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Molecular heterogeneity of RET loss of function in Hirschsprung's disease
F Carlomagno1, G De Vita, M T Berlingieri
1Centro di Endocrinologia ed Oncologia Sperimentale del CNR, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli, Italy.
Abstract:
The RET proto-oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non-neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non-neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprung's disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild-type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972-->Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32-->Leu) and HSCR393 (Phe393-->Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.
Insights
Mutations in the RET proto-oncogene cause Hirschsprung's disease (HSCR) by impairing RET protein function. These HSCR mutations lead to loss of RET kinase activity or improper protein maturation and cell surface transport.
Area of Science:
- Oncology
- Genetics
- Cell Biology
Background:
- The RET proto-oncogene is crucial for development and implicated in various cancers and Hirschsprung's disease (HSCR).
- RET mutations drive thyroid tumors and MEN syndromes, while germline mutations cause HSCR, a congenital disorder.
Purpose of the Study:
- To investigate the functional impact of HSCR-associated RET mutations.
- To elucidate the molecular mechanisms underlying RET dysfunction in HSCR.
Main Methods:
- Introduction of three HSCR missense mutations into wild-type and oncogenic RET alleles (RET(MEN2A), RET(MEN2B)).
- Functional assays in NIH 3T3 and PC12 cell systems to assess RET activity.
- Analysis of protein maturation and cell surface transport.
Main Results:
- All three HSCR mutations resulted in a loss of RET function in cellular models.
- The HSCR972 mutation impaired RET tyrosine kinase activity.
- Extracellular HSCR32 and HSCR393 mutations disrupted RET protein maturation and cell surface transport.
Conclusions:
- HSCR-associated RET mutations lead to loss-of-function through distinct molecular mechanisms.
- Understanding these mechanisms is vital for comprehending HSCR pathogenesis and developing therapeutic strategies.