We are here live at the Fourth BHD Symposium, at the University of Cincinnati. The University of Cincinnati has not only been called one of the world’s most beautiful campuses (by Forbes magazine), but also has an illustrious history in medical research, including the development of the first antihistamine and the first oral polio vaccine.

The Symposium began on Wednesday evening, when attendees met for a reception in the Kingsgate Conference Centre. The reception gave everyone the chance to meet and catch up before the scientific and family sessions kicked off on Thursday.

After an introduction by the chair of the scientific organising committee, Professor Maria Czyzyk-Krzeska, the Thursday morning session began with an overview of the treatment and management of BHD kidney cancer, given by Dr W. Marston Linehan of the National Cancer Institute at the NIH. The rest of the morning focused on clinical talks given by presenters from around the world. Dr Koga from the Chiba University School of Medicine in Japan spoke about pulmonary cysts in 14 BHD families, and Dr Fred Menko of the VU Medical Center in Amsterdam spoke about familial multiple discoid fibromas (FMDF), a novel syndrome with similar skin symptoms to BHD.

The morning session also included an interesting and dynamic panel discussion, led by researchers representing different health systems and approaches. Topics such as the age at which to begin kidney screening and the management of lung symptoms were discussed.

After the panel discussion, the scientific sessions focused on more basic science, with invited talks from Professor George Thomas of the University of Cincinnati and Professor Arnim Pause of McGill University, Montreal. The families attended a separate session, led by Lindsay Middleton, a genetic counsellor at the NIH.

All attendees came together at the end of the day for the poster session, followed by a dinner at the Verdin Bell Event Centre. The Centre was originally St. Paul’s church, built in 1850 and decommissioned in 1975. Bought in 1981 by The Verdin Company, the oldest bell and clock company in North America, it has now been carefully restored and contains a wonderful collection of historic bells and clocks.

Many more exciting talks are still to come on the final day of the Symposium. Remember to watch out for our full reports from the Symposium over the next few weeks, covering the scientific and family sessions. Don’t forget to keep following the live Symposium news, as well as all other BHD syndrome news on Twitter, hashtag #BHDSyndrome.

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

The Fourth BHD Symposium is less than a week away, and this year it will be held in Cincinnati, USA, on 28^th to 30^th March. Registration is still open and more information regarding the entire Symposium can be found here.

In particular, the Fourth BHD Symposium agenda and Patient and family sessions schedule are now available. There will also be a social gathering to welcome everyone to the Symposium on Wednesday 28^th March from 6 pm at the Kingsgate Marriott.

From Thursday 29^th March, there will be a wide variety of clinical and basic science talks, including those from Dr Marston Linehan (National Cancer Institute, USA) and Professor Angela Christiano (Columbia University Medical Center, USA). As with previous years, there will be a poster session introducing yet more BHD research. In addition, patients and family members will be able to meet each other and experts in the BHD field during the Patient and family sessions.

Please do follow the meeting on our Twitter feed, hashtag #BHDSyndrome. Next week’s blog post will also be a live report from the Symposium, and there will be further highlights from Cincinnati in the weeks to come. We hope to see you there!

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

Recent advances in DNA sequencing have led to the discovery of genes and mutations which drive tumourigenesis. In this blog, we have previously described papers by Varela et al. (2011), Dalgliesh et al. (2011) and Guo et al. (2011) which all used exome sequencing to identify genes involved in kidney cancer (described here, here and here respectively). It is hoped that by understanding more about the cancer genome, effective targeted therapies can be developed.

A recent study by Xu et al. (2012) performed sequencing analysis on one ccRCC kidney tumour sample, this time by using single-cell exome sequencing. Exome sequencing was first used on a sample of ccRCC tissue to analyse the mutational status of VHL and PBRM1, the main drivers of ccRCC tumourigenesis. Three low frequency mutations in PBRM1 were identified, but no mutations were found in VHL.

Single-cell exome sequencing was then performed on 20 cells from the tumour sample and 5 cells from noncancerous adjacent tissue, which were used as controls. In total, 260 somatic mutations were identified in the cancer cells, compared with 12 somatic mutations in the healthy control cells. Mutations in 94 genes were identified.

The mutation pattern for each individual cell was compared and the results were found to be significantly different from each other. The authors therefore suggest that the tumour does not contain subpopulations of cells. Additionally, more than 70% of the mutations were classed as cell-specific, meaning they only occurred in a small fraction of the cells. Both of these results demonstrate the high complexity and heterogeneity of this tumour sample.

