People with Birt-Hogg-Dubé syndrome (BHD) have a 25% chance of developing at least one spontaneous pneumothorax (collapsed lung) in their lifetime and many people will have several. A pneumothorax occurs when air enters the lining of the lung (known as the pleural cavity) causing the lung to be compressed. The size of the pneumothorax usually determines the clinical presentation and required intervention ranging from a conservative watch and wait approach to surgery. Pneumothoraces in BHD patients are primarily thought to be caused by the lung cysts (which are seen in 80% of BHD patients) bursting. However, it has been suggested that this is not the only cause (1).  Okamoto et al., conducted the first study assessing the effect of folliculin haploinsufficiency (one mutated folliculin gene) on pleural mesothelial cells (PMCs) (2). PMCs are a single layer of cells that blanket the lung and chest wall acting as a protective barrier. If this layer is disrupted it can result in a pneumothorax.  Studying these cells increases our understanding of how BHD causes pneumothoraces and potential drug targets to prevent them.    

Okamoto et al., collected PMCs from 32 patients who developed spontaneous pneumothoraces: 12 BHD patients and 20 other patients who had no underlying lung conditions (control group). The purpose was to compare the cells in both cohorts to see if there were any differences. The cells were examined under a microscope and they found that PMCs in BHD patients were cuboidal in shape compared with the cobblestone appearance of the control group.

Next, Okamoto et al., looked at the expression of genes involved in cell adhesion (binding of cells together). The expression of E-cadherin, which is involved in cell adhesion was reduced in BHD PMCs. To investigate the effects of reduced E-cadherin on BHD PMCs they assessed cell adhesion in Petri dishes. A significantly greater number of cells detached in the BHD PMCs compared to the control and then underwent anoikis (programmed cell death due to loss of attachment to other cells). In addition to reduced cell adhesion and anoikis, Okamoto et al., demonstrated delayed cell growth and reduced migration in BHD PMCs compared with the control group.  PMCs are involved in maintaining the health of the lining of the lung and repairing any disruption, so need to be able to move and grow. Altogether this suggests that folliculin haploinsufficiency causes PMCs to be disrupted through loss of adhesion and cell death and the repair process hindered resulting in pneumothoraces.  Okamoto et al., suggest these changes to PMCs function may be due to reduced activation of the E-Cadherin-LKb1- AMPK pathway which is a known pathway affected by folliculin loss (3).

Altogether Okamoto et al, found that folliculin haploinsufficiency causes changes in the shape of PMCs in BHD patients and impairs the function of the cells, potentially through the E-Cadherin-LKb1- AMPK pathway. This is important as the disruption of PMCs may be a cause of pneumothoraces in BHD. Further research is required to determine how exactly PMC disruption causes pneumothoraces and whether the pathways affected could be targeted to prevent them.

References

1.            Ohata M, Suzuki H. Pathogenesis of spontaneous pneumothorax. With special reference to the ultrastructure of emphysematous bullae. Chest [Internet]. 1980 Jun 1 [cited 2021 Jun 8];77(6):771–6. Available from: http://journal.chestnet.org/article/S0012369215456168/fulltext

2.            Okamoto S, Ebana H, Kurihara M, Mitani K, Kobayashi E, Hayashi T, et al. Folliculin haploinsufficiency causes cellular dysfunction of pleural mesothelial cells. Sci Rep [Internet]. 2021 Dec 24 [cited 2021 Jun 8];11(1):10814. Available from: http://www.nature.com/articles/s41598-021-90184-9

3.            Goncharova EA, Goncharov DA, James ML, Atochina-Vasserman EN, Stepanova V, Hong SB, et al. Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival through E-Cadherin, LKB1, and AMPK. Cell Rep [Internet]. 2014 Apr 24 [cited 2021 Jun 8];7(2):412–23. Available from: https://pubmed.ncbi.nlm.nih.gov/24726356/

This month’s blog posts are focusing on Birt-Hogg-Dubé Syndrome (BHD) and the lung, in line with our Meet the Expert session happening later this month (see here for more details and to sign up for the event). Here, we present 2 recent studies focusing on diagnosing BHD from CT imaging of the lungs, and 1 case report of a woman presenting with a symptom not normally associated with BHD.

