The BHD International Registry (BIRT) – a first step to unravelling the intricacies of BHD

We are delighted to announce that we will be launching the BHD International Registry (BIRT) in March, and hope that as many of the BHD community as possible can join us to help drive research forward in the field.

A registry is a large database, containing information provided by patients on their condition. We hope in the initial stages to gather enough information to help inform the management of BHD, as well as provide information to help researchers.

One of the most common queries we receive at the BHD Foundation is whether other cancers are linked with BHD. There have been suggestions that this may be the case, but conclusive findings haven’t been made. This is largely because studies have been small – as a rare disease it is hard for clinicians to have enough patients to conduct large scale studies. We are hoping the new registry will overcome these obstacles and that research will benefit from this larger-scale approach.

This is a truly collaborative project. We are very grateful to our working group (clinicians and patients) for their continued help and advice.

To make the BIRT registry a success we need the BHD community to join and enter their data. We launch in March, and will have a dedicated zoom meeting to answer questions and help with any technical issues. It is an exciting time at the BHD Foundation, and we hope you can help to make this a success!

More information can be found here, and don’t forget to sign up for updates to be the first to hear about this and other projects we have planned.

April Newsletter

The April 2021 BHD newsletter is out! We discuss the highlights from this month, including our first Meet the Experts event, the European International Kidney Conference 2021, the latest research and the patients’ perspective of being diagnosed with Birt-Hogg-Dubé syndrome.

Read it now

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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.


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.


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.


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.

  • 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

The PI3K/mTOR inhibitor GSK2126458 is effective for treating TSC solid renal tumours

Tuberous sclerosis (TSC) is an inherited tumour syndrome that shares clinical similarities with Birt-Hogg-Dube Syndrome. It is caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR, affecting multiple organs, including the kidney and lung. In the kidney, lesions such as multiple renal cysts and renal cell carcinoma (RCC) can occur. Tumour reduction in TSC patients after treatment with rapamycin, an inhibitor of mTOR, is partial and reversible probably due to feedback activation of Akt. In their new study, Narov et al. (2017) test the efficacy of GSK2126458, an inhibitor of PI3K/mTOR, in comparison to rapamycin, for treatment of renal tumours in genetically engineered Tsc2+/- mice, that spontaneously develop various lesions in the kidneys. Both GSK2126458 and rapamycin caused significant reduction in number and size of solid renal tumours. GSK2126458 inhibited both PI3K and mTOR while rapamycin exerted stronger inhibitory effect on mTORC1 in renal tumours. Both GSK2126458 and rapamycin suppressed proliferation of tumour cells. However, GSK2126458 increased apoptosis of solid tumours but rapamycin did not. Further investigations are needed to test whether rapamycin in combination with GSK2126458 can improve anti-tumour therapy.

The kidney lesions of adult Tsc2+/- mice had aberrant activation of the mTOR complex 1 (mTORC1) and mTORC2. The MAPK pathway was also activated in these lesions. To test the anti-tumour efficacy of GSK2126458, the authors first determined the maximum tolerated dose (MTD) of GSK2126458 in Tsc2+/- mice. After this, adult mice were treated for two months with vehicle, the MTD dose of GSK2126458 or rapamycin. Both GSK2126458 and rapamycin significantly reduced total number, size and cellular area of solid renal tumours and other lesion types. Rapamycin caused greater reduction than GSK2126458 in number, size and cellular area of all types of lesions but the difference in reduction of solid tumour burden was not significant. IHC of kidney sections was used to investigate PI3K/Akt/mTOR signalling. GSK2126458 reduced phosphorylation of Akt at T308 but rapamycin did not. It is not known whether this reduction in Akt phosphorylation contributes to antitumour efficacy. The phosphorylation of a part of the Erk1/2 signalling pathway, was reduced in GSK2126458 treated solid renal tumours. The Erk1/2 signalling was inhibited by rapamycin in solid renal tumours. In contrast, reduced phosphorylation of mTOR at S2481, an indicator of mTORC2 activation, was detected in rapamycin treated solid tumours. Ki67 staining was used to assess proliferation of renal tumour cells on treated mice. Both GSK2126458 and rapamycin markedly reduced the median percentage of Ki67-positive cells. Rapamycin inhibited proliferation of tumour cells to a greater extent than GSK2126458. Active caspase 3 was used to test whether treatment induced apoptosis in tumour cells. Interestingly, GSK2126458 significantly increased the median total area of active caspase 3-positive tumour cells but rapamycin did not. Similar results were observed when other apoptosis marker was analysed. To investigate the mechanism behind the increased apoptosis associated with GSK2126458 treatment, expression of p53 and phosphorylation of MDM2 at S166 were analysed by IHC. Renal lesions had a lower level of p53 and decreased phosphorylation of MDM2 in rapamycin treated mice, but not GSK2126458 treated mice, compared to vehicle treated mice. Similar results were observed by Western analysis.

In conclusion, the authors demonstrated that GSK2126458 was effective for treating solid renal tumours. A Phase I clinical trial has recently reported that GSK2126458 is well tolerated in patients treated for multiple solid malignancy types and tumour responses and disease stabilization were observed (Munster et al., 2016). The authors found that GSK2126458 inhibited both mTORC1 and mTORC2 in all types of renal lesions in Tsc2+/- mice but the inhibitory effect of GSK2126458 on mTORC1 was weaker than that of rapamycin. Both GSK2126458 and rapamycin reduced proliferation of tumour cells. However, GSK2126458 increased apoptosis of solid tumours but rapamycin did not.

