FNIP1 negatively regulates AMPK activity

Birt-Hogg-Dubé (BHD) syndrome is caused by mutations of the folliculin (FLCN) gene. FLCN is a tumour-suppressor protein which associates with two homologous proteins, folliculin-interacting protein (FNIP)1 and FNIP2. Previous studies have reported that the FLCN/FNIP complex acts as a positive regulator of the AMP-activated protein kinase (AMPK) complex (Hasumi et al., 2015), while other studies suggest the opposite (Reyes et al., 2015). To address this issue, a new report from Siggs et al. (2016) uses a new mouse model of FNIP1 deficiency to find that Fnip1 mutation leads to B-cell deficiency and development of cardiomyopathy with gain-of-function mutations in AMPK supporting the idea that FNIP acts as a negative regulator of AMPK.

Using whole-genome sequencing in mouse mutants with an autosomal recessive B-cell deficiency phenotype the authors identified that this was associated with a splice donor variant mutation in Fnip1. By flow cytometry they showed that B cells were absent from the peritoneum and spleen of Fnip1 homozygous mutants. In the bone marrow (BM) there was an absence of IgM+ or IgD+ cells, although B-cell precursors were still present. Heterozygous mutants revealed a reduced frequency of the marginal zone B-cell population. Because the antiapoptotic protein BCL2 is important for B-cell survival the effects of its overexpression on B-cell development in Fnip1 mutants were measured. Unlike previously reported (Baba et al., 2012), BCL2 overexpression only partially restored B-cell values in the BM, peritoneum and spleen. Examination of the hearts of homozygous Fnip1 mutants revealed that they were enlarged due to hypertrophy and that skeletal muscle was darker than heterozygous or wild-type mice. Histology and biochemical analysis revealed that the mutant myocardium had a marked accumulation of glycogen. Homozygous Fnip1 mutant mice also showed high expression of Nppa, Nppb and Acta1, markers of cardiac stress. Due to the known interaction between FNIP1 and AMPK (Baba et al., 2006) and the cardiac phenotype observed, authors assessed the effect of the Fnip1 mutation on cardiac AMPK activity. A basal activation of the γ2 subunit-containing AMPK complexes but reduced AMP responsiveness, in homozygous Fnip1 mutants, was observed in cardiac tissue but not in the liver. This suggests that Fnip1 and Fnip2 may be functionally redundant in the liver as they are in the kidney (Hasumi et al., 2015). One of the roles of AMPK is to promote autophagy via different pathways including the unc-51-like autophagy activating kinase 1 (ULK1) (Gwinn et al., 2008; Egan et al., 2011) which is activated via phosphorylation at serine 555. In the present study, S555 p-ULK1 is higher in Fnip1 mutant BM cells suggesting that autophagy is enhanced. This was supported by the staining of LC3, an autophagosome marker, in Fnip1 mutant B cells. Overexpression of BLC2 reduced LC3 staining in mutant and wild type cells indicating that BLC2 could oppose the enhanced autophagy in Fnip1 mutant B cells while increasing survival (Cash et al., 2011). AMPK promotes cell-cycle arrest by stabilizing p53 (Jones et al., 2005), p53 is known to enhance autophagy and apoptosis in response to cellular stress (Crighton et al., 2006). However, no differences in B-cell numbers were observed between p53-sufficient or deficient Fnip1 mutants in this study. Lack of Fnip1 caused a block in B-cell development and although autophagy was increased, its inhibition, via deletion of the autophagy protein Atg3, failed to rescue the phenotype, suggesting that enhanced autophagy alone is not sufficient to block B-cell development.

In summary, the study supports other reports showing a non-redundant role for FNIP1 in B-cell development (Baba et al., 2012), skeletal muscle position (Reyes et al., 2015) and cardiac function (Hasumi et al., 2015) and it provides evidence that FNIP1 is an endogenous negative regulator of AMPK. Supporting this idea, previous studies have reported that FLCN repressed AMPK in C. elegans and mammalian cells (Possik et al., 2014; Yan et al., 2014). In these models, absence of FLCN resulted in AMPK activation, which induced autophagy. However, these models oppose the suggestion that autophagy is reduced in BHD-associated tumour tissue (Dunlop et al., 2014).

