Figure 1. Novel BRAF alterations in pilocytic astrocytoma.

a, Schematic representation of the RNF130:BRAF fusion gene in ICGC_PA112 resulting from a translocation between chromosomes 5 and 7. A similar fusion was observed in ICGC_PA96. The cDNA sequence at the fusion breakpoint and resulting exon and protein structures are indicated. A reciprocal fusion between RUN and FYVE domain containing 1 (RUFY1) and transmembrane protein 178B (TMEM178B) on the derivative chromosome 5 in ICGC_PA112 was also found to be expressed by RNA-seq analysis (not shown). RPM, reads per million; KD, kinase domain.

b, Computational modeling of two BRAF monomers (light/dark grey) with a VLR insertion (blue/magenta) between Arg506 and Lys507 (green), as identified in ICGC_PA65. The PDB structure 4E26 was used as a template. Val600, a mutational hotspot, is shown in yellow. A novel dimer interface is formed between the protomers, with hydrogen bonds from the new arginine side chains (dashed lines) and a hydrophobic interaction between the leucine side chains (magenta).

c, Western blot analysis of NIH3T3 cells transfected with empty vector (EV), BRAF^WT, BRAF^V600E and BRAF^insVLR. The newly identified insVLR mutant results in elevation of ERK1/2 phosphorylation (pERK1/2) at a similar level to that seen with the known oncogenic V600E form.

d, Co-immunoprecipation assay with pull-down of HA-tagged BRAF^insVLR, demonstrating that this novel mutant forms homodimers with co-transfected AU1-tagged insVLR, but does not appear to form a strong heterodimer with wild-type BRAF.