1: CT Findings of Pulmonary Cysts
Arango-Díaz et al.,(1) reviewed the CT imaging findings of diffuse cystic lung diseases (DCLD) to provide a practical approach for the evaluation of lung cysts. DCLD are a heterogeneous group of disorders, including BHD, that can be diagnosed based on CT. However, recognising the specific underlying cause of cysts can be challenging as many of these diseases have similar features. CT imaging enables definition of the cysts, including their relationship with the bronchovascular structure and distribution of the cysts within the lung which can be informative for diagnosis. The most common diagnoses are lymphangioleiomyomatosis (LAM) and Langerhans cell histiocytosis (LCH). BHD, Lymphocytic interstitial pneumonia (LIP), and the fungal pathogen Pneumocystis jiroveci can also be the cause of lung cysts and are described in this review. Arango-Díaz et al., summarise the main CT findings of lung cysts and identify features, or combination of features, that are unique to each disorder. They state the importance of taking into account the whole picture to get a true diagnosis, including other physical symptoms (e.g. the fibrofolliculomas and renal tumours associated with BHD), other factors (e.g. smoking, a major cause of LCH), and the distribution of the cysts within the lung. This is particularly crucial to the diagnosis of BHD, as the size and wall thickness of the cyst alone is not sufficient for diagnosis. Cysts in patients with BHD are found to affect the basal and paramediastinal regions of both lungs, a feature that is unique within DCLD.

2: Quantitative Analysis of Cystic Lung Diseases by Use of Paired Inspiratory and Expiratory CT
As discussed above, differential diagnosis of the underlying cause of cystic lung diseases can be done based on CT imaging alone, however, this can be very challenging when the number of cysts is relatively small. Furthermore, early diagnosis of the correct disorder is critical for ensuring that appropriate care is given on time.  The authors proposed to evaluate the ‘dynamic properties’ of cysts during respiration as another diagnostic tool and have devised an index called the cyst-airway communicating index (CACI)(2). The CACI takes into consideration the total lung volume (TLV) and the low-attenuation area volume (LAAV, areas that appear less intense on CT which are assumed cysts) at both inspiration and expiration. 71 patients (15 with BHD, 43 with LAM, and 13 ‘other’) were examined. No difference in the TLV was observed between the different groups, however the difference in LAAV between inspiration and expiration was statistically smaller for BHD compared to LAM or the other group. The CACI was also statistically smaller for BHD compared to LAM/other suggesting there is less communication between the cysts and the airway in BHD. There are limitations to this study which the authors discussed, including the small study size, particularly for the BHD and other groups, and the lack of pathological confirmation that the LAAV truly represented cysts, a major caveat of the study. The CACI may also not be applicable to patients with a large number of cysts, however it does appear to be a useful tool for differentiating BHD from other cystic lung diseases with mild pulmonary symptoms.

3: Birt-Hogg-Dubé Syndrome presenting with chronic progressive dyspnea
This case report describes a 42-year-old female with no history of smoking and a past medical history of asthma, sleep obstructive apnea, and gastroesophageal reflux disease(3). She reported wheezing and dyspnea (shortness of breath) and CT revealed DCLD for which a presumptive diagnosis of bullous emphysema was given. However, her age and lack of smoking prompted further investigation. A physical exam revealed numerous skin-coloured bumps on the face and neck which were later confirmed to be fibrofolliculomas. High-resolution CT imaging of the lungs showed many cysts predominantly in the lower lung. Upon genetic testing, a truncation mutation of folliculin was discovered and a diagnosis of BHD was given. Despite an optimised treatment regime for asthma, the patient continued to experience dyspnea and as such a decision to start continuous positive airway pressure (CPAP) was made.

In the context of BHD, this can be a difficult decision as there is a slightly increased risk of pneumothorax with CPAP treatment, however the benefits outweighed the risks in this circumstance and the patient was given detailed precautions regarding the symptoms of pneumothorax. There are currently no guidelines on the risk of pneumothorax with CPAP therapy in BHD and further research is required to address this issue. The authors also state the importance of considering BHD as a differential diagnosis for DCLD as BHD is not normally associated with significant pulmonary dysfunction, however the early diagnosis of BHD is very beneficial for the screening and early detection of renal tumours.

References

1. Arango-Dí­az A, Martí­nez-de-Alegrí­a-Alonso A, Baleato-González S, García-Figueiras R, Ecenarro-Montiel A, Trujillo-Ariza M V., et al. CT findings of pulmonary cysts. Clinical Radiology. 2021
This paper isn’t freely available but please contact us if you have any questions.
2. Suzuki K, Seyama K, Ebana H, Kumasaka T, Kuwatsuru R. Quantitative Analysis of Cystic Lung Diseases by Use of Paired Inspiratory and Expiratory CT: Estimation of the Extent of Cyst-Airway Communication and Evaluation of Diagnostic Utility. Radiol Cardiothorac Imaging. 2020
Full paper here
3. Reilly D, Pourzand L, Chima-Melton C. Birt-Hogg-Dubé Syndrome presenting with chronic progressive dyspnea. Respir Med Case Reports. 2021
Full paper here

Guest Blog by Neil Pearson

Birt-Hogg-Dubé syndrome (also known as BHD) is a dominant hereditary condition caused by variants (mutations) in the Folliculin (FLCN) gene that results in a loss of function causing skin lesions, lung cysts, collapsed lungs, and kidney cancer. As researchers uncover how FLCN achieves these functions we will understand, and hopefully find ways to treat, BHD.

A collaborative project involving researchers in the USA, Japan and Singapore showed there was an increase in activated phagocytes in FLCN mutant mice that mimic human BHD(1). Phagocytes are cells in our bodies that “eat” substances like bacteria, damaged or infected cells and protect the body from infection and damage. Phagocytes contain structures called lysosomes which store acids and chemicals that destroy the substances “eaten” by the macrophage. Lysosomes are also important in sensing and responding to low nutrient and energy levels in the cell by recycling material inside the cells for energy and repair. Loss of FLCN results in increased lysosome function, glycogen accumulating in the cytoplasm and phagocytes no longer functioning properly. The accumulation of glycogen has also been observed in cancer cells taken from the kidneys of BHD patients suggesting a potential link between accumulation of glycogen in phagocytes and cancer in BHD.

How this causes damage to organs and contributes to cancer in BHD is not yet known but there are two likely possibilities. One is that glycogen is required for generating fats and oils, too much of which can damage cells and tissues. The other possibility is that cells which can survive in the absence of accurate nutrient sensing are more likely to become cancerous. Both scenarios could potentially explain the high frequency of kidney cancer in BHD patients. More research is required to investigate these possible scenarios, but it could be an important development in understanding FLCN function and BHD.

Another exciting finding in this paper was that deleting TFE3 in FLCN deficient mice reduced the accumulation of glycogen in phagocytes. TFE3 is a transcription factor meaning it controls the expression of other genes. FLCN is known to prevent TFE3 from entering the nucleus where it controls expression(2). The researchers showed that TFE3 increases the expression of two genes that are involved in making glycogen, Gys1 and Gyg. TFE3, Gys1 and Gyg are potential drug targets for treating BHD as reducing their amount or activity could have therapeutic benefits. Although there needs to be further confirmation that the observed lysosomal effects are actively contributing to BHD symptoms in patients and mouse models, these initial findings are very promising and provide more evidence that FLCN is a key player in regulating nutrient sensing and lysosomal function.

References

1.       Endoh M, Baba M, Endoh T, Schmidt LS, Linehan WM, Suda T. A FLCN-TFE3 Feedback Loop Prevents Excessive Glycogenesis and Phagocyte Activation by Regulating Lysosome Activity. CellReports [Internet]. 2020 [cited 2021 May 27];30:1823-1834.e5. Available from: https://doi.org/10.1016/j.celrep.2020.01.042

2.       Hong SB, Oh H Bin, 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 [Internet]. 2010 [cited 2021 May 27];5(12). Available from: https://pubmed.ncbi.nlm.nih.gov/21209915/
Read our blog to find out more about FLCN and TFE3

The prevalence of Birt-Hogg-Dubé (BHD) is currently unknown and is particularly difficult to ascertain considering the wide range and variability of symptoms associated with BHD, even within the same family. Furthermore, the only definitive way of diagnosing BHD is through genetic testing for folliculin mutations, therefore many people go undiagnosed. Around one in four people with BHD experience one or more spontaneous pneumothorax (SP) in their life and it is thought that BHD is the cause of 5-10% of SP. To determine the prevalence of BHD, researchers from the University of Lausanne conducted a meta-analysis of previously published studies on the prevalence of SP among BHD patients and the general population using a statistical method called Bayes equation¹. To estimate the prevalence of BHD they calculated the following 3 components:

Firstly, Muller et al., determined the prevalence of BHD in apparent primary SP (PSP) to be 9%. The calculation of the second component, prevalence of PSP in the general population, was a little more complicated as this factor is not directly measurable. Instead, they used a formula to estimate the prevalence based on the incidence and average duration of PSP. The overall incidence rate of PSP was 8.69 per 100,000 person-years. From this, Muller et al., determined the overall prevalence of PSP in the general population (using all available data and a duration of 30 days) as 0.77/100,000 people. Finally, the third component identifying the prevalence of PSP in BHD individuals, was determined to be 43%.

Combining the above components in the Bayes equation, and assuming the most accurate data came from studies post-2000, they estimated the prevalence of BHD to be around 2 cases per million people, with no difference in gender.

This is the first study of its kind to determine the prevalence of BHD, a statistic the field would greatly benefit from. However, this study is not without caveats and limitations, some of which the authors addressed in their discussion. There are only four articles in the literature, with a total of 827 cases of PSP, where BHD has been identified.  However, this does suggest that BHD isn’t rare in PSP and that lung abnormalities and a genetic cause in PSP should be investigated, especially in cases where there is a family history.

It is also important to note that the prevalence of BHD estimated here is based entirely on a single symptom of BHD, pneumothorax. This is a major caveat of this study as not every BHD patient experiences a pneumothorax.

Although this study does provide useful information on the prevalence of BHD based on pneumothorax, the limitations and caveats in this study, combined with the wide range of symptoms an individual with BHD can present with implies that prevalence of BHD will be higher than the figure estimated here. Further investigation into the prevalence of BHD that would encompass all patients with BHD, such as genetic screening for mutations in folliculin, is warranted and would be of great interest to researchers, clinicians and patients.

References

1.        Muller, M.-E., Daccord, C., Taffé, P. & Lazor, R. Prevalence of Birt-Hogg-Dubé Syndrome Determined Through Epidemiological Data on Spontaneous Pneumothorax and Bayes Theorem. Front. Med. 8, (2021).

The symptoms of Birt-Hogg-Dubé syndrome (BHD) have been widely described in Europe and the United States (USA) with approximately 90% of individuals developing skin lesions, 30% developing kidney cancer, 80% developing lung cysts and 25% developing pneumothoraces. However, studies looking at BHD in the East Asian population suggest a difference in prevalence of these characteristics. Guo et al., published a study to provide further information about the clinical characteristics of BHD in the East Asian population to aid diagnosis and ensure early intervention (1).

Guo et al., reviewed the current literature on BHD in Japan, China and Korea and enrolled 166 patients from these papers for their study. In addition, they gathered data from 10 BHD patients from Xiangya Hospital in China. The patients’ age, sex, BHD symptoms, genetics and family history were recorded and analysed. They found that skin lesions were less common in the East Asian BHD population with only 36.7% patients recorded to have them, though the most common lesions were fibrofolliculomas and trichodiscomas as seen in the European/USA population. Prevalence of kidney cancer were also lower with 7.2% of patents developing kidney cancer. However, there were more incidences of lung cysts (87.3%) and pneumothoraces (74.7%) in the East Asian BHD population.

The reason for these differences in characteristics is not clear. Interestingly Guo et al., found no significant difference in folliculin mutations found in the East Asian BHD population compared with other countries.  This supports research by Schmidt et al., which found no link between folliculin mutations and BHD characteristics and suggests difference in characteristics may be the result of other genetic or environmental factors(2). Guo et al., also suggest that the reduced incidences of skin lesions could be due to misdiagnosis, highlighting that there are differences in medical habits in different countries which could contribute to the difference in BHD characteristics being diagnosed.

Altogether this study suggests BHD will present differently in the East Asian population, with the biggest difference seen in prevalence of pneumothoraces (25% of cases in the Europe/USA population compared with 74.7% in the East Asian population).  This is important as a pneumothorax is more likely to be the only characteristic of BHD in the East Asian population and therefore it is essential that clinicians consider the diagnosis of BHD in patients who have spontaneous pneumothoraces in the absence of any other BHD characteristics.    

1.          Guo T, Shen Q, Ouyang R, Song M, Zong D, Shi Z, et al. The clinical characteristics of East Asian patients with Birt-Hogg-Dubé syndrome. Ann Transl Med [Internet]. 2020 Nov [cited 2021 May 12];8(21):1436–1436. Available from: http://dx.doi.org/10.21037/atm-20-1129

2.          Schmidt LS, Nickerson ML, Warren MB, Glenn GM, Toro JR, Merino MJ, et al. Germline BHD-Mutation Spectrum and Phenotype Analysis of a Large Cohort of Families with Birt-Hogg-Dubé Syndrome. Vol. 76, Am. J. Hum. Genet. 2005.

---

Birt-Hogg-Dubé syndrome (BHD) is caused by a mutation in a gene called Folliculin (FLCN). FLCN is involved in numerous cellular process throughout the body including cell growth and proliferation, stress resistance, and autophagy (controlled cell death), however we still have a lot to uncover. An interesting study by Wang et al., suggests FLCN is also involved in iron homeostasis(1).

FLCN was recently found to be involved in the Rab11a pathway, which transports and recycles proteins within a cell(2). During this research they incidentally found that FLCN also bound to Transferrin Receptor 1 (TfR1) a protein involved in iron homeostasis. Iron is a mineral that the body requires to stay healthy. It is involved in oxygen delivery, energy production and DNA metabolism. Too little or too much iron can result in harmful side effects and therefore the body has mechanisms in place to ensure iron levels are tightly controlled including TfR1. TfR1 sits on the surface of cells, acting as a sentry, waiting to bind passing iron and bring it into the cell cytoplasm. Once the iron is released within the cytoplasm, TfR1 is recycled and returns to its role as sentry. If iron levels are low, Hypoxic-Inducible Factor (HIF) proteins, increase the transcription of TfR1 to recruit more to the cell surface and increase iron levels.

Wang et al., investigated the relationship between TfR1 and FLCN using human kidney cancer cells in vitro. They found that FLCN increased the binding of TfR1 and Rab11a and suggested that FLCN may act as a scaffold to facilitate transportation. They then knocked down FLCN, reducing its expression in the kidney cancer cells.  This caused a reduction in TfR1 recycling and therefore less TfR1 was present on the cell surface to capture iron, leading to iron deficiency. Additionally Wang et al., hypothesized that the increase in HIF, which has been suggested to be linked with tumor formation in BHD(3), may be caused by iron deficiency. They discovered that HIF proteins increased in the FLCN knockdown cells and that this increase could be reversed with iron supplementation, supporting the idea that it is linked with iron deficiency.  Lastly, Wang et al., used an animal model for BHD in the fruit fly Drosophila and found that an iron rich diet reversed the BHD phenotype.

Altogether Wang et al., demonstrated that FLCN is involved in iron homeostasis in kidney cancer cells and provide further evidence that folliculin is involved in multiple cellular processes. Future studies are required in other cell types and animal models to determine if iron homeostasis is a conserved function of FLCN throughout the body and whether proteins involved in iron homeostasis could be targeted therapeutically to inhibit HIF activation and therefore tumour growth in BHD.

References

1.          Wang X, Wu H, Zhao L, Liu Z, Qi M, Jin Y, et al. FLCN regulates transferrin receptor 1 transport and iron homeostasis. JBC [Internet] 2021 [cited 2021 May 5]; Available from: https://doi.org/10.1016/j.jbc.2021.100426

2.         Zhao L, Ji X, Zhang X, Li L, Jin Y, Liu W. FLCN is a novel Rab11A-interacting protein that is involved in the Rab11A-mediated recycling transport [Internet]. Vol. 131, Journal of Cell Science. Company of Biologists Ltd; 2018 [cited 2021 May 7]. Available from: https://pubmed.ncbi.nlm.nih.gov/30446510/

3.          Preston RS, Philp A, Claessens T, Gijezen L, Dydensborg AB, Dunlop EA, et al. Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene [Internet]. 2011 Mar 10 [cited 2021 May 6];30(10):1159–73. Available from: /pmc/articles/PMC3787473/

To read this blog in another language please click on the globe on the top right side of the page. We also have a French translation available here.

Jim was diagnosed with Birt-Hogg-Dubé syndrome (BHD) in 2005. He has a medical history of pneumothoraces (the first of which occurred at the age of 13), fibrofolliculomas and a family history of colon cancer. His mother, also believed to be a carrier of the mutated folliculin gene, was diagnosed with colon cancer in her 60’s.  Many people wonder whether BHD is associated with colon cancer and whether BHD patients such as Jim should be screened regularly.

