Folliculin regulates fluid stress-induced mTORC1 suppression in primary cilia.

Birt-Hogg-Dubè can be thought of as a ciliopathy. Ciliopathies are a set of rare diseases resulting from defects in cilia; membrane-protruding organelles that provide either propulsion (motile cilia) or a sensory function (primary cilia) in mammalian cells. Like BHD, cystic lesions in the kidneys are a major manifestation of ciliopathies such as polycystic kidney disease and Bardet-Biedl syndrome, which result from mutations in the ciliary proteins that regulate cilia formation and retraction, a process known as ciliogenesis. In 2013 Luijten et al., reported that FLCN localizes to the primary cilium and influences various parameters of ciliogenesis. Furthermore, knock-down of FLCN in polarized epithelial cells of the kidney interferes with canonical Wnt signaling, an important cilia-associated pathway, and distorts the ability of these cells to organize into 3D spheroid cultures.

In 2016, Zhong et al. published an interesting study that further examined the role of FLCN in cilia, this time in the context of flow stress and mTOR signaling. Primary cilia of kidney cells experience flow stress when fluid passes over the cell surface causing the cilia to bend, and this is transduced into a cellular response by the suppression of mTORC1 signaling in an AMPK-dependent manner (Boehlke et al., 2013). Excitingly, the paper by Zhong et al., implicates a role for FLCN in this process, thereby providing another mechanism by which mTOR signaling could be dysregulated in BHD.

Zhong et al. first established that an interaction between FLCN with KIF3A  (a subunit of the cilia motor protein Kinesin-2) occurred in a cilia-dependent manner and was absent in cell populations lacking cilia, and therefore, all subsequent experiments were conduct on ciliated cells. This is consistent with the findings of Dodding et al., 2011, which predicted that FLCN houses a WD motif that enables it to bind Kinesisn-1, a cytoplasmic motor protein that is associated with intracellular transport. Next the study sought to confirm the observation that flow stress on ciliated cells causes a suppression of mTOR signaling by measuring the levels of P-S6, a product of mTOR phosphorylation. In normal cells, flow stress caused a down-regulation of P-S6, which is consistent with a reduction of mTOR activity, however, in FLCN knockout (UOK-257) and knock-down cells (siRNA) this P-S6 down-regulation failed to occur in response to the stress. The investigators also looked at the P-AMPK signaling that is upstream of the pathway and that acts as a repressor of mTOR signaling. In normal cells, the flow stress increased the expression of P-AMPK (which in turn decreases mTOR activity) but this increase was less apparent in the FLCN deficient knockdown cells. This behavior was also mirrored by P-TSC2, a signaling molecule down stream of P-AMPK (see diagram). Taken together, this is strong evidence that FLCN is required to down-regulate mTORC1 in flow stress signals, the very pathway strongly implicated in FLCN tumor suppressor role (Tsun et al., 2013).

Next, using another series of intricate co-immunoprecipitation experiments, the authors demonstrated that FLCN recruits the protein LKB1  to activate AMPK (see diagram below) This is consistent with the findings of Goncharova et al. 2014, who report that FLCN controls AMPK activity via LKB1 for cell survival processes in the lung alveoli. Like previous experiments, this process was cilia dependent and only occurred in response to fluid stress. When FLCN was knocked down the interaction of LKB1 and AMPK was significantly reduced. These experiments were nicely complemented with imaging and FLCN knockdown experiments that demonstrated that FLCN was required for the localisation of both p-AMPK and LKB-1 to the basal bodies, the microtubule structure present at the base of the cilia.

In summary, Zhong et al., have identified a novel role for FLCN in the mechanosensing function of cilia when exposed to fluid stress. A reduction in FLCN levels interferes with the fluid stress–induced suppression of mTORC1 and causes prolonged activation of this pathway. Notably, hyperexcitation of mTOR pathways is well documented in cancer biology and this study is consistent with of a wealth of recent literature that implicates a role for cilia within cancer and tumorigenesis. Unfortunately, the paper did not look to rescue the effects of FLCN knockdown/knockout on fluid stress signaling, which would have given them a good opportunity to test the effects of FLCN mutations on the process. Nevertheless, the study opens some interesting follow-up opportunities, including understanding if the process of mechanosensing involved in BHD cyst formation and tumourigenesis .

Image from Zhong et al., 2016.

