title: Four novel RET germline variants in exons 8 and 11 display an oncogenic potential in vitro.
abstract: BACKGROUND
  Most germline-activating mutations of the RET proto-oncogene associated with inherited medullary thyroid cancer (MTC) are localized in exons 10, 11 and 13-15. Four novel RET variants, located in the extracellular domain (p.A510V, p.E511K and p.C531R) coded by exon 8 and in the intracellular juxtamembrane region (p.K666N) coded by exon 11, were identified on the leukocyte DNA from apparently sporadic cases.
  
  
  METHODS
  Plasmids carrying Ret9-wild-type (Ret9-WT), Ret9-C634R and all Ret9 variants were transfected, and the phosphorylation levels of RET and ERK were evaluated by western blot analyses. The transforming potentials were assessed by the focus formation assay.
  
  
  RESULTS
  The p.A510V, p.E511K and p.C531R variants were found to generate RET and ERK phosphorylation levels and to have a transforming activity higher than that of Ret9-WT variant, but lower than that of Ret9-C634R variant. Differently, the p.K666N variant, located immediately downstream of the transmembrane domain, and involving a conserved residue, displayed high kinase and transforming activities. Computational analysis predicted non-conservative alterations in the mutant proteins consistent with putative modifications of the receptor conformation.
  
  
  CONCLUSIONS
  The molecular analyses revealed an oncogenic potential for all the novel germline RET variants. Therefore, the prevalence of exon 8 genomic variations with an oncogenic potential may be higher than previously thought, and the analysis of this exon should be considered after the exclusion of mutations in the classical hotspots. In addition, on the basis of these functional data, it is advisable to extend the genetic screening to all the first-degree relatives of the MTC patients, and to perform a strict follow-up of familial carriers.
authors.affiliation: Dipartimento di Scienze Mediche, Università degli Studi di Milano, 20122 Milan, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Muzza
authors.forename: Daniela
authors.initials: D
authors.lastname: Cordella
authors.forename: Johny
authors.initials: J
authors.lastname: Bombled
authors.forename: Brigitte
authors.initials: B
authors.lastname: Bressac-de Paillerets
authors.forename: Fabiana
authors.initials: F
authors.lastname: Guizzardi
authors.forename: Zelia
authors.initials: Z
authors.lastname: Francis
authors.forename: Paolo
authors.initials: P
authors.lastname: Beck-Peccoz
authors.forename: Martin
authors.initials: M
authors.lastname: Schlumberger
authors.forename: Luca
authors.initials: L
authors.lastname: Persani
authors.forename: Laura
authors.initials: L
authors.lastname: Fugazzola
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D018276:Carcinoma, Medullary / Q000235:genetics* / Q000378:metabolism; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005091:Exons; D005260:Female; D020022:Genetic Predisposition to Disease; D018095:Germ-Line Mutation; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D004273:DNA, Neoplasm; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret