Mutations in VHL are known to be associated with the development of clear cell renal cell carcinoma (ccRCC), and recent sequencing studies have identified several genes involved in chromatin regulation that are also frequently mutated in ccRCC (Dalgliesh et al., 2010; Varela et al., 2011 – as discussed in our previous blogs here and here). A new sequencing study by Guo et al. (2011) has now identified 12 more genes linked to ccRCC, many of which appear to be involved in the ubiquitin-mediated proteolysis pathway (UMPP).

The authors sequenced and analysed the whole exomes of 10 primary ccRCCs (with matched controls) from institutions linked to the Urinogenital Cancer Genomics Consortium in China. They also performed a screen of approximately 1,100 cancer/RCC-associated genes (from the Cancer Gene Census and the Catalogue Of Somatic Mutations In Cancer) in 88 additional samples of ccRCC, with their corresponding controls. In total, 412 non-silent somatic mutations were identified in these 98 cases of ccRCC, of which a proportion were confirmed by genotyping and Sanger sequencing.

Guo et al. then used statistical approaches to show that 23 genes were specifically mutated at elevated levels in these ccRCCs. However, 12 of these genes were not previously known to be associated with ccRCC. For example, the tumour suppressor gene TSC1 is known to be involved in tuberous sclerosis complex, but was also mutated in 3% of the ccRCCs studied. The tumour suppressor gene BAP1 was also mutated in 8% of the cases, and it encodes a component of the UMPP, a pathway which is involved in the ubiquitin-mediated degradation of proteins within cells.

Subsequent pathway analysis of the mutated genes in the whole-exome study showed that the UMPP was the most frequently altered system within these 10 tumours. Sequencing of all the genes in the UMPP in the remaining 88 samples also showed that the UMPP genes were mutated at a significantly higher frequency than background.

Since the UMPP gene VHL is known to have an important role in ccRCC tumourigenesis through its effects on HIF signalling, this particular system was analysed further. Using immunohistochemical analysis, Guo et al. noted that tumours with mutations in the UMPP had a significant overexpression of HIF1α and HIF2α. In particular, it could be seen that tumours with alterations in VHL were significantly associated with HIF1α overexpression (and not HIF2α). In contrast, tumours with alterations in non-VHLUMPP genes were significantly associated with HIF2α overexpression (and not HIF1α). Consequently, in addition to VHL, a variety of UMPP genes may be involved in the regulation HIF signalling. Furthermore, many other signalling networks could be affected by alterations in these genes in both a HIF-dependent and independent fashion.

Finally, no significant mutations were found in FLCN, which is not surprising as clear-cut cases of ccRCC are relatively uncommon in BHD syndrome. However, it would be of interest to see if these UMPP genes play a role in BHD-associated tumourigenesis, as FLCN has also been linked to HIF signalling by Preston et al. (2010).

• Dalgliesh, G., Furge, K., Greenman, C., Chen, L., Bignell, G., Butler, A., Davies, H., Edkins, S., Hardy, C., Latimer, C., Teague, J., Andrews, J., Barthorpe, S., Beare, D., Buck, G., Campbell, P., Forbes, S., Jia, M., Jones, D., Knott, H., Kok, C., Lau, K., Leroy, C., Lin, M., McBride, D., Maddison, M., Maguire, S., McLay, K., Menzies, A., Mironenko, T., Mulderrig, L., Mudie, L., O’Meara, S., Pleasance, E., Rajasingham, A., Shepherd, R., Smith, R., Stebbings, L., Stephens, P., Tang, G., Tarpey, P., Turrell, K., Dykema, K., Khoo, S., Petillo, D., Wondergem, B., Anema, J., Kahnoski, R., Teh, B., Stratton, M., & Futreal, P. (2010). Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes Nature, 463 (7279), 360-363. DOI:10.1038/nature08672
• Guo G, Gui Y, Gao S, Tang A, Hu X, Huang Y, Jia W, Li Z, He M, Sun L, Song P, Sun X, Zhao X, Yang S, Liang C, Wan S, Zhou F, Chen C, Zhu J, Li X, Jian M, Zhou L, Ye R, Huang P, Chen J, Jiang T, Liu X, Wang Y, Zou J, Jiang Z, Wu R, Wu S, Fan F, Zhang Z, Liu L, Yang R, Liu X, Wu H, Yin W, Zhao X, Liu Y, Peng H, Jiang B, Feng Q, Li C, Xie J, Lu J, Kristiansen K, Li Y, Zhang X, Li S, Wang J, Yang H, Cai Z, Wang J. Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma. Nat Genet. 2011 Dec 4. doi: 10.1038/ng.1014. PMID: 22138691
• 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 (2011). Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene, 30 (10), 1159-73. PMID: 21057536
• Varela I, Tarpey P, Raine K, Huang D, Ong CK, Stephens P, Davies H, Jones D, Lin ML, Teague J, Bignell G, Butler A, Cho J, Dalgliesh GL, Galappaththige D, Greenman C, Hardy C, Jia M, Latimer C, Lau KW, Marshall J, McLaren S, Menzies A, Mudie L, Stebbings L, Largaespada DA, Wessels LF, Richard S, Kahnoski RJ, Anema J, Tuveson DA, Perez-Mancera PA, Mustonen V, Fischer A, Adams DJ, Rust A, Chan-on W, Subimerb C, Dykema K, Furge K, Campbell PJ, Teh BT, Stratton MR, & Futreal PA (2011). Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature, 469 (7331), 539-42. PMID: 21248752

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

This week we would like to introduce Dr Seung-Beom Hong, a post-doctoral researcher in the lab of Professor Vera Krymskaya at the University of Pennsylvania.

Dr Hong was part of the group at the NIH that helped to identify and characterise FNIP1 and FNIP2, which directly links FLCN with the key cellular energy sensor AMPK (Baba et al., 2006; Hasumi et al., 2008). This work also showed that FLCN is associated with mTOR signalling, which is also a major regulator of growth, proliferation and metabolism in cells.

In addition to this, Dr Hong’s extensive experience with mouse models of VHL (Ma et al., 2003; Hong et al., 2006) and BHD (Baba et al., 2008; Hasumi et al., 2009) places him in an ideal position to further decipher the downstream targets of FLCN. Accordingly, his current research has shown that FLCN plays an essential role in the regulation of TGF-β signalling (Hong et al., 2010a), as well as the nucleo-cytoplasmic transcription factor TFE3 (Hong et al., 2010b). These findings have been discussed further in our blog posts from June 2010 and January 2011 respectively, and are of particular interest as they implicate yet more factors and signalling pathways in the pathogenesis of BHD syndrome. Ultimately, it is hoped that such work can not only identify novel therapeutic targets, but may also lead to the elucidation of effective biomarkers for the varied symptoms of BHD.

To hear more about Dr Hong’s work, watch his video interview from the Third BHD Symposium (with its accompanying transcript and audio only file). Alternatively, the following publications may also be of interest:

• Hong SB, Oh H, Valera VA, Baba M, Schmidt LS, Linehan WM. Inactivation of the FLCN tumor suppressor gene induces TFE3 transcriptional activity by increasing its nuclear localization. PLoS One. 2010 Dec 29;5(12):e15793. PMID: 21209915
• Hong SB, Oh H, Valera VA, Stull J, Ngo DT, Baba M, Merino MJ, Linehan WM, Schmidt LS. Tumor suppressor FLCN inhibits tumorigenesis of a FLCN-null renal cancer cell line and regulates expression of key molecules in TGF-beta signaling. Mol Cancer. 2010 Jun 23;9:160. PMID: 20573232
• Hasumi Y, Baba M, Ajima R, Hasumi H, Valera VA, Klein ME, Haines DC, Merino MJ, Hong SB, Yamaguchi TP, Schmidt LS, Linehan WM. Homozygous loss of BHD causes early embryonic lethality and kidney tumor development with activation of mTORC1 and mTORC2. Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18722-7. Epub 2009 Oct 22. PMID: 19850877
• Hasumi H, Baba M, Hong SB, Hasumi Y, Huang Y, Yao M, Valera VA, Linehan WM, Schmidt LS. Identification and characterization of a novel folliculin-interacting protein FNIP2. Gene. 2008 May 31;415(1-2):60-7. Epub 2008 Mar 4. PMID: 18403135
• Baba M, Furihata M, Hong SB, Tessarollo L, Haines DC, Southon E, Patel V, Igarashi P, Alvord WG, Leighty R, Yao M, Bernardo M, Ileva L, Choyke P, Warren MB, Zbar B, Linehan WM, Schmidt LS. Kidney-targeted Birt-Hogg-Dube gene inactivation in a mouse model: Erk1/2 and Akt-mTOR activation, cell hyperproliferation, and polycystic kidneys. J Natl Cancer Inst. 2008 Jan 16;100(2):140-54. Epub 2008 Jan 8. PMID: 18182616
• Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, Zbar B. 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. 2006 Oct 17;103(42):15552-7. Epub 2006 Oct 6. PMID: 17028174
• Hong SB, Furihata M, Baba M, Zbar B, Schmidt LS. Vascular defects and liver damage by the acute inactivation of the VHL gene during mouse embryogenesis. Lab Invest. 2006 Jul;86(7):664-75. Epub 2006 May 1. PMID: 16652107
• Ma W, Tessarollo L, Hong SB, Baba M, Southon E, Back TC, Spence S, Lobe CG, Sharma N, Maher GW, Pack S, Vortmeyer AO, Guo C, Zbar B, Schmidt LS. Hepatic vascular tumors, angiectasis in multiple organs, and impaired spermatogenesis in mice with conditional inactivation of the VHL gene. Cancer Res. 2003 Sep 1;63(17):5320-8. PMID: 14500363

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

Many studies into BHD syndrome have investigated the prevalence of pneumothorax and renal cell carcinoma amongst FLCN mutation carriers. Previous estimations of the RCC risk have varied from 6.5% to 34% (Toro et al., 2008), and for pneumothorax, from 24% to 38% (Schmidt et al., 2005; Toro et al., 2007; Toro et al., 2008). This variation is likely to be due to ascertainment bias. Recently, a study by Houweling et al. (2011) investigated 115 FLCN mutation carriers from 35 families to reassess the risk of RCC and pneumothorax in BHD syndrome. The study also evaluated the histological subtypes of renal cancer commonly found in BHD patients. The majority of patients were referred by dermatologists and so it is important to remember that this study may also have an ascertainment bias.

From the 115 FLCN mutation carriers assessed in this study, 28 (24%) had a history of pneumothorax, 8 of which were recurrent. The mean age of the first pneumothorax was found to be 36 (range 18 – 74). CT examination of 12 FLCN mutation carriers showed that 5 patients (42%) had multiple lung cysts. After incorporating data on relatives who did not undergo DNA testing for FLCN mutations, the estimated prevalence for the first episode of spontaneous pneumothorax was found to be 29% at 70 years.

The same analysis was carried out to determine the risk of RCC amongst BHD patients. 14 out of the 115 FLCN mutation carriers (12%) had RCC, 5 of which had metastasised. Three BHD patients had multiple cases of renal cancer. Taking all the data together, including the non-tested BHD family members, the prevalence for renal cancer was 16% at age 70.

Previous reports found that the most prevalent RCC histology identified in BHD syndrome was a hybrid chromophobe and oncocytic tumour (Pavlovich et al., 2002). However, several other histologies have also been identified. The histology of the renal tumours in this cohort was determined and it was found that the majority of tumours had characteristics of both chromophobe and clear cell carcinoma. The tumours also resembled those seen in sporadic RCC. Knowing the histological pattern expected in BHD tumours could aid early diagnosis.