The transcriptional landscape of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease that causes a range of symptoms. Like BHD, it can cause benign growths in several organs including the lungs, kidney and skin. However, TSC patients can also suffer from neurological problems, including intellectual disability, and it is a leading genetic cause of autism and epilepsy. The disease is caused by mutations in the TSC1 and TSC2 genes, which are components of the mTOR pathway. Mutations in TSC1 and TSC2 cause dysregulation of mTOR signaling and this is thought to contribute to the pathophysiology of TSC (Franz et al., 2017).

Seizures in TSC patients are thought to be partly caused by cortical tubers (brain lesions with distorted cellular architectures) and these are surgically removed in TSC cases where the epilepsy is unresponsive to medication. A recent paper from Mills et al., 2017, examined the gene expression profiles of several cortical tubers from TSC patients to look for clues into the mechanisms that underlie the neuropathology of the disease. The authors measured levels of RNA in cortical tubers from TSC patients (10 surgically removed and 2 autopsied) and cortical samples from age-matched controls who died from unrelated illnesses. Excitingly, the authors simultaneously measured both the protein-coding RNA and non-coding small RNAs (RNA that is not translated but regulates gene expression) using RNA Seq, to produce the first parallel coding and non-coding transcriptome analysis for TSC.

Mills et al. found 438 protein-encoding genes were differentially expressed in cortical tubers (measured by fold change); 269 over-expressed and 169 under-expressed. The authors noted that many of the more highly-transcribed genes were associated with the adaptive and innate immune responses including genes coding for the complement system. This is consistent with other studies that have implicated inflammation genes and the inflammatory response in the pathophysiology of TSC and that have made an association between the activation of the complement system and seizures (Vezanni et al., 2013). Interestingly, no significant differences in gene expression were observed between patients with TSC1 (4 samples) and TSC2 (8 samples) mutations. However, since the analysis was performed on a mixed population of cells, in both the TSC and control samples, it does not reveal which cell types within the tubers are driving these changes. Therefore, the investigators next looked to see which of the differentially expressed genes were exclusive to certain cell types of the cortex. By examining the 269 more highly-transcribed genes in healthy human cortex, it was found that 23 were exclusive to microglia, 3 to oligodendrocytes, 5 to neurons and 8 to astrocytes. The 32 genes that were specific to the astrocytes and microglia were components of the complement system, which is consistent with the role of these cells in neuroinflammation. The study did not report any RNA-seq comparisons between the different cell types of TSC and controls samples, which would have considerably raised the resolution of this analysis.

Next, the authors analysed the small non-coding RNAs and identified differential expression of 991 transcripts, including many different small RNA species. They found that members of the microRNA (miRNA) 34 family were amongst the most over-transcribed in the TSC samples. miRNAs silence gene expression by targeting protein coding mRNA transcripts and labeling them for degradation instead of allowing them to translate into proteins. In situ hybridization targeting of the miR-34 family was performed on TSC and control tissues and, consistent with the RNA-Seq data, showed an increase in expression of miR-34a-5p and miR-34b-5p in the TSC tubers.

To unite the RNA-Seq results of both the coding and small non-coding RNAs, the investigators applied a weighted gene co-expression network approach (WGCNA) to the datasets. This data mining method seeks to examine the implications of gene expression changes in the context of gene modules (a network of genes with related or interconnected functions) instead of at the individual gene level. The WGCNA analysis showed that many of the 169 genes that were transcribed less in the TSC coding RNA-Seq, and whose relevance could not be determined at an individual level, were predominantly involved in neurogenesis or glutamate receptor signaling. Moreover, several miR34 targets were also predicted to be involved in these two processes, suggesting that they could be affected by miRNA species that are differentially expressed in TSC. With this in mind, the authors sought to briefly explore the effect of miR-34 overexpression on neurogenesis in an in vitro hippocampal neuronal culture assay. Here, mimic miR-34b-5p molecules were overexpressed in the neurons and when compared to a non-targeting control, the miR-34 mimic samples had longer and more numerous neurites and larger cell somas. Accordingly, the authors concluded that miR-34 expression could be a contributing factor to the neuropathology of cortical tubers.

Figure from Mills et al., 2017.

Mills et al, 2017 is an ambitious study that uses powerful transcriptome analysis to highlight the complexity of gene regulation processes involved in the pathophysiology of TSC. The analysis of different RNA species, alongside the application of advanced bioinformatic techniques, has much potential as an ‘epigenetic-driven’ strategy for identifying new targets for therapies. The study of rare diseases such as BHD, would greatly benefit from such insight.

