Rare Disease Day 2015: Living with a Rare Disease

There are over 6000 rare diseases known worldwide. Whilst individually these diseases only affect a small number of people it is estimated that, assessed cumulatively, 1 in 17 people will be affected by a rare disease in their lifetime. To raise awareness of these diseases, every year since 2008, at the end of February there has been a Rare Disease Day.

The theme for Rare Disease Day 2015 is focused on the daily lives of patients, families and caregivers who are Living with a Rare Disease. Limited access to healthcare professionals knowledgeable about these rare and typically complex diseases often results in family members and friends becoming the main source of support and care. Rare Disease Day 2015 is a tribute to all the family and friends whose lives are affected by rare disease and who stand day-by-day and hand-in-hand with rare disease patients.

The lack of specific knowledge from local care authorities can lead to the patients and families becoming relative experts in their disease, often providing the information to new medical professionals when required. One invaluable source of information and support for rare disease patients is a disease-specific patient organisation. Although these can vary in the level of expertise and resources, many families find comfort in knowing that they are not alone in their diagnosis.

The BHD foundation website was established to help patients, families and doctors understand BHD from a more scientific stance, including the dissemination of new research. It also provides a primary contact for new BHD patients needing information on testing or local doctors. Complementary to this are the active facebook BHD groups which continue to be a wonderful source of mutual support and information from families affected by BHD regarding testing, symptoms, treatments and day-to-day issues associated with having a rare disease.

BHD has only been recognised as a disease since 1977. However thanks to the continuing work of a small, but increasing, number of clinicians and researchers worldwide, it has been possible to identify the gene associated with BHD, Follciulin, and to begin to understand its functions in the cell. Other research has allowed for the identification of more BHD patients and saved lives by allowing earlier treatment. The more we can understand about BHD and the interactions of folliculin in both normal and pathological situations the closer we will come to alleviating BHD symptoms and develop targeted treatments. They may not know every individual BHD patient in the world but our BHD researchers and clinicians are standing with them to try and make a difference.

There are Rare Disease Day events happening around the world, but if you are planning your own event and want help raising awareness get in touch with the Rare Disease Day team. You can also help spread awareness across social media by joining in with the Rare Disease Day Thunderclap.

Clicking on the images below will take you to Rare Disease Day Events homepages

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Characterisation of renal tumours in patients with Birt-Hogg-Dubé Syndrome

Due to mutations in their folliculin (FLCN) gene Birt-Hogg-Dubé (BHD) syndrome patients have a greater risk of developing renal cell carcinomas (RCC) than others (Zbar et al., 2002, Houweling et al., 2011). Unlike sporadic cases of RCC, where the majority are classified as clear cell RCC (ccRCC), studies of FLCN-related tumours have found that the majority are either chromophobe RCCs (34%) or hybrid oncocytoma/chromophobe tumours (HOCTs, 50%) with fewer cases of ccRCC (9%) and only rare occurrences of renal oncocytomas or papillary RCCs (Pavlovich et al., 2002). It is also common for multiple tumours of different subtypes to develop on the same kidney. Using current histology methods diagnosing a tumour as being related to BHD rather than sporadic can be difficult, especially in undiagnosed BHD cases where the genetic background will be unknown.

A new paper by Iribe et al., (2015) has investigated if the immunohistochemical profile of BHD-related tumours is sufficiently distinct from sporadic RCC tumours to be of help in classification. They suggest that a panel of markers including Carbonic Anhydrase IX (CA-IX), Kidney specific cadherin (Ksp-cadherin), Cytokeratin 7 (CK7) and CD82 could help in the screening for FLCN-related RCCs and enable the correct classification of some different subtypes.

In total 32 tumours, from 17 BHD patients, were analysed by in situ hybridisation. The tumours had already been classified as chromophobe RCCs (n=14), HOCTs (n=15) or ccRCC (n=3). Expression of the markers in these samples was compared to sporadic chromophobe RCC, ccRCCs and oncocytomas – the presence of FLCN mutations in these controls was not excluded but, based on a lack of other BHD pathologies, was assumed.

Most of the FLCN-related chromophobe RCC and HOCTs analysed were Ksp-cadherin+, CD82+ and CA-IX-, a profile similar to sporadic chromophobe RCCs. Unfortunately the results reported in this paper indicate that it would be difficult to distinguish between sporadic and FLCN-associated chromophobe RCC using this marker panel. However sporadic chromophobe RCC samples showed significantly higher expression of CK7 than FLCN-associated HOCTs. Additionally FLCN-associated HOCTs showed significantly greater expression of Ksp-cadherin and CD82 compared to sporadic oncocytomas. These differences indicate that the suggested panel of markers would be useful for distinguishing FLCN-associated HOCTs from sporadic chromophobe RCCs and oncocytomas.

All of the studied ccRCC samples, both BHD-associated and sporadic, stained positive for CA-IX but negative for the other markers. Therefore the panel suggested would be unsuitable for distinguishing FLCN-associated ccRCC from sporadic ccRCC. It would however be useful in determining if the clear cell-looking foci seen in some FLCN-associated RCCs are true ccRCC based on CA-IX+ expression or in fact Ksp-cadherin+, CK7+ and CD82+ HOCT or chromophobe cells.

Although the presence of HOCTs is more classically related to BHD, ccRCC and chromophobe RCC tumours also develop in BHD patients and therefore it is important to be able to distinguish between sporadic cases and those associated with FLCN mutations. A FLCN mutation will increase the likelihood of more tumours developing and the need for continual monitoring. The markers suggested by Iribe et al., are suitable for distinguishing FLCN-associated HOCTs from sporadic tumours but are insufficient to make this distinction for other RCC subtypes. Additional work from the group (Fuyura et al., 2015) will be able to add to the screening protocol and will be the topic of this blog in a few weeks.