Authors are unsure how the lack of Fnip1 causes a block in B-cell development and it remains to be investigated if another AMPK-regulated pathway or a different pathway is responsible for the phenotype. It is therefore crucial to pursue future investigations on the role of AMPK and the FLCN/FNIP complex in BHD.

  • Siggs OM, Stockenhuber A, Deobagkar-Lele M, Bull KR, Crockford TL, Kingston BL, Cornall RJ. (2016). Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity. Proceedings of the National Academy of Sciences of the United States of America. PMID: 27303042


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FLCN activates mTORC1 by maintaining lysosomal leucine level

The intracellular amino acid pool within the lysosome has been shown to activate the mTORC1 signaling pathway (Zoncu et al., 2011; Jewell et al., 2013). However, how the sequester of the signaling molecules within the lysosome occurs remains poorly understood. New research from Wu et al. (2016) shows that the suppression of FLCN, a tumour suppressor gene associated with the Birt-Hogg-Dubé (BHD) syndrome, controls mTORC1 activity by modulating the lysosomal leucine levels. FLCN exerts this new function by regulating the accumulation of the amino acid transporter PAT1 on the lysosome surface.

Previous work by the authors using a Drosophila model showed that fly FLCN mutants had a growth defects and did not reach adulthood. Interestingly, the growth defects were rescued by supplementation with high levels of leucine, but not other amino acids, in the medium. Adding an inhibitor of mTOR to the leucine-rich medium failed to rescue the slow growth phenotype (Liu et al., 2013). In mammalian cell systems, FLCN was necessary to anchor mTORC1 and this function was performed at the lysosome (Petit et al., 2013; Tsun et al., 2013). In the present study, Wu et al. (2016) show in human embryonic kidney (HEK) 293 cells, that depletion of FLCN suppresses mTORC1 activity. Addition of high amounts of leucine, but not other tested amino acids, to the medium reverses this down-regulation of mTORC1 in FLCN-depleted cells. Starving cells and then adding either basal or high levels of leucine to the medium shows that depletion of FLCN sensitizes cells to the available leucine for mTORC1 induction. The authors confirm work by previous groups (Petit et al., 2013; Tsun et al., 2013) showing that FLCN is localized on the lysosome in a nutrient-dependent manner. They show by western blotting that leucine is reduced in lysosomes from cells lacking FLCN and that this effect is reversed by supplementation with high levels of leucine in the medium. This suggests that FLCN plays an important role keeping the leucine level in lysosome but this can alternatively be achieved by increasing the amount of leucine supplied to the environment to keep the leucine signal strong enough to activate mTORC1. To test if ectopic FLCN would sequester more leucine to the lysosome, the authors generate a cell line overexpressing FLCN (FLCN-HA). In starving conditions, leucine levels and mTORC1 activation are decreased in wild-type cells. In FLCN-HA cells this reduction is much less pronounced suggesting that ectopic FLCN promotes the accumulation of leucine within the lysosome, enabling cells to resist to leucine shortages in the environment and facilitating mTORC1 activation. Similar results were shown in a Drosophila model. FLCN is located on the lysosome and it seems to regulate the efflux of leucine. This process requires it to be loaded onto membrane-anchored transporters. PAT1, a lysosome-associated membrane protein whose overexpression promotes the release of amino acids from lysosome and inhibits mTORC1 (Zoncu et al., 2011) seemed a good candidate to be involved in the FLCN-mediated mTORC1 signaling. After confirming that overexpression of PAT1 in HEK293 cells inhibits mTORC1 the authors show that both the addition of high levels of leucine to the medium and the overexpression of FLCN prevent this inhibitory effect. In addition, depletion of PAT1 using siRNA inhibits the starvation-induced decrease of mTORC1 activation and this effect is counteracted by co-depletion of FLCN. These results led to the conclusion that FLCN and PAT1 antagonize each other to control mTORC1 pathway. In starvation, cells recruit PAT1 to the lysosome to recycle the luminal storage, resulting in a decreased leucine level. However, authors show that this recruitment of PAT1 can be inhibited by ectopic FLCN resulting in the maintenance of the lysosome level. Immunoprecipitation experiments show that FLCN may regulate the localization of PAT1 through direct interactions.

