The modifier role of RET-G691S polymorphism in hereditary medullary thyroid carcinoma: functional characterization and expression/penetrance studies

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Abstract

Background

Hereditary medullary thyroid carcinoma (MTC) is caused by germ-line gain of function mutations in the RET proto-oncogene, and a phenotypic variability among carriers of the same mutation has been reported. We recently observed this phenomenon in a large familial MTC (FMTC) family carrying the RET-S891A mutation. Among genetic modifiers affecting RET-driven MTC, a role has been hypothesized for RET-G691S non-synonymous polymorphism, though the issue remains controversial. Aim of this study was to define the in vitro contribution of RET-G691S to the oncogenic potential of the RET-S891A, previously shown to harbour low transforming activity.

Methods

The RET-S891A and RET-G691S/S891A mutants were generated by site-directed mutagenesis, transiently transfected in HEK293T cells and stably expressed in NIH3T3 cells. Their oncogenic potential was defined by assessing the migration ability by wound healing assay and the anchorage-independent growth by soft agar assay in NIH3T3 cells stably expressing either the single or the double mutants. Two RET-S891A families were characterised for the presence of RET-G691S.

Results

The functional studies demonstrated that RET-G691S/S891A double mutant displays a higher oncogenic potential than RET-S891A single mutant, assessed by focus formation and migration ability. Moreover, among the 25 RET-S891A carriers, a trend towards an earlier age of diagnosis was found in the MTC patients harboring RET-S891A in association with RET-G691S.

Conclusions

We demonstrate that the RET-G691S non-synonymous polymorphism enhances in vitro the oncogenic activity of RET-S891A. Moreover, an effect on the phenotype was observed in the RET-G691S/S891A patients, thus suggesting that the analysis of this polymorphism could contribute to the decision on the more appropriate clinical and follow-up management.

Electronic supplementary material

The online version of this article (doi:10.1186/s13023-015-0231-z) contains supplementary material, which is available to authorized users.

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Most cited references 31

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RET tyrosine kinase signaling in development and cancer.

Elena Arighi, Maria Grazia Borrello, Hannu Sariola (2015)

The variety of diseases caused by mutations in RET receptor tyrosine kinase provides a classic example of phenotypic heterogeneity. Gain-of-function mutations of RET are associated with human cancer. Gene rearrangements juxtaposing the tyrosine kinase domain to heterologous gene partners have been found in sporadic papillary carcinomas of the thyroid (PTC). These rearrangements generate chimeric RET/PTC oncogenes. In the germline, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Both MEN 2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, activate the RET downstream targets. Loss-of-function mutations of RET cause Hirschsprung's disease (HSCR) or colonic aganglionosis. A deeper understanding of the molecular signaling of normal versus abnormal RET activity in cancer will enable the development of potential new treatments for patients with sporadic and inherited thyroid cancer or MEN 2 syndrome. We now review the role and mechanisms of RET signaling in development and carcinogenesis.

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Multiple endocrine neoplasia type 2 syndromes (MEN 2): results from the ItaMEN network analysis on the prevalence of different genotypes and phenotypes.

Cristina Romei, Augusto Taccaliti, Isabella Ceccherini … (2010)

Multiple endocrine neoplasia type 2 (MEN 2) is a genetic disease characterized by medullary thyroid carcinoma (MTC) associated (MEN 2A and 2B) or not familial MTC (FMTC) with other endocrine neoplasia due to germline RET gene mutations. The prevalence of these rare genetic diseases and their corresponding RET mutations are unknown due to the small size of the study population. We collected data on germline RET mutations of 250 families with hereditary MTC followed in 20 different Italian centres. The most frequent RET amino acid substitution was Val804Met (19.6%) followed by Cys634Arg (13.6%). A total of 40 different germline RET mutations were present. Six families (2.4%) were negative for germline RET mutations. The comparison of the prevalence of RET germline mutations in the present study with those published by other European studies showed a higher prevalence of Val804Met and Ser891Ala mutations and a lower prevalence of Leu790Phe and Tyr791Phe (P<0.0001). A statistically significant higher prevalence of mutations affecting non-cysteine codons was also found (P<0.0001). Furthermore, the phenotype data collection showed an unexpected higher prevalence of FMTC (57.6%) with respect to other MEN 2 syndromes (34% MEN 2A and 6.8% of MEN 2B). In conclusion, we observed a statistically significant different pattern of RET mutations in Italian MEN 2 families with respect to other European studies and a higher prevalence of FMTC phenotype. The different ethnic origins of the patients and the particular attention given to analysing apparently sporadic MTC for RET germline mutations may explain these findings.