100,000 genomic insights into cancer and rare diseases

A haploid human genome comprises of 23 chromosomes, roughly 20,000 genes and is encoded by approximately 3 million base pairs (the building blocks of DNA – A, T, C and G). The full code for life’s blueprint for making a human, was revealed back in 2003 through an international collaboration known as The Human Genome Project (HGP). Considered as one of the greatest scientific achievements of the century, the HGP gave scientists never-before-seen insight into human genetics and sparked a wider interest in the study of human genomics.

Taken alone, a single human genome provides limited diagnostic information for a disease of unknown cause: the power of human genomic studies lies in being able to make comparisons. In 2012, another international effort known as the 1000 Genomes Project was completed, this time with many more genomes to study, and the project gave scientists a fresh look at the extent of human genetic variation (Nature, 2012). The 1000 Genomes project greatly benefited from faster and cheaper DNA sequencing technology (Mardis, 2011), a field that is still advancing today (Scott, 2016). Whereas the HGP took 13 years and nearly $2.7 billion to obtain the sequence of one mosaiced human genome (samples were provided by multiple donors), today a person’s genome can be sequenced for around $1,000 and in about a day (Scott, 2016). The same year the 1000 Genomes Project announced it had completed 1092 genome sequences, another exciting and ambitious genomics initiative was launched, this time entirely in the UK; the 100,000 Genomes Project.

The 100,000 Genome Project is funded by the Department of Health and it aims to annex a genomic medicine program to the NHS while also providing a platform for UK genomic scientific investigation. At this point, you may be wondering what the 100,000 genomes project has to do with BHD? Well, the 100,000 genomes project largely focuses on two areas, cancer and rare diseases, two fields that are relevant to the BHD community. In fact, one of the criteria for rare disease patients wanting to volunteer is familial (family-history associated) primary spontaneous pneumothorax. Recurrent pneumothorax can be suggestive of BHD especially if it runs in the family, and BHD patients are believed to be 50 times more likely to experience a pneumothorax than the general population (Zbar et al. 2002).

Unfortunately, anyone who is known to have folliculin (FLCN) mutations will not be able to sign up to the 100,000 Genome Project because it is only recruiting patients with primary pneumothorax, (whereas BHD patients suffer from secondary pneumothorax associated with an underlying pulmonary cause). There is a requirement that prospective volunteers with suspected familial pulmonary primary pneumothorax be screened for FLCN mutations prior to entering the study, to rule out BHD as the potential cause. The BHD Foundation thinks that there are some positive messages to be taken away from this. Firstly, BHD awareness has clearly infiltrated the study of rare diseases well, which is important given that it is so rare, even by rare disease standards. Currently there are roughly 600 families worldwide with a BHD diagnosis (published BHD families) but BHD is certainly underdiagnosed, perhaps significantly. Secondly, the requirement of FLCN screening before entering the study will hopefully increase the diagnosis rate of people with a FLCN mutation: quicker and earlier diagnosis of BHD is important because it allows for better management and surveillance for all possible BHD symptoms, including renal cell carcinoma. 

The GAP in FLCN’s tumour suppressor function addressed in research paper.

It is well known that mutations in the folliculin gene (FLCN) cause BHD syndrome; a disorder that can result in fibrofolliculomas, lung and kidney cysts, pneumothorax and renal cancers. The FLCN protein has long been suspected of acting as a tumour suppressor and has been identified as a modulator of the AMPK-mTOR pathway (Baba et al. 2006), a signaling cascade involved in cell metabolic processes and cancer. Unfortunately, the precise mechanism by which FLCN exerts its tumour suppressor role remains unknown. This is addressed by a recent research paper by Laviolette et al. 2017, which finds that FLCN acts as a GTPase activating protein (GAP) for RAB7A and proposes that normal FLCN-RAB7A interactions may suppress tumour growth by modulating the receptor tyrosine kinase (RTK) EGFR (epidermal growth factor receptor).

Elevated EGFR signaling has already been heavily associated with cancer mechanisms (reviewed in Jones et al. 2014) and consistent with this, the paper demonstrates increased levels of EGFR and EFGR signaling in FLCN defective cells (human follicular thyroid carcinoma cell line) relative to FLCN wildtype cells. Upon stimulation with EGF ligand, higher levels of phosphorylated EGFR (and of phosphorylated downstream effector molecules pERK and pS6) were detected in both FLCN deficient cells and those expressing tumour-associated FLCN mutations. In addition to this, high levels of EGFR-activated signaling molecules were detected (via immunohistochemistry) in the renal cysts and tumours from a conditional FLCN knockout mouse and in various types of renal cell carcinomas biopsied from BHD patients. Taken together, this is evidence for the involvement of heightened EGFR signaling in the tumorigenesis associated with FLCN dysfunction. The implications of this finding are that a loss of FLCN function may result in increased mTOR activity (which is downstream of the EGFR pathway) and the paper states that this is contrary to studies claiming FLCN activates mTOR (Petit et al. 2013). However, the authors acknowledge that many of those experiment were performed under different stimulating conditions (amino acid vs EGF excitation) and raises the hypothesis that the interactions and dynamics of FLCN could vary according to the stimulus.

This study identifies a novel interaction between FLCN and GTPase RAB7A by co-immunoprecipitation experiments and mass spectrometry. The FLCN-RAB7A interaction was investigated in more detail using RAB7A proteins locked in several conformations, including a constitutively active GTP-bound form (induced by a Q67L mutation) and a dominant negative form (T22N mutation). Here, preferential precipitation of both the constitutively active and wildtype forms of RAB7A suggested that FLCN could be acting as a GTPase activating protein (GAP) that promotes hydrolysing activity in RAB7A. This hypothesis was confirmed in a functional assay that revealed FLCN enhances the GTPase activity of RAB7A and that GAP activity was lost in FLCN when it housed the tumour-associated mutation K508R.

FLCN is known to regulate the spatial distribution of lysosomes via regulating Rab-RILP interactions in a process that is dependent on nutrient status (Starling et al. 2016). Furthermore, Rab7A is known to regulate the endocytic recycling and lysosomal degradation of EGFRs (Ceresa et al. 2006, Rush et al. 2013). Therefore, the authors of this paper looked to examine how FLCN, as a Rab7A GAP, effects EGFR trafficking and signaling. Intricate imaging experiments showed that FLCN-deficient cells (renal carcinoma cell line) had slower endocytic trafficking of EGFRs compared to normal cells and this manifested as an accumulation of EGFR in early endosomes, alongside a delayed transition into late endosomes. The authors noted that delayed endocytic trafficking could result in more recycled and less degraded EGFR, causing an increase and prolonging of EGFR signaling. Accordingly, this provides a potential explanation for the observed heightened EGFR signaling in FLCN deficient cells and BHD tumours (see above).

Having implicated the involvement of Rab7A and EGFR signaling in FLCN’s tumour suppressor role, the authors examined the mechanism in vivo. Afatinib, an inhibitor of EGFR signaling, was used to treat FLCN-negative induced tumour xenografts in mice. Consistent with the general findings of this paper, that there is heighten EGFR signaling in FLCN defective cells, the study found that Afatinib significantly hindered the growth of these tumours compared to the control-treated mice but, notably, there was no tumour regression. Although the focus of this paper was on EGFR signaling, the authors highlight the importance of investigating the role of other RTK pathways in FLCN-associated renal cancers. Furthermore, it would be interesting to see with further research and study, whether or not Afatinib holds any promise as a future treatment of BHD.

A case report by Takegahara et al., 2017

Pulmonary cysts are a frequent clinical manifestation of Birt-Hogg-Dubé syndrome and are assumed to be the cause of pneumothorax in BHD patients (Johannesma et al. 2014f). Pneumothorax is when lungs collapse due to air leaking into the pleural cavity and in BHD patients the number of cysts is strongly correlated to the incidence of recurrent pneumothorax (Johannesma et al., 2014f). Since many lung diseases carry an increased risk of pneumothorax, it is hard to identify potential BHD cases when pulmonary symptoms present alone.

Takegahara et al. 2017, reports on a 40-year-old man who had experienced recurrent bilateral pneumothorax from the age of 22. At the time of this report, X-ray showed that the patient had a pneumothorax in his left lung, although it came to light that he had previously experienced three pneumothoraces on his right side. His case of pneumothorax did not subside following thoracostomy tube insertion and the decision was made to perform bilateral thoracoscopic surgery after a chest CT revealed the presence of cysts in both lungs. This enabled surgeons to remove the larger cyst suspected to be responsible for the problem, seal any lesions and performed a pleural covering with a polyglycolic sheet with the intention of reducing the recurrence of pneumothorax.

Despite the patient only exhibiting pneumothorax, clinicians suspected there to be an underlying genetic cause such as BHD, because of several observations including the patient’s family history of pneumothorax (mother and maternal grandmother). Subsequent genetic testing of the affected family members revealed a duplication mutation (c.1285dupC) in exon 11 of the FLCN gene, confirming them to be cases of BHD syndrome. Further examinations revealed no fibrofolliculomas or renal symptoms but long-term monitoring was recommended including for further pneumothoraces. Such a diagnosis is important because it can lead to early recognition and the better treatment of life-threatening BHD symptoms such as renal cell carcinoma, should they develop at a later age.