The presence of germline FLCN mutations was confirmed in the Iribe et al., cohort however there was no identified pattern between mutation and tumour subtypes. Based on the Knudson two-hit hypothesis, tumourgenesis only occurs if both alleles of a tumour suppressor gene are mutated. Previous work by Vocke et al., (2005) identified such second-hit mutation in 70% of BHD-RCC tumours analysed. Such analysis was not completed in this study but it could be that larger analysis of such secondary mutations could provide clues to RCC subtype development in different patients. This could help with our understanding of tumourgenesis in BHD but also impact on the development of more targeted treatments.

  • Furuya M, Hong SB, Tanaka R, Kuroda N, Nagashima Y, Nagahama K, Suyama T, Yao M, Nakatani Y. Distinctive Expression Patterns of GPNMB and Folliculin in Renal Tumors in Patients with Birt-Hogg-Dubé Syndrome. Cancer Sci. 2015 Jan 8. doi: 10.1111/cas.12601. [Epub ahead of print] PubMed PMID: 25594584.
  • Houweling AC, Gijezen LM, Jonker MA, van Doorn MB, Oldenburg RA, van Spaendonck-Zwarts KY, Leter EM, van Os TA, van Grieken NC, Jaspars EH, de Jong MM, Bongers EM, Johannesma PC, Postmus PE, van Moorselaar RJ, van Waesberghe JH,  Starink TM, van Steensel MA, Gille JJ, Menko FH. Renal cancer and pneumothorax risk in Birt-Hogg-Dubé syndrome; an analysis of 115 FLCN mutation carriers from 35 BHD families. Br J Cancer. 2011 Dec 6;105(12):1912-9. doi: 10.1038/bjc.2011.463. PubMed PMID: 22146830.
  • Iribe Y, Kuroda N, Nagashima Y, Yao M, Tanaka R, Gotoda H, Kawakami F, Imamura Y, Nakamura Y, Ando M, Araki A, Matsushima J, Nakatani Y, & Furuya M (2015). Immunohistochemical characterization of renal tumors in patients with Birt-Hogg-Dubé Syndrome. Pathology international PMID: 25597876
  • Pavlovich CP, Walther MM, Eyler RA, Hewitt SM, Zbar B, Linehan WM, Merino MJ. Renal tumors in the Birt-Hogg-Dubé syndrome. Am J Surg Pathol. 2002 Dec;26(12):1542-52. PubMed PMID: 12459621.
  • Vocke CD, Yang Y, Pavlovich CP, Schmidt LS, Nickerson ML, Torres-Cabala CA, Merino MJ, Walther MM, Zbar B, Linehan WM. High frequency of somatic frameshift BHD gene mutations in Birt-Hogg-Dubé-associated renal tumors. J Natl Cancer Inst. 2005 Jun 15;97(12):931-5. Erratum in: J Natl Cancer Inst. 2005 Jul 20;97(14):1096. PubMed PMID: 15956655.
  • Zbar B, Alvord WG, Glenn G, Turner M, Pavlovich CP, Schmidt L, Walther M, Choyke P, Weirich G, Hewitt SM, Duray P, Gabril F, Greenberg C, Merino MJ, Toro J, Linehan WM. Risk of renal and colonic neoplasms and spontaneous pneumothorax in the Birt-Hogg-Dubé syndrome. Cancer Epidemiol Biomarkers Prev. 2002 Apr;11(4):393-400. PubMed PMID: 11927500.


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Pulmonologists should be more aware of Birt-Hogg-Dubé Syndrome

When BHD was first described in 1977 it was based on the presence of characteristic skin lumps (Birt et al., 1977). In addition BHD is now known to be associated with increased risk of renal cancer, the development of lung cysts and an associated increased risk in pneumothorax. In 2002, the association of BHD with mutations in the folliculin (FLCN) gene provided the definitive mechanism for BHD diagnosis (Nickerson et al., 2002). As such all patients suspected of having BHD should undergo genetic testing to confirm this diagnosis.

Pneumothorax in BHD patients is associated with the development of pulmonary cysts. Numerous research and clinical groups have confirmed high prevalence (up to 90%) of lung cysts in BHD patients (Toro et al., 2008, Agarwal et al., 2011). Whilst the development of lung cysts or blebs is not limited to BHD patients – pulmonary blebs are also associated with other pulmonary disease and smoking – the distribution and formation of the cysts seen in BHD is quite distinct. In BHD patients it is typical to find multiple, thin-walled, elliptical or lentiform, well-defined but with no internal structure, air-filled cysts. These cysts are also predominantly found in the medial or basal sections of the lung and are often subpleural (Tobino et al., 2009, Johannesma et al., 2014d). Toro et al., (2007) reported that BHD patients with a family history of spontaneous pneumothorax and more pulmonary cysts were significantly more likely to develop pneumothoraces.

BHD is underdiagnosed due to a lack of awareness in the medical profession of such a rare disease in combination with its variable presentation and onset. Only a small percentage of BHD diagnoses are suspected based on the presentation of pulmonary pathologies alone. However over the last few years there have been increasing numbers of such case studies where individuals have presented with spontaneous pneumothorax and genetic testing was used to confirm a BHD diagnosis (Predina et al., 2011, Auerbach et al., 2014, Kilinceret al., 2014, Johannesma et al., 2013, Johannesma et al., 2014c, Johannesma et al., 2014fArdilouze et al., 2015).

It has been estimated that BHD patients have a 50-times higher risk of developing pneumothorax than non-BHD individuals (Zbar et al., 2002). Two large cohort studies also looked at the incidence of BHD in cases of primary spontaneous pneumothorax (PSP) and found that approximately 5-10% of PSP patients carried FLCN mutations (Ren et al.,2008, Johannesma et al., 2014f). PSP incidence is 1.2-6/100,000 for women and 7.4-18/100,000 for men, annually (Luh, 2010)) and the global population is 7×109.  This suggests that the total number of PSP annually is 300,000-800,000, with 15,000-80,000 of those patients carrying FLCN mutations.  These suggested figures are vastly more than the current number of diagnosed BHD patients, which might  be  a result of unintentional cohort sampling bias or due to the difficultly of establishing accurate statistics when dealing with such a small sample population as BHD patients. The true number of un- or mis-diagnosed BHD patients is difficult to estimate however it is clear that many BHD patients are being misdiagnosed as PSP patients. This will continue to be the case unless there is more awareness of BHD as a potential cause of spontaneous pneumothorax. The variation in BHD presentation makes it is highly important, especially with younger patients, to obtain a detailed family history with regards to BHD pathologies as these can contain additional differential diagnostic information.

New diagnosis guidelines suggested by Gupta et al., (2013) would allow diagnosis of BHD to be based on the presence of “characteristic BHD-cysts” on a high resolution CT scan in combination with fibrofolliculomas, BHD-related renal cancer, a first or second degree relative with confirmed BHD, or a positive genetic test. Whilst the numerous individual case and cohort studies suggest that the presence of characteristic lung cysts is indicative of BHD, it is only via genetic testing that the diagnosis can be confirmed. Identifying these patients, and any other affected family members, as early as possible is important as it will impact on future monitoring and treatment. Therefore it is of great importance that awareness of BHD and its pulmonary presentation is increased in pulmonologists and associated professions.

  • Agarwal PP, Gross BH, Holloway BJ, Seely J, Stark P, Kazerooni EA. Thoracic CT findings in Birt-Hogg-Dube syndrome. AJR Am J Roentgenol. 2011 Feb;196(2):349-52. PubMed PMID: 21257886.
  • Ardilouze P, Jacquin J, Ait Ali T, Schneider S. Birt-Hogg-Dubé syndrome: A little known cause of pulmonary cysts. Diagn Interv Imaging. 2015 Jan;96(1):99-101. Epub 2014 Jul 9. PubMed PMID: 25022726.
  • Auerbach A, Roberts DH, Gangadharan SP, Kent MS. Birt-Hogg-Dubé syndrome in a patient presenting with familial spontaneous pneumothorax. Ann Thorac Surg. 2014 Jul;98(1):325-7 PubMed PMID: 24996715.
  • Birt AR, Hogg GR, Dubé WJ. Hereditary multiple fibrofolliculomas with trichodiscomas and acrochordons. Arch Dermatol. 1977 Dec;113(12):1674-7. PubMed PMID: 596896.
  • Gupta N, Seyama K, McCormack FX. Pulmonary manifestations of Birt-Hogg-Dubé syndrome. Fam Cancer. 2013 Sep;12(3):387-96. Review. PubMed PMID:23715758.
  • Johannesma PC, Thunnissen E, Postmus PE. Lung cysts as indicator for Birt-Hogg-Dubé syndrome. Lung. 2013; Epub 2013 Oct 22. PubMed PMID: 24146214.
  • Johannesma PC, van den Borne BE, Gille JJ, Nagelkerke AF, van Waesberghe JT, Paul MA, van Moorselaar RJ, Menko FH, Postmus PE. Spontaneous pneumothorax as indicator for Birt-Hogg-Dubé syndrome in paediatric patients. BMC Pediatr. 2014c Jul 3;14:171. PubMed PMID: 24994497.
  • Johannesma PC, Houweling AC, van Waesberghe JH, van Moorselaar RJ, Starink TM, Menko FH, Postmus PE. The pathogenesis of pneumothorax in Birt-Hogg-Dubé syndrome: a hypothesis. Respirology. 2014d Nov;19(8):1248-50. PubMed PMID: 25302759.
  • Johannesma PC, Reinhard R, Kon Y, Sriram JD, Smit HJ, van Moorselaar RJ, Menko FH, Postmus PE; on behalf of the Amsterdam BHD working group. Prevalence of Birt-Hogg-Dubé syndrome in patients with apparently primary spontaneous pneumothorax. Eur Respir J. 2014f Dec 23. pii: ERJ-01969-2014. [Epub ahead of print] PubMed PMID: 25537564.
  • Kilincer A, Ariyurek OM, Karabulut N. Cystic lung disease in birt-hogg-dubé syndrome: a case series of three patients. Eurasian J Med. 2014 Jun;46(2):138-41. PubMed PMID: 25610314.
  • Nickerson ML, Warren MB, Toro JR, Matrosova V, Glenn G, Turner ML, Duray P, Merino M, Choyke P, Pavlovich CP, Sharma N, Walther M, Munroe D, Hill R, Maher E, Greenberg C, Lerman MI, Linehan WM, Zbar B, Schmidt LS. Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome. Cancer Cell. 2002 Aug;2(2):157-64. PubMed PMID: 12204536.
  • Ren HZ, Zhu CC, Yang C, Chen SL, Xie J, Hou YY, Xu ZF, Wang DJ, Mu DK, Ma DH, Wang Y, Ye MH, Ye ZR, Chen BF, Wang CG, Lin J, Qiao D, Yi L. Mutation analysis of the FLCN gene in Chinese patients with sporadic and familial isolated primary spontaneous pneumothorax. Clin Genet. 2008 Aug;74(2):178-83. Epub 2008 May 25. PubMed PMID: 18505456.
  • Predina JD, Kotloff RM, Miller WT, Singhal S. Recurrent spontaneous pneumothorax in a patient with Birt-Hogg-Dubé syndrome. Eur J Cardiothorac Surg.  2011 Mar;39(3):404-6.. Epub 2010 Aug 6. PubMed PMID: 20692178.
  • Tobino K, Gunji Y, Kurihara M, Kunogi M, Koike K, Tomiyama N, Johkoh T, Kodama Y, Iwakami S, Kikkawa M, Takahashi K, Seyama K. Characteristics of pulmonary cysts in Birt-Hogg-Dubé syndrome: thin-section CT findings of the chest in 12 patients. Eur J Radiol. 2011 Mar;77(3):403-9 Epub 2009 Sep 25. PubMed PMID: 19782489.
  • Toro JR, Pautler SE, Stewart L, Glenn GM, Weinreich M, Toure O, Wei MH, Schmidt LS, Davis L, Zbar B, Choyke P, Steinberg SM, Nguyen DM, Linehan WM. Lung  cysts, spontaneous pneumothorax, and genetic associations in 89 families with Birt-Hogg-Dubé syndrome. Am J Respir Crit Care Med. 2007 May 15;175(10):1044-53.  Epub 2007 Feb 22. PubMed PMID: 17322109
  • Toro JR, Wei MH, Glenn GM, Weinreich M, Toure O, Vocke C, Turner M, Choyke P, Merino MJ, Pinto PA, Steinberg SM, Schmidt LS, Linehan WM. BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dubé syndrome: a new series of 50 families and a review of published reports. J Med Genet. 2008 Jun;45(6):321-31. Epub 2008 Jan 30. Review. PubMed PMID:18234728.
  • Zbar B, Alvord WG, Glenn G, Turner M, Pavlovich CP, Schmidt L, Walther M, Choyke P, Weirich G, Hewitt SM, Duray P, Gabril F, Greenberg C, Merino MJ, Toro J, Linehan WM. Risk of renal and colonic neoplasms and spontaneous pneumothorax in the Birt-Hogg-Dubé syndrome. Cancer Epidemiol Biomarkers Prev. 2002 Apr;11(4):393-400. PubMed PMID: 11927500.
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The development of targeted therapy for renal cell carcinoma

Over 85% of kidney and renal pelvis cancers are renal cell carcinomas (RCC); a disease with multiple subtypes including clear cell, papillary, chromophobe and oncocytomic. The variable nature of RCC makes one blanket treatment impossible, and therefore targeted therapies may be more effective at enhancing survival. A recent review from Randall et al., (2014) summarises the current knowledge on RCC genetic aberrations and their effects, as well as discussing current and future treatment options.

The majority (>80%) of sporadic RCC cases are clear cell RCC (ccRCC), often with increased risk of metastatic spread, associated with mutations in the VHL, PBRM, SETD2 and BAP1 genes although mutations in over 30 other genes have also been found includingTP53, PTEN and occasionally FLCN. The loss of VHL leads to a reduction in HIF repression and subsequent changes in transcription, including overexpression of VEGF, PDGF-β, TGF-α and erythropoietin, results in vascularised tumour growth. Disruption in HIF signalling and changes in cellular metabolism have also been identified as a result of FLCN loss (Preston et al., 2011).

Currently the molecular drivers of papillary RCC (10-15% of cases), chromophobe RCC (5%) and renal oncocytomas (3-5%) are less well understood. These subtypes generally have a better prognosis as they are slower growing, chromophobe RCCs in particular.

Cases of inherited RCC are seen in BHD patients although the histology of these RCC tumours is highly variable, and dissimilar to sporadic RCC. ccRCC is seen in comparatively fewer cases (~9%), and in addition hybrids of clear cell/papillary and clear cell/chromophobe RCCs have been reported. Such tumours of a clear cell nature have been shown to metastasise in BHD patients and often result in increased mortality. The majority of BHD-RCC tumours however are slow growing chromophobe RCCs (34%) or have a hybrid chromophobe/oncocytoma histology (50%), and can be surgically managed. It is also not uncommon for tumours of several different subtypes can be found within the same kidney (Hudon et al., 2010, Kuroda et al., 2014).

Increasing understanding of the molecular pathways perturbed in RCCs can enhance the use of targeted therapies. The majority of ccRCC cases are currently treated with VEGF-inhibitors such as the tyrosine kinase inhibitor sunitinib. However in cases where there is an additional disruption to the PI3K-AKT-mTOR signalling pathways there has been some success using mTOR inhibitors such as everolimus (Hudes et al., 2007).

Future development of effective RCC treatments would be improved with patient selection based on molecular analysis of the tumours. Previous trials have shown marked differences in survival between unselected cohorts and subsets of patients with the same germline mutations (Chouieri et al., 2013). Even with well-established drugs it has been shown that the likelihood of a response can be predicted based on molecular expression patterns (Dornbusch et al., 2013). Beuselinck et al., 2015 recently reported that multi-omic analysis of ccRCC tumours could be used to predict response to sunitinib, with significant variation in progression-free survival and overall survival between subgroups.

BHD-RCC tumours are morphologically similar to sporadic chromophobe RCCs and renal oncocytomas and both types show an increase in expression of mitochondrial and oxidative phosphorylation associated genes (Klomp et al., 2010,). In BHD this is associated with an increase in PGC1α-activation (discussed in an alternative context in last week’s blog) and aberrant activation of the mTOR pathway. As a result rapamycin can be used to treat local recurrence and metastases in BHD patients (Kuroda et al., 2014). Further information on the molecular pathogenesis in both sporadic and inherited RCCs will make it possible to develop more targeted treatments in the future.

