvement occurred in 5 out of our 22 ptients (five ptients were reluctnt to do the test), 4 hd renl cysts, nd two hd hmrtom. Considering pulmonry lesions, only two ptients did not hve CTdetectble pulmonry cysts, but they both hd history of pneumothorx. Discussion When BHD ws described in 1977, its incidence ws unknown. A study in Lncet Oncology in 2009 reported tht pproximtely 200 fmilies hd been identified worldwide; but the number of ffected fmilies now fr Fig. 2 Muttion spectrum of the FLCN gene responsible for BHD syndrome. Top of this figure: muttions identified in this study; Bottom of this figure: muttions identified in other studies s reviewed in Schmidt et l. [11], Furuy et l. [14], Zhng et l. [24], Rossing et l. [25]. Definitions of bbrevitions: FS = frmeshift; MS = missense; NS = nonsense; AAΔ, mino cid deletion infrme; pmet1? = proposed deletion of inititor codon; SS = splice site. ATG = inititor codon. CpG = puttive promoter region. E8 = whole exon 8 loss. Blue br, intrgenic deletion; Brown br, intrgenic dupliction

5 Liu et l. Orphnet Journl of Rre Diseses (2017) 12:104 Pge 5 of 8 Tble 2 Clinicl mnifesttions of BHD ptients Ptient Number Sex Age Pneumothorx history Smoking history Fmily history Skin involved Kidney involved Lung cysts B1 F 63 Y N N N N Lrge B2 M 55 Y Y Y Skin fibrofolliculom NA Medium B3 F 35 N N N Srcom cutis N Lrge B4 F 46 Y N N N N Lrge B5 F 41 Y NA Y N Bilterl renl cysts Lrge B6 F 34 N N Y N NA Medium B7 F 34 Y N Y N NA Lrge B8 F 54 Y N Y N N Lrge B9 F 29 Y N Y N N Medium B10 F 57 Y N Y N NA Lrge B11 F 64 N N Y N N Smll B12 F 65 Y NA Y N N Lrge B13 F 39 Y N Y Skin fibrofolliculom NA Lrge B14 M 32 Y Y Y N N N B15 F 50 Y N Y N N Smll B16 F 63 Y N N N N N B17 F 50 N N Y N Bilterl renl cysts Medium B18 F 43 Y N N N N Lrge B19 F 55 N N Y N Left renl hmrtom Medium B20 F 39 Y N Y N N Lrge B21 F 47 Y N Y N N Medium B22 F 41 Y N Y N N Medium B23 M 44 Y N Y N N Medium B24 F 65 N N Y N Left renl hmrtom, Lrge bilterl renl cysts B25 F 68 N N Y N Bilterl renl cysts Medium B26 F 58 Y N Y N N Medium B27 F 33 Y N Y N N Lrge Definitions of bbrevitions: F femle, M mle, Y yes, N no, NA not vilble outnumbers tht report [16]. It ws not until 2008 tht BHD ws recognized by physicins in Chin [15]. No epidemiologicl dt bout the incidence rte of BHD in the Chinese popultion hs been vilble until now. This is the first report of systemtic muttion screening nlysis of FLCN in comprbly lrge cohort of Chinese ptients with BHD. In 27 of the 51 ptients with symptoms suggestive of BHD, we found 20 muttions in FLCN, including 14 novel nd 6 lredy reported muttions. Two novel smll deletions of nucleotides, c.933delt nd c.214dela, cused frmeshift muttion, resulting in premture termintion codons or triggering nonsense-medited mrna decy (NMD). These muttions would generte possible structurl nd functionl chnges in folliculin or mrna degrdtion; further functionl studies were wrrnted. Three novel muttions involving the splicing sites of exons hve been found in this study, c.780-1g > T, c G > T nd c _1179-8deltcc. The first two could chnge the cceptor site of intron 7 or the donor site of intron 4, respectively, which would generlly cuse exon skipping. The third one might crete n intronic ESE site of intron 11 s predicted by Humn Splicing Finder; however, ESE finder didn t get the sme prediction. Considering the typicl clinicl mnifesttions of BHD nd lso the positive fmily history (both her fther nd brother ffected) of this ptient, the ity of this vrint might be strong. However, further functionl tests re needed to vlidte this prediction. Additionlly, six novel nonsense muttions, p.gln220*; p.gln53*, p.gln339*, p.glu389*, p.trp553* nd p.glu49*, hve been discovered; the resulting premture termintion codons will indeed cuse truncted protein production nd/or NMD. One missense muttion, c1067t > C; p.leu356- Pro, in exon 10 ws noted. PolyPhen2, Muttion Tster,

