Birt-Hogg-Dubé (BHD) syndrome is characterised by skin lesions; lung cysts and predisposition to pneumothoraces; and predisposition to kidney cancer. It is caused by inactivating mutations in the FLCN gene, and is inherited in an autosomal dominant manner.
Although BHD is caused by the loss of just a single FLCN allele, the mechanism of how this leads to pathogenesis is unknown. It is thought that FLCN acts as a tumour suppressor in the kidney, as somatic loss of the wildtype allele is seen in a high proportion of tumours (Vocke et al., 2005). Currently there is no evidence to suggest that FLCN has such tumour suppressor activity in the skin or the lungs, suggesting that its behaviour may vary in different cell types. Indeed, van Steensel et al. (2007) found no evidence of somatic mutation or loss of heterozygosity in the fibrofolliculomas of three confirmed BHD patients. Thus, it is possible that FLCN is a haploinsufficient gene, meaning that both gene copies are required in order for FLCN to fully function, or it is possible that the mutant form of FLCN exerts a dominant negative effect on the wildtype FLCN protein, preventing its function, meaning that these cells are functionally null for FLCN despite having a full coding copy of the gene.
93% of all pathogenic FLCN mutations are truncating (LOVD FLCN mutation database, accessed on 08/08/2013), while the remaining 7% of mutations are missense. A truncating mutation is one that shortens the wildtype protein and it is thought that the majority of such aberrant RNA transcripts are degraded by the nonsense mediated decay surveillance pathway, before they are translated into proteins, or that any aberrant proteins would be unstable and quickly degraded. Missense mutations substitute one amino acid for another, leaving the rest of the protein code intact, meaning that their effect is more variable and must be determined on a case-by-case basis. However, missense mutations that are seen in families with BHD syndrome must be deleterious to FLCN function or stability in order to cause disease symptoms to develop.
If mutant FLCN transcripts are degraded, BHD syndrome is caused by haploinsufficiency of the FLCN protein. A study by Nahorski et al. (2011) showed that eight of ten mutant FLCN proteins showed reduced stability in vitro, suggesting that the majority of mutant FLCN proteins are degraded.
For a mutant protein to be able to exert a dominant negative effect on its wildtype counterpart, the aberrant transcript or protein must evade rapid degradation. Indeed, a number of studies have shown mutant FLCN proteins to be expressed. Menko et al. (2012) reported that a tumour resected from a BHD patient showed robust immunohistochemical staining for FLCN protein expression, despite the tumour carrying truncating mutations in both FLCN alleles; Luijten et al. (2013) saw the expression of four BHD-associated mutant FLCN proteins, albeit at low levels, in FLCN-null HK-2 cells; and Laviolette et al. (2013) show that two mutations (c.1408_1418del11 and c.469_471del3) are expressed in UOK-257 cells with reduced stability. Both the Luijten et al. and Laviolette et al. studies show that the disease-associated K508R missense mutation is expressed at levels similar to wildtype FLCN.
It is also interesting to note that the FLCN gene is part of a region of chromosome 17 that is deleted in Smith-Magenis Syndrome (SMS) (Lucas et al., 2002). However, SMS patients do not seem to develop any of the symptoms of BHD, except in one reported case of a patient who suffered three pneumothoraces as a child (Truong et al., 2010). Cases where partial gene deletion has more severe consequences than whole gene deletion is a hallmark of a dominant negative gene. However, a recurrant FLCN mutation seen in BHD patients is the deletion of exon 1, which includes the promoter region (Benhammou et al., 2011). Deletion of this exon ablates transcription of this allele altogether, meaning that no protein would be produced, and yet patients carrying this mutation do develop symptoms, which is a hallmark of haploinsufficiency (Benhammou et al., 2011).
Taken together, it seems likely that although up to 80% of mutant FLCN proteins are probably unstable (Nahorski et al., 2011), a few might be stably expressed – for example the missense mutation K508R – either with altered or dominant negative function. As well as being mutation specific, it is possible that how mutant FLCN proteins interact with wildtype FLCN may be context or cell-specific. Indeed, FLCN seems to behave recessively with respect to kidney cancer, with inactivation of the wildtype allele through loss of heterozygosity or somatic mutation seen in a high proportion of tumours (Vocke et al., 2005).