Lung ultrasound limitations in rare cystic lung diseases

Lung ultrasound (LUS) is a fast and non-invasive examination procedure to diagnose and monitor conditions in the lungs. It is used to identify interstitial syndrome (IS) and pleural thickening related to diffuse parenchymal lung disease (DPLD) and has shown significant correlations with ground glass opacity (GGO) on high-resolution computed tomography (HRCT). However, the applicability of LUS in patients with rare cystic lung diseases, a DPLD subtype, has not been addressed. In their new study, Davidsen et al. (2017) investigate if distinctive LUS findings could be found in patients with lymphangioleiomyomatosis (LAM), pulmonary Langerhans cell histiocytosis (PLCH), and Birt-Hogg-Dubé syndrome (BHD). LUS findings were compared to findings on HRCT and the results revealed that LUS has limitations as a diagnostic tool in patients with LAM, PLCH, and BHD.

LUS is a radiation-free procedure that can be performed relatively rapidly, with minimal discomfort and is a procedure which is easy to assimilate. Therefore, LUS may be preferable to diagnose and monitor DPLDs in some settings compared to HRCT. LUS was performed by two experienced physicians. The LUS records for each patient were independently reviewed by two pulmonologists. The latest available HRCT prior to examination date for each patient was evaluated and scored according to a modified version of Belmaati et al. (2009) which specifically aims to identify dichotomised presence of findings such as GGO, among others.

In total 12 patients with rare cystic lung diseases participated, six were diagnosed with LAM, three with PLCH, two with BHD, and one with uncharacteristic cystic lung disease. All LAM patients were females and two patients had tuberous sclerosis complex (TSC) associated LAM. The two patients with BHD were men and they also presented renal cysts. The three patients with PLCH were smokers. BHD patients and the patient with unclassified cystic lung disease had normal ventilation and diffusion parameters. Patients with LAM and PLCH exhibited a moderately reduced obstructive ventilation and diffusion capacity.

In general, all patients had normal LUS findings. In three (two with LAM and one with PLCH) patients pleural thickening was found in one zone, and observed only in the inferior lateral zones. Lung consolidation was observed in two patients. One had sonomorphologic characteristics of bilateral pneumonia, and the other had uncharacteristic findings. HRCTs prior to individual examination date were performed between 2005–2015. HRCT findings consistent with interstitial syndrome (IS) and pleural thickening showed low scores coherent with no or almost no observation of these findings. High mean scores for GGO were present in nine patients (75%) and in only two of the patients’ HRCTs were no GGOs observed.

In summary, this is the first study to investigate the applicability of LUS in a population of patients with rare cystic lung diseases. IS and pleural thickening, which are characteristic LUS findings in other subtypes of DPLD, were not significantly present in these patients with rare cystic lung diseases. Despite severe cystic formation on HRCTs (showed by the high GGO scores) the LUS results were normal. One possible explanation for the contradicting HRCT-GGO and LUS-IS findings is a possible time-bias, since recently performed HRCTs were not available. However, the chronic nature of these cystic lung diseases reduces the impact of time bias, and it is unlikely that the results of the study would have changed if more recent HRCT examinations were available. The applicability and diagnostic value of LUS in patients with rare cystic lung diseases such as LAM, PLCH, and BHD seems limited as normal LUS findings did not rule out severe cystic lung disease.

Early onset RCC in BHD patient

Most patients diagnosed with Birt-Hogg-Dube (BHD) syndrome exhibit fibrofolliculomas and pneumothorax, but only 30–45% of them develop renal cell carcinoma (RCC) with a median age of diagnosis at 48. The earliest reported onset of RCC in a BHD patient has been age 20 (Benusiglio et al., 2014). In a new case study, Schneider et al. (2017) report the case of a 14-year-old patient with a FLCN mutation leading to an early-onset RCC that could not be classified according to typical histology. This is the youngest patient with reported BHD-related RCC. This early onset presentation supports genetic testing of at-risk patients and initiation of imaging surveillance for RCC at an early age.

BHD-associated renal tumours can present with different histological subtypes and for BHD patients, baseline imaging of the kidneys is recommended at age 20 (Menko et al., 2009). This new case reports a 14-year-old girl presenting with a renal cell carcinoma (RCC) of the left kidney that had been histologically unclassified. Apart from the tumour, physical examination was unremarkable, including the absence of skin lesions. Her past medical history included only a surgically repaired patent ductus arteriosus. Family history was unremarkable for RCC. An abdominal CT demonstrated a large 21 cm solid mass arising from the inferior pole of the left kidney causing severe hydronephrosis. The patient underwent a unilateral nephrectomy without complications. Histological examination showed that the viable tumour cells formed tubular, trabecular and focally follicular structures. The differential diagnosis based on routine histology included mucinous tubular spindle cell carcinoma (MTSC) and thyroid-like follicular carcinoma. In the patient’s age group, consideration was also given to metanephric adenoma and MiTF family translocation RCC. The tumour showed no morphological features to suggest MiTF translocation carcinoma. MTSC was not supported by the histology or protein markers expression. In addition, the thyroid follicles were too focal to suggest thyroid-like follicular carcinoma. Wilms’ tumour and metanephric adenoma were also not supported by marker expression. No neuroendocrine marker expression was found to suggest carcinoid tumour. On the basis of the morphological features and immunophenotype, the tumour did not fit into any of the typical diagnostic categories. Genetic testing was offered to the patient and a panel of kidney cancer susceptibility genes was examined for mutations. The results revealed a germline FLCN p.Gly84_Glu-132del mutation of exon 5 and the patient was diagnosed with BHD syndrome. To assess the functional significance of this novel in-frame mutation on FLCN function the authors stably re-introduced wild type and mutant FLCN gene into the FTC133 FLCN-null cell line and used a xenograft mouse assay to show that the in-frame deletion inactivates the tumour suppressor effect of the encoded protein. Testing of both parents revealed that her father also carried the same mutation. An abdominal MRI examination was offered to the father and no renal cysts or masses were identified. Neither the patient nor the father had fibrofolliculomas or history of spontaneous pneumothorax.

The authors describe a case of early-onset BHD syndrome caused by a novel inherited deletion of exon 5 of the FLCN gene. This case is unusual in the young age of onset of initial manifestation of disease and the atypical histology. Deletion of just exon 5 has not been reported so far to cause BHD syndrome and very little is known about a possible correlation between the genotype and the risk to develop RCC. It is becoming more evident that there is a wide clinical variability in BHD syndrome and this case report demonstrates the importance of genetic testing especially in patients with an uncommon clinical presentation and histology. In the paediatric population, RCC constitutes a small fraction of renal tumours and up to 25% are considered histologically unclassified. This raises the question of whether FLCN mutations are especially under diagnosed in this population. This study highlights the need to revisit the appropriate age to initiate surveillance for RCC in BHD patients.

Spontaneous pneumothorax in diffuse cystic lung diseases

Diffuse cystic lung diseases (DCLDs) are a group of disorders with different mechanisms that are characterized by the presence of air-filled lung cysts. These cysts are prone to rupture, leading to the development of recurrent spontaneous pneumothoraces. In their new review, Cooley et al., 2017 give an overview of the epidemiology, clinical features, and management of spontaneous pneumothoraces in patients with DCLDs, with a focus on Birt-Hogg-Dube (BHD) syndrome, lymphangioleiomyomatosis (LAM) and pulmonary Langerhans cell histiocytosis (PLCH). The understanding of spontaneous pneumothoraces in DCLDs can help clinicians to find an early diagnosis and appropriately manage the disorders.

BHD syndrome

Patients with BHD have a 50-fold greater likelihood of spontaneous pneumothorax as compared with controls. Recently, studies have shown that BHD can be the underlying cause of 5–10% of apparent primary spontaneous pneumothoraces (Johannesma et al., 2015). The prevalence of spontaneous pneumothorax in BHD varies between reports. Pneumothorax prevalence rates of 22.5-38% are reported by renal/skin-focused centres, while prevalence rates of 42–76% are reported from patients in pulmonary cohorts. Pneumothoraces due to BHD usually occur in the patient’s mid-late 30s; however, they have been reported in pediatric and geriatric patients. Lung cysts and cyst burden are associated with increased risk of pneumothorax. Interestingly, there are reports of pneumothorax in patients without apparent cysts on CT imaging. Family history of pneumothorax is associated with higher risk of pneumothorax but smoking and the presence or severity of renal tumours or fibrofolliculomas are not. Pneumothorax recurrence rates in BHD are estimated to be 75–80%. Patients with BHD-associated pneumothorax experience an average of 3.6 total episodes of pneumothorax. Given the high recurrence rate, pleurodesis should be performed following the first pneumothorax rather than waiting for a recurrence since pleurodesis can halve the recurrence rates of ipsilateral pneumothorax in BHD patients.

