Functional characterization of the T1799-1801del and A1799-1816ins BRAF mutations in papillary thyroid cancer.

This study was supported by NIH RO-1 grant CA113507-01. The RAS → RAF → MEK → MAP kinase/ERK signaling pathway (MAP kinase pathway) is a conserved protein kinase cascade that regulates cell growth, proliferation, and differentiation in response to growth factors, cytokines, and hormones.1 Constitutive activation of this pathway is common in human cancers. Activating mutations in the gene for the B-type RAF or BRAF were identified in many human cancers.2 Over 40 missense mutations have been identified in the BRAF gene, the majority of which clusters in exons 11 and 15 and affects residues located within the kinase domain of the protein.3 The T1799A point BRAF mutation in exon 15, resulting in a V600E substitution (i.e., valine replaced by glutamic acid) in BRAF protein accounts for about 90% of all the oncogenic BRAF mutations.3 Numerous studies have shown frequent occurrence of this mutation in papillary thyroid cancer (PTC), with a prevalence of 44% on average.4 PTC is the most common thyroid cancer, accounting for 80% of thyroid malignancies.5 A few other BRAF mutations were also observed in PTC, including BRAFK601E (formerly named BRAFK600E) and BRAF599ins mutations, and their oncogenic function were welldocumented.6,7 By direct genomic DNA sequencing, we identified two cases of PTC samples from a BRAF mutation study published previously,8 whose DNA sequencing chromatogram showed an overlapping pattern of nucleotide sequence starting at base position T1799 in the BRAF gene (Fig. 1). These could represent either deletion or insertion of nucleotides in the affected alleles mixed with wild alleles. To distinguish the two possibilities, we subcloned the PCR products from tissue samples into pGEM-T vector (Promega Corporation, Madison, WI, USA) and amplified and sequenced the subcloned DNA to delineate single alleles of the BRAF gene. As illustrated in Figure 1, we confirmed two different new mutations, T1799-1801del and A1799-1816ins, in exon 15 of the BRAF gene (Fig. 1). The T1799-1801del mutation, originating from a deletion of three nucleotides (TGA) at the base position 1799-1801, resulted in a deletion of amino acid K601and a V600E amino acid substitution in the resultant BRAF protein while preserving the translation frame of the coding region of the gene (Fig. 1A). This BRAF mutation was previously found in 3/8 lymph node-metastasized PTC.9 We did not have matched lymph node tissues to analyze this mutation in the present study. The A1799-1816ins mutation originated from an insertion of eighteen nucleotides at the base position 1799-1816, resulting in the insertion of six amino acids in the BRAF protein (Fig. 1B). The inserted sequence was identical to the sequence of T1781-1798 in the BRAF gene. This is a novel and previously unknown BRAF mutation, representing an interesting and unusual genetic mutation with a repeat of a relatively long DNA sequence in a functionally critical region of the gene. This mutation resulted in a V600D amino acid substitution (i.e., valine replaced by aspartic acid), followed by addition of five amino acids (Phe-Gly-Leu-AlaThr) in the resultant BRAF protein. We blasted this inserted sequence in GenBank but found no additional gene or DNA sequence that had homology to this insertion. Although the T1799-1801del BRAF mutation was reported previously in PTC,8 its function consequence has not been characterized. It would be particularly interesting to know whether the V600E substitution associated with the deletion of its neighbor K601 in the amino acid sequence of BRAF protein still confers the BRAF kinase constitutive activation and oncogenic function. It would be similarly interesting to know whether the D600 followed by five new amino acids in the BRAF mutant from the A1799-1816ins mutation was functional. To examine their functions, we generated the two BRAF mutations by in vitro mutagenesis using wild BRAF in pEFm6 (which, together with the BRAFV600E construct, was a gift from Dr. Richard Marais) as the template and appropriately designed primers and the Quick-Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). For T1799-1801del BRAF mutation, the mutagenesis primers used were 5'-GGT GAT TTT GGT CTA GCT ACA GAA TCT CGA TGG AGT GGG TCC