If FLCN behaves haploinsufficiently, the best therapeutic approach would be to increase the amount of FLCN protein, or introduce a small molecule FLCN mimic, in heterozygous cells. However, if mutant FLCN proteins have a dominant negative function, one could aim to specifically inhibit the mutant FLCN protein, thus allowing the remaining wildtype protein to function normally. It is of course possible that some specific FLCN mutations have a dominant negative effect, and also that wildtype FLCN behaves haploinsufficiently: in this case both therapeutic approaches could be adopted. Therefore, determining the mechanism by which a FLCN mutation causes BHD symptoms to develop, and whether this mechanism varies in different organs and cell types, is vital to inform the development of appropriate therapies.
- Benhammou JN, Vocke CD, Santani A, Schmidt LS, Baba M, Seyama K, Wu X, Korolevich S, Nathanson KL, Stolle CA, & Linehan WM (2011). Identification of intragenic deletions and duplication in the FLCN gene in Birt-Hogg-Dubé syndrome. Genes, chromosomes & cancer, 50 (6), 466-77 PMID: 21412933
- Laviolette LA, Wilson J, Koller J, Neil C, Hulick P, Rejtar T, Karger B, Teh BT, & Iliopoulos O (2013). Human Folliculin Delays Cell Cycle Progression through Late S and G2/M-Phases: Effect of Phosphorylation and Tumor Associated Mutations. PloS one, 8 (7) PMID: 23874397
- Lucas RE, Vlangos CN, Das P, Patel PI, & Elsea SH (2001). Genomic organisation of the approximately 1.5 Mb Smith-Magenis syndrome critical interval: transcription map, genomic contig, and candidate gene analysis. European journal of human genetics : EJHG, 9 (12), 892-902 PMID: 11840190
- Luijten MN, Basten SG, Claessens T, Vernooij M, Scott CL, Janssen R, Easton JA, Kamps MA, Vreeburg M, Broers JL, van Geel M, Menko FH, Harbottle RP, Nookala RK, Tee AR, Land SC, Giles RH, Coull BJ, & van Steensel MA (2013). Birt-Hogg-Dube syndrome is a novel ciliopathy. Human molecular genetics PMID: 23784378
- Menko FH, Johannesma PC, van Moorselaar RJ, Reinhard R, van Waesberghe JH, Thunnissen E, Houweling AC, Leter EM, Waisfisz Q, van Doorn MB, Starink TM, Postmus PE, Coull BJ, van Steensel MA, & Gille JJ (2012). A de novo FLCN mutation in a patient with spontaneous pneumothorax and renal cancer; a clinical and molecular evaluation. Familial cancer PMID: 23264078
- Nahorski MS, Reiman A, Lim DH, Nookala RK, Seabra L, Lu X, Fenton J, Boora U, Nordenskjöld M, Latif F, Hurst LD, & Maher ER (2011). Birt Hogg-Dubé syndrome-associated FLCN mutations disrupt protein stability. Human mutation, 32 (8), 921-9 PMID: 21538689
- Truong HT, Dudding T, Blanchard CL, & Elsea SH (2010). Frameshift mutation hotspot identified in Smith-Magenis syndrome: case report and review of literature. BMC medical genetics, 11 PMID: 20932317
- van Steensel MA, Verstraeten VL, Frank J, Kelleners-Smeets NW, Poblete-Gutiérrez P, Marcus-Soekarman D, Bladergroen RS, Steijlen PM, & van Geel M (2007). Novel mutations in the BHD gene and absence of loss of heterozygosity in fibrofolliculomas of Birt-Hogg-Dubé patients. The Journal of investigative dermatology, 127 (3), 588-93 PMID: 17124507
- Vocke CD, Yang Y, Pavlovich CP, Schmidt LS, Nickerson ML, Torres-Cabala CA, Merino MJ, Walther MM, Zbar B, & Linehan WM (2005). High frequency of somatic frameshift BHD gene mutations in Birt-Hogg-Dubé-associated renal tumors. Journal of the National Cancer Institute, 97 (12), 931-5 PMID: 15956655
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.