Determining the prevalence of pneumothorax and RCC risk in BHD syndrome is important for planning the correct surveillance programme for FLCN mutation carriers and their families. The guidelines proposed by Menko et al. (2009) suggest beginning renal cancer surveillance for BHD patients at age 20 and performing annual MRI scans. Although the reported prevalence in this study for both RCC and pneumothorax is lower than that in some of the previous studies, the need for surveillance and an awareness of BHD symptoms is as important as ever.

• Schmidt LS, Nickerson ML, Warren MB, Glenn GM, Toro JR, Merino MJ, Turner ML, Choyke PL, Sharma N, Peterson J, Morrison P, Maher ER, Walther MM, Zbar B, Linehan WM. Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dubé syndrome. Am J Hum Genet. 2005 Jun;76(6):1023-33. Epub 2005 Apr 25. PMID:15852235
• 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. 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. PMID:18234728
• 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. PMID:22146830
• 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. PMID:12459621
• Menko FH, van Steensel MA, Giraud S, Friis-Hansen L, Richard S, Ungari S, Nordenskjöld M, Hansen TV, Solly J, Maher ER; European BHD Consortium. Birt-Hogg-Dubé syndrome: diagnosis and management. Lancet Oncol. 2009 Dec;10(12):1199-206. PMID:19959076

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

The RARE 2011 conference was an exceptional opportunity to focus on the development of effective policies and infrastructure for rare diseases. Speakers, including representatives from Orphanet, public bodies, funding organisations, patient associations, research centres, and industry, analysed the obstacles and achievements in European management of rare diseases.

One of the greatest obstacles is lack of reliable, accessible and well-organised knowledge. Firstly, there is little known medically and scientifically about many rare diseases. For example, two common situations for a rare disease, a wide and sparsely spread population and a large range of symptoms, mean that meagre amounts of data are available on the progression of a given disease. While certainly more research is required, a practical tool emphasised throughout the meeting was the patient registry. A patient registry, open to and well-maintained by an international community of researchers, would allow greater insight into epidemiology, genotype-phenotype correlations and so on. A patient registry would also make it easier to run a good clinical trial, because, for example, researchers could show the effect of a treatment more clearly.

While the obstacles are significant, recent achievements and new opportunities promise an exciting future for tackling rare diseases. One of these is the International Rare Disease Consortium (IRDiRC), a body jointly established by the European Commission, the National Institutes of Health in the United States and the Canadian Institutes for Health Research. IRDiRC’s goals for 2020 are to produce diagnostic tests for most rare diseases, and to develop 200 new therapies for rare diseases. Also of significant note is the creation of National Foundation for Rare Diseases in France (Fondation des Maladies Rares). Part of the Foundation’s plans is the establishment of more centres of excellence, standardised protocols and a national bank for rare diseases. Orphanet is also continuing to develop reliable, well-organised knowledge and to raise the visibility of rare diseases in all medical settings by contributing to the International Classification of Diseases (ICD). The ICD, managed by the World Health Organisation, is an international system for organising/coding diseases on medical records, and is used in a wide variety of studies such as gathering disease statistics. All previous versions of the ICD do not include codes for rare diseases. Many processes in managing a rare disease are made more complex by this exclusion, from gathering epidemiological information to including a rare disease diagnosis in a patient’s medical record.

Emerging over the meeting was a sense of anticipation built on considerable accomplishment. With the creation of IRDiRC and inclusion of rare diseases in the new ICD, it seems rare diseases are gaining much more of a public health presence. As new initiatives and programmes begin to act and bear fruit in 2012, there is much hope for substantial change in the current ways of understanding, diagnosing, managing and treating rare diseases.