In summary, this study shows a new role for FLCN in modulating the leucine level in the lysosome and how important this mechanism is to regulate mTORC1 signaling pathway. Previous work by Preston et al. (2011) on the FLCN deficient BHD patient-derived renal tumour cell line (UOK257) showed that these cells have a higher dependency on glucose metabolism demonstrating a loss of ‘metabolic flexibility’. The present work sheds light into how cells sense the available nutrients in their environment and helps to appreciate the context-dependent relations between FLCN and mTORC1, crucial for the advancement in BHD syndrome research where targeting metabolic pathways may be used therapeutically to treat BHD-associated kidney lesions.

Wu X, Zhao L, Chen Z, Ji X, Qiao X, Jin Y, & Liu W (2016). FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1. PloS one, 11 (6) PMID: 27280402

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BHD syndrome and thyroid conditions

Dong et al., (2016) have recently reported two BHD syndrome patients also affected with papillary thyroid cancer. Lesions were bilateral and multifocal and small lymph node metastases occurred. Due to the small number of patients in the study the authors are unsure whether thyroid cancer in BHD patients is susceptible to exhibiting bilaterally and lymph node metastasis. However, they suggest considering thyroidectomy and prophylactic lymph node dissection for thyroid cancer patients with BHD. They also strongly recommend neck ultrasound for BHD patients and their families and suggest a large-scale investigation be conducted to evaluate the prevalence of thyroid cancer or nodules in patients with BHD.

So far, there is no evidence associating BHD and thyroid conditions.

The original BHD patients were found in a family in which six of nine siblings had hereditary thyroid medullary carcinoma (Birt et al. 1977). However, it was subsequently shown that these six patients also displayed a mutation in the RET proto-oncogene which might have caused the thyroid carcinoma (Toro et al., 1999). Several cases of thyroid cancer have been reported in BHD patients in the literature. Gunji et al. (2007) identify thyroid carcinoma in one of five unrelated BHD cases studied where the patient also had a familial history of thyroid cancer. Fahmy et al. (2007), Toro et al. (2008), Kunogi et al. (2010) and Yamada et al. (2014) describe another five cases of thyroid cancer in BHD patients. In all cases the authors did not attribute the thyroid tumour to BHD or investigate a link between the two. Benusiglio et al., 2014 describe a BHD patient with thyroid cancer where a FLCN mutation was identified both in the patient’s blood DNA and thyroid tumour DNA, this provides a molecular basis for this association, however, alternative genetic lesions or causes of thyroid cancer were not ruled out, so no association between the patient’s BHD and thyroid cancer could be shown. In a previous study Warren et al. (2004) did not find any FLCN mRNA expression in normal thyroid.

In another report, mutation of FLCN, TSC2 and TP53 were found in a case of anaplastic thyroid cancer treated with Everolimus that had a near-complete response for 18 months, at which point the tumour acquired resistance to treatment due to mutation in the mTOR gene (Wagle et al., 2014). This suggests that somatic FLCN mutation, and other genes that activate mTOR, can cause additional types of cancer to renal cell carcinoma (RCC) and make tumours resistant to treatments with mTOR inhibitors. A population-based study in the USA shows that patients with thyroid cancer have an increase in prevalence of subsequent RCC, and in patients with RCC, there is an increase in the prevalence of subsequent thyroid cancer (Van Fossen et al., 2013). This bidirectional association could be explained by shared genetic and/or environmental risk factors or treatment effects. Thyroid cancer has also been found with a greater frequency in the Cowden syndrome, familial adenomatous polyposis, and in multiple endocrine neoplasia type 2A (Cetta et al., 2000; Liaw et al., 1997; Biscolla et al., 2004).

