Phenotypic expansion of retinal abnormalities in folliculin (FLCN) variant-related pathology (Birt–Hogg–Dubé syndrome)

The Birt-Hogg-Dube (BHD) autosomal dominantly inherited syndrome has clinical features that include the development of skin fibrofolliculomas, lung cysts, and renal neoplasia. Renal tumors occur in approximately one third of individuals with BHD. The gene responsible for BHD syndrome is Folliculin (FLCN), which encodes a 68kD phosphoprotein (FLCN). One allele of the FLCN gene is mutated in the germline of >90% of BHD patients and somatic mutations or loss of the wild type FLCN allele are observed in the renal tumors of patients with BHD, suggesting that FLCN acts as a tumor suppressor protein. The human renal cell carcinoma cell line UOK257, derived from a patient with a germline mutation in the FLCN gene, harbors a truncated version of the FLCN protein. Reconstitution of the wild type FLCN protein into the FLCN-deficient UOK257 cells decreased their proliferation rate. Here we tested whether this difference in cell proliferation rate may be partly attributed to the effect of FLCN on cell cycle progression. We synchronized FLCN-deficient UOK257 cells and their isogenic, wild type or mutant FLCN counterparts, by double thymidine block and followed cell cycle progression after release. UOK257 cells expressing wild type FLCN exhibited a slower progression through the late S and G2/M-phases of the cell cycle compared to the vector only controls (FLCN-deficient). The reintroduction of tumor-associated FLCN mutants failed to delay cell cycle progression in UOK257 cells, indicating that the effect of wild type FLCN on cell cycle progression may contribute to its tumor suppressor function. FLCN phosphorylation on serines S62 and S73 fluctuates throughout the cell cycle and peaks in cells arrested at the G2/M boundary following treatment with nocodazole. In keeping with this observation, the reintroduction of a FLCN S62/73 phosphomimetic mutant into the UOK257 cell line resulted in faster progression through the cell cycle compared to those expressing the wild type FLCN protein. These findings suggest that orderly changes in FLCN phosphorylation during the cell cycle are linked to and may be required for its effect on cell cycle progression and possibly its activity as a tumor suppressor protein. We expect that these studies will provide insight into the role of FLCN in kidney tumorigenesis and will identify putative new targets for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2171. doi:1538-7445.AM2012-2171

Purpose To report a rare case of Birt-Hogg-Dubé Syndrome (BHD) with progressive chorioretinopathy. Methods Case report. Results A 55-year-old woman presented with longstanding nyctalopia attributed to a congenital retinal dystrophy, but no prior genetic testing. Her posterior pole examination demonstrated retinal pigment epithelium (RPE) mottling with extensive macular drusen and paracentral chorioretinal atrophy, consistent with a fleck retinopathy. Her past medical history was remarkable for nephrectomy for unilateral renal malignancy, parotid tumors and thyroid nodules. Dark adaptation time was prolonged, and electroretinography (ERG) revealed abnormal waveforms with depressed amplitudes. Genetic testing confirmed a deletion mutation in the folliculin (FLCN) gene and was negative for other relevant mutations, including EFEMP1 responsible for autosomal dominant macular and peripapillary drusen in Doyne honeycomb retinal dystrophy and TIMP3 responsible for Sorsby Fundus Dystrophy. Conclusion BHD is a rare autosomal-dominant disorder with multi-systemic clinical manifestations caused by a mutation in the FLCN gene. Affected individuals are prone to renal and pulmonary cysts, renal cancer, and fibrofolliculomas. Reports on ocular manifestations of BHD include eyelid fibrofolliculomas, flecked chorioretinopathy, choroidal melanoma, choroidal melanoma with sector melanocytosis, and retinal pigment epithelial micro-detachments. In this case of BHD, we note a fleck retinopathy with bilateral chorioretinal atrophy, displaying a phenotype of extensive chorioretinopathy associated with impaired dark adaptation and ERG abnormalities. Abbreviations BHD: Birt-Hogg-Dubé syndrome; FLCN: Folliculin. RPE: retinal pigment epithelium; OD: Oculus dexter (right eye); OS: Oculus sinister (left eye). OU: Oculus uterque (both eyes); ERG: electroretinogram; mfERG: multifocal electroretinography. ffERG: full-field electroretinography; FAF: fundus autofluorescence; OCT: optical coherence tomography; FA: fluorescein angiography; DA: dark-adapted; LA: light-adapted; mTOR: mammalian target of rapamycin; EFEMP1: epithelial growth factor-containing fibulin-like extracellular matrix protein 1; VPS13B: Vacuolar Protein Sorting 13 Homolog B; AGBL5: AATP/GTP-Binding Protein Like 5; ALMS1: Alstrom Syndrome 1; COL1BA1: Collagen Type I Beta, Alpha Chain 1; PDE6A: Rod Phosphodiesterase 6-alpha; USH2A: Usherin 2a; VCAN: Versican; RP: Retinitis pigmentosa; AR: Autosomal recessive.