LAM

55–73% of patients with LAM experience a pneumothorax in their lifetime, with pneumothorax leading to the diagnosis of LAM in approximately 40% of patients. On average, patients experience 2.2 pneumothoraces before the diagnosis of LAM is made. Patients with larger cyst size on HRCT and those with a history of smoking are more likely to develop a pneumothorax. Given the high rates of recurrence, pleurodesis should be performed after the first spontaneous pneumothorax in patients with LAM. In one study, the ipsilateral pneumothorax recurrence rate was 66% if managed conservatively, but this was reduced to 27% with chemical pleurodesis and 32% with surgical pleurodesis.

 PLCH

Approximately 15–20% of patients with PLCH experience a pneumothorax. Nearly 63% of those patients will have more than one pneumothorax. Simultaneous bilateral pneumothorax can occur and may be fatal. Due to high rate of recurrence, pleurodesis should be performed following the initial episode of pneumothorax. A study found a recurrence rate of 58% when managed conservatively, compared with 0% following surgical pleurodesis. Treatment of PLCH must involve smoking cessation. The effect of steroids or chemotherapeutics on disease course, and occurrence of pneumothorax, is unclear.

Management

Air travel and diving may lead to cyst expansion and predispose patients with DCLDs to a higher risk of pneumothorax. The risk of pneumothorax associated with air travel has been studied in LAM and, more recently, BHD as discussed in a previous blog. The risk of flight-related pneumothorax is currently being evaluated for patients with PLCH. Studies have concluded that it is safe for most patients with DCLDs to undertake air travel but patients should be educated about the signs and symptoms of a pneumothorax and counselled prior to boarding an airplane. Limited data are available regarding the safety of diving in patients with DCLDs but the authors recommend that patients with DCLDs avoid diving in accordance with the British Thoracic Society guidelines.

Recent data suggest that BHD, LAM, and PLCH likely cause approximately 10% of apparent primary spontaneous pneumothoraces. Current guidelines do not recommend screening CT for first-time pneumothoraces. However, in a recent study, performing a screening HRCT to facilitate early diagnosis of LAM, BHD, or PLCH followed by pleurodesis was found to be cost-effective. Based on this, the authors recommend that all patients with an apparent primary spontaneous pneumothorax be screened with HRCT for the presence of underlying DCLDs.

In summary, DCLDs are being recognized as the cause of spontaneous pneumothoraces. The impact of pneumothoraces on these diseases, the efficacy of alternative techniques to reduce recurrence risk, the impact of targeted pharmacologic therapy on pneumothoraces and the molecular pathways behind them, are some of the major unanswered questions to be addressed.

Pneumothorax BHD Awareness campaign

BHD Syndrome is the genetic cause of 10% of primary spontaneous pneumothorax. Unfortunately, it is not widely known that spontaneous pneumothorax can be a symptom of BHD. Therefore, many people presenting with pneumothorax go undiagnosed for BHD syndrome. An accurate diagnosis would allow patients to be screened for kidney cancer enabling them to receive timely treatment, and would also give their family the chance to be tested for Folliculin mutation.

The BHD Foundation (with the Myrovlytis Trust) is promoting an awareness campaign that tackles this issue.

Pneumothorax BHD Awareness campaign is a campaign to raise awareness among pulmonologists, radiologists, thoracic surgeons, respiratory nurses, technicians and medical students of the fact that pneumothorax can be a symptom of BHD syndrome, and that BHD should be considered as a diagnosis for patients presenting with pneumothorax. Another aim of the campaign is to inform as many people as possible, who have had an unexplained pneumothorax, of BHD as a potential cause.

Initially, we are aiming to have a presence (personally and in combination with oral or poster presentations, leaflets and exhibition stands) at respiratory conferences, and possibly submitting a collaborative multi-author piece to a respiratory journal.

This week we are at the JointCHESTSGP2017 in Basel, Switzerland, a joint congress organised by the American College of Chest Physicians (CHEST) and the Swiss Pneumology Society (SGP) with almost 1000 chest specialists. We have been talking to doctors, students, nurses and imaging experts about BHD Syndrome, unknown to most of them.

A social media engagement plan is also being developed to create an online movement around the campaign. We hope that patients, professional and patient societies will help to promote the campaign through their social channels and we will provide social media content to support with this.