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

HLRCC, caused by mutations in FH, predisposes patients to develop papillary renal cell carcinoma. FH-deficient cells have an accumulation of fumarate, which leads to the stabilisation of HIF-α subunits and therefore an increase in HIF-α levels. An increased expression of HIF target genes is believed to drive tumourigenesis. Along with FH, increased levels of HIF are also seen in renal cell carcinomas with VHL or SDH mutations. However, unlike HLRCC, these tumours are predominantly of the clear cell histology, suggesting a different mechanism of tumourigenesis in HLRCC.

Two independent studies, published recently in the same journal, have now identified a HIF-independent pathway for FH-mediated tumourigenesis. Adams et al. (2011) used mice models with both Fh1 and either Hif-1α or Hif-2α inactivation, to demonstrate that cyst formation in HLRCC is HIF independent. Interestingly, specifically deleting both Fh1 and Hif-1α actually accelerated cyst formation.

In the other study, Ooi et al. (2011) performed gene expression profiling using FH^-/- fibroids to identify additional pathways deregulated in HLRCC. The most highly upregulated gene was the aldo-keto reductase family 1 member B10 gene (AKR1B10). Ooi and colleagues next set out to identify the transcription factors regulating this gene. Promoter sequence analysis identified the antioxidant response element as a regulator of AKR1B10, which is bound by the transcription factor NRF2. NRF2 levels were shown to increase with the accumulation of fumarate, suggesting NRF2 drives the expression of AKR1B10.

Adams et al. also performed gene expression profiling and found that the NRF2-mediated antioxidant pathway was the most upregulated in Fh1^-/- mice. NRF2 target genes also had increased expression in Fh1^-/- mouse kidneys. Interestingly, Hmox1, the haem oxygenase gene shown to be synthetically lethal in HLRCC (Frezza et al., 2011 – see this previous blog post) is a target gene of NRF2 and was shown to be upregulated in this study. Adams et al. used mouse models to confirm that the upregulation of NRF2 is HIF-independent.

Both studies found that the increase in NRF2 levels was due to inactivation of KEAP1, a negative regulator of NRF2 which binds to NRF2 and mediates its ubiquitination. It was found that in FH-deficient cells, KEAP1 is succinated by fumarate, leading to its inactivation and therefore the stabilisation of NRF2 and the expression of antioxidant genes.

How the increased level of NRF2 drives tumourigenesis in HLRCC is still unclear and warrants further investigation. These studies also highlight the need for more research into the effects of increased HIF activity in BHD syndrome: although HIF-1α activity has been shown to be increased in FLCN-null cells (Preston et al., 2011), does it contribute to tumourigenesis or are HIF-independent mechanisms involved?

• Adam J, Hatipoglu E, O’Flaherty L, Ternette N, Sahgal N, Lockstone H, Baban D, Nye E, Stamp GW, Wolhuter K, Stevens M, Fischer R, Carmeliet P, Maxwell PH, Pugh CW, Frizzell N, Soga T, Kessler BM, El-Bahrawy M, Ratcliffe PJ, Pollard PJ. Renal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 Signaling. Cancer Cell. 2011 Oct 18;20(4):524-37. PMID:22014577
• Ooi A, Wong JC, Petillo D, Roossien D, Perrier-Trudova V, Whitten D, Min BW, Tan MH, Zhang Z, Yang XJ, Zhou M, Gardie B, Molinié V, Richard S, Tan PH, Teh BT, Furge KA. An Antioxidant Response Phenotype Shared between Hereditary and Sporadic Type 2 Papillary Renal Cell Carcinoma. Cancer Cell. 2011 Oct 18;20(4):511-23. PMID:22014576
• Frezza C, Zheng L, Folger O, Rajagopalan KN, MacKenzie ED, Jerby L, Micaroni M, Chaneton B, Adam J, Hedley A, Kalna G, Tomlinson IP, Pollard PJ, Watson DG, Deberardinis RJ, Shlomi T, Ruppin E, Gottlieb E. Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Nature. 2011 Aug 17;477(7363):225-8. PMID:21849978
• 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. PMID:21057536

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