Birt-Hogg-Dubé (BHD) syndrome is characterised by a triad of benign skin tumours, pulmonary cysts with an attendant risk of pneumothorax, and renal cancer. BHD is caused by heterozygous pathogenic variants in folliculin (FLCN), a tumour suppressor gene.

Radiologically Indeterminate Pulmonary Cysts in Birt-Hogg-Dubé Syndrome

SAT-097 HEREDITARY RENAL CANCER PREDISPOSING SYNDROMES IN TASMANIA

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Introduction: Birt–Hogg–Dubé (BHD) syndrome is an autosomal dominant disorder that predisposes to fibrofolliculomas, pulmonary cysts, spontaneous pneumothorax and renal neoplasia. BHD is characterized by germline mutations in the tumor suppressor gene folliculin (FLCN) gene. Inactivation of the remaining FLCN allele in kidney cells drives tumorigenesis. Novel FLCN-interacting proteins, FNIP1 and FNIP2, were identified. Studies with FLCN-deficient in vitro and in vivo models support a role for FLCN in modulating AKT-mechanistic target of rapamycin (mTOR) signaling. Emerging evidence suggests that FLCN may interact in a number of pathways/processes. Identification of FLCN’s major functional roles will provide the basis for developing targeted therapies for BHD patients.Areas covered: This review covers BHD diagnostic criteria, clinical manifestations and genetics, as well as molecular consequences of FLCN inactivation. Recommended surveillance practices, patient management and potential therapeutic options are discussed.Expert opinion: In the decade since FLCN was identified as causative for BHD, we have gained a greater understanding of the clinical spectrum and genetics of this cancer syndrome. Recent studies have identified interactions between FLCN and a variety of signaling pathways and cellular processes, notably AKT-mTOR. Currently, surgical intervention is the only available therapy for BHD-associated renal tumors. Effective therapies will need to target primary pathways/processes deregulated in FLCN-deficient renal tumors and fibrofolliculomas.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder characterized by fibrofolliculomas, renal tumors, and pulmonary cysts with recurrent pneumothorax. Multiple pulmonary cysts and pneumothorax are the key signs for diagnosing BHD syndrome. The pathologic features of BHD pulmonary cysts, however, are poorly understood. This disorder is caused by mutations in the gene that encodes folliculin (FLCN). FLCN is regarded as a tumor suppressor; it mediates cellular activities by interacting with the mammalian target of rapamycin (mTOR). In this study, we investigated the lungs of 11 patients from 9 BHD families. The majority of patients consulting doctors were women between 30 and 60 years of age who had pulmonary cysts and repeated pneumothoraces. Genomic DNA testing revealed 5 different mutation patterns. Histopathologic examination found that the inner surface of cysts was lined by epithelial cells, sometimes with a predominance of type II pneumocyte-like cuboidal cells. The cysts occasionally contained internal septa consisting of alveolar walls or showed an "alveoli within an alveolus" pattern. The cells constituting the cysts stained positive for phospho-S6 ribosomal protein expression, suggesting activation of the mTOR pathway. Although BHD pulmonary cysts are frequently misdiagnosed as nonspecific cystic diseases, they are distinctly different in histopathology from other bullous changes. Mechanical stress such as rupture and postrupture remodeling allows mesothelial invagination and fibrosis. Such modified BHD pulmonary cysts are virtually indistinguishable from nonspecific blebs and bullae. We propose a new insight, namely, that the BHD syndrome-associated pulmonary cyst may be considered a hamartoma-like cystic alveolar formation associated with deranged mTOR signaling.

