FindZebra: a specialised search engine for rare diseases

When you hear hoof beats think of horses, not zebras

Dr Theodore Woodward


Every doctor is taught to think of the common causes of a problem before contemplating the rare ones. This works for the majority of patients but occasionally the cause is something unusual – a “zebra” – and isn’t so easy to find. For rare disease patients a specific diagnosis can be elusive with 46% receiving at least one incorrect diagnosis and 20% waiting over five years for a final diagnosis (Rare Disease UK, 2010).

Medical experts, and non-experts, use the internet for health related searches. The ability to mine the increasing quantity of available information can help clinicians to diagnose difficult cases. It is also common for patients to research their symptoms to try and find a diagnosis. However the standard search engines are not optimised to find results relevant to rare diseases. Search engines generally rank results based on popularity and can return irrelevant and untrustworthy results.

FindZebra is a specialist search engine designed specifically to help doctors diagnose rare diseases (Dragusin et al., 2013). FindZebra only mines for relevant information from reputable rare disease sources – OMIM, Orphanet, GARD, NORD and other smaller rare disease databases – to limit irrelevant results and reduce the time required for assessment. A search can be based on multiple phenotypes and reference points, and FindZebra will return near match results as well as exact matches to account for missing or non-specific phenotypes. A recent addition to FindZebra is a separate list of genes associated with the search term. This provides rapid access to relevant genes for future genetic testing or alternatively allows for the identification of diseases associated with mutation in a particular gene.

Dragusin et al. (2013) compared the ability of FindZebra and Google to find disease-relevant information based on the symptoms of 56 difficult diagnostic cases. Within the top 20 results FindZebra produced a relevant result in 68% of cases, compared to only 32% when using Google. Limiting Google to the same datasets used by FindZebra produced relevant information in only 11% of cases demonstrating the unsuitability of the Google ranking system for this this kind of query. FindZebra is also significantly better with long search queries, such as complex medical histories, than Google which is optimised for short queries.

A Google search for individual BHD symptoms does not produce any immediately relevant results on the first page. The exception is a search for “fibrofolliculomas” but as a highly BHD-specific term this is unlikely to be a common differential diagnostic search term. Combinations of BHD phenotypes yield more prominant relevant results. In comparison a search on FindZebra for “spontaneous pneumothorax” listed BHD in the top results, and has FLCN as the most relevant gene. On FindZebra, as with Google, a search for “renal cell carcinoma” is not specific enough to identify BHD from other hereditary RCC diseases such as VHL or TSC. However the addition of “hybrid” or “multifocal” to the search puts BHD and FLCN in the top results. Again combinations of phenotypes result in BHD being a top result.

One of the biggest differences between the results produced by Google and FindZebra is the ease of reading and assessment. A Google search result involves visiting new pages and sometimes finding the relevant information can be difficult. The FindZebra system shows the detailed result, with the disease in the title, alongside the initial search results and highlights relevant information making it easier to quickly scan for relevance and compatibility.

FindZebra was launched in 2013 and produced over 1,000,000 diagnostic hypotheses for 30,000 unique visitors in the first five weeks (Dragusin et al., 2013b) although no data has been published yet regarding the accuracy of these hypotheses. As more clinicians become aware of the benefits of using FindZebra undoubtedly its user group will grow. Such a tailored search engine has great potential to aid in the rapid diagnosis of numerous rare disease patients.

FindZebra is undoubtedly one of the fastest and most reliable ways to link crucial symptoms to causal genes… [and] has rapidly become an integrated part of our diagnostic set up for rare diseases.

Finn Cilius Nielsen, Center for Genomic Medicine, University of Copenhagen


  • Dragusin R, Petcu P, Lioma C, Larsen B, Jørgensen HL, Cox IJ, Hansen LK, Ingwersen P, Winther O (2013a). FindZebra: a search engine for rare diseases. Int J Med Inform. Jun;82(6):528-38 PubMed PMID: 23462700.
  • Dragusin R, Petcu P, Lioma C, Larsen B, Jørgensen HL, Cox IJ, Hansen LK, Ingwersen P, Winther O (2013b). Specialized tools are needed when searching the web for rare disease diagnoses. Rare Diseases, May 16;1:e25001. PubMed PMID: 25002998.
  • Rare Disease UK (2010). Experiences of Rare Disease: An Insight from Patients and Families.
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Rhabdomyomas: an additional BHD hamartoma phenotype?

Hamartomas are benign, focal malformations formed by an excess of normal tissue growing in a disorganised fashion. Several hamartoma syndromes have been linked to aberrant mTOR signalling including BHD and Tuberous Sclerosis Complex (TSC). In addition to the predisposition of BHD patients to develop hair follicle hamartomas or fibrofolliculomas (Birt et al., 1977), Fuyura et al., (2012) propose that the pulmonary cysts in BHD patients are hamartoma-like cystic alveolar formations. The benign nature of these BHD growth phenotypes, in comparison to the potentially malignant growth of BHD renal cell carcinomas, shows that folliculin (FLCN) haploinsufficiency gives a less severe pathology than FLCN loss-of-function.

