The germline FLCN missense mutations H255Y (Hasumi et al., 2009) and K508R (Toro et al., 2008) have been identified in patients with bilateral multifocal (BMF) kidney tumours and other clinical symptoms of Birt-Hogg-Dube (BHD) syndrome, or with BMF kidney tumours as the only manifestation. Building on their previous work identifying the H255Y mutation in human BHD kidney tumour, Hasumi et al. (2016) investigated whether these mutations have an impact on FLCN function. The authors evaluated the FLCN missense mutations, H255Y and K508R in genetically engineered mice, and demonstrated that they both are pathogenic mutations that promote aberrant kidney cell proliferation to different degrees, with the potential for malignancy. In addition, the phenotypic effect of the Flcn K508R mutant expression in mice with wild-type Flcn by expressing the Flcn K508R mutant transgene in heterozygous Flcn knockout mice was examined.
The authors reported three patient cases. one patient had germline FLCN missense mutation H255Y andwas clinically diagnosed with BHD; the other two patients had germline FLCN missense mutation K508R and presented with kidney neoplasia but without cutaneous or pulmonary BHD manifestations. To evaluate the pathogenicity of these FLCN missense mutations, the authors looked for a somatic second hit mutation in the remaining FLCN allele in tumour samples from these patients. In a tumour that developed in the patient carrying H255Y mutation, a somatic intron 12 splice site mutation was identified, suggesting that the loss of the wild type FLCN allele led to kidney tumorigenesis. None of the renal tumours that developed in the patients with the FLCN K508R mutation had second hit somatic mutations in FLCN suggesting that haploinsufficiency of FLCN may be sufficient for tumour development in patients with the FLCN K508R mutation.
Using BAC transgenic technology, the authors established genetically engineered mouse models introducing each of the missense mutant transgenes into their kidney-targeted Flcn knockout mouse model, which develops enlarged polycystic kidneys (Baba et al., 2008), in order to determine if expression of the Flcn missense mutant protein could rescue the polycystic kidney phenotype.Expression of the Flcn H255Y mutant in the kidney-targeted Flcn knockout mouse model could not rescue the polycystic kidney phenotype of mice with Flcn deficient kidneys, mirroring the phenotype of kidney-targeted homozygous Flcn deletion.
Expression of the Flcn K508R mutant protein in the kidney-targeted Flcn knockout mouse model partially abrogated the Flcn-deficient phenotype but eventually lead to highly cystic kidneys, suggesting that both Flcn H255Y and K508R mutant proteins were functionally impaired resulting in aberrant kidney cell proliferation. The FLCN K508R missense mutation is responsible for the kidney tumour phenotype in patients with this mutation, but can be classified as ‘weak’ compared to the H255Y mutation.
Heterozygous (Het) Flcn knockout mice begin to develop renal cysts after 10 months of age but these mice do not display any phenotype in other organs (Hasumi et al., 2009). Expression of the Flcn K508R mutant in Het Flcn knockout mice caused the development of renal cystis and cardiac hypertrophy in 26 and 7% of mice, respectively, by 9 months of age, indicating that the Flcn K508R mutant protein may have a potential dominant negative effect on the function of wild-type Flcn to suppress cell growth of kidney cells and cardiomyocytes. These results suggest that increased copy number of FLCN K508R mRNA may predispose to cardiac hypertrophy suggesting that cardiac follow up should be considered in FLCN K508R patients.
In summary, germline missense mutations H255Y and K508R in the FLCN gene were associated with kidney tumours in patients with and without BHD cutaneous manifestations. Based on phenotypes resulting from their expression in kidney-targeted Flcn-deficient mouse models, FLCN H255Y and K508R are predicted to be pathogenic mutations that promote aberrant kidney cell proliferation. The FLCN K508R mutant protein may have a dominant negative effect on wild-type FLCN in kidney and heart. These findings provide further insight into the role of FLCN in regulating kidney tumourigenesis and will add knowledge to the development of novel therapies for kidney cancer.
- Hasumi H, Hasumi Y, Baba M, Nishi H, Furuya M, Vocke CD, Lang M, Irie N, Esumi C, Merino MJ, Kawahara T, Isono Y, Makiyama K, Warner AC, Haines DC, Wei MH, Zbar B, Hagenau H, Feigenbaum L, Kondo K, Nakaigawa N, Yao M, Metwalli AR, Marston Linehan W, & Schmidt LS (2016). H255Y and K508R missense mutations in tumour suppressor folliculin (FLCN) promote kidney cell proliferation. Human molecular genetics PMID: 28007907