  • Beuselinck B, Job S, Becht E, Karadimou A, Verkarre V, Couchy G, Giraldo N, Rioux-Leclercq N, Molinié V, Sibony M, Elaidi R, Teghom C, Patard JJ, Méjean A, Fridman WH, Sautès-Fridman C, de Reyniès A, Oudard SM, Zucman-Rossi J. Molecular Subtypes of clear cell renal cell carcinoma are associated to sunitinib response in the metastatic setting. Clin Cancer Res. 2015 Jan 12. pii: clincanres.1128.2014. [Epub ahead of print] PubMed PMID: 25583177.
  • Choueiri TK, Vaishampayan U, Rosenberg JE, Logan TF, Harzstark AL, Bukowski RM, Rini BI, Srinivas S, Stein MN, Adams LM, Ottesen LH, Laubscher KH, Sherman L, M Dermott DF, Haas NB, Flaherty KT, Ross R, Eisenberg P, Meltzer PS, Merino MJ, Bottaro DP, Linehan WM, Srinivasan R. Phase II and biomarker study of the dual MET/VEGFR2 inhibitor foretinib in patients with papillary renal cell carcinoma. J Clin Oncol. 2013 Jan 10;31(2):181-6. doi: 10.1200/JCO.2012.43.3383. Epub 2012 Dec 3. PubMed PMID: 23213094
  • Dornbusch J, Zacharis A, Meinhardt M, Erdmann K, Wolff I, Froehner M, Wirth MP, Zastrow S, Fuessel S. Analyses of potential predictive markers and survival data for a response to sunitinib in patients with metastatic renal cell carcinoma. PLoS One. 2013 Sep 27;8(9):e76386. doi: 10.1371/journal.pone.0076386. eCollection 2013. PubMed PMID: 24086736
  • Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A, Staroslawska E, Sosman J, McDermott D, Bodrogi I, Kovacevic Z, Lesovoy V, Schmidt-Wolf IG, Barbarash O, Gokmen E, O’Toole T, Lustgarten S, Moore L, Motzer RJ; Global ARCC Trial. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med. 2007 May 31;356(22):2271-81. PubMed PMID:17538086.
  • Hudon V, Sabourin S, Dydensborg AB, Kottis V, Ghazi A, Paquet M, Crosby K, Pomerleau V, Uetani N, Pause A. Renal tumour suppressor function of the Birt-Hogg-Dubé syndrome gene product folliculin. J Med Genet. 2010 Mar;47(3):182-9. doi: 10.1136/jmg.2009.072009. Epub 2009 Oct 19. PubMed PMID: 19843504.
  • Klomp JA, Petillo D, Niemi NM, Dykema KJ, Chen J, Yang XJ, Sääf A, Zickert P, Aly M, Bergerheim U, Nordenskjöld M, Gad S, Giraud S, Denoux Y, Yonneau L, Méjean A, Vasiliu V, Richard S, MacKeigan JP, Teh BT, Furge KA. Birt-Hogg-Dubé renal tumors are genetically distinct from other renal neoplasias and are associated with up-regulation of mitochondrial gene expression. BMC Med Genomics. 2010 Dec 16;3:59. doi: 10.1186/1755-8794-3-59. PubMed PMID:21162720
  • Kuroda N, Furuya M, Nagashima Y, Gotohda H, Kawakami F, Moritani S, Ota S, Hora M, Michal M, Hes O, Nakatani Y. Review of renal tumors associated with Birt-Hogg-Dubé syndrome with focus on clinical and pathobiological aspects. Pol J Pathol. 2014 Jun;65(2):93-9. Review. PubMed PMID: 25119168.
  • Preston RS, Philp A, Claessens T, Gijezen L, Dydensborg AB, Dunlop EA, Harper KT, Brinkhuizen T, Menko FH, Davies DM, Land SC, Pause A, Baar K, van Steensel MA, Tee AR. Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene. 2011 Mar 10;30(10):1159-73. doi: 10.1038/onc.2010.497. Epub 2010 Nov 8. PubMed PMID: 21057536
  • Randall JM, Millard F, & Kurzrock R (2014). Molecular aberrations, targeted therapy, and renal cell carcinoma: current state-of-the-art. Cancer metastasis reviews, 33 (4), 1109-24 PMID: 25365943
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Fnip1 regulates skeletal muscle fibre type specification, fatigue resistance, and suspectibility to muscular dystrophy

Folliculin (FLCN) and the associated folliculin-interacting proteins FNIP1 and FNIP2 have been shown to play a role in cell metabolism through regulation of the AMPK-mTOR pathways. Previously Hasumi et al. (2012) reported that selective deletion of Flcn in mouse skeletal muscle resulted in an increase in mitochondrial biogenesis and muscle fibre specification (discussed in this blog post).

Reyes et al. (2014) have now contributed more to the understanding of muscle fibre specification using a Fnip1-/- mouse (described in Park et al. 2012) which also shows altered muscle fibre specification from type IIb (fast twitch) to type I (slow twitch). Usually Fnip1 protein is expressed in type IIb muscle fibres but not in type I fibres. However in theFnip1 null mice there was an increased proportion of type I fibres indicated by an increase in mitochondrial gene transcripts, an increase in oxygen consumption indicative of an increase of functional mitochondria, and type I fibre specific physiology.

FLCN and FNIP1/2 interact with each other but also directly interact with the metabolicAMPK – a key regulator in energy homeostasis and mitochondrial biogenesis but also skeletal muscle fibre specification in response to exercise (Hardie, 2011). AMPK activation results in activation of the transcriptional regulators PGC1α and PGC1β and subsequent downstream programmes for mitochondrial biogenesis and oxidative metabolism. Within skeletal muscle activation of AMPK and PGC1 α, often associated with endurance exercise, also increases type I fibre specification (Hambrecht et al., 1997).

Fnip1 interaction inhibits AMPK activity and subsequently, as seen in this paper, a loss of Fnip1 results in increased phosphorylation of AMPK and increased levels of downstream pathway components, such as PGC1α, as detected by qPCR and western blot. This suggests that perturbed Fnip1 activity, as seen with FLCN loss, results in an increase in basal AMPK activity which in turn leads to changes in PGC1α expression and activity.

Fnip1-/- PGC1a-/- double null mice show a reduction in aberrant muscle specification, suggesting that the muscle fibre specification changes seen in the Fnip1-/- mice are dependent on PGC1α induction and as such the authors conclude that PGC1α is an essential mediator of fibre specification. Although the exact connection between Fnip1 loss and an increase in PGC1α activity is not fully understood it is likely to be mediated by AMPK activity. The authors suggest that under normal conditions Flcn and Fnip1 work as a complex to inhibit AMPK thereby reducing PGC1α expression and oxidative metabolism.

