Human artificial chromosomes (HACs) offer an alternative system which does not suffer from these disadvantages. In particular, these vectors have a functional centromere which enables the HAC to be stably maintained as a single-copy episome, without the risk of integration into the host genome. They also have a large cloning capacity, which allows entire genomic loci (including all their regulatory elements) to be used. A recent paper by Kim et al. (2011) has now used such a vector in a VHL-null human kidney cancer cell line (786-O) to demonstrate that it can correct for its VHL deficiency.

The authors first isolated the VHL gene from human genomic DNA using transformation-associated recombination cloning. The vectors were then constructed and transfected into CHO cells, and RT-PCR and fluorescence in situ hybridisation (FISH) were used to confirm the presence of the VHL-containing HAC within these cells.

Using microcell-mediated chromosome transfer, the VHL-HAC was transferred to VHL-null 786-O cells. FISH showed that this construct propagated autonomously without any integration into host chromosomes, and RT-PCR confirmed the expression of wild-type VHL within these cells. Moreover, when compared to controls, this HAC downregulated the protein levels of HIF2α, Cyclin D1 and Cdk1, which are known targets of VHL. Additionally, VHL-deficient 786-O cells have a more branched and invasive phenotype when supplemented with either hepatocyte growth factor or foetal bovine serum, but these phenotypes and their migration were substantially suppressed with the VHL-HAC.

This study made use of a HAC in which the centrosome can be inactivated, resulting in the loss of the HAC. This feature provides a useful phenotypic control for any HAC-associated alterations within the cells. Accordingly, centrosome inactivation and HAC loss led to the increased expression of HIF2α, Cyclin D1 and Cdk1, back to their original levels. Altogether, these results suggest that the VHL gene expressed from the HAC produces a functional protein which can correct for the VHL deficiency within these 786-O cells.

Much more work is necessary before HACs can be developed as a treatment for VHL, but a similar system could be tested in FLCN-null cells. Nevertheless, a number of BHD-associated gene therapy projects are currently in progress – to learn more, please do read our articles about Dr Justin Roth, Dr Richard Harbottle, Dr Laura Denby and Dr Suet-Ping Wong. Alternatively, join us at 4^th BHD Symposium in Cincinnati on 28^th-30^th March to hear more about the current state of BHD research in general. Registration and abstract submission are open, and further information about the Patient and Family sessions can also be found here.

• Kim JH, Kononenko A, Erliandri I, Kim TA, Nakano M, Iida Y, Barrett JC, Oshimura M, Masumoto H, Earnshaw WC, Larionov V, & Kouprina N (2011). Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells. Proceedings of the National Academy of Sciences of the United States of America, 108 (50), 20048-53 PMID: 22123967

Dr Cash’s work focused on identifying the biological role of FLCN. In 2011, Cash et al. showed that FLCN was involved in the regulation of apoptosis through the TGF-β signalling pathway. Cells that lacked FLCN were resistant to cell death and this was thought to be due to the dysregulation of pro-apoptotic proteins. This paper has been reviewed in a previous blog post. Lim et al. (2012) have recently found FLCN, FNIP2 and AMPK to be involved in MNU-induced apoptosis, further highlighting the link between FLCN and cell death. Read more about this paper and how apoptosis could be implicated in the pathogenesis of BHD syndrome in last week’s blog post.

In addition to TGF-β signalling, Dr Cash worked on the link between FLCN and mTORC1 activity, demonstrating that downregulation of FLCN leads to mTOR inhibition (Hartman et al., 2009). Furthermore, renal tumours in mice carrying a FLCN mutation had low levels of phosphorylated S6, suggesting low levels of mTOR activity and adding to the debate on how FLCN affects mTOR signalling, which is described in more detail here.

Dr Cash submitted his PhD thesis, entitled ‘Molecular mechanisms of the Birt-Hogg-Dubé tumor suppressor’, in 2010 and has now moved to a postdoctoral position at the CNIO in Madrid where he is focusing on cellular aging and metabolism. Watch our video interview, filmed at the Third BHD Symposium, to learn more about Dr Cash and his BHD work. A transcript and audio-only file are also available for this interview.

To learn more about current BHD research and to meet researchers working on BHD syndrome, attend the Fourth BHD Symposium, to be held in Cincinnati on 28^th-30^th March 2012. The meeting will include sessions for patients and researchers. Registration and abstract submission are now open and more information can be found here. We hope to see you in Cincinnati!

• Cash TP, Gruber JJ, Hartman TR, Henske EP, & Simon MC (2011). Loss of the Birt-Hogg-Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription. Oncogene, 30 (22), 2534-46 PMID: 21258407
• Hartman TR, Nicolas E, Klein-Szanto A, Al-Saleem T, Cash TP, Simon MC, & Henske EP (2009). The role of the Birt-Hogg-Dubé protein in mTOR activation and renal tumorigenesis. Oncogene, 28 (13), 1594-604 PMID: 19234517
• Lim TH, Fujikane R, Sano S, Sakagami R, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2011). Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA. DNA repair PMID: 22209521

Co-immunoprecipitation experiments using mouse YT102 Mgmt^-/- cells, which lack the enzyme MGMT that repairs alkylated DNA, confirmed that FNIP2 interacts with both FLCN and AMPK. The authors then showed that siRNA knockdown of FLCN and AMPKα in this cell line suppressed their apoptotic response to MNU when compared to siRNA controls. They subsequently used compound C (a specific inhibitor of AMPK) to show that it also suppressed MNU-induced apoptosis when compared to untreated YT102 cells. AMPK is usually activated after MNU treatment, which is associated with an increased level of AMPKα phosphorylation. However, less of this activation was observed after compound C treatment.

This result was investigated further by treating YT102 cells with MNU, and assessing the levels of phosphorylated AMPKα over 72 hours. Here, Lim et al. (2011) saw that the levels of phosphorylated AMPKα gradually increased over time, but that this increase was abrogated by the siRNA-mediated knockdown of FLCN. In addition, KH101 Mgmt^-/- Fnip2^+/- cells, which were shown to be resistant to MNU-induced apoptosis by Komori et al. (2009), also showed no increase in phosphorylated AMPKα after MNU treatment.

To confirm that AMPK activation is involved in the induction of apoptosis, AICAR (a specific activator of AMPK) was added to YT102 and KH101 cells. This treatment increased the levels of AMPKα phosphorylation, as well as the amount of cell death detected by trypan blue staining in YT102 cells. However, no such increases were observed in the FNIP2-defective KH101 cells after treatment. To see if this cell death was attributable to apoptosis, mitochondrial membrane depolarisation (which is known to occur during apoptosis) was measured in these cells, and it could be seen that this depolarisation took place in the YT102 cells (but not the KH101 cells) after the addition of AICAR. Similar results were obtained with YT102 cells treated with AICAR and FLCN-siRNA, where less cell death, mitochondrial membrane depolarisation and AMPKα phosphorylation were observed when compared to siRNA controls.

Together, these results suggest that FLCN and FNIP2 play a role in AMPK activation and the induction of apoptosis by MNU. However, the exact mechanism by which this occurs is still unknown, and the elucidation of this process should help us to further understand the pathophysiology of BHD syndrome.

• Komori K, Takagi Y, Sanada M, Lim TH, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2009). A novel protein, MAPO1, that functions in apoptosis triggered by O6-methylguanine mispair in DNA. Oncogene, 28 (8), 1142-50 PMID: 19137017
• Lim TH, Fujikane R, Sano S, Sakagami R, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2011). Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA. DNA repair PMID: 22209521

The symposium aims to be the best BHD meeting to date, with stimulating scientific discussion between experts from around the world. There will be a mixture of talks and posters, as well as time to exchange ideas and discuss collaborations with other members of the BHD community. A social dinner will take place on the evening of Thursday 29^th March. Highlights from last year’s meeting can be read here.

Invited speakers will include Dr W. Marston Linehan of the National Cancer Institute at the National Institutes of Health, Professor George Thomas of the University of Cincinnati,  Professor Kuniaki Seyama of Juntendo University, Professor Arnim Pause of McGill University and Professor Dr Maurice van Steensel of the University Hospital Maastricht. Talks will focus on all aspects of BHD syndrome, including the skin, lungs and kidneys, as well as both clinical and basic science. To find out more about these speakers, see our video interviews with Dr Linehan, Professor Pause and Professor Dr van Steensel.

Abstract submission is now open and the deadline for abstracts is 15^th February 2012. Abstracts may be submitted for both basic and clinical science presentations and researchers working on related conditions are also encouraged to submit an abstract. Abstracts can be submitted here and more details, including instructions for abstract submission, can be found here.

As with previous symposia, there will also be sessions for patients and family members. These will be led by a genetic counsellor who is an expert in BHD syndrome. Previous patient sessions have been very informative, allowing patients to learn more about BHD, meet other people affected by the syndrome, as well as talk to experts about their symptoms. Read a review of last year’s patient session here.

Registration and hotel information will be available soon so please check back. We will also update the webpage as more information on the programme is released. Don’t forget to get your abstracts in before 15^th February! We are looking forward to welcoming you to the Fourth BHD Symposium in March.