The results from this study were compared to the recent sequencing study by Guo et al. (2011). Four of the 94 genes identified here were also mutated in the cohort of 98 patients studied by Guo et al. One gene of particular interest is AHNAK, which was mutated in 5 of the 99 patients from both studies. AHNAK activates protein kinase C and it is also involved in chromatin remodelling, a process in which related genes appear to be frequently mutated in RCC. Furthermore, protein-protein prediction methods used in this study suggest an interaction between AHNAK and HIF1A.

With regards to BHD syndrome, no FLCN mutations were identified in the RCC cells studied here. However, it would be interesting to perform a similar study using cells from a BHD tumour to identify other mutations which could contribute to tumourigenesis in BHD syndrome. The authors of this study suggest that ccRCC is more genetically complex than previously thought. Perhaps the same is true for BHD-related tumours and that there are other, as yet, unidentified mutations which promote tumourigenesis.

• Xu X, Hou Y, Yin X, Bao L, Tang A, Song L, Li F, Tsang S, Wu K, Wu H, He W, Zeng L, Xing M, Wu R, Jiang H, Liu X, Cao D, Guo G, Hu X, Gui Y, Li Z, Xie W, Sun X, Shi M, Cai Z, Wang B, Zhong M, Li J, Lu Z, Gu N, Zhang X, Goodman L, Bolund L, Wang J, Yang H, Kristiansen K, Dean M, Li Y, Wang J. Single-cell exome sequencing reveals single-nucleotide mutation characteristics of a kidney tumor. Cell. 2012 Mar 2;148(5):886-95. PMID: 22385958
• 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. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature. 2011 Jan 27;469(7331):539-42. PMID: 21248752
• Dalgliesh GL, 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 PJ, Forbes S, Jia M, Jones D, Knott H, Kok CY, Lau KW, Leroy C, Lin ML, McBride DJ, 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 PS, Turrell K, Dykema KJ, Khoo SK, Petillo D, Wondergem B, Anema J, Kahnoski RJ, Teh BT, Stratton MR, Futreal PA. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature. 2010 Jan 21;463(7279):360-3. PMID: 20054297
• 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;44(1):17-9. PMID: 22138691

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

Our lab-profile for this month introduces the work of Professor Arnim Pause, an Associate Professor in the Department of Biochemistry and a Canada Research Chair in Molecular Oncology at McGill University, Canada.

Much of Professor Pause’s early work involved researching eukaryotic translation initiation factors, such as eIF-4A and eIF-4E, which play an important role in protein biosynthesis (Dostie et al., 2000). Concurrent work focussed on the function of VHL, which was discovered to be involved in the ubiquitination of proteins and their degradation by the proteasome (Iwai et al., 1999).

Currently, the Pause lab continues to characterise the function of the VHL protein (Kurban et al., 2008), as well as investigating the role of a His domain-containing protein tyrosine phosphatase encoded by the tumour suppressor gene candidate PTPN23 (Gingras et al., 2009). Professor Pause also studies the role of several proteins in the life cycle of the Hepatitis C virus (Tedbury et al., 2011).

Another major research interest of the Pause lab is the functional characterisation of the FLCN protein. Using mouse models and human cell lines, Hudon et al. (2010) noted that a loss of FLCN expression resulted in both elevated and reduced levels of mTOR signalling, which may account for the differing results observed in previous studies (see here for more information). The Pause lab also collaborated with Dr Andrew Tee’s group at Cardiff University to show that FLCN is associated with HIF signalling (Preston et al., 2011), which has been discussed in more detail in a previous blog post and lab-profile. Further studies have also been planned that make use of a transparent nematode called C. elegans. This multicellular model organism facilitates systematic molecular and genetic approaches, while still maintaining relevance to other eukaryotes such as humans.

To hear more about the research of the Pause lab, and why rare disease research is particularly important for medical research in general, please do watch our video interview (with its accompanying transcript and audio file). Alternatively, join us at the 4^th BHD Symposium on 28^th – 30^th March in Cincinnati, USA. Registration is still open and more information about the patient and family sessions can be found here.

• Dostie J, Ferraiuolo M, Pause A, Adam SA, Sonenberg N. A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5′ cap-binding protein, eIF4E. EMBO J. 2000 Jun 15;19(12):3142-56. PMID: 10856257
• Gingras MC, Zhang YL, Kharitidi D, Barr AJ, Knapp S, Tremblay ML, Pause A. HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain. PLoS One. 2009;4(4):e5105. Epub 2009 Apr 2. PMID: 19340315
• 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. Epub 2009 Oct 19. PMID: 19843504
• Iwai K, Yamanaka K, Kamura T, Minato N, Conaway RC, Conaway JW, Klausner RD, Pause A. Identification of the von Hippel-lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex. Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12436-41. PMID: 10535940
• Kurban G, Duplan E, Ramlal N, Hudon V, Sado Y, Ninomiya Y, Pause A. Collagen matrix assembly is driven by the interaction of von Hippel-Lindau tumor suppressor protein with hydroxylated collagen IV alpha 2. Oncogene. 2008 Feb 7;27(7):1004-12. Epub 2007 Aug 13. PMID: 17700531
• 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. Epub 2010 Nov 8. PMID: 21057536
• Tedbury P, Welbourn S, Pause A, King B, Griffin S, Harris M. The subcellular localization of the hepatitis C virus non-structural protein NS2 is regulated by an ion channel-independent function of the p7 protein. J Gen Virol. 2011 Apr;92(Pt 4):819-30. Epub 2010 Dec 22. PMID: 21177929

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

Hypoxia Inducible Factor (HIF) regulates key processes within the cell and its dysregulation is involved in the development of renal cell carcinoma (RCC), including those associated with VHL, HLRCC, TSC and also BHD syndrome. HIF is a potential therapeutic target for RCC and examples of HIF inhibitors have been described in this previous blog. Despite this, the regulation of HIF and its involvement in tumourigenesis remains poorly understood.

A recent paper by Schietke et al. (2012) has investigated the expression of HIF subunits within the human kidney and the contribution each subunit makes towards tumourigenesis. HIF is a heterodimeric transcription factor composed of a HIF-β subunit bound to either a HIF-1α subunit or a HIF-2α subunit. Both α subunits are regulated by VHL but they control transcription of different genes, and have distinct expression patterns in rat kidneys (Rosenberger et al., 2002). Previously, the HIF-2α subunit, rather than HIF-1α, has been described as the driving force for tumourigenesis (Raval et al., 2005).

Schietke et al. used immunohistochemistry to show that the expression pattern of the HIF-1α and HIF-2α protein in human kidneys matched that seen in the rat kidney. Under hypoxic conditions, HIF-1α was expressed exclusively in tubular epithelial cells, whereas HIF-2α was expressed in interstitial cells and glomeruli. HIF-2α was never seen in the tubular cells. See this page for images describing the location of different cell types within the kidney.

The authors then knocked out VHL in mouse tubular cells and repeated the expression profiling. The HIF-1α expression pattern did not change, but surprisingly, HIF-2α was now strongly expressed in the tubular cells, suggesting VHL specifically represses HIF-2α in this region. A mouse model with HIF-2α overexpressed in renal tubular cells was used to study the effect of this expression. These mice had impaired renal function, fibrosis and cysts in the kidney, however RCC did not develop. The authors suggest that HIF-2α overexpression may be an important early step in tumourigenesis but it is not sufficient to induce RCC.

The levels and activity of HIF-2α have been investigated in BHD syndrome by Preston et al. (2011). Here, the authors found no change in the protein level of HIF-2α after FLCN inactivation, but saw an increase in its activity. Perhaps this increase in activity mimics the increased expression seen in VHL-deficient kidneys, both leading to renal dysfunction. Despite this, Adams et al. (2011) and Ooi et al. (2011) recently identified a HIF-independent mechanism of cyst development in HLRCC (described here). It is clear that more research is required to fully understand the role of HIF in RCC, including that associated with BHD syndrome.

• Schietke RE, Hackenbeck T, Tran M, Günther R, Klanke B, Warnecke CL, Knaup KX, Shukla D, Rosenberger C, Koesters R, Bachmann S, Betz P, Schley G, Schödel J, Willam C, Winkler T, Amann K, Eckardt KU, Maxwell P, Wiesener MS. Renal Tubular HIF-2α Expression Requires VHL Inactivation and Causes Fibrosis and Cysts. PLoS One. 2012;7(1):e31034. Epub 2012 Jan 27. PMID:22299048
• Rosenberger C, Mandriota S, Jürgensen JS, Wiesener MS, Hörstrup JH, Frei U, Ratcliffe PJ, Maxwell PH, Bachmann S, Eckardt KU. Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys. J Am Soc Nephrol. 2002 Jul;13(7):1721-32. PMID:12089367
• 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
• 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
• Raval RR, Lau KW, Tran MG, Sowter HM, Mandriota SJ, Li JL, Pugh CW, Maxwell PH, Harris AL, Ratcliffe PJ. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol. 2005 Jul;25(13):5675-86. PMID:15964822