title: The modifier role of RET-G691S polymorphism in hereditary medullary thyroid carcinoma: functional characterization and expression/penetrance studies.
abstract: 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.
authors.affiliation: Department of Clinical Sciences and Community Health, University of Milan, and Endocrine Unit, Fondazione IRCCS Ca' Granda, Milan, Italy. carla.colombo1@unimi.it.
authors.forename: Carla
authors.initials: C
authors.lastname: Colombo
authors.affiliation: Molecular Mechanisms Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. emanuela.minna@istitutotumori.mi.it.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Minna
authors.affiliation: Molecular Mechanisms Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. mariagrazia.rizzetti@istitutotumori.mi.it.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Rizzetti
authors.affiliation: Molecular Mechanisms Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. paola.romeo@istitutotumori.mi.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Romeo
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. daniele.lecis@istitutotumori.mi.it.
authors.forename: Daniele
authors.initials: D
authors.lastname: Lecis
authors.affiliation: Department of Clinical Sciences and Community Health, University of Milan, and Division of Endocrine and Metabolic Diseases, Ospedale San Luca, IRCCS Istituto Auxologico Italiano, Milan, Italy. luca.persani@unimi.it.
authors.forename: Luca
authors.initials: L
authors.lastname: Persani
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. piermonde@alice.it.
authors.forename: Piera
authors.initials: P
authors.lastname: Mondellini
authors.affiliation: Scientific Directorate, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. marco.pierotti@istitutotumori.mi.it.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Pierotti
authors.affiliation: Molecular Mechanisms Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. angela.greco@istitutotumori.mi.it.
authors.forename: Angela
authors.initials: A
authors.lastname: Greco
authors.affiliation: Department of Pathophysiology and Transplantation, Endocrine Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, University of Milan, Milan, Italy. laura.fugazzola@policlinico.mi.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Fugazzola
authors.affiliation: Molecular Mechanisms Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. mariagrazia.borrello@istitutotumori.mi.it.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Borrello
mesh_terms: D000818:Animals; D018276:Carcinoma, Medullary / Q000151:congenital* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation / Q000502:physiology; D023281:Genomics; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018813:Multiple Endocrine Neoplasia Type 2a / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D009154:Mutation; D041681:NIH 3T3 Cells; D019683:Penetrance; D011110:Polymorphism, Genetic; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism*; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human