The only study that attempts to identify a link between BHD and thyroid conditions is a five-year prospective study of twenty-two patients from ten unrelated French families with BHD (Kluger et al. 2010).  The authors identified thyroid nodules or cysts by ultrasonography in thirteen of twenty BHD patients. None of the affected individuals had thyroid carcinomas or a familial history of thyroid cancer. Overall, nine of the ten families affected by BHD with germline FLCN mutations included individuals with thyroid nodules. The high prevalence of thyroid nodules in the BHD patients in this study is suggestive, but the lack of a control group limits assessment of the significance of the results. Benhammou et al. (2011) also identified thyroid pathology in four of eleven BHD patients studied: three had hypothyroidism and one had a thyroid nodule. Mikesell et al., (2014) report another case of a BHD patient with thyroid nodules. Hypothyroidism and Hashimoto’s thyroiditis were noted in two other cases (Nadershahi et al., 1997; Khoo et al., 2002). There are also reports of multinodular goiter in association with BHD (Drummond et al., 2002; Welsch et al., 2005; Zeibek et al,. 2013; Mikesell et al., 2014). A parathyroid adenoma was diagnosed in one BHD patient of the Kluger et al. (2010) study, and previously in another BHD patient by Chung et al. (1996).

There is currently insufficient evidence to associate thyroid cancer and other thyroid conditions with BHD. However, all the studies mentioned suggest a possible link between the two that should be considered for future research.

Dong L, Gao M, Hao WJ, Zheng XQ, Li YG, Li XL, & Yu Y (2016). Case Report of Birt-Hogg-Dubé Syndrome: Germline Mutations of FLCN Detected in Patients With Renal Cancer and Thyroid Cancer. Medicine, 95 (22) PMID: 27258496

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Anonymising and sharing patient data

Patient data is extremely valuable for biomedical and healthcare research. Collecting and sharing patient data globally can lead to several benefits such as better understanding diseases, identifying patterns in public health and disease, developing and monotoring drugs and treatments, allowing researchers to build on the work of others efficiently and finding suitable candidates to take part in clinical trials. However, concerns about privacy have been a barrier for making patient data available. Data custodians are able to legally share patient data for research via (a) consent and (b) anonymization. It is difficult and time-consuming to rely on consent as the primary method for sharing data as it is not practical to obtain consent from several patients and there is evidence of consent bias (Kho et al., 2009). Ethics boards allow the sharing of patient data without consent for research purposes if it is anonymised (Willison et al., 2008). However, there is an expectation that this anonymization is adequate and used for legitimate purposes. Studies show that, when concerned about how their health data is used, patients adopt defensive privacy behaviours, such as giving inaccurate information and not seeking care (Malin et al., 2013). It is therefore crucial to increase public confidence by creating clear governance frameworks for accessing patient data and developing methods to safeguard patient identification.

The concept of anonymous or non-identifiable data can be ambiguous. In an effort to clarify inconsistencies, El Eman et al. (2015) describe in their article the key principles for anonymising health data while ensuring it remains suitable for relevant analysis. The group explains that ensuring anonymity technically means ensuring that there is a very small probability of assigning a correct identity to a record in a dataset. Existing guidelines divide the variables in a dataset into two groups: direct identifiers and quasi-identifiers. Direct identifiers allow direct recognition or communication with the corresponding individuals (e.g. names, addresses and social insurance numbers) while quasi-identifiers can indirectly identify individuals (e.g. date of birth, postal code and ethnicity). Both groups must be addressed during anonymization. The acceptable probability of re-identification of a record varies accordingly to how the data is being shared. For a public data release the probability needs to be low because there are no other controls in place. For non-public data, a higher probability is acceptable because other security and contractual controls would be already in place. If the probability of re-identification is high, perturbation techniques can be applied to reduce it. One of the simplest and quite often used ways to perturb data is to reduce its precision through generalisation, for example, generalizing a date of birth into a month and year of birth. Better and more complex computational methods of perturbation have been reviewed by Gkoulalas-Divanis et al. (2014), these methods can reduce the amount of distortion and produce higher data quality. However, knowing when to stop perturbing the data is important to balance privacy protection and data utility and to avoid that inadequate anonymization techniques slow down research, something that already happens by introducing other disproportionate measures on data protection. Anonymization is usually time limited to account for advances in technology and for availability of other data that can be used to re-identification.

