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