6 Liu et l. Orphnet Journl of Rre Diseses (2017) 12:104 Pge 6 of 8 Fig. 3 MLPA result of ptient B17 showing loss of one copy of exon 8 of FLCN FATHMM nd PROVEAN gve probbly dmging/ disese cusing/ dmging/ deleterious results, nd its REVEL score is All the prediction tools supported its ity. In the future, however, functionl tests of this missense vrint will be needed to further provide solid evidence. In ddition to the identifiction of single nucleotide vrints nd intrgenic smll indels mentioned bove, exon deletion ws detected by the MLPA test. A whole exon 8 loss ws identified in one ptient, nd truncted protein or NMD re highly likely to result from the loss of this exon. All of the vrints identified in this study were from ExAC dtbse except for the recurrent muttion hot spot (c.1285dupc/delc); they were ll clssified s with some mnul djustments such s checkinging PVS1, PM2, PP3 nd/or PP4 ( bsed on ACMG/AMP 2015 guidelines (Tble 1). Similr to the muttion hot spots reported previously in Cucsins [23], 5 dupliction nd 4 deletion muttions t c.1285, hypermutble C8 trct in exon 11, were found in our study, ccounting for 33.3% (9/27) of ll cses nd suggesting tht this muttion hot spot does not differ between ethnic popultions. No other significnt muttions were found to be potentil hot spot in the Chinese popultion. Nevertheless, 14 of 27 (>50% of totl cses) FLCN positive ptients were reported to hve novel muttions, thus demonstrting the diversity of muttion spots long the gene nd drmticlly brodening the muttion spectrum of FLCN. Thus, the dt might suggest rce differences in the muttion spots between Chinese nd Cucsins. However, considering the inctivtion role of FLCN in the etiology of BHD, it is not surprising to see novel ones spreding ll over the genes. More studies with lrger popultions of Chinese ptients with BHD re needed to investigte this topic further. The first erly onset symptom in substntil proportion of our BHD ptients ws pneumothorx (74%, 20/ 27), nd similr clinicl pttern ws reported in Jpnese Asin popultions in 2016 [14]. Typicl skin fibrofolliculoms were only detected in two ptients (Fig. 4); probble explntion for the low detection rte ws tht the ppules were inconspicuous nd symptomtic. Fig. 4 A picture showing Skin fibrofolliculinom from one ptient with BHD syndrome

7 Liu et l. Orphnet Journl of Rre Diseses (2017) 12:104 Pge 7 of 8 Fig. 5 CT imge showing renl cyst (rrow) in Ptients B24 Furthermore, no renl cell crcinom ws dignosed in our BHD ptients, except for 1 with hmrtoms nd 4 with renl cysts (Fig. 5). Ptients enrolled in this study were mostly from respirtory clinic, which could explin the low renl cell crcinom rte. Though no significnt ssocition between FLCN muttion sttus nd lung cyst prmeters ws stted, Dr. Toro et l. discovered tht BHD muttions in exon 9 were ssocited with more lung cysts thn other muttion loci [22]. Nevertheless, tht correltion ws not identified in our study, nd no significnt correltions between the extent of clinicl mnifesttions severity nd types of gene muttions were noted. Additionlly, five of our ptients did not undergo renl rdiologicl screening becuse of the ptients reluctnce. Therefore, we cnnot confirm low prevlence of renl lesions in our group. The smll number of ptients my lso hve contributed to lower rte of renl cncer in this study, lthough this study hs reltively lrge smple size compred with other studies of BHD in Chin. There re severl limittions of our study. Pulmonry cysts exist not only in ptients with BHD but lso in those with DCLD, such s lymphngioleiomyomtosis nd Lngerhns cell histiocytosis [5]. FLCN, FBN1, COL3A1, CBS, SERPINA1 nd TSC1/TSC2 were involved in different