Rare diseases

When it comes to rare diseases the anonymising and sharing of patient data becomes even more important. The rarity of diseases makes it difficult to gather information, to develop treatments and to conduct large clinical trials. Data sharing has such value in these cases that it is almost a necessity for the progress of rare disease research. Presence of a rare disease does not necessarily make data impossible to anonymise. If the dataset is a sample from a population of patients with that disease the probability of re-identification may still be very small (Eguale et al., 2005).

The VHL Alliance, in collaboration with the NORD and partially funded by the Myrovlytis Trust, has developed the CGIP Databank to collect detailed medical information on patients all over the world with BHD, VHL, HLRCC, SDH and other related tumours to help scientists and clinicians to discover possible factors contributing to disease progression, to help evaluate efficacy of novel therapies and to enable a more complete advice to patients. Therapies for these diseases are emerging and there is a need to go through clinical trials to evaluate their effectiveness. The databank may also be used to contact patients about trials, including those investigating new treatments, for which they may be eligible. Participation in a clinical trial will be based upon the voluntary consent of the patient.

The CGIP Databank is maintained on a secure server, and only authorised researchers and clinicians will be able to access to an anonymised dataset.

If you have BHD, VHL, HLRCC or SDHB and would like to find out more information or if you want to join the databank and help advance research please click here. If you have any questions or thoughts, please contact the VHL alliance on databank@vhl.org.

  • Eguale T, Bartlett G & Tamblyn R (2005). Rare visible disorders/ diseases as individually identifiable health information. AMIA Annu Symp Proc PMID: 16779234
  • El Eman K, Rodgers S & Malin BA (2015). Anonymising and sharing individual data. BMJ PMID: 25794882
  • Gkoulalas-Divanis A, Loukines G & Sun J (2014). Publishing data from electronic health records while preserving privacy: a survey of algorithms. J Biomed Inform. PMID: 24936746
  • Kho ME, Duffett M, Willison DJ, Cook DJ & Brouwers MC (2009). Written informed consent and selection bias in observational studies using medical records: systematic review. BMJ PMID: 19282440
  • Malin BA, El Emam K & O’Keefe CM (2013). Biomedical data privacy: problems, perspectives, and recent advances. J Am Med Inform Assoc PMID: 23221359
  • Willison DJ, Emerson C, Szala-Meneok KV, Gibson E, Schwartz L, Weisbaum KM Fournier F, Brazil K & Coughlin MD (2008). Access to medical records for research purposes: varying perceptions across research ethics boards. J Med Ethics PMID: 18375687
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Starvation-induced FLCN association with lysosomes via a Rab34–RILP complex

Dynamic positioning of lysosomes in the cytoplasm plays an important role in their function and is, in part, regulated by cellular nutrient status. The FLCN/FNIP complex is known to be active on the lysosome surface, where it interacts with Rag GTPases, supports the nutrient‐dependent recruitment and activation of mTORC1, and regulates the localisation of lysosome associated transcription factors (Petit et al., 2013; Tsun et al., 2013). New research from Starling et al. (2016) now shows that folliculin (FLCN) also controls the dynamic cytoplasmic position of the lysosome itself.

Lysosomal positioning coordinates cellular nutrient responses (Korolchuk et al., 2011), and is affected by several components, including the GTPase Rab34 that can promote lysosome clustering in the peri-nuclear region (Wang et al., 2002). Nutrient starvation, which suppresses mTORC1 activity, can also promote peri-nuclear clustering of lysosomes in HeLa cells, while nutrient-abundance and high mTORC1 activity leads to dispersion and accumulation of lysosomes at the cell periphery. mTORC1 is activated on the lysosomal surface, via a signaling network composed of Rag GTPases, the FLCN/FNIP complex and other protein complexes. FLCN/FNIP complex receives signaling inputs from metabolic pathways via phosphorylation, upon activation of mTORC1 and AMPK (Baba et al., 2006).

Starling et al. (2016) present strong evidence for a model where starvation‐induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34‐positive peri-nuclear membranes, by promoting the association of Rab34 with its effector RILP. This restricts lysosome motility and thus promotes their retention in the peri-nuclear region of the cell.

Figure obtained from Starling et al. (2016)


The group shows that FLCN/FNIP complex is required for starvation‐induced peri-nuclear lysosome clustering.  Depletion of FLCN or of both FNIP1 and FNIP2 proteins using siRNA strongly affects lysosome positioning under starvation conditions, suggesting a functional connection between FLCN/FNIP-lysosome association and lysosome dynamics. As small GTPases are known to play a role in lysosome dynamics the group considered, among other GTPases, Rab34 and its effector protein RILP. Rab34 itself contributes to starvation‐induced peri‐nuclear clustering of lysosomes. However, in HeLa cells, depletion of FLCN significantly suppresses this ability.  FLCN was shown to associate with mitochondrial targeted Rab34 and the FLCN C‐terminal DENN domain is necessary for this association. Pull down experiments show that the FLCN‐DENN domain directly promotes the formation of an active Rab34–RILP complex.

The same type of experiments examining Rab34 and lysosome distribution were conducted in the BHD kidney cancer cell line UOK257 (FLCN deficient) and UOK2572 (FLCN restored) with results showing reduction in lysosome dynamics in the FLCN expressing cells in a DENN domain‐dependent way through Rab34/RILP.  The results in UOK257 cells are nutrient-independent, which the authors suggest might be due to the complete long term loss of full FLCN compared with acute depletion, or perhaps due to metabolic changes in the UOK257 cells.

Overall, the study shows how, in HeLa cells, FLCN couples the lysosomal nutrient signalling network to the cellular machinery that controls the intracellular distribution of the lysosome itself. The functional relevance of the study is supported by similar results, although nutrient-independent, in the BHD kidney cancer cell line, suggesting that this pathway may play a role in the pathogenesis of BHD syndrome. Since UOK257 cells do not show large deficiencies in mTORC1 activity (Baba et al., 2006), authors suggest that expanding these studies to other BHD-relevant epithelial cell types to understand how FLCN/Rab34‐dependent changes in lysosome motility may contribute to BHD syndrome.

In summary, given the complex relationship between lysosome positioning, autophagy and mTORC1 activity (Korolchuk et al., 2011), and the emerging connections between FLCN and the same pathways (Petit et al., 2013; Tsun et al., 2013), the group suggests that the dysregulation of lysosome dynamics by disruption of FLCN may contribute to the dysregulated autophagy and mTORC1 activity phenotypes found in various BHD model systems studied. The study sheds light on the mechanisms of lysosome dysregulation and can be exploited to develop therapies for kidney cancer therapies.

  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins III RF, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR, Linehan WM, Schmidt LS, Zbar B. (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc Natl Acad Sci U S A. 103(42):15552-7. PMID: 17028174
  • Korolchuk VI, Saiki S, Lichtenberg M, Siddiqi FH, Roberts EA, Imarisio S, Luca Jahreiss L, Sarkar S, Futter M, Menzies FM, O’Kane CJ, Deretic V, Rubinsztein DC. (2011). Lysosomal positioning coordinates cellular nutrient responses. Nat Cell Biol. 13(4): 453–460. PMCID: PMC3071334
  • Petit CS, Roczniak-Ferguson A, Ferguson SM. (2013). Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases. J Cell Biol. 202(7):1107-22. PMID: 24081491
  • Starling GP, Yip YY, Sanger A, Morton PE, Eden ER, Dodding MP. (2016). Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes. EMBO Rep. PMID: 27113757
  • Tsun ZY, Bar-Peled L, Chantranupong L, Zoncu R, Wang T, Kim C, Spooner E, Sabatini DM. (2013). The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1. Mol Cell. 52(4):495-505 PMID: 24095279
  • Wang T, Hong W. (2002). Interorganellar Regulation of Lysosome Positioning by the Golgi Apparatus through Rab34 Interaction with Rab-interacting Lysosomal Protein. Mol Biol Cell. 13(12): 4317–4332. PMCID: PMC138636


