Table 1.
Clinical data of the PA cases.
Fig 1.
(A) Axial (left) and sagittal (right panel) MR images of PA3 tumor (T1-weighted, contrast enhanced) showing a cerebellar tumor (fat arrow), 4,5 x 3 x 3 cm, partly cystic with irregular contrast enhancement. In sella turcica, a 1.5 cm in diameter intensely contrast enhancing round tumor with a 0.5 cm cystic component is seen (slim arrow). The S-prolactin level was elevated, 1510 mIU