Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2007) 14 P117

ECE2007 Poster Presentations (1) (659 abstracts)

A novel activating germline mutation in the RET gene (Y606C) in a patient with medullary thyroid carcinoma

Michaela Luconi , Tonino Ercolino , Adriana Lombardi , Lucia Becherini , Elisabetta Piscitelli , Maria Sole Gaglianò , Mario Serio & Massimo Mannelli

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University of Florence, Dept. Clinical Physiopathology, Florence, Italy.


Germline mutations in the RET gene cause MEN2, an inherited cancer syndrome associated with medullary thyroid carcinoma (MTC). We performed genetic analysis on DNA from whole blood of a 58 yr old female affected by a multifocal MTC. Exons 10, 11, 13, 15 and 16 of RET gene were amplified by PCR using specific primers and characterised by direct automatic sequencing. Here, we report a new RET point mutation: a heterozigous missense mutation Y606C, a G to A nucleotide substitution leading to a Tyrosine (Y) to Cysteine (C) amino acid change in exon 10. We approached the functional effects of such a mutation in an in vitro system by cloning the wild-type RET, the Y606C mutation as well as the C620Y mutation, previously described as less strong RET oncogene associated with MTC, in an expression vector and transiently transfecting NIH3T3 fibroblasts. All mutations were obtained by site-directed mutagenesis. We first demonstrated by western blot analysis using a specific antibody an increased tyrosine phosphorylation in the Y905 residue in the RET/Y606C, corresponding to receptor activation. Since RET activation results in an intracellular signalling cascade leading to extracellular signal regulated kinases (ERKs), we investigated ERK activity in our transfected cells. Results demonstrate a significant increase in ERK2 phosphorylation/activation in the RET/Y606C versus the wild type and RET/C620Y. We finally showed by gel electrophoresis of transfected cell lysates in non reducing conditions that the introduction of a C due to the Y606C mutation results in an increased dimerization of the receptor. All these findings suggest that the Y606C mutation confers constitutive activation of RET signalling.

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