Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer

Abstract

BackgroundRenal cell carcinoma is among the most prevalent malignancies. It is generally sporadic. However, genetic studies of rare familial forms have led to the identification of mutations in causative genes such as VHL and FLCN. Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé syndrome, a rare tumor syndrome which is characterized by the combination of renal cell carcinoma, pneumothorax and skin tumors.MethodsUsing Sanger sequencing we identify a heterozygous splice-site mutation in FLCN in lymphocyte DNA of a patient suffering from renal cell carcinoma. Furthermore, both tumor DNA and DNA from a metastasis are analyzed regarding this mutation. The pathogenic effect of the sequence alteration is confirmed by minigene assays and the biochemical consequences on the protein are examined using TALEN-mediated transgenesis in cultured cells.ResultsHere we describe an FLCN mutation in a 55-year-old patient who presented himself with progressive weight loss, bilateral kidney cysts and renal tumors. He and members of his family had a history of recurrent pneumothorax during the last few decades. Histology after tumor nephrectomy showed a mixed kidney cancer consisting of elements of a chromophobe renal cell carcinoma and dedifferentiated small cell carcinoma component. Subsequent FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. We demonstrate skipping of exon 11 to be the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. Interestingly, the truncated protein was still expressed both in cell culture and in tumor tissue, though it was strongly destabilized and its subcellular localization differed from wild-type FLCN. Both, altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation.ConclusionsIdentification of disease-causing mutations in BHD syndrome requires the analysis of intronic sequences. However, biochemical validation of the consecutive alterations of the resulting protein is especially important in these cases. Functional characterization of the disease-causing mutations in BHD syndrome may guide further research for the development of novel diagnostic and therapeutic strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-017-0416-5) contains supplementary material, which is available to authorized users.

Genotypic characteristics of Chinese patients with BHD syndrome and functional analysis of FLCN variants

“…Interestingly, the wild-type pCAS2-FLCN-E10–13 minigene produced an additional transcript with exon 11 skipped (Fig. 4a, Group 4), which has previously been observed in normal human cDNA and the product of another reported minigene containing FLCN exon 11 [15]. Moreover, the positive control minigene carrying variant c.249 + 1G > A produced an aberrant transcript (Fig.…”

Pathology of Birt–Hogg–Dubé syndrome: A special reference of pulmonary manifestations in a Japanese population with a comprehensive analysis and review

“…Intronic splice‐site mutations are expected to result in premature protein truncation. Most splice‐site mutations involve the 3′‐end of the affected intron (acceptor site), and are predicted to cause exon skipping in FLCN mRNAs . Fewer cases involve the 5′‐end of the affected intron (donor site), which are predicted to cause partial translation of intron regions …”

Folliculin variants linked to Birt-Hogg-Dubé syndrome are targeted for proteasomal degradation

“…The absence of nuclear-localized FLCN has been observed for an unstable FLCN splice variant before (Bartram et al, 2017), and considering the unstable nature of these proteins, this could suggest that the observed increased protein turnover is restricted to the pool of nuclear FLCN. However, repeating the localization experiments in the presence of bortezomib did not result in any increased nuclear localization of the unstable variants or did otherwise affect the subcellular distribution of the FLCN variants ( Fig.…”