Abstract
BackgroundHereditary 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.MethodsThe 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.ResultsThe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0231-z) contains supplementary material, which is available to authorized users.
Highlights
Hereditary medullary thyroid carcinoma (MTC) is caused by germ-line gain of function mutations in the rearranged during transfection (RET) proto-oncogene, and a phenotypic variability among carriers of the same mutation has been reported
Hereditary forms account for 25% of cases and include multiple endocrine neoplasia syndromes type 2A (MEN2A), type 2B (MEN2B) and familial MTC (FMTC), caused by mutations in the rearranged during transfection (RET) proto-oncogene
Agar colonies size, calculated as mean diameter, was determined by the count/size tool in Image-Pro Plus 7.0.1 software analyzing the agar colonies images the day Transforming activity of RET-S891A and RET-G691S/S891A mutants To assess the contribution of the RET-G691S polymorphism to the in vitro oncogenic potential of RET-S891A mutant, both localized in the intracellular domains of RET protein (Figure 1A), we generated RET-S891A single and RET-G691S/S891A double mutants by site-directed mutagenesis of recombinant plasmids carrying the RET-wt long isoform
Summary
In vitro analyses Construction of the RET mutants All RET mutants were obtained by mutagenesis of RET51wt construct (pCDNA3 vector expressing the proto-RET gene long isoform). Transfected cells were grown in DMEM with 10% serum and selected in the presence of G418 antibiotic (650 μg/ml) to obtain G418-resistant colonies indicative of transfection efficiency; transformed foci were obtained in DMEM with 5% serum in the presence of chronic stimulation with the RET ligand GDNF at a concentration of 10 ng/ml (AlomoneLabs, Jerusalem, Israel). Genomic DNA was extracted from peripheral blood leucocytes of FMTC families members and the presence of RET-G691S polymorphism was assessed by PCR and direct sequencing as previously described [23]. All patients gave their informed consent, approved by the local Ethical Committee, to the genetic characterization and to the analysis of data. Agar colonies size, calculated as mean diameter, was determined by the count/size tool in Image-Pro Plus 7.0.1 software analyzing the agar colonies images the day
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