title: In silico and in vitro analysis of rare germline allelic variants of RET oncogene associated with medullary thyroid cancer.
abstract: Germline and somatic RET oncogene mutations are found in 98% hereditary and 40% sporadic medullary thyroid carcinomas. Our aim was to analyse by in silico and in vitro assays the transforming activity of six rare RET mutations (T338I, V648I, M918V, A883T, S904F and M848T). Six known RET mutations were used as controls. The in silico analysis showed the highest score value (i.e. 65) for S904F, M848T, M918T and C634R, whereas L790F, G691S, T338I and V648I had 0 score. Intermediate score values were obtained by A883T (score=55), M918V, V804M and Y791F (score=15). The in vitro focus formation assay showed that cells transfected with S904F, M918T, M848T or C634R generated the largest number of focus formation units (FFU). Intermediate numbers of FFU were observed in cells transfected with M918V, V804M, Y791F or A883T, while cells transfected with L790F, G691S, T338I or V648I showed a number of FFU similar to control cells. A positive correlation between the in silico score and in vitro FFU was found (P=0.0005). Only cells transfected with M918T or C634R grew faster and generated higher number of colonies in soft agar than control cells. However, the cells that were transfected with V804M produced an intermediate number of colonies. In conclusion, two of the six rare RET mutations, S904F and M848T possessed a relatively high transforming activity but a low aggressiveness; the other four mutations T338I, V648I, M918V and A883T were low or non-transforming, and their ability to induce tumoural transformation might be related to particular genetic conditions.
authors.affiliation: Department of Endocrinology and Metabolism, High Technology Center for the Study of the Environmental Damage of the Endocrine and Nervous Systems, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Cosci
authors.forename: A
authors.initials: A
authors.lastname: Vivaldi
authors.forename: C
authors.initials: C
authors.lastname: Romei
authors.forename: F
authors.initials: F
authors.lastname: Gemignani
authors.forename: S
authors.initials: S
authors.lastname: Landi
authors.forename: R
authors.initials: R
authors.lastname: Ciampi
authors.forename: A
authors.initials: A
authors.lastname: Tacito
authors.forename: E
authors.initials: E
authors.lastname: Molinaro
authors.forename: L
authors.initials: L
authors.lastname: Agate
authors.forename: V
authors.initials: V
authors.lastname: Bottici
authors.forename: V
authors.initials: V
authors.lastname: Cappagli
authors.forename: D
authors.initials: D
authors.lastname: Viola
authors.forename: P
authors.initials: P
authors.lastname: Piaggi
authors.forename: P
authors.initials: P
authors.lastname: Vitti
authors.forename: A
authors.initials: A
authors.lastname: Pinchera
authors.forename: R
authors.initials: R
authors.lastname: Elisei
mesh_terms: D016475:3T3 Cells; D000328:Adult; D000483:Alleles; D000818:Animals; D018276:Carcinoma, Medullary / Q000235:genetics*; D003114:Colony-Forming Units Assay; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005260:Female; D014644:Genetic Variation; D018095:Germ-Line Mutation / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013964:Thyroid Neoplasms / Q000235:genetics*; D014162:Transfection
chemical_list: D004273:DNA, Neoplasm; D051096:Proto-Oncogene Proteins c-ret