There have been multiple cases of colon cancer reported in BHD patients, however a link between the two has never been established. Previous studies have not included a large enough number of patients to be able to make conclusions. A recent study in the Netherlands compared a cohort of 399 BHD patients with 382 family members who did not have a folliculin mutation, to try and answer this question and to ensure patients with BHD are receiving the correct screening(1).   

We spoke to Dr Arjan Houweling and MD Irma van de Beek at Amsterdam University Medical Centre about their research.

What prompted you to initiate this research?
Patients with an inherited form of cancer are often interested in the risk for other types of cancer. Patients with Birt-Hogg-Dubé syndrome are at a significantly increased risk for kidney cancer, but less information is available about other types of cancer. In one of the earliest publications on BHD, one of the patients also suffered from colon polyps(2). Colon polyps are benign, but can be a precursor for colon cancer. Following that early publication, many patients with both BHD and colon cancer have been reported. However, it was not clear whether these occurred together accidentally or whether they were really related to each other. This can only be assessed by studying larger groups of patients with BHD. One study from 2002 by Zbar and colleagues did not find significantly more colon polyps and colon cancer in patients with BHD compared to family members without BHD (3). However, even after publication of this study clinicians apparently were still not completely convinced, since colon screening for BHD patients was still advised in several guidelines. We hoped to gather more information about risks for colon polyps and colon cancer in patients with BHD, so we could adjust our guidelines if necessary.

What did you find out?
We compared data from the Dutch national pathology database between two groups: 399 patients with BHD and 382 of their family members without BHD. There was no significant difference between the number of colon cancers that had occurred in both groups. There were significantly more patients with BHD who had had removal of colon polyps. We did anticipate this, since a part of the BHD patients was advised to undergo colon surveillance based on their BHD diagnosis, while their family members without BHD do not undergo colon surveillance. Since colon polyps are common in middle- and older-aged individuals, it was to be expected that more BHD patients had colon polyps removed. Analyzing these polyps in more detail, there was no significant difference between the two groups in the median number of polyps per individual, the location of the polyps, the subtype of the polyps and the number of advanced polyps.

Our study had some limitations. We anonymized all patient records, therefore we could not be sure whether all extra detected polyps in patients with BHD were actually the result of extra colon surveillance. Furthermore, the removal of colon polyps may prevent the development of colon cancer. The extra surveillance in patients with BHD might therefore have led to a lower number of BHD patients with colon cancer. Despite these limitations, we believe that our data are a clear indication that the risk for colon polyps and cancer in patients with BHD is low, in line with the observations by Zbar et al(3).

What message would you give to BHD patients who are concerned about developing colon cancer.
There is no evidence for an increased risk for colon polyps or colon cancer in BHD. Even if the risk is increased, it is probably only slightly increased and does not need extra surveillance based on current knowledge. Most countries offer population screening for colon cancer, which should be sufficient for patients with BHD.

Altogether this study suggests that the risk of developing colorectal cancer if you have BHD is not significantly different from the general population and that additional colon screening is not required.

The BHD Foundation sincerely thanks Dr Arjan Houweling and MD Irma van de Beek for taking part in this interview and sharing their insights on the study. In addition, thank you to Jim Laycock, member of the BHD community for sharing his background and contributing to this blog.

Read the freely available research article here.

References

1.          van de Beek I, Glykofridis IE, Wolthuis RMF, Gille HJJP, Johannesma PC, Meijers-Heijboer HEJ, et al. No evidence for increased prevalence of colorectal carcinoma in 399 Dutch patients with Birt-Hogg-Dubé syndrome. Br J Cancer [Internet]. 2020 Feb 18 [cited 2021 Apr 27];122(4):590–4. Available from: https://doi.org/10.1038/s41416-019-0693-1

2.          Hornstein OP, Knickenberg M. Perifollicular fibromatosis cutis with polyps of the colon-a cutaneo-intestinal syndrome sui generis. Arch Dermatological Res [Internet]. 1975 Jan [cited 2021 Apr 27];253(2):161–75. Available from: https://pubmed.ncbi.nlm.nih.gov/1200700/