DCLD. Here, we only focused on the FLCN gene nd BHD syndrome. Zhng et l. developed new detecting method using rpid next genertion sequencing-bsed (NGS) pnel to differentite FLCN gene muttions in ptients with PSP [24]. In future efforts, trgeted NGS pnel or whole exome sequencing nlysis on FLCN-negtive cses might be necessry. Nevertheless, here, we provide further genetic chrcteriztions of ptients with symptoms of BHD. Conclusion In conclusion, this study reports 14 novel muttions of FLCN in 27 ptients with BHD nd is the first study to demonstrte the muttion spectrum of FLCN in Chinese study popultion. The muttion spectrum in the Chinese popultion is even more extensively distributed over the entire FLCN gene thn tht in Cucsins. These genetic findings provide stronger evidence for the clinicl moleculr dignosis of BHD in Chin. Our results suggest tht muttion nlysis of the FLCN gene should be systemticlly conducted in ptients with cystic lung diseses. Additionl files Additionl file 1: Tble S1. Primers of Exon 4 to 14 in the FLCN gene. (XLSX 8 kb) Additionl file 2: Tble S2. Clinicl dignosis for those ptients with FLCN-negtive results. (XLSX 8 kb) Abbrevitions BHD: Birt-Hogg-Dubé syndrome; DCLD: Diffuse cysts lung diseses; FLCN: Folliculin; MLPA: Multiplex ligtion-dependent probe mplifiction; mtor: mmmlin trget of rpmycin; NGS: Next genertion sequencing; NMD: Nonsense-medited mrna decy; PUMCH: Peking Union Medicl College Hospitl Acknowledgments We thnk ll of the ptients nd fmilies for their contributions to this work. Funding The reserch ws supported by the Ntionl Nturl Science Foundtion of Chin ( ) to Yping Liu; Beijing Municipl Science nd Technology Commission (Z ),CAMS Innovtion Fund for Medicl Sciences (2016-I2M-1-002) to Xue Zhng; nd The Ntionl Key Reserch nd Development Progrm of Chin (No. 2016YFC ) to Ki-Feng Xu.

8 Liu et l. Orphnet Journl of Rre Diseses (2017) 12:104 Pge 8 of 8 Avilbility of dt nd mterils Not pplicble. Dt shring not pplicble to this rticle s no dtsets were generted or nlyzed during the current study. Authors contributions XZ, K-FX nd XT designed the study; YL nd ZX prticipted in moleculr genetic studies; YL, ZX nd XT wrote the mnuscript; XT, JW, GL, XL, XH nd K-FX collected the ptient smples; XT, YZ nd ZX nlyzed the clinicl dt; XT nd WZ suggested the dignosis of BHD from CT scn. RF reviewed ll the pthologicl slides of the ptients who hd lung pthologicl smples. All uthors red nd pproved the finl mnuscript. Competing interests The uthors declre tht they hve no competing interests. Consent for publiction Not pplicble. Ethics pprovl nd consent to prticipte The protocol of this study ws pproved by the Institutionl Review Bord committee t PUMCH. Signed informed consent ws obtined from the ptients for the moleculr genetic study. Publisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in published mps nd institutionl ffilitions. Author detils 1 McKusick-Zhng Center for Genetic Medicine, Stte Key Lbortory of Medicl Moleculr Biology, Institute of Bsic Medicl Sciences, Chinese Acdemy of Medicl Sciences nd Peking Union Medicl College, Beijing , Chin. 2 Deprtment of Internl Medicine, Peking Union Medicl College Hospitl, Beijing, Chin. 3 Deprtment of Pthology, Peking Union Medicl College Hospitl, Beijing, Chin. 4 Deprtment of Respirtory Medicine, Chinese Acdemy of Medicl Sciences, Peking Union Medicl College Hospitl, Beijing , Chin. 5 Deprtment of Respirtory Medicine, Nnfng Hospitl, Southern Medicl University, Gungzhou, Chin. 6 Deprtment of Rdiology, Peking Union Medicl College Hospitl, Beijing, Chin. 7 Deprtment of Respirtory Medicine, Anhui Provincil Hospitl Affilited to Anhui Medicl University, Hefei, Chin. Received: 27 Februry 2017 Accepted: 16 My 2017 References 1. 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