Novel BRAFI599Ins Mutation Identified in a Follicular Variant of Papillary Thyroid Carcinoma: A Molecular Modeling Approach

Abstract

Objective: BRAF mutations are the most common genetic alteration found in papillary thyroid carcinoma (PTC). Approximately, 90% correspond to BRAFV600E, although other less common BRAF mutations have been described. The aim of this study was to describe a new mutation on BRAF gene discovered on the previous thyroid cytology of a patient diagnosed with a follicular variant of PTC (FV-PTC). Methods: The mutation was identified by independent cloning of the 2 alleles and direct sequencing in the previous cytology and tumor tissue samples from a patient diagnosed with FV-PTC. To elucidate the effect of the mutation on the structure and hence on the activating mechanism of the protein, the structures of BRAFI599Ins, BRAFT599Ins, BRAFV599Ins and BRAFV600E were modeled by using the reconstructed wild-type BRAF (BRAFWT) crystal structure. Results: The novel mutation in BRAF consisted in the in-frame insertion of 3 nucleotides (TAA) after nucleotide 1795, resulting in the incorporation of an extra isoleucine residue at position 599 (BRAFI599Ins) of the protein. The structural comparison of BRAFI599Ins, BRAFT599Ins, BRAFV599Ins with BRAFWT, and BRAFV600E models revealed that the overall shape of the kinase was conserved in the protein produced by this novel mutation, except for the displacement of the activation loop (A-loop), as a direct consequence of the increase in loop size, and the exposition of 1 of the 2 residues involved in BRAF activation (T599), probably facilitating its phosphorylation. Conclusion: BRAFI599Ins mutation constitutes a new BRAF mutation affecting the length of the A-loop, which most likely facilitates BRAF activation by altering the A-loop conformation.