Although AMPK activation has been reported, via activation of TSC, which inhibits mTOR activity, thereby minimising ATP consumption and cell growth (Gowans et al. 2014), the Fnip1-/- muscle showed an increase in mTOR activity. The changes in mTOR activity were determined not to play a role in muscle fibre specification but may indicate a novel role for Fnip1 in coupling AMPK to mTOR. It is also possible that the increase in intracellular ATP has, through a negative feedback loop, resulted in AMPK inactivation thereby disrupting mTOR regulation as suggested previously in cardiac hypertrophy associated with a loss of FLCN (Hasumi et al 2014).

The loss of FLCN resulting in hyperactivity of AMPK and PGC1α, increased mitochondrial biogenesis and changes to energy levels has been previously reported (Yan et al. 2014). In BHD an increase in mitochondrial respiration has been linked to increased activation of the HIF pathway known to increase tumourigenic potential with increased expression of PGC1α and HIF1 having been identified in BHD renal carcinoma samples (Klomp et al. 2010, Preston et al. 2011). Whilst disruptions to the AMPK-mTOR regulatory pathways are of great importance in BHD research, the exact role played FNIP1 is not entirely understood. What Reyes et al. indicate however, based on the loss of Fnip1 being sufficient to induce an increase in AMPK activity and mitochondrial biogenesis, is that FNIP1 does play an important role that requires further investigation.

Interestingly the group also report a reduction in muscle damage in the Dmdmdx-4CV mouse model of Duchenne Muscular Dystrophy (DMD) when Fnip1 is absent. As the specific overexpression of PGC1α in skeletal muscle has a similarly protective effect (Chan et al. 2014) it suggests that the reduction in muscle loss seen with the inhibition of Fnip1 may, in part, be through induction of PGC1α.

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  • Gowans GJ, Hardie DG. AMPK: a cellular energy sensor primarily regulated by AMP. Biochem Soc Trans. 2014 Feb;42(1):71-5. doi: 10.1042/BST20130244. PubMed PMID: 24450630
  • Hambrecht R, Fiehn E, Yu J, Niebauer J, Weigl C, Hilbrich L, Adams V, Riede U, Schuler G. Effects of endurance training on mitochondrial ultrastructure and fiber type distribution in skeletal muscle of patients with stable chronic heart failure. J Am Coll Cardiol. 1997 Apr;29(5):1067-73. PubMed PMID: 9120161
  • Hardie DG. Energy sensing by the AMP-activated protein kinase and its effects on muscle metabolism. Proc Nutr Soc. 2011 Feb;70(1):92-9. doi: 10.1017/S0029665110003915. Epub 2010 Nov 11. Review. PubMed PMID:21067629
  • Hasumi H, Baba M, Hasumi Y, Huang Y, Oh H, Hughes RM, Klein ME, Takikita S, Nagashima K, Schmidt LS, Linehan WM. Regulation of mitochondrial oxidative metabolism by tumor suppressor FLCN. J Natl Cancer Inst. 2012 Nov 21;104(22):1750-64. doi: 10.1093/jnci/djs418. Epub 2012 Nov 12. PubMed PMID: 23150719
  • Hasumi Y, Baba M, Hasumi H, Huang Y, Lang M, Reindorf R, Oh HB, Sciarretta S, Nagashima K, Haines DC, Schneider MD, Adelstein RS, Schmidt LS, Sadoshima J, Marston Linehan W. Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation. Hum Mol Genet. 2014 Nov 1;23(21):5706-19. doi: 10.1093/hmg/ddu286. Epub 2014 Jun 6. PubMed PMID: 24908670
  • Klomp JA, Petillo D, Niemi NM, Dykema KJ, Chen J, Yang XJ, Sääf A, Zickert P, Aly M, Bergerheim U, Nordenskjöld M, Gad S, Giraud S, Denoux Y, Yonneau L, Méjean A, Vasiliu V, Richard S, MacKeigan JP, Teh BT, Furge KA. Birt-Hogg-Dubé renal tumors are genetically distinct from other renal neoplasias and are associated with up-regulation of mitochondrial gene expression. BMC Med Genomics. 2010 Dec 16;3:59. doi: 10.1186/1755-8794-3-59. PubMed PMID: 24908670
  • Reyes NL, Banks GB, Tsang M, Margineantu D, Gu H, Djukovic D, Chan J, Torres M, Liggitt HD, Hirenallur-S DK, Hockenbery DM, Raftery D, & Iritani BM (2015). Fnip1 regulates skeletal muscle fiber type specification, fatigue resistance, and susceptibility to muscular dystrophy. Proceedings of the National Academy of Sciences of the United States of America, 112 (2), 424-9 PMID: 25548157
  • Park H, Staehling K, Tsang M, Appleby MW, Brunkow ME, Margineantu D, Hockenbery DM, Habib T, Liggitt HD, Carlson G, Iritani BM. Disruption of Fnip1 reveals a metabolic checkpoint controlling B lymphocyte development. Immunity. 2012 May 25;36(5):769-81. doi: 10.1016/j.immuni.2012.02.019. Epub 2012 May 17. PubMed PMID: 22608497
  • Yan M, Gingras MC, Dunlop EA, Nouët Y, Dupuy F, Jalali Z, Possik E, Coull BJ, Kharitidi D, Dydensborg AB, Faubert B, Kamps M, Sabourin S, Preston RS, Davies DM, Roughead T, Chotard L, van Steensel MA, Jones R, Tee AR, Pause A. The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation. J Clin Invest. 2014 Jun 2;124(6):2640-50. doi: 10.1172/JCI71749. Epub 2014 Apr 24. PubMed PMID: 24762438
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BHD Research Blog: 2014 Annual Review

With the New Year upon us, we thought we would use this week’s blog to review the studies we’ve particularly enjoyed writing about, and to revisit emerging themes.

During the summer-autumn period, there were a number of interesting studies that shed light on the molecular function of FLCN. Firstly, Goncharova et al. reported that FLCN activates AMPK signaling via LKB1 and E-cadherin, in Alveolar Type II (ATII) cells. Conversely, both Yan et al. and Possik et al. showed that FLCN inhibits AMPK signaling in MEFs and C.elegans nematodes, and that loss of FLCN leads to tumorigenic metabolic changes consistent with the Warburg Effect. These conflicting findings were resolved in August, when Khabibullin et al. published data showing that FLCN function is highly cell-specific.