Folliculin gene-negative Birt-Hogg-Dube syndrome: a case report

Germline loss‐of‐function BHD mutations cause cystic lung disease and hereditary pneumothorax, yet little is known about the impact of BHD mutations in the lung. Folliculin (FLCN), the product of the Birt–Hogg–Dube (BHD) gene, has been linked to altered cell–cell adhesion and to the AMPK and mTORC1 signaling pathways. We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho‐S6, a marker of mTORC1 activation, highlighting the cell type–dependent functions of FLCN. Cell–cell adhesion forces were significantly increased in FLCN‐deficient HBE cells, consistent with prior findings in FLCN‐deficient human kidney‐derived cells. To determine how these altered cell–cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd+/− mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg. Our results indicate that FLCN regulates the AMPK and mTORC1 pathways and cell–cell adhesion in a cell type–dependent manner. FLCN deficiency may impact the physiologic response to inflation‐induced mechanical stress, but further investigation is required. We hypothesize that FLCN‐dependent effects on signaling and cellular adhesion contribute to the pathogenesis of cystic lung disease in BHD patients.

IntroductionOur basic understanding of renal cell carcinoma (RRC) in the general population is limited. To gain a better understanding of how RRC develops, I took advantage of a genetic model of kidney cancer that mirrors a genetic disease called Birt-Hogg-Dubé (BHD) syndrome. BHD patients have an increased risk of developing kidney cancer, and unlike other genetic disorders with a predisposition to RRC, BHD patients are prone to all tumour subtypes. These patients are born with inactivating mutations within a gene called Folliculin (FLCN), a tumour suppressor protein of unknown function that could be involved in monitoring DNA damage.Material and methodsTo better define the tumour suppressor role of FLCN, I carried out a protein-protein interaction using FLCN as bait, which revealed that FLCN interacts with the DNA-damage response machinery. To further explore FLCN involvement in DNA damage, I used RNAi to generated FLCN knockdown in human proximal tubule kidney cells – which are thought to be the origin cells of BHD associated renal tumours – and performed basic cellular experiment such as western blot and flow cytometry.Results and discussionsI discovered that FLCN interacts with DNA-PKcs, a DNA damage component that is responsible for repairing double strand DNA (dsDNA) breaks. This is an important advancement, as it now implicates FLCN in the maintenance of DNA. The association of FLCN with DNA-PKcs weakens when cells are subjected to DNA damage. As a consequence of FLCN loss of function, kidney cells accumulate DNA damage and the activity of DNA damage response pathways are elevated. I also have evidence showing that a perturbed G1/S check point in FLCN-deficient cells which is a known driver of cancer progression.ConclusionUltimately, my work highlights a novel role of FLCN within renal cell tumourgenesis, suggesting it functions to prevent genomic instability. Genetic conditions, such as BHD that predispose individuals to cancer, while rare themselves, provide valuable insight into somatic tumour development. By using BHD syndrome as a model of genetic instability, further work will be carried out to mechanistically establish FLCN’s role in DNA integrity and will provide valuable insight into sporadic renal cancer within the general population.

Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival through E-Cadherin, LKB1, and AMPK

BackgroundPulmonary cysts and spontaneous pneumothorax are presented in most patients with Birt-Hogg-Dubé (BHD) syndrome, which is caused by loss of function mutations in the folliculin (FLCN) gene. The pathogenic mechanisms...

Ciliopathy is a general term applied to diseases that originate from ciliary dysfunction, which often coincide with renal cyst development. Any syndrome displaying renal cysts, such as folliculin (FLCN) in Birt-Hogg-Dube (BHD) syndrome, might therefore be suspected as a novel ciliopathy. Indeed, FLCN localizes to the primary cilium. To study ciliopathies in detail, we set up a renal 3D culture system using IMCD3 cells that physiologically mimick polarized renal tubuli. SiFlcn was essayed in this system, identifying several affected processes; 1) Reduction of ciliation in vitro and in vivo. 2) Increase in cell volume. 3) Increase of mis-oriented cell divisions. These results suggest Flcn functions in cilia stability/ciliogenesis and planar cell polarity (PCP) regulation. PCP signaling is a form of non-canonical Wnt signaling subjected to ciliary regulation Accordingly, whereas β-catenin is normally present in the axoneme, this is never observed upon Flcn depletion and Wnt signaling appears increased as downstream effector Axin2 is stabilized. This suggests a switch from PCP to canonical signaling. Notably, upon ectopic GFP-FLCN expression, but not the non-functional allele p.L508R, cilia become stabilized, accompanied by an accumulation of both GFP-FLCN and β-catenin in the cilium. The second most common mutant FLCN allele is p.T463X; forced read-through with pharmaceutical agent PTC124, targeting nonsense-mediated decay, was tested and showed a robust response as protein expression appears fully restored. We suggest that BHD regulates ciliary function in a 3D polarized cell assay, and PTC124 might be a simple therapy for the second most frequent allele in BHD carriers.

FLCN: The causative gene for Birt-Hogg-Dubé syndrome