3.          Zbar B, Alvord WG, Glenn G, Turner M, Pavlovich CP, Schmidt L, et al. Risk of renal and colonic neoplasms and spontaneous pneumothorax pneurnothorax in the Birt-Hogg-Dubé syndrome. Cancer Epidemiol Biomarkers Prev [Internet]. 2002 Apr 1 [cited 2021 Apr 27];11(4):393–400. Available from: http://intl-cebp.aacrjournals.org/cgi/content/full/11/4/393

Although everyone experiences Birt-Hogg-Dubé syndrome (BHD) differently, a common feature that people share is that healthcare professionals often have not heard of the condition. An article by Dazé et al not only described a case of BHD but designed a quiz, based on the case, as an education tool for healthcare professionals.

The article describes a 54-year-old man with a 10-year history of white- and skin-coloured papules across his face and neck. Biopsies from the papules revealed them to be trichodiscomas, which are benign skin lesions similar to fibrofolliculomas – the difference being these are skin coloured rather than white. This finding led to imaging of his abdomen and pelvis, which showed lung cysts and bilateral kidney cysts.  Genetic testing confirmed he had a mutation in his folliculin gene, and hence a diagnosis of BHD was obtained.

The quiz takes this scenario and asks three questions. Firstly, it asks for a diagnosis based on this case.  Secondly it asks about the cancer associated with the syndrome and finally it asks about the histology of fibrofolliculomas. Although short this article allows healthcare professionals to work through a case of BHD and pinpoint exactly the features of BHD to aid future diagnosis.  

Read the freely available article (Includes photos) and take the quiz!

References

Dazé R, Fronek L, Moon S, Farsi M, Miller R. Masquerading case of a lumpy bumpy face. JAAD Case Rep. Oct 2020 16;6(12):1261-1263. doi: 10.1016/j.jdcr.2020.10.014.

Smith-Magenis Syndrome (SMS) is a neurodevelopmental condition most commonly caused by a deletion of an area called 17p11.2 on chromosome 17. The gene which predominantly causes the neurodevelopmental symptoms exhibited in SMS is called Retinoic Acid Induced 1 (RAI1), however in most cases of SMS multiple genes on this region of chromosome 17 are affected including Folliculin (FLCN). Variants in Folliculin including deletions cause Birt-Hogg-Dubé syndrome (BHD). Interestingly there have been very few reports of BHD symptoms occurring in SMS. A recent study by Finucane et al., collected data from 117 SMS patients asking if they had any of the three core symptoms associated with BHD: fibrofolliculomas, pneumothorax and kidney cancer. Five people reported pneumothoraces and two reported fibrofolliculoma. Four of the pneumothoraces occurred in childhood, three being under the age of 4. This differs to individuals with BHD syndrome, who most commonly develop their first spontaneous pneumothoraces in their 20’s or 30’s.  It must be noted that unexpectedly one of the participants who developed a pneumothorax only had a mutation in their RAI1 gene and not Folliculin . It remains to be determined if this was an unrelated spontaneous event or if there is a relationship between RAI1 and Folliculin.

Although more research is required to fully understand the relationship between BHD and SMS, this study suggests that patients with SMS may be at greater risk of manifesting symptoms of BHD and it is important for families to be aware of the association so that they can recognize symptoms of BHD early. 

PRISM is a nonprofit, advocacy, education, and support organization for individuals with SMS and their families. They provide advice on their website regarding BHD and recommendations for kidney screening.  

References

Finucane B, Savatt JM, Shimelis H, Girirajan S, Myers SM. Birt-Hogg-Dubé symptoms in Smith-Magenis syndrome include pediatric-onset pneumothorax. Am J Med Genet A 2021. doi: 10.1002/ajmg.a.62159.

By Katie Nightingale

Mutations in the tumour suppressor gene Folliculin (FLCN) cause Birt-Hogg-Dubé syndrome (BHD). Although the loss of a single allele of FLCN is sufficient to induce fibrofolliculomas and lung cysts, it is thought that a secondary mutation in the wild-type allele of FLCN is required for the development of renal cancer. Despite the wide variety of cellular processes FLCN has been connected to, including cell growth and proliferation, stress resistance, and autophagy, the mechanism by which loss of FLCN can induce tumourigenesis remains largely unknown. A recent study by Glykofridis et al., investigated the effect of loss of FLCN in renal tubular epithelial cells (RPTEC), a widely accepted in vitro model of human kidney function. They demonstrated that loss of FLCN led to the upregulation of several interferon (IFN) responsive genes in addition to activation of the transcription factor TFE3, resulting in the upregulation of genes involved in lysosome function and autophagy.

