title: Functional characterization of the MTC-associated germline RET-K666E mutation: evidence of oncogenic potential enhanced by the G691S polymorphism.
abstract: Activating mutations of RET, a gene encoding two isoforms of a tyrosine kinase receptor physiologically expressed in several neural crest-derived cell lineages, are associated with the inherited forms of medullary thyroid carcinoma (MTC). The identification and characterization of novel RET mutations involved in MTC is valuable, as RET gene testing plays a crucial role in the management of these patients. In an MTC patient, we have identified a germline c.1996A>G transition in heterozygosis leading to K666E substitution. In addition, the conservative S904S (c.2712C>G) and the non-conservative functional G691S (c.2071G>A) polymorphisms have been identified. Through functional studies, we demonstrate for the first time that K666E is a gain-of-function mutation with oncogenic potential, based on its ability to transform NIH3T3 cells. It was not possible to define whether K666E is a de novo or inherited RET variant in the patient, as the family history was negative for MTC, and the carrier status of family members could not be tested. Our results, together with a recent report of co-segregation of the mutation in three MTC families, suggest that K666E is a causative MTC mutation. As we have shown that the same patient allele carries both K666E and G691S variants, the latter known to increase downstream RET signaling, a possible role for the G691S polymorphism has also been investigated. We have demonstrated that, although RET-G691S is not oncogenic per se, it enhances the transforming activity of the RET-K666E mutant, thus suggesting a modifier role for this functional polymorphism.
authors.affiliation: Operative Unit Molecular Mechanisms, Department of Experimental Oncology, IRCCS Istituto Nazionale dei Tumori Foundation, Milan, Italy. mariagrazia.borrello@istitutotumori.mi.it
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Borrello
authors.forename: Antonella
authors.initials: A
authors.lastname: Aiello
authors.forename: Bernard
authors.initials: B
authors.lastname: Peissel
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Rizzetti
authors.forename: Piera
authors.initials: P
authors.lastname: Mondellini
authors.forename: Debora
authors.initials: D
authors.lastname: Degl'Innocenti
authors.forename: Veronica
authors.initials: V
authors.lastname: Catalano
authors.forename: Morena
authors.initials: M
authors.lastname: Gobbo
authors.forename: Paola
authors.initials: P
authors.lastname: Collini
authors.forename: Italia
authors.initials: I
authors.lastname: Bongarzone
authors.forename: Marco A
authors.initials: MA
authors.lastname: Pierotti
authors.forename: Angela
authors.initials: A
authors.lastname: Greco
authors.forename: Ettore
authors.initials: E
authors.lastname: Seregni
mesh_terms: D000818:Animals; D015153:Blotting, Western; D018276:Carcinoma, Medullary / Q000151:congenital; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D018095:Germ-Line Mutation / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D051379:Mice; D008875:Middle Aged; D018813:Multiple Endocrine Neoplasia Type 2a; D041681:NIH 3T3 Cells; D009386:Neoplastic Syndromes, Hereditary / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics* / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
chemical_list: D004273:DNA, Neoplasm; D051096:Proto-Oncogene Proteins c-ret