A recently published study from Bondavalli et al., (2015) was the first report of a cardiac rhabdomyoma (hamartoma) in an infant carrying a FLCN mutation. Cardiac rhabdoyomas are the most common cardiac tumour in children and can be sporadic or syndromic. Syndromic cardiac rhabdomyomas are associated with TSC and mutations in the TSC1 and TSC2 genes, however no such mutations were found in the infant.

A diagnosis of BHD in the extended family subsequently led to identification of a three base pair deletion in FLCN (c.469_471delTTC) in the infant, his mother and grandmother. Although genetic testing for BHD, typically an adult onset disease, is not always recommended for children it was deemed necessary in this case to determine if the child require further monitoring for TSC-associated phenotypes. Examination of the extended family identified a history of fibrofolliculomas but only one case of pneumothorax and one case of RCC.

Two other rhabdomyomas have been reported in BHD patients to date. A laryngeal rhabdomyoma was reported among the cohort assessed in Toro et al (2008). The second patient was diagnosed with BHD and multiple endocrinopathoes and found to have an adult rhabdomyoma in a presumed parathyroid adenoma (Mikesell et al., 2014). In addition cardiac rhabdomyomas have been reported in the Nihon rat model of BHD (n=15/125, Kouchi et al., 2009).

Cardiac rhabdomyomas in TSC patients have been linked to aberrant mTOR signalling (Kotulska et al., 2009) through the loss of regulation by the hamartin (TSC1) and tuberin (TSC2) complex. The mechanism by which FLCN regulates mTOR signalling is not fully understood and appears to be context dependent, but its dysregulation has been linked to the acknowledged BHD pathologies (Baba et al., 2006, Fuyura et al., 2012). Further research is required to determine if FLCN haploinsufficiency in muscle tissue also affects mTOR signalling and can subsequently be linked to rhabdomyoma formation in BHD patients and models.

Bondavalli et al. raise the possibility that mutations in FLCN could be the cause of cardiac rhabdomyomas and argue for BHD to be included in the differential diagnosis of these hamartomas. However they also believe that routine cardiac monitoring of children from BHD is not required. It is possible that the presence of FLCN mutations in patients with these hamartomas is more common but a limited knowledge of BHD, especially among paediatric cardiologists, is resulting in cases going unrecognised. These few reports, whilst insufficient to causatively link BHD and rhabdomyomas, could suggest an additional source of hamartomas associated with a haploinsufficiency of FLCN.


  • 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. PubMed PMID: 17028174.
  • Birt AR, Hogg GR, Dubé WJ. Hereditary multiple fibrofolliculomas with trichodiscomas and acrochordons. Arch Dermatol. 1977 Dec;113(12):1674-7. PubMed PMID: 596896.
  • Bondavalli D, White SM, Steer A, Pflaumer A, Winship I. Is cardiac rhabdomyoma a feature of Birt Hogg Dubé syndrome? Am J Med Genet A. 2015 Apr;167(4):802-4. PubMed PMID: 25655561.
  • Furuya M, Tanaka R, Koga S, Yatabe Y, Gotoda H, Takagi S, Hsu YH, Fujii T, Okada A, Kuroda N, Moritani S, Mizuno H, Nagashima Y, Nagahama K, Hiroshima K, Yoshino I, Nomura F, Aoki I, Nakatani Y. Pulmonary cysts of Birt-Hogg-Dubé syndrome: a clinicopathologic and immunohistochemical study of 9 families. Am J Surg Pathol. 2012 Apr;36(4):589-600. PubMed PMID: 22441547.
  • Kotulska K, Larysz-Brysz M, Grajkowska W, Jóźwiak J, Włodarski P, Sahin M, Lewin-Kowalik J, Domańska-Pakieła D, Jóźwiak S. Cardiac rhabdomyomas in tuberous sclerosis complex show apoptosis regulation and mTOR pathway abnormalities. Pediatr Dev Pathol. 2009 Mar-Apr;12(2):89-95. Epub 2007 Jul 30. PubMed PMID: 17990907.
  • Kouchi M, Okimoto K, Matsumoto I, Tanaka K, Yasuba M, Hino O. Natural history of the Nihon (Bhd gene mutant) rat, a novel model for human Birt-Hogg-Dubé syndrome. Virchows Arch. 2006 Apr;448(4):463-71. PubMed PMID: 16447066.
  • Luijten MN, Basten SG, Claessens T, Vernooij M, Scott CL, Janssen R, Easton JA, Kamps MA, Vreeburg M, Broers JL, van Geel M, Menko FH, Harbottle RP, Nookala RK, Tee AR, Land SC, Giles RH, Coull BJ, van Steensel MA. Birt-Hogg-Dube syndrome is a novel ciliopathy. Hum Mol Genet. 2013 Nov 1;22(21):4383-97. PubMed PMID: 23784378.
  • Mikesell KV, Kulaylat AN, Donaldson KJ, Saunders BD, Crist HS. A rare soft tissue tumor masquerading as a parathyroid adenoma in a patient with birt-hogg-dubé syndrome and multiple cervical endocrinopathies. Case Rep Pathol. 2014; 2014:753694. PubMed PMID: 25610687.
  • 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. Review. PubMed PMID: 18234728.
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Intragenic deletions in folliculin identified and mapped

The phenotypes associated with BHD – fibrofolliculomas, pulmonary cysts with an increased risk of pneumothorax, and an increased risk of renal cell carcinoma (RCC) – show variable penetrance between and within families, making diagnosis difficult when only one symptom is present. Studies have found that up to 10% of patient cohorts diagnosed with Primary Spontaneous Pneumothorax (PSP) have folliculin (FLCN) mutations (Ren et al., 2008, Johannesma et al., 2014).

Patients suspected of having BHD undergo genetic testing via DNA sequencing to detect FLCN mutations. However, for a minority patients with clinical BHD no mutation can be detected using these techniques. Large intragenic deletions and duplications have been identified in such patients (Kunogi et al., 2010, Sempau et al., 2010, Benhammou et al., 2011). New work from Ding et al., (2015) has identified and mapped three large intragenic deletions in the FLCN genes of 40 patients from nine Chinese families with a family history of PSP and lung cysts.

Ding et al., (2015) analysed 12 families in total including five families identified in Ren et al., (2008) that were found not to have small mutations detectable by DNA sequencing. The families showed reduced fibrofolliculoma penetrance and no RCC. The families were identified through a proband admitted after a spontaneous pneumothorax, so this selection bias might make the reduction in fibrofolliculomas and RCC less significant. Larger comparative studies are required to determine if there are significant difference in phenotype penetrance in different ethnic groups.‎

Ding et al., used Multiplex Ligation-Dependent Probe Amplification (MLPA) and breakpoint analysis to identify a large deletion across exons 9-14 (c.872-492_1740+1763del) in five families, a deletion across exon 14 (c.1539-536_1740+1701del) in two families and a large deletion across exons 1-3 (c.-504-1303_-25+845del) in two families. The FLCN start codon is in exon 4 therefore deletion of exons 1-3 would disrupt the promotor reducing expression (Benhammou et al., 2011). The other deletions would prematurely truncate FLCN and as several identified functions rely on the C-terminal (Baba et al., 2006) these mutations would be pathogenic.

A 5.5Mb disease haplotype was identified around the exon 9-14 deletion suggesting a founder mutation. In addition all five families shared a point mutation in exon 5. This deletion was estimated to have occurred approximately 16 generations ago. Haplotypes around the exon 14 and exon 1-3 deletions were also found, however estimating the age was not possible from two families.

To date one large intragenic duplication and 11 large intragenic deletions (Figure 1) have been identified in 20 BHD families and 2 sporadic patients. Two of the deletions identified by Ding et al., have previously been reported (Kunogi et al., 2010) but not mapped in detail. The three deletions mapped in this work and several of the other deletions have Alu repeat sequences (black arrows in Figure 1) on either side. Interestingly the FLCN gene has a high density of Alu repeats compared to the genome in general and this increases the risk of homologous recombination resulting in deletions and duplications.

This work supports the conclusion that a percentage of PSP patients are actually undiagnosed BHD patients, and that the 10% previously suggested could be an underestimation if only DNA sequencing is used to detect FLCN mutations. MLPA analysis would therefore be an important and useful additional DNA testing tool for detecting mutations in suspected BHD patients.

FLCN mutations


Figure 1: Intragenic deletions and duplications identified in FLCN 
Previously identified deletions and duplications are shown in blue and the three mutations found in Ding et al., (2015) are shown in red. Alu repeats are represented by black arrows. Taken from Ding et al., 2015, American Journal of Medical Genetics Part A 21 MAR 2015 DOI: 10.1002/ajmg.a.36979


  • 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. PubMed PMID: 17028174.
  • Benhammou JN, Vocke CD, Santani A, Schmidt LS, Baba M, Seyama K, Wu X, Korolevich S, Nathanson KL, Stolle CA, Linehan WM. Identification of intragenic deletions and duplication in the FLCN gene in Birt-Hogg-Dubé syndrome. Genes Chromosomes Cancer. 2011 Jun;50(6):466-77. PubMed PMID: 21412933.
  • Ding Y, Zhu C, Zou W, Ma D, Min H, Chen B, Ye M, Pan Y, Cao L, Wan Y, Zhang W, Meng L, Mei Y, Yang C, Chen S, Gao Q, Yi L. FLCN intragenic deletions in Chinese familial primary spontaneous pneumothorax. Am J Med Genet A. 2015 Mar 21. PubMed PMID: 25807935.
  • Johannesma PC, Reinhard R, Kon Y, Sriram JD, Smit HJ, van Moorselaar RJ, Menko FH, Postmus PE; Amsterdam BHD working group. Prevalence of Birt-Hogg-Dubé syndrome in patients with apparently primary spontaneous pneumothorax. Eur Respir J. 2015 Apr;45(4):1191-4. PubMed PMID: 25537564.
  • Kunogi M, Kurihara M, Ikegami TS, Kobayashi T, Shindo N, Kumasaka T, Gunji Y, Kikkawa M, Iwakami S, Hino O, Takahashi K, Seyama K. Clinical and genetic spectrum of Birt-Hogg-Dube syndrome patients in whom pneumothorax and/or multiple lung cysts are the presenting feature. J Med Genet. 2010 Apr;47(4):281-7. PubMed PMID: 20413710.
  • 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. PubMed PMID: 18505456.
  • Sempau L, Ruiz I, González-Morán A, Susanna X, Hansen TV. [New mutation in the Birt Hogg Dube gene]. Actas Dermosifiliogr. 2010 Sep;101(7):637-40. Spanish. PubMed PMID: 20858390.
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Recent developments in treatments for kidney cancer

There are 338,000 new cases of kidney cancer per year worldwide, and the incidence has increased in recent years (Ferley et al., 2012). Although the majority of Renal Cell Carcinoma (RCC) cases are sporadic and affect those over 50 years old, 2-3% of cases are caused by inherited conditions such as BHD, VHL, HLRCC and TSC and are associated with an earlier onset (Randall et al., 2014). These inherited forms of RCC have provided great insights into the genetics of sporadic cancer – for example 75% of RCC cases are associated with mutations in the VHL gene.

The majority of small local RCC tumours can be surgically removal. However these treatments are not without risk and complete nephrectomies leave patients with severely reduced kidney function. The development of selective drug treatments that target only cancerous cells could therefore increase patient quality of life.

Current treatments for advanced or metastasised RCC are focused on counteracting the metabolic and angiogenic changes associated with the VHL/HIF pathway: Tyrosine Kinase Inhibitors (TKI) sunitinib, sorafenib and axitinib inhibit VEGF and PDGF signalling, increased as a result of aberrant HIF signalling, to limit angiogenesis; and the mTOR inhibitor everolimus, and derivatives, reduce mTOR and downstream HIF signalling to minimise tumour growth and the metabolic shift from oxidative phosphorylation to glycolysis. Despite these advances only half of kidney cancer patients are currently expected to survive past 10 years (Chow et al., 2010), so more advanced and effective treatments are sorely required.

This week’s blog is a brief review of several recent reports on the development of new treatments for RCC and updates on current treatments.

  • Englerin A, a purified molecule from Phyllanthus engleri bark, selectively kills cancer cells (Ratnayake et al., 2009) by increasing intracellular calcium levels (Sulzmaier et al., (2012). Akbulut et al., (2015) now report that activation of the Transient Receptor Potential cation channels TRPC4 and TRPC5 induces calcium influx resulting in rapid death of tumourigenic cells, thereby identifying novel drug targets for the selectively treatment of cancerous kidney cells.
  • Hall et al., (2014) reported that TRPM3 cation channel activation promotes clear cell RCC tumour growth by activating autophagy. Increased intracellular calcium levels remove miR-214-inhibition of autophagy, however this pathway can be blocked by mefenamic acid (MFA). The identified drug targets could form the basis of an effective combination treatment targeting the upregulation of autophagy and tumourigenic angiogenesis.
  • Additional targets identified through genetic and biochemical studies of tumours and tumour cell lines include HIF2α, chromatin regulators SETD2, BAP1 and PBRM, MET in papillary type I tumours, and inhibitors that can limit glucose uptake in tumours that have switched to aerobic glycolysis as the main source of energy (reviewed in Srinivasan et al., 2015).

In addition to this new research, preliminary data from several clinical trials has recently been released.

  • A trial assessing a combination of dalantercept and axitinib for treatment of advanced RCC reported encouraging results. In part 1 of the phase II trial dalantercept, and ALK-1 inhibitor, in combination with the multiple TKI axitinib, showed a partial response and prolonged disease control in patients. A larger phase II part 2 trial will assess if the combination therapy enhances progression free survival more than treatment with axitinib alone.
  • The ASSURE clinical trial reported that adjuvant use of sorafenib and sunitinib did not significant improve disease free survival or overall survival, nor reduction time to disease recurrence compared to a placebo in patients following surgical tumour removal. Due to significant side effects both drugs were individually titrated to reduce the high discontinuation rate.
  • Phase II trial results results show lenvatinib, a multi-TKI, alone or in combination with everolimus, prolongs progression free survival significantly more than everolimus treatment alone in patients with advanced or metastatic RCC. The combination therapy inhibits multiple tumourigenic signalling pathways simultaneously reducing the risk of developed drug resistance.
  • A combinatorial trial of Bevacizumab and Erlotinib in HLRCC and sporadic papillary RCC patients reported a 65% and 29% response rate in HLRCC patients and sporadic patients respectively, with the majority of tumours shrinking or remaining stable and extended survival. The enhanced need of these papillary tumours for high levels of glucose makes them particularly susceptible to treatments that impair glucose delivery.

As RCC is really a collection of distinct diseases that occur in the same organ, it is unlikely that any one single therapy will be suitable for all patients. Instead through increased understanding of the tumourigenic pathways and processes in different patient cohorts more personalised targeted therapies (as discussion in this blog post) can be developed.


  • Akbulut Y, Gaunt HJ, Muraki K, Ludlow MJ, Amer MS, Bruns A, Vasudev NS, Radtke L, Willot M, Hahn S, Seitz T, Ziegler S, Christmann M, Beech DJ, Waldmann H. (-)-Englerin A is a Potent and Selective Activator of TRPC4 and TRPC5 Calcium Channels. Angew Chem Int Ed Engl. 2015 Mar 16;54(12):3787-91. PubMed PMID: 25707820.
  • Chow WH, Dong LM, Devesa SS. Epidemiology and risk factors for kidney cancer. Nat Rev Urol. 2010 May;7(5):245-57. Review. PubMed PMID: 20448658.
  • Ferlay J, Soerjomataram I, Ervik M, et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2013. Available from:, accessed March 2015.
  • Hall DP, Cost NG, Hegde S, Kellner E, Mikhaylova O, Stratton Y, Ehmer B, Abplanalp WA, Pandey R, Biesiada J, Harteneck C, Plas DR, Meller J, Czyzyk-Krzeska MF. TRPM3 and miR-204 establish a regulatory circuit that controls oncogenic autophagy in clear cell renal cell carcinoma. Cancer Cell. 2014 Nov 10;26(5):738-53. PubMed PMID: 25517751.
  • 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
  • Ratnayake R, Covell D, Ransom TT, Gustafson KR, Beutler JA. Englerin A, a selective inhibitor of renal cancer cell growth, from Phyllanthus engleri. Org Lett. 2009 Jan 1;11(1):57-60. PubMed PMID: 19061394.
  • Srinivasan R, Ricketts CJ, Sourbier C, Linehan WM. New strategies in renal cell carcinoma: targeting the genetic and metabolic basis of disease. Clin Cancer Res. 2015 Jan 1;21(1):10-7. PubMed PMID:25564569.
  • Sulzmaier FJ, Li Z, Nakashige ML, Fash DM, Chain WJ, Ramos JW. Englerin a selectively induces necrosis in human renal cancer cells. PLoS One. 2012;7(10):e48032. PubMed PMID: 23144724.
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Differential effects of HIF-α isoforms on apoptosis in renal carcinoma cell lines

Under hypoxic conditions the activation of HIF transcription factors enables cells to alter their metabolism and avoid stress-induced apoptosis. Aberrant HIF activity in the inherited renal cancers BHD, VHL, HLRCC and TSC, is linked to the expression of growth and pro-angiogenic factors that are important in tumour growth. A new report from Doonachar et al., (2015) focuses on the differential effects of the HIF-1α and HIF-2α isoforms on stress-induced apoptosis in two VHL-deficient renal cell carcinoma (RCC) cell lines.

Under normoxic conditions VHL protein forms part of the E3 ligase complex that targets HIF for degradation thereby limiting the production of HIF-target genes; HIF-1α primarily upregulates glycolytic genes and HIF-2α upregulates angiogenic and growth factors. Homozygous loss-of-function mutations in VHL, both inherited and sporadic, in renal cells results in an increase in either HIF-1α and HIF-2α activity or only HIF-2α activity, and subsequent tumour growth. HIF-2α is therefore seen as a major driver of renal tumourigenesis following the loss of a tumour suppressor such as VHL (Kondo et al., 2003).

VHL has also been shown to help protect renal cells from apoptosis following a range of stresses (Schoenfeld et al., 2000). Doonachar et al., aimed to determine any differential effects of HIFα isoform on apoptosis in VHL-defective cells following cellular stress.

In the VHL-deficient 786-O cell line the transgenic expression of mutant VHL, compared to transgenic wild type VHL, induced expression of HIF-2α (the only isoform this line expresses) and apoptosis following UV exposure. Inhibiting HIF-2α translation with shRNAs, confirmed through reduced target gene GLUT1 expression, marginally reduced apoptosis suggesting a partial protection from UV-induced apoptosis. The loss of HIF-2α in this cell line did alter the rate of cell death induced by glucose starvation or serum withdrawal. Under all stress conditions the expression of functional VHL drastically reduced 786-O cell death confirming it as anti-apoptosis factor.