FLCN’s role in autophagy was further elucidated by Dunlop et al. who reported that FLCN modulates autophagy through its interactions with ULK1 and GABARAP; FLCN was shown to be important for cardiomyocyte development (Hasumi et al., 2014); and a hitherto unknown role for FNIP1 in iNKT cell development was reported by Park et al.

The results from the trial of Rapamycin as a treatment for fibrofolliculomas were published this year, and reported that Rapamycin was not effective (Gijezen et al., 2014).

A cohort study of 33 BHD patients with kidney cancer showed that the majority of tumours were of oncocytic, choromophobe or mixed histology, and that the median age at the diagnosis of the first tumour was 46 (Benusiglio et al., 2014). This corresponds well with a study by Schuch et al. which found that patients with a genetic predisposition to kidney cancer developed tumours nearly 25 years earlier than patients with sporadic tumours. Thus, Schuch et al. recommend that doctors should consider germline genetic testing in kidney cancer patients under the age of 46.

A number of papers regarding the lung symptoms of BHD have been published this year. In July, it was reported that BHD can cause pneumothoraces in children in rare cases (Johannesma et al., 2014a), and in September, 1 in 16 BHD patients were found to be at risk of developing a pneumothorax within 30 days of taking a commercial flight (Postmus et al. 2014). Two separate groups hypothesized that defective cell-cell adhesion made cyst walls more likely to burst under mechanical stress, leading to an accumulation of air in the pleural space and potentially causing a pneumothorax (Johannesma et al., 2014b, Kumasaka et al., 2014).

Finally, two case studies have reported that somatic FLCN mutations contributed to the development of sporadic tumours (Sirintrapun et al., 2014, Wagle et al., 2014). These studies indicate that FLCN mutations may play a wider role in disease than just causing BHD Syndrome, suggesting that BHD is a fundamental disease.

These papers are just a selection of those published in 2014, and we at the BHD Foundation are very much looking forward to seeing how the field develops in 2015. We wish all our readers a very Happy New Year.


  • Benusiglio, P., Giraud, S., Deveaux, S., Méjean, A., Correas, J., Joly, D., Timsit, M., Ferlicot, S., Verkarre, V., Abadie, C., Chauveau, D., Leroux, D., Avril, M., Cordier, J., & Richard, S. (2014). Renal cell tumour characteristics in patients with the Birt-Hogg-Dubé cancer susceptibility syndrome: a retrospective, multicentre study Orphanet Journal of Rare Diseases, 9 (1) DOI: 10.1186/s13023-014-0163-z
  • Dunlop EA, Seifan S, Claessens T, Behrends C, Kamps MA, Rozycka E, Kemp AJ, Nookala RK, Blenis J, Coull BJ, Murray JT, van Steensel MA, Wilkinson S, & Tee AR (2014). FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation. Autophagy, 10 (10) PMID: 25126726
  • Gijezen LM, Vernooij M, Martens H, Oduber CE, Henquet CJ, Starink TM, Prins MH, Menko FH, Nelemans PJ, & van Steensel MA (2014). Topical rapamycin as a treatment for fibrofolliculomas in birt-hogg-dubé syndrome: a double-blind placebo-controlled randomized split-face trial. PloS one, 9 (6) PMID: 24910976
  • 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 reports, 7 (2), 412-23 PMID: 24726356
  • Hasumi Y, Baba M, Hasumi H, Huang Y, Lang M, Reindorf R, Oh HB, Sciarretta S, Nagashima K, Haines DC, Schneider MD, Adelstein RS, Schmidt LS, Sadoshima J, & Marston Linehan W (2014). Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation. Human molecular genetics PMID: 24908670
  • Johannesma PC, van den Borne BE, Gille JJ, Nagelkerke AF, van Waesberghe JT, Paul MA, van Moorselaar RJ, Menko FH, & Postmus PE (2014a). Spontaneous pneumothorax as indicator for Birt-Hogg-Dubé syndrome in paediatric patients. BMC pediatrics, 14 PMID: 24994497
  • Johannesma PC, Houweling AC, van Waesberghe JH, van Moorselaar RJ, Starink TM, Menko FH, & Postmus PE (2014b). The pathogenesis of pneumothorax in Birt-Hogg-Dubé syndrome: A hypothesis. Respirology (Carlton, Vic.), 19 (8), 1248-50 PMID: 25302759
  • 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. Physiological reports, 2 (8) PMID: 25121506
  • Kumasaka T, Hayashi T, Mitani K, Kataoka H, Kikkawa M, Tobino K, Kobayashi E, Gunji Y, Kunogi M, Kurihara M, & Seyama K (2014). Characterization of pulmonary cysts in Birt-Hogg-Dubé syndrome: histopathologic and morphometric analysis of 229 pulmonary cysts from 50 unrelated patients. Histopathology PMID: 24393238
  • Park H, Tsang M, Iritani BM, & Bevan MJ (2014). Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development. Proceedings of the National Academy of Sciences of the United States of America, 111 (19), 7066-71 PMID: 24785297
  • Possik E, Jalali Z, Nouët Y, Yan M, Gingras MC, Schmeisser K, Panaite L, Dupuy F, Kharitidi D, Chotard L, Jones RG, Hall DH, & Pause A (2014). Folliculin regulates ampk-dependent autophagy and metabolic stress survival. PLoS genetics, 10 (4) PMID: 24763318
  • Postmus PE, Johannesma PC, Menko FH, & Paul MA (2014). In-Flight Pneumothorax: Diagnosis May Be Missed because of Symptom Delay. American journal of respiratory and critical care medicine, 190 (6), 704-5 PMID: 25221882
  • Shuch B, Vourganti S, Ricketts CJ, Middleton L, Peterson J, Merino MJ, Metwalli AR, Srinivasan R, & Linehan WM (2014). Defining early-onset kidney cancer: implications for germline and somatic mutation testing and clinical management. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 32 (5), 431-7 PMID: 24378414
  • Sirintrapun SJ, Geisinger KR, Cimic A, Snow A, Hagenkord J, Monzon F, Legendre BL Jr, Ghazalpour A, Bender RP, & Gatalica Z (2014). Oncocytoma-like renal tumor with transformation toward high-grade oncocytic carcinoma: a unique case with morphologic, immunohistochemical, and genomic characterization. Medicine, 93 (15) PMID: 25275525
  • Wagle N, Grabiner BC, Van Allen EM, Amin-Mansour A, Taylor-Weiner A, Rosenberg M, Gray N, Barletta JA, Guo Y, Swanson SJ, Ruan DT, Hanna GJ, Haddad RI, Getz G, Kwiatkowski DJ, Carter SL, Sabatini DM, Jänne PA, Garraway LA, & Lorch JH (2014). Response and acquired resistance to everolimus in anaplastic thyroid cancer. The New England journal of medicine, 371 (15), 1426-33 PMID: 25295501
  • Yan M, Gingras MC, Dunlop EA, Nouët Y, Dupuy F, Jalali Z, Possik E, Coull BJ, Kharitidi D, Dydensborg AB, Faubert B, Kamps M, Sabourin S, Preston RS, Davies DM, Roughead T, Chotard L, van Steensel MA, Jones R, Tee AR, & Pause A (2014). The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation. The Journal of clinical investigation PMID: 24762438