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BHD pulmonary cysts: The stretch hypothesis

The majority of BHD patients develop pulmonary cysts and approximately 1 in 3 will suffer a pneumothorax. Although BHD pulmonary cysts have defining characteristics compared to other cystic lung diseases (as discussed in recent reviews), the underlying pathogenesis is not yet clearly understood. A recent review from Kennedy, Khabibullin & Henske (2016) summarises the current understanding of BHD pulmonary pathology relative to the stretch hypothesis for cyst formation.

The stretch hypothesis is based on reports of FLCN interacting with PKP4/p0071 to regulate cell-cell adhesion, with the loss of either protein increasing adhesive strength (Medvetz et al., 2012, Nahorski et al., 2012, Khabibullin et al., 2014). During respiration, due to less negative intrapleural pressure, the alveoli in the basal regions of the lungs undergo a greater change in volume than those in the apical regions. The stretch hypothesis suggests that defects in cell-cell adhesion in the areas of the lung repeatedly subjected to higher inspiration stretch-forces, including anchor points to the pleura, lead to failure of the septal wall and subsequent cyst formation. This is supported by the location of BHD pulmonary cysts, which are predominantly basilar and frequently abut pleura and blood vessels.

The loss of the FLCN-PKP4 complex could be impacting cell-cell adhesion in several ways. Abnormal expression and organisation of E-cadherin and Claudin-1, components of adherens and tight junctions respectively, were seen in mouse IMCD-3 renal epithelial cells after FLCN knockdown (Nahorski et al., 2012), and increased desmosome production was reported in FLCN-null human UOK-257 renal cells (Medvetz et al., 2012). This was associated with reduced transepithelial electrical resistance and disrupted cell polarity, resulting in additional cellular stress. E-cadherin expression was also reduced in primary mouse airway epithelial cells lacking Flcn (Goncharova et al, 2014) suggesting that the same disruptions could be contributing to pulmonary pathology.

It is still unclear whether pulmonary cystogenesis in BHD patients is due to haploinsufficiency or if a loss of the second FLCN allele is required, as with renal tumours. Furuya et al. (2012) reported expression of FLCN in BHD patient cysts, but a heterozygous Flcn+/- mouse model showed no airspaces enlargement at five months (Khabibullin et al. 2014).  Interestingly alveolar enlargement was seen in mice where total Flcn loss was induced only in SP-C+ epithelial lung cells (Goncharova et al, 2014). This suggests a specific role for these cells in cyst formation, further supported by reports of SP-C+ epithelial cells lining BHD patient cysts (Furuya et al., 2012, Furuya & Nakatani, 2013) – however these cells still expressed some FLCN protein.

There are several other aspects of pulmonary pathogenesis in BHD patients that also require more research: when and how rapidly the cysts develop; why the risk of pneumothorax decreases with age; whether cystogenesis is due to inflammatory destruction, aberrant proliferation or both; and whether it is due to defects only in epithelial cells or if there is a role for mesenchyml cells. A clearer understanding of the complete pathogenic basis of cystogenesis in BHD patients could enable development of a treatment to reduce cyst formation thereby reducing the risk of pneumothorax.