In a second kidney tumour cell line, RCC10, that expresses both HIFα isoforms, the presence of only mutated VHL resulted in increased expression of HIF-1α and HIF-2α and a greater rate of apoptosis following UV exposure. Inhibiting transcription of either HIF-1α or HIF-2α via shRNA reduced respective protein levels but did not impact on GLUT1 expression suggesting transcriptional redundancy. shRNA-inhibition of HIF-1α increased cell death following UV exposure and glucose starvation suggesting that HIF-1α has an anti-apoptotic role. In contrast shRNA-inhibition of HIF-2α reduced cell death following UV exposure and serum withdrawal, indicative of a pro-apoptotic role. However, as there was also a, potentially compensatory, increase in HIF-1α expression following HIF-2α shRNA-inhibition, it could also be attributed to an increase in HIF-1α anti-apoptotic activity. There was no comparative increase in HIF-2α expression detected following shRNA-inhibition of HIF-1α.

Doonachar et al., conclude that HIFα isoforms seem to have different roles following cellular stress; HIF-1α is generally anti-apoptotic and HIF-2α pro-apoptotic. Further work is needed to determine if differences in isoform function are also relevant in tumour formation and if they could be exploited in treatments.

In contrast to VHL studies, Flcn-null cell lines show no increase in HIF-1α or HIF-2α expression despite a marked increase in activity determined by increased expression of target genes (Preston et al., 2010, Yan et al., 2014). The increase in HIF activity reported in BHD RCCs is thought to be mediated through AMPK and PGC1α activity. Yan et al., propose that FLCN loss increases AMPK and PCG1α activity leading to increased mitochondrial biogenesis, increased ROS production and subsequent activation of HIF signalling. Mutations in FLCN have also been linked to aberrant mTOR activity (Baba et al., 2006), an upstream activator of HIF signalling in tumour cells (Hudson et al., 2002). It is possible that these mechanisms, alongside increased HIF protein stability resulting from reduced VHL-dependent ubiquitination and degradation, drive enhanced HIF signalling in VHL-RCCs.

Increased HIF activity has also been reported in BHD pulmonary cyst epithelial cells. Nishii et al., (2013) detected a mild increase in HIF-1α and VEGF levels, and increased angiogenesis in BHD-lung cysts but not control tissues. As there is no evidence of second hit mutations in lung tissue any changes are more likely due to haploinsufficiency that a complete loss of function. The resulting milder changes in HIF activity could explain the lack of tumourigenic cell proliferation in pulmonary cysts.

Aberrant HIF activity in renal tumours suggests an important role in tumourigenesis. Increased understanding of the expression and targets of HIF-α isoforms under normal and renal tumourigenic conditions will enable identification of new drug targets. In addition, as HIF signalling appears to play a role in other aspects of BHD pathology, such treatments could be applicable to these systems.


  • 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 PubMed PMID: 17028174.
  • Doonachar A, Gallo MD, Doukas D, Pasricha R, Lantsberg I, Schoenfeld AR. Differential effects of HIF-α isoforms on apoptosis in renal carcinoma cell lines. Cancer Cell Int. 2015 Feb 22;15:23. doi: 10.1186/s12935-015-0175-3. eCollection 2015. PubMed PMID: 25729330.
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  • Kondo K, Kim WY, Lechpammer M, Kaelin WG Jr. Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth. PLoS Biol. 2003 Dec;1(3):E83. PubMed PMID: 14691554.
  • Nishii T, Tanabe M, Tanaka R, Matsuzawa T, Okudela K, Nozawa A, Nakatani Y, Furuya M. Unique mutation, accelerated mTOR signaling and angiogenesis in the pulmonary cysts of Birt-Hogg-Dubé syndrome. Pathol Int. 2013 Jan;63(1):45-55. PubMed PMID: 23356225.
  • 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 PubMed PMID: 21057536.
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  • 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;124(6):2640-50. PubMed PMID: 24762438.
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Drug Repositioning for Rare Diseases

A recent Findacure meeting focused on the importance and progression of drug repositioning in rare diseases. Dr Bruce Bloom, Dr Mike Briggs and Dr Farid Khan, discussed ongoing repositioning work, methods to identify new drugs and targets and the need for collaborations to drive new research and trials. Cures Within Reach are launching a new interactive platform CureAccelerator to help form such collaborations. The session’s presentations are available here.

There are over 6000 rare diseases1 recognised worldwide, and as the development of a new drug is thought to take between 10-15 years2 and cost approximately $1.2 billion USD3, the limited market appeal of rare disease or orphan drugs reduces investment incentive.  The Orphan Drug Act of 1983 was passed in the US to increase incentive and, to date, 486 orphan drugs have been approved4.  The remaining 90-95% of rare diseases do not have any specific treatments.

An alternative approach is to reposition (or repurpose) some of the 4000 existing drugs approved for human use worldwide (Huang et al., 2011). Existing early phase safety and efficacy data for these drugs means repositioning studies can be cheaper, safer and faster than conventional drug development.

BHD is a rare disease for which no preventative treatments are available and lists only three trials for BHD. Two long running studies at the National Cancer Institute are looking at the clinical, genetic and molecular basis of BHD and all heritable urologic disorders, and a third, funded by the Myrovlytis Trust, assessed the use of topical rapamycin to treat fibrofolliculomas (discussed here).

Rapamycin, an mTOR inhibitor, is a repositioned drug: first licenced as an immunosuppressant it is now used as a treatment for a range of diseases including cancers including advanced kidney cancer in BHD patients. The role of mTOR activity in fibrofolliculoma pathology is less understood. This could explain why the clinical trial did not find evidence of topical rapamycin as an effective treatment for BHD fibrofolliculomas (Gijezan et al., 2014).

FLCN loss also disrupts other pathways including HIF signalling; increased HIF signalling is seen in FLCN-null cell lines (Preston et al., 2011) as a result of altered AMPK activity, and could be a driving force in the development of renal tumours. HIF-1 hyperactivity is also seen in sporadic renal tumours which lead to the development of HIF-1 inhibitors (Onnis et al., 2009, Welsh et al., 2013). If BHD pathologies are linked to aberrant HIF-1 activity these drugs may be able to provide the basis for new treatments.

A drug screen, funded by the Myrovlytis Trust, using a BHD-kidney cell line (Yang et al., 2008) identified Mithramycin, an antineoplastic antibiotic, as selectively toxic to FLCN null cells (Lu et al., 2011). Used to treat testicular cancer, leukaemia and Paget’s disease, Mithramycin could potentially, after further research, be a viable drug for repositioning trials in BHD.

The pathology of BHD fibrofolliculomas and pulmonary cysts is less well understood than FLCN-associated renal tumours. This potentially limits the development of new treatments based on aberrant biological pathways or activity. Further research into the mechanisms through which a reduction in FLCN leads to these phenotypes will hopefully enable new drug targets to be identified.

Drug screens, incidental findings, bioscience research and computational biology can all provide insights into potential new treatments for specific diseases or pathways. Greater understanding of the pathways affected in BHD and their roles in pathology will enable researchers to identify more likely drug targets. Repositioning of known drugs could then prove useful in discovering safe and effective treatments for BHD and other rare disease patients in a shorter time span than conventional methods.

  • Gijezen LM, Vernooij M, Martens H, Oduber CE, Henquet CJ, Starink TM, Prins MH, Menko FH, Nelemans PJ, van Steensel MA. Topical rapamycin as a treatment for fibrofolliculomas in Birt-Hogg-Dubé syndrome: a double-blind placebo-controlled randomized split-face trial. PLoS One. 2014 Jun 9;9(6):e99071. PubMed PMID: 24910976.
  • Huang, R., Southall, N., Wang, Y., Yasgar, A., Shinn, P., Jadhav, A., … Austin, C. P. (2011). The NCGC Pharmaceutical Collection: A comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics.Science Translational Medicine3(80). PMID: 21525397
  • Lu X, Wei W, Fenton J, Nahorski MS, Rabai E, Reiman A, Seabra L, Nagy Z, Latif F, Maher ER. Therapeutic targeting the loss of the birt-hogg-dube suppressor gene. Mol Cancer Ther. 2011 Jan;10(1):80-9. PubMed PMID: 21220493.
  • Onnis B, Rapisarda A, Melillo G. Development of HIF-1 inhibitors for cancer therapy. J Cell Mol Med. 2009 Sep;13(9A):2780-6. PubMed PMID: 19674190.
  • 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. PubMed PMID:21057536.
  • Welsh SJ, Dale AG, Lombardo CM, Valentine H, de la Fuente M, Schatzlein A, Neidle S. Inhibition of the hypoxia-inducible factor pathway by a G-quadruplex binding small molecule. Sci Rep. 2013 Sep 30;3:2799. PubMed PMID: 24165797.
  • Yang Y, Padilla-Nash HM, Vira MA, Abu-Asab MS, Val D, Worrell R, Tsokos M, Merino MJ, Pavlovich CP, Ried T, Linehan WM, Vocke CD. The UOK 257 cell line: a novel model for studies of the human Birt-Hogg-Dubé gene pathway. Cancer Genet Cytogenet. 2008 Jan 15;180(2):100-9. PubMed PMID: 18206534.


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Distinctive expression patterns of FLCN and GPNMB in BHD renal tumours

As discussed on this blog previously, developing histological screening techniques for renal cell carcinomas (RCCs) associated with BHD is important for early diagnosis. Individuals with folliculin (FLCN) mutations are more likely to develop multiple bilateral renal tumours (Zbar et al., 2002, Pavlovich et al., 2002). A misdiagnosis of sporadic RCC may compromise future treatment and wellbeing of the patient and other affected family members. Currently there are no known histological markers to distinguish between all subtypes of sporadic and FLCN-associated tumours.

A new report by Furuya et al., (2015) addressed this by analysed expression of FLCN and one of its downstream targets GlycoProtein Non-Metatastic B (GPNMB) in normal and neoplastic tissue to determine if they could be used to aid differential diagnosis in RCC samples. FLCN is expressed in normal kidney tissue, including in those who carry heterozygous FLCN mutations, but is not detectable in BHD tumours (Warren et al., 2004). In comparison GPNMB is not typically expressed in kidney tissue but is expressed in a range of neoplastic tissues (Hong et al. 2010).

Furuya et al. analysed 27 tumours from 18 unrelated, except for a parent and child pair, Japanese BHD patients: 12 chromophobe RCCs; six hybrid oncocytoma/chromophobe tumours (HOCTs); three papillary RCCs; and two clear cell RCCs (ccRCC). For seven of these tumours sections of non-neoplastic kidney were also frozen for comparison. Expression of FLCN and GPNMB in the BHD-associated tumours was compared to 62 sporadic renal tumours from an unreported number of patients. All patients were medically examined for the classical BHD symptoms – pulmonary cysts, pneumothorax, fibrofolliculomas and multifocal/hybrid RCC – and a family history obtained. One of the sporadic group also had pulmonary cysts so a diagnosis of BHD was ruled out by genetic testing. The rest of this group did not show any BHD manifestations or have any family history so were assumed to not carry FLCN mutations.

FLCN expression in the tumour samples was assessed by western blot and immunohistochemistry using a monoclonal (D14G9) and previously unpublished polyclonal (ab93196) antibody respectively. The western blot results showed a clear loss of FLCN in BHD-associated but not sporadic tumours or non-neoplastic BHD tissue. In addition the typical strong nuclear staining for FLCN was seen in the majority of sporadic tumours whereas the majority of BHD-associated tumours showed only cytoplasmic or no staining. It is suggested that the presence of cytoplasmic staining should be considered a potential BHD indicator.

The presence of GPNMB protein was confirmed by western blot and immunohistochemistry, but mRNA levels were also quantified by quantitative RT-PCR. The markedly higher expression (up to 23-fold) detected in BHD tumours by qRT-PCR correlated with the intensity of the western band seen. GPNMB was barely detectable in the sporadic tumour samples and non-neoplastic BHD renal tissue. Immunostaining showed positive staining in BHD-associated tumours excluding the ccRCC samples and one papillary sample. The majority of sporadic samples were in contrast negative with the exception of 50% of sporadic chromophobe RCC samples which showed weak GPNMB staining.

It was also possible to detect low expression of GPNMB in small nodules of “non-neoplastic” BHD kidney tissue which could indicate the sites of future tumour development. As the formation of multiple small tumours is characteristic of BHD this could also help in differential diagnosis.

The germline FLCN mutations for each patient was determined from a peripheral blood sample; the most common (50%) mutation was duplication in the hypermutable cytosine tract in exon 11 and four patients had the same GATG deletion in exon 13. In addition Furuya et al., attempted to detect secondary somatic mutations in a number of the tumour samples. A second mutation was found in six individual tumours and a loss of heterozygosity in a further two. Only one of these secondary mutations has so far been reported as a germline mutation in a BHD patient. The other mutations may therefore not be pathogenic but as all would result in a frameshift it is more likely they are new unique mutations.

The results from this work suggest that staining for both a lack of FLCN and a gain of GPNMB could help to distinguish sporadic oncocytomas, chromophobe and papillary RCC from BHD-associated HOCTS, chromophobe and papillary RCCs. It would not be sufficient however to definitively classify a ccRCC as FLCN-related, but a lack of nuclear FLCN staining could identify cases appropriate for genetic testing.

  • Furuya M, Hong SB, Tanaka R, Kuroda N, Nagashima Y, Nagahama K, Suyama T, Yao M, & Nakatani Y (2015). Distinctive expression patterns of glycoprotein non-metastatic B and folliculin in renal tumors in patients with Birt-Hogg-Dubé syndrome. Cancer science PMID: 25594584
  • 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. doi:10.1371/journal.pone.0015793. PMID: 21209915.
  • 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.
  • Warren MB, Torres-Cabala CA, Turner ML, Merino MJ, Matrosova VY, Nickerson ML, Ma W, Linehan WM, Zbar B, Schmidt LS. Expression of Birt-Hogg-Dubé gene mRNA in normal and neoplastic human tissues. Mod Pathol. 2004 Aug;17(8):998-1011. PMID: 15143337.
  • 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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