The clinical similarities between BHD and TSC suggest that FLCN and TSC proteins may function within a common pathway. It may be worthwhile investigating whether combination of GSK2126458 with rapamycin could improve anti-tumour therapy through increased tumour cell death in different disease models.

  • Narov, K., Yang, J., Samsel, P., Jones, A., Sampson, J., & Hong Shen, M. (2017). The dual PI3K/mTOR inhibitor GSK2126458 is effective for treating solid renal tumours in Tsc2+/- mice through suppression of cell proliferation and induction of apoptosis Oncotarget DOI: 10.18632/oncotarget.17215

Characterization of a FLCN mutation associated with RCC

Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé (BHD) syndrome, a rare disease characterized by renal cell carcinoma (RCC), pneumothorax and fibrofolliculomas. In their new study, Bartram et al. (2017) identify a heterozygous mutation in the FLCN gene in a patient with RCC. DNA from tumour and a metastasis was analysed and the authors demonstrated skipping of exon 11 as the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. The FLCN protein was still expressed but it was strongly destabilized and had a different subcellular localization. Both altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation.

In this study, a 55-year-old patient presented with weight loss, bilateral kidney cysts and tumours. He and family members had a history of recurrent pneumothorax. Histology after kidney tumour nephrectomy showed elements of a chromophobe RCC (chRCC) and a small cell carcinoma component. In addition, CT scan showed liver and spleen enlargement, and several lung cysts and pulmonary nodules. Open surgery revealed peritoneal metastases.

Histology of one metastasis showed features of the small cellular tumour component, suggesting that these cells might contribute to the aggressive tumour phenotype in the patient. After surgery, hemodialysis was initiated due to renal failure. Unfortunately, the patient died shortly afterwards as a consequence of the advanced stage of the metastatic tumour disease.

The co-occurrence of chromophobe RCC with familial recurrent pneumothorax lead to the suspicion of BHD syndrome.

BHD-syndrome associated RCC normally show a benign nature and rarely metastasize. Here, the patient suffered from metastases and pulmonary lesions. The metastases did not show the classical characteristics of the chRCC but rather a small-cell morphology. Since the chRCC showed different levels of dedifferentiation towards the areas of the small cell tumour component the authors speculate that the small cell carcinoma arose from the chRCC by acquiring further genetic alterations.

FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. In silico analyses by bioinformatic tools predicted this variant to be likely pathogenic. FLCN MLPA analyses were consistent with deletion of the second FLCN allele in both tumour tissues.

The metastasis appears to be linked to BHD since it showed loss-of-heterozygosity in the FLCN gene. It will be interesting to see in future cases whether this entity is associated with BHD syndrome.

To validate the mutation’s impact on splicing of the FLCN transcript the authors generated minigene constructs containing either the FLCN  wild-type (WT) or the mutant sequence. These minigene constructs showed that the deletion indeed abrogated the acceptor splice site of exon 11, leading to skipping of exon 11 and fusion of exon 10 to exon 12 which generates a frameshift and premature stop codon.

To investigate whether the predicted FLCN protein is expressed the authors analysed protein expression in HEK293T cells. Overexpression of a FLAG-tagged cDNA revealed that the mutant protein is expressed, however, at lower levels than the WT protein. A commercially available anti-FLCN antibody detected the overexpressed mutant protein in Western Blots and this antibody was used to analyse the endogenous expression in patient tumour tissue with a clear signal being obtained by immunohistochemistry. To confirm this the authors generated transgenic cell lines using the TALEN technology that expresses GFP-fused versions of either the WT or the mutant protein and mimic the physiological situation. WT FLCN was detected by western blot but the mutant protein again showed markedly lower protein levels. This effect was partially reversed by treatment with MG-132, a proteasome inhibitor and chloroquine, suggesting that inhibition of both lysosomal and proteasomal degradation stabilized the mutant protein.

Additionally, fluorescence imaging revealed an altered subcellular localization of mutant FLCN comparing to the WT protein. WT FLCN localized to both cytoplasm and nucleus and the mutant protein was restricted to the cytoplasm. Treatment with MG-132 not only stabilized the mutant protein but also led to a nuclear localization shift making it more similar to the WT. This may be the consequence of accumulation of ubiquitinated mutant FLCN, since ubiquitination has been shown to be a key regulator of subcellular localization of different proteins. It remains to be elucidated whether a ubiquitination or similar approach may have any therapeutic implication in the treatment of BHD – once stabilized and correctly localized – to fix the molecular function required to prevent tumorigenesis.

While this manuscript was under revision a different group characterized the same mutation in two sisters with RCC and found similar results regarding splicing of WT and mutant FLCN (Rossing et al., 2017).

In summary, this study shows that the functional characterization of the pathogenic mutations in BHD syndrome may shed light into further research for the development of novel diagnostic and therapeutic strategies.

  • Bartram MP, Mishra T, Reintjes N, Fabretti F, Gharbi H, Adam AC, Göbel H, Franke M, Schermer B, Haneder S, Benzing T, Beck BB, & Müller RU (2017). Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer. BMC medical genetics, 18 (1) PMID: 28499369