Background
Hereditary medullary thyroid carcinoma (MTC) is caused by germ-line gain of function mutations in the RET proto-oncogene, and a phenotypic variability among carriers of the same mutation has been reported. We recently observed this phenomenon in a large familial MTC (FMTC) family carrying the RET -S891A mutation. Among genetic modifiers affecting RET -driven MTC, a role has been hypothesized for RET -G691S non-synonymous polymorphism, though the issue remains controversial. Aim of this study was to define the in vitro contribution of RET -G691S to the oncogenic potential of the RET -S891A, previously shown to harbour low transforming activity.
Methods
The RET -S891A and RET -G691S/S891A mutants were generated by site-directed mutagenesis, transiently transfected in HEK293T cells and stably expressed in NIH3T3 cells. Their oncogenic potential was defined by assessing the migration ability by wound healing assay and the anchorage-independent growth by soft agar assay in NIH3T3 cells stably expressing either the single or the double mutants. Two RET -S891A families were characterised for the presence of RET -G691S.
Results
The functional studies demonstrated that RET -G691S/S891A double mutant displays a higher oncogenic potential than RET -S891A single mutant, assessed by focus formation and migration ability. Moreover, among the 25 RET -S891A carriers, a trend towards an earlier age of diagnosis was found in the MTC patients harboring RET -S891A in association with RET -G691S.
Conclusions
We demonstrate that the RET -G691S non-synonymous polymorphism enhances in vitro the oncogenic activity of RET -S891A. Moreover, an effect on the phenotype was observed in the RET -G691S/S891A patients, thus suggesting that the analysis of this polymorphism could contribute to the decision on the more appropriate clinical and follow-up management.