Endocrine Abstracts (2006) 11 P480

An in-frame complex germline mutation in the juxtamembrane intracellular domain causing RET activation in familial medullary thyroid carcinoma

L Fugazzola1, D Cordella2, M Muzza1, L Alberti2, P Travaglini3, P Colombo3, P Beck-Peccoz1 & L Persani2

1Institute of Endocrine Sciences, Fondazione Policlinico IRCCS, Milan, Italy; 2Institute of Endocrine Sciences, Istituto Auxologico Italiano, Milan, Italy; 3Istituto Clinico Humanitas, Milan, Italy.

Activating mutations of the RET proto-oncogene, encoding a tyrosine kinase receptor, are associated with inherited syndromes, MEN2A and MEN2B, and with both familial and sporadic medullary thyroid cancer (MTC). Single base-pair missense mutations in the extracellular cysteine-rich domain are responsible for the majority of MEN2A and familial MTC (FMTC) cases. Rarely, somatic deletions and germline duplications of variable segments of the gene have been reported in sporadic MTC and in FMTC. We report the detection and functional studies of a deletion/insertion (c.2646delGinsTTCT) in exon 11 associated with FMTC. This in-frame complex rearrangement leads to the substitution of an Asparagine for a Lysine (Lys666Asn) and to a Serine insertion. The mutation was found in the proband, who had a diagnosis of metastatic MTC at 41 years, and in her son, who presented diffuse C-cells hyperplasia at 4 years of age. After site-directed mutagenesis, pRC-CMV constructs were transiently transfected in 293T cells. Immunoprecipitation and Western blot with anti-Ret and anti-Ptyr specific antibodies demonstrated that the Ret9-delGinsTTCT mutant protein was significantly more phosphorylated than the wild-type. Therefore, even in the absence of ligands, Ret9-delGinsTTCT can be phosphorylated. Computational analysis performed by means of the PredictProtein server predicted significant changes into the protein folding as a result of this in-frame mutation. In particular, the transmembrane α-helical acquires a different conformation, which could impair the flexibility of the receptor at that level. Moreover, the content and distribution of β-strands is modified and the proportion of solvent-exposed residues decreases drastically.

In conclusion, the functional studies on a complex germline RET mutation lying in the juxtamembrane region of the receptor are reported. This mutation is predicted to significantly change the secondary structure of the protein and can activate Ret in a ligand independent manner.

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