TFE3 directs the expression of genes involved in autophagy and stress tolerance under growth restrictive conditions and has been associated with the development of renal cell carcinoma in children. To investigate the localisation of TFE3 in the context of FLCN loss, they used CRISPR-Cas9 to generate a FLCN knock-out (FLCN-KO) RPTEC cell line. Comparing this to control RPTEC cells, in which TFE3 remains in the cytoplasm under normal growth conditions, they showed that TFE3 localised to the nucleus in FLCN-KO cells, indicating constitutive activation of TFE3.

To examine the effect of FLCN loss in a wider cellular context, they subsequently performed transcriptomic and proteomic analyses comparing FLCN-KO RPTEC cells to wild-type. They observed the induction of a large number of genes upon FLCN loss, which led them to identify a subset of 115 known TFE targets upregulated in the FLCN-KO cells. As many of these genes function in autophagy and lysosome regulation they next assessed mTOR signalling by looking at the phosphorylation status of the mTORC1 targets 43-BP1 and AKT and, contrary to previous reports, found no evidence for altered mTOR signalling or nutrient sensing in the FLCN-KO RPTEC cells. Analysis of the promoter regions of upregulated genes led to the discovery that the majority of upregulated genes could be assigned to 2 different promoter motifs: the E-box motif (regulated by TFE) and the interferon-stimulated response element (ISRE) motif. The upregulation of many of these genes was validated by quantitative PCR. Moreover, they confirmed the elevated expression of these genes in two independent BHD kidney tumours by mass spectrometry.

FLCN forms a complex with its interacting partners FNIP1 and FNIP2. To investigate the role of these proteins in the context of gene expression, they generated an FNIP1/FNIP2-KO RPTEC cell line and confirmed that upregulation of E-box or ISRE genes is dependent on the FLCN-FNIP1/FNIP2 axis. Re-introduction of FLCN to the FLCN-KO cell lines completely reverted the gene expression observed and established that upregulation of ISRE genes was entirely dependent on STAT1 and STAT2 through siRNA knockdown of these proteins. They could not, however, find a role for FLCN in the canonical IFN signalling pathway and thus concluded FLCN loss in RPTEC leads to non-canonical activation of STAT1/2. An unusual phenotype of FLCN-KO RPTEC is the severely reduced proliferation of these cells. This is perhaps unexpected as the loss of a tumour suppressor gene such as FLCN should result in increased proliferation. Indeed, constitutive activation of TFE3 in an FLCN-independent manner promoted uncontrolled proliferation. They hypothesised that the STAT2-mediated IFN signature observed in FLCN-KO RPTEC results in growth inhibition, which could counteract the hyperproliferative effects of TFE3 activation.

Relating these data to clinical outcomes for BHD patients the authors commented that this database of target genes provides a foundation for further investigation into the discovery of biomarkers for early-stage tumourigenesis as well as therapeutic strategies to prevent renal cell carcinoma in BHD patients. Of particular interest, the authors noted that GPNMB, a TFE3 target gene and protein frequently upregulated in a wide variety of tumours, was strongly upregulated in the absence of FLCN. Although it is unclear whether overexpression of GPNMB alone is sufficient to drive tumourigenesis, GPNMB can be targeted therapeutically using antibody-drug conjugates (such as glembatumumab vedotin) which are currently in clinical trials for cancer therapy warranting the investigation of this in the treatment of BHD tumours. In summary, Glykofridis et al., performed a comprehensive analysis of the transcriptome and proteome of cells lacking FLCN however, to truly carry this work forwards into a clinically relevant setting, analysis of further tumours from BHD patients would have to be carried out to confirm their findings.

Reference:

Glykofridis IE, Knol JC, Balk JA, Westland D, Pham TV, Piersma SR, Lougheed SM, Derakhshan S, Veen P, Rooimans MA, van Mil SE, Böttger F, Poddighe PJ, van de Beek I, Drost J, Zwartkruis FJ, de Menezes RX, Meijers-Heijboer HE, Houweling AC, Jimenez CR, Wolthuis RM. Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells. Elife. 2021 Jan 18;10:e61630.