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Multiple endocrine neoplasia type 2 RET protooncogene database: repository of MEN2-associated RET sequence variation and reference for genotype/phenotype correlations.

Todd R Seamons, Fernanda R O Calderon, Linda Margraf … (2009)

Multiple endocrine neoplasia type 2 (MEN2) is an inherited, autosomal-dominant disorder caused by deleterious mutations within the RET protooncogene. MEN2 RET mutations are mainly heterozygous, missense sequence changes found in RET exons 10, 11, and 13-16. Our group has developed the publicly available, searchable MEN2 RET database to aid in genotype/phenotype correlations, using Human Genome Variation Society recommendations for sequence variation nomenclature and database content. The MEN2 RET database catalogs all RET sequence variation relevant to the MEN2 syndromes, with associated clinical information. Each database entry lists a RET sequence variation's location within the RET gene, genotype, pathogenicity classification, MEN2 phenotype, first literature reference, and comments (which may contain information on other clinical features, complex genotypes, and additional literature references). The MEN2 phenotype definitions were derived from the International RET Mutation Consortium guidelines for classification of MEN2 disease phenotypes. Although nearly all of the 132 RET sequence variation entries initially cataloged in the database were from literature reports, novel sequence variation and updated phenotypic information for any existing database entry can be submitted electronically on the database website. The database website also contains links to selected MEN2 literature reviews, gene and protein information, and RET reference sequences. The MEN2 RET database (www.arup.utah.edu/database/MEN2/MEN2_welcome.php) will serve as a repository for MEN2-associated RET sequence variation and reference for RET genotype/MEN2 phenotype correlations. (c) 2009 Wiley-Liss, Inc.

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Author and article information

Contributors

Carla Colombo: carla.colombo1@unimi.it

Emanuela Minna: emanuela.minna@istitutotumori.mi.it

Maria Grazia Rizzetti: mariagrazia.rizzetti@istitutotumori.mi.it

Paola Romeo: paola.romeo@istitutotumori.mi.it

Daniele Lecis: daniele.lecis@istitutotumori.mi.it

Luca Persani: luca.persani@unimi.it

Piera Mondellini: piermonde@alice.it

Marco A Pierotti: marco.pierotti@istitutotumori.mi.it

Angela Greco: angela.greco@istitutotumori.mi.it

Laura Fugazzola: laura.fugazzola@policlinico.mi.it

Maria Grazia Borrello: mariagrazia.borrello@istitutotumori.mi.it

Journal

Journal ID (nlm-ta): Orphanet J Rare Dis

Journal ID (iso-abbrev): Orphanet J Rare Dis

Title: Orphanet Journal of Rare Diseases

Publisher: BioMed Central (London )

ISSN (Electronic): 1750-1172

Publication date (Electronic): 1 March 2015

Publication date PMC-release: 1 March 2015

Publication date Collection: 2015

Volume: 10

Electronic Location Identifier: 25

Affiliations

[ ]Department of Clinical Sciences and Community Health, University of Milan, and Endocrine Unit, Fondazione IRCCS Ca’ Granda, Milan, Italy

[ ]Molecular Mechanisms Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

[ ]Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

[ ]Department of Clinical Sciences and Community Health, University of Milan, and Division of Endocrine and Metabolic Diseases, Ospedale San Luca, IRCCS Istituto Auxologico Italiano, Milan, Italy

[ ]Scientific Directorate, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

[ ]Department of Pathophysiology and Transplantation, Endocrine Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, University of Milan, Milan, Italy

Article

Publisher ID: 231

DOI: 10.1186/s13023-015-0231-z

PMC ID: 4373282

PubMed ID: 25887804

SO-VID: c4903d7c-9d6a-4475-ae0f-39ee846e2f3e

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.