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

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A shower of second hit mutations causes bilateral, multifocal kidney cancer in TSC patients

Tuberous sclerosis complex (TSC) is caused by autosomal dominant inactivating mutations in either the TSC1 or TSC2 genes, and patients are predisposed to developing tumours in the brain, eyes, heart, skin, lungs and kidneys throughout their lifetime. While more than 80% of TSC patients develop benign renal angiomyolipoma, only 3% of TSC patients develop renal cell carcinoma (Yang et al., 2014).

Tyburczy et al., (2014) describe two TSC patients who presented with bilateral multifocal renal cell carcinoma.

The first patient received a left nephrectomy to remove 12 tumours at the age of 24. Three years later, a right nephrectomy was required to remove three tumours. Histological analysis showed all tumours to be of TSC-associated papillary renal cell carcinoma (RCC) (Yang et al., 2014).

Fresh frozen tissue was available from four left and one right kidney tumour. Direct sequencing and microsatellite marker analysis showed loss of heterozygosity (LOH) at the TSC2 allele in one tumour sample, but not in the other four tumours. Targeted next generation sequencing found different second hit somatic TSC2 mutations in each of the other four tumours: two nonsense, one frameshift and one missense mutation. Whole exome sequencing of DNA from all five tumours showed an average of four somatic mutations in other genes in each tumour, and no gene was mutated in more than one sample. Furthermore, no copy number changes were observed in any tumour. This suggests that biallelic inactivation of TSC2 is the driving tumorigenic mutation in all five tumours.

The second patient was found to have bilateral, multifocal tumours, and received partial nephrectomy of both kidneys at the age of 36. All tumours showed the same TSC-associated papillary RCC histology. Analysis of three tumours showed no evidence of LOH at the TSC2 allele. Only one tumour sample yielded sufficient DNA for sequencing analysis, and showed a somatic splicing mutation in TSC2, again suggesting that somatic second hits in TSC2 are driving tumorigenesis in this patient.

Both patients carried the same missense R905Q TSC2 mutation, which is associated with a milder presentation of TSC (Jansen et al., 2006). As this mutation is present in fewer than 1% of TSC patients, and only 3% of TSC patients develop RCC, it seems likely this this mutation may predispose patients to developing RCC. Indeed, in a cohort of 19 TSC patients who developed RCC, germline sequencing data was available for three patients who also developed TSC-associated papillary RCC. While none carried the R905Q mutation, all three had non-truncating TSC2 mutations – two missense and one in-frame deletion. This suggests that patients with non-truncating mutations may be at a higher risk of developing RCC (Yang et al., 2014).

Tyburczy et al. calculate the likelikhood of the first patient developing 15 tumours through 15 independent second hit events to be 1 in 3.6 trillion. This suggests that all tumours arose following a single event, which led to a shower of second hit TSC2 mutations occuring in these patients’ kidney tissue. However, the disease mechanism that leads to multiple second hit mutations in in a single gene is currently unknown.


  • Jansen AC, Sancak O, D’Agostino MD, Badhwar A, Roberts P, Gobbi G, Wilkinson R, Melanson D, Tampieri D, Koenekoop R, Gans M, Maat-Kievit A, Goedbloed M, van den Ouweland AM, Nellist M, Pandolfo M, McQueen M, Sims K, Thiele EA, Dubeau F, Andermann F, Kwiatkowski DJ, Halley DJ, & Andermann E (2006). Unusually mild tuberous sclerosis phenotype is associated with TSC2 R905Q mutation. Ann Neurol, 60 (5), 528-39 PMID: 17120248
  • Tyburczy ME, Jozwiak S, Malinowska IA, Chekaluk Y, Pugh TJ, Wu CL, Nussbaum RL, Seepo S, Dzik T, Kotulska K, & Kwiatkowski DJ (2014). A shower of second hit events as the cause of multifocal renal cell carcinoma in Tuberous Sclerosis Complex. Hum Mol Genet PMID: 25432535
  • Yang P, Cornejo KM, Sadow PM, Cheng L, Wang M, Xiao Y, Jiang Z, Oliva E, Jozwiak S, Nussbaum RL, Feldman AS, Paul E, Thiele EA, Yu JJ, Henske EP, Kwiatkowski DJ, Young RH, & Wu CL (2014). Renal cell carcinoma in tuberous sclerosis complex. Am J Surg Pathol, 38 (7), 895-909. PMID: 24832166

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

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