  • Furuya M, Tanaka R, Koga S, Yatabe Y, Gotoda H, Takagi S, Hsu YH, Fujii T, Okada A, Kuroda N, Moritani S, Mizuno H, Nagashima Y, Nagahama K, Hiroshima K, Yoshino I, Nomura F, Aoki I, Nakatani Y (2012). Pulmonary cysts of Birt-Hogg-Dubé syndrome: a clinicopathologic and immunohistochemical study of 9 families. Am J Surg Pathol. 36(4):589-600. PMID: 22441547.
  • Furuya M, Nakatani Y (2013). Birt-Hogg-Dube syndrome: clinicopathological features of the lung. J Clin Pathol. 66(3):178-86. PMID: 23223565.
  • Goncharova EA, Goncharov DA, James ML, Atochina-Vasserman EN, Stepanova V, Hong SB, Li H, Gonzales L, Baba M, Linehan WM, Gow AJ, Margulies S, Guttentag S, Schmidt LS, Krymskaya VP (2014). Folliculin controls lung alveolar enlargement and epithelial cell survival through E-cadherin, LKB1, and AMPK. Cell Rep. 7(2):412-23. PMID: 24726356.
  • Kennedy JC, Khabibullin D, & Henske EP (2016). Mechanisms of Pulmonary Cyst Pathogenesis in Birt-Hogg-Dube Syndrome: The Stretch Hypothesis. Seminars in cell & developmental biology PMID: 26877139.
  • Khabibullin D, Medvetz DA, Pinilla M, Hariharan V, Li C, Hergrueter A, Laucho Contreras M, Zhang E, Parkhitko A, Yu JJ, Owen CA, Huang H, Baron RM, Henske EP (2014). Folliculin regulates cell-cell adhesion, AMPK, and mTORC1 in a cell-type-specific manner in lung-derived cells. Physiol Rep. 2(8). pii: e12107. PMID: 25121506.
  • Medvetz DA, Khabibullin D, Hariharan V, Ongusaha PP, Goncharova EA, Schlechter T, Darling TN, Hofmann I, Krymskaya VP, Liao JK, Huang H, Henske EP (2012). Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion. PLoS One. 7(11):e47842. PMID: 23139756.
  • Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, Maher ER (2012). Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Hum Mol Genet. 21(24):5268-79. PMID: 22965878.
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Rare Disease Day 2016: The Patient Voice

International Rare Disease Day is celebrated on the last day of February to raise awareness of rare diseases amongst the general public, researchers, healthcare professionals and policymakers. Now in its ninth year Rare Disease Day is celebrated in over 80 different countries with events in hundreds of cities. The theme for Rare Disease Day 2016 is the Patient Voice with a campaign inviting a wider audience to join us in making the voice of rare diseases heard.

Increasing awareness of rare diseases in the public and political domains ensures they remain an international health priority, helping patients access high quality care. Patients and their advocacy groups have crucial roles in raising awareness and can help others understand patient needs. In addition they can help instigate changes in policy and healthcare that can improve the lives of patients, family members and carers.

Patients and family members are often experts in their own disease and care, which can be very important when there is a lack of general medical knowledge. Patients can offer their personal experiences and identify their most pressing needs to help researchers and industry develop effective treatments and care strategies. Additionally expert patient involvement in regulatory reviews can help ensure patients can access the most critical treatments and interventions earlier.

Several training opportunities are provided by major rare disease organisations to support patients and advocacy groups that want to be representatives at a national and international level. In the UK, Findacure supports both established and developing patient advocacy groups, running regular training workshops and providing online guides. Similar patient advocacy support is available in other countries from organisations including NORD in the USA and CORD in Canada. Patient advocates interested in training related to clinical research and regulatory affairs can also attend the EURORDIS ExPRESS summer school.

The BHD Foundation provides support to BHD patients and researchers from around the world, and represents the BHD community at numerous international conferences helping to raise awareness. As well as promoting research, we encourage patients to share their stories and expertise through the website and forums including a patient-run Facebook group. Being able to seek advice and reassurance from others in a similar situation can help rare disease patients feel less isolated and more confident to discuss their condition. The organisations above can offer assistance for those wishing to create new support groups but communities can also be create on global platforms like RareConnect.

It is estimated that 1 in 17 people will be affected by a rare disease in their lifetime, meaning that cumulatively rare diseases are not actually uncommon. Rare disease day is a chance to further raise awareness of the impact of rare diseases and the need for representation in research and healthcare. Find out how you can help spread awareness by visiting the Rare Disease Day website, joining the Thunderclap campaign on social media and sharing your own story. You can also find events near you and share the details of your own events online.

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