Functional characterization of the novel BRAF complex mutation, BRAF V600delinsYM, identified in papillary thyroid carcinoma

摘要

An activating mutation in the BRAF gene is the most common genetic alteration in papillary thyroid carcinomas (PTCs). The mutation in PTCs is almost a c.1799TA transversion, resulting in a p.V600E amino acid substitution (BRAFV600E). Here, we report a novel complex BRAF mutation identified in 4/492 Japanese PTC cases (0.81%). The mutation was comprised of one nucleotide substitution at position 1798, followed by an in-frame insertion of three nucleotides, c.1798delinsTACA in Exon 15, resulting in p.V600delinsYM. In silico three-dimensional protein structure prediction implied altered kinase activity of this mutant. In vitro kinase assay and western blotting revealed that this mutation conferred high kinase activity on the BRAF protein, leading to constitutive activation of the MAPK signaling pathway. The mutation also showed high transforming ability in focus formation assay using NIH3T3 cells. The degree of all the functional characteristics was comparable to that of BRAFV600E, and treatment with a BRAF inhibitor Sorafenib was also equally effective in this mutant. These findings suggest that the novel BRAF mutation, BRAFV600delinsYM, is a gain-of-function mutation and plays an important role in PTC development. What's new? Most papillary thyroid cancer results from mutation in the BRAF gene. Commonly, oncogenic mutations disrupt the inactive form of BRAF, causing it to become "on" all the time, activating the MAPK cascade when it shouldn't. The authors identified a new mutation in BRAF, and they show that this one also ramps up the protein's kinase activity, indiscriminately switching on the MAPK cascade. They also demonstrate that the new mutation transforms cells, suggesting that it could lead to papillary thyroid cancer formation.