In the future, we are considering contacting radiology, pulmonary, thoracic clinics/hospital services with a questionnaire about pneumothorax and BHD and sending informational e-mails about pneumothorax and BHD that is clear, credible and attracts attention. Also, creating an eLearning module about BHD (possibly combined with LAM and other diffuse cystic lung diseases) and producing “Hospital information packs” about BHD to be sent to staff at hospitals.

Help us spread the word! Connect with us on social media and take our Clinical Introduction to BHD and our Lung Symptoms leaflets to your doctor and lung specialist to make them consider BHD.

Three new BHD case reports

Marous et al. (2017) present the case of a 38-year-old white man with confirmed BHD, manifesting cutaneous fibrofolliculomas and trichodiscomas on the face and upper chest, bilateral spontaneous pneumothorax and a choroidal nevus in the left eye. Later, the patient experienced photopsia and decreased visual field in the left eye. Microscopy revealed subtle iris melanocytosis in the left eye. Fundus evaluation in the left eye disclosed a choroidal melanoma arising from patchy inferotemporal sectoral choroidal melanocytosis. In addition, subtle pinpoint retinal pigment epithelium (RPE) microdetachments were noted. Fluorescein angiography confirmed hyperfluorescence of the melanoma and also highlighted multifocal pinpoint RPE defects in each eye.

One ophthalmic study previously reported two patients with BHD with RPE alterations, and another report described choroidal melanoma with no documentation of melanocytosis (Walter et al., 1997; Fontcuberta et al., 2011). The relationship of these findings with BHD remains unclear. It would be interesting to examine a cohort of BHD patients to investigate the prevalence of RPE alterations and choroidal melanocytic features.

Rato et al. (2017) describe two BHD patients with previously-undescribed FLCN mutations, and one of them with a type of adrenal gland tumour associated for the first time with BHD. The first case was a 60-year-old caucasian man with multiple asymptomatic skin lesions of the face, scalp and neck. The patient denied any respiratory signs or symptoms. His medical history was consistent with hypertension, diabetes mellitus, dyslipidemia and benign prostatic hypertrophy. Physical examination revealed multiple, dome-shaped, whitish and erythematous papules scattered over the scalp, face and upper neck. Biopsy of two papules showed features consistent with fibrofolliculoma. A genetic test revealed a new heterozygotic mutation in exon 6 of the FLCN gene (p.Lys192Argfs*31). Computed tomography (CT) scan of the thorax and abdomen showed numerous bilateral lung cysts without suspicious kidneys lesions. Patient had no history of spontaneous pneumothorax. Pulmonary function tests, thyroid ultrasonography and colonoscopy were normal.

The second case was a 39-year-old caucasian man with a history of epilepsy and asthma presenting with multiple asymptomatic skin lesions located on the face and neck. Similar dermatological findings were present in the patient’s family. Physical examination found numerous firm, whitish papules on the face and upper neck. Biopsy of two papules was performed and histopathological analysis was compatible with fibrofolliculoma. The patient was assessed for FLCN gene mutations and a new heterozygotic mutation was detected in exon 9 of the FLCN gene (c.1015C>T), responsible for the introduction of a premature stop codon at position 339 amino acid (p.Gln339*). This mutation has not been described previously but it can be considered pathogenic and so responsible for BHD in the patient. CT scan of the thorax and abdomen showed a lung cyst in the right middle lobe and a nodular formation. Laparoscopic left adrenalectomy was performed and histopathological examination revealed a malignant perivascular epithelioid cell tumour (malignant PEComa). Pulmonary function tests and thyroid ultrasonography were normal. This case represents the first malignant PEComa diagnosed in a patient with BHD. Some types of PEComa are seen at high frequency in tuberous sclerosis complex (TSC), like renal angiomyolipoma, as well as pulmonary lymphangioleiomyomatosis. A possible association between BHD and the occurrence of malignant PEComa in this patient is interesting but purely speculative.

These new cases reinforce the message that the management of Birt-Hogg-Dube patients should be multidisciplinary.

  • Marous CL, Marous MR, Welch RJ, Shields JA, & Shields CL (2017). CHOROIDAL MELANOMA, SECTOR MELANOCYTOSIS, AND RETINAL PIGMENT EPITHELIAL MICRODETACHMENTS IN BIRT-HOGG-DUBÉ SYNDROME. Retinal cases & brief reports PMID: 28541267
  • Rato M, Monteiro AF, Parente J, Aranha J, & Tavares E (2017). Birt-Hogg-Dubé Syndrome – report of two cases with two new mutations. Journal of dermatological case reports, 11 (1), 12-15 PMID: 28539984