Dr Suet-Ping Wong, working in the lab of Dr Richard Harbottle at Imperial College London, was nominated for the Fairbairn young investigator award and gave a talk on FLCN gene therapy using S/MAR vectors (for more information see our lab profile). The Harbottle lab have successfully developed vectors which express FLCN, and have shown re-expression of FLCN in UOK257 cells using these vectors. Additionally, the corrected cells have been xenografted into mice, with promising results. It is hoped that future in vivo experiments will continue to develop this therapeutic product as a potential treatment for BHD syndrome.

The difficulties facing the development of gene therapy products for rare diseases were highlighted by Dr Janneke de Wal from Amsterdam Molecular Therapeutics. The small population of affected individuals means data sets are small and large geographical areas need to be covered in order to gain enough participants for rare disease clinical trials. In Dr de Wal’s work developing Glybera, a gene therapy product for the rare disease lipoprotein lipase deficiency, more academics were involved than clinical trial participants.

Dr Marco Ranzani from the San Raffaele Telethon Institute for Gene Therapy in Milan gave another interesting talk on using insertional mutagenesis to identify cancer genes. Although insertional mutagenesis is seen as an adverse effect of gene therapy, it can be exploited for cancer gene discovery. Dr Ranzani showed that vectors which target and disrupt the Pten and Cdkn2a genes induced hepatocellular carcinoma (HCC) in mice models. Using this technique, the Braf, Fign, Sos1 and Dlk1-Dio3 genes were found to be associated with HCC development. Insertional mutagenesis using gene therapy could be a valuable technique to identify cancer-associated genes that could be potential therapeutic targets.

Information on upcoming meetings that may be of interest to BHD researchers can be found on the Conferences and Events page. More information on the upcoming 4th BHD Symposium, to be held in Cincinnati, USA, 28^th-30^th March 2012, will also be available soon.

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-VHL UMPP 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 (2011). Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma. Nature genetics, 44 (1), 17-9 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

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 (2010). Inactivation of the FLCN tumor suppressor gene induces TFE3 transcriptional activity by increasing its nuclear localization. PloS one, 5 (12) PMID: 21209915
• Hong SB, Oh H, Valera VA, Stull J, Ngo DT, Baba M, Merino MJ, Linehan WM, & Schmidt LS (2010). Tumor suppressor FLCN inhibits tumorigenesis of a FLCN-null renal cancer cell line and regulates expression of key molecules in TGF-beta signaling. Molecular cancer, 9 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 (2009). Homozygous loss of BHD causes early embryonic lethality and kidney tumor development with activation of mTORC1 and mTORC2. Proceedings of the National Academy of Sciences of the United States of America, 106 (44), 18722-7 PMID: 19850877
• Hasumi H, Baba M, Hong SB, Hasumi Y, Huang Y, Yao M, Valera VA, Linehan WM, & Schmidt LS (2008). Identification and characterization of a novel folliculin-interacting protein FNIP2. Gene, 415 (1-2), 60-7 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 (2008). Kidney-targeted Birt-Hogg-Dube gene inactivation in a mouse model: Erk1/2 and Akt-mTOR activation, cell hyperproliferation, and polycystic kidneys. Journal of the National Cancer Institute, 100 (2), 140-54 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 (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America, 103 (42), 15552-7 PMID: 17028174
• Hong SB, Furihata M, Baba M, Zbar B, & Schmidt LS (2006). Vascular defects and liver damage by the acute inactivation of the VHL gene during mouse embryogenesis. Laboratory investigation; a journal of technical methods and pathology, 86 (7), 664-75 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 (2003). Hepatic vascular tumors, angiectasis in multiple organs, and impaired spermatogenesis in mice with conditional inactivation of the VHL gene. Cancer research, 63 (17), 5320-8 PMID: 14500363

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 (2005). Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dubé syndrome. American journal of human genetics, 76 (6), 1023-33 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 (2007). Lung cysts, spontaneous pneumothorax, and genetic associations in 89 families with Birt-Hogg-Dubé syndrome. American journal of respiratory and critical care medicine, 175 (10), 1044-53 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 (2008). BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dubé syndrome: a new series of 50 families and a review of published reports. Journal of medical genetics, 45 (6), 321-31 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 (2011). Renal cancer and pneumothorax risk in Birt-Hogg-Dubé syndrome; an analysis of 115 FLCN mutation carriers from 35 BHD families. British journal of cancer, 105 (12), 1912-9 PMID: 22146830
• Pavlovich CP, Walther MM, Eyler RA, Hewitt SM, Zbar B, Linehan WM, & Merino MJ (2002). Renal tumors in the Birt-Hogg-Dubé syndrome. The American journal of surgical pathology, 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 (2009). Birt-Hogg-Dubé syndrome: diagnosis and management. The lancet oncology, 10 (12), 1199-206 PMID: 19959076

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.

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 (2011). 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, 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 (2011). An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma. Cancer cell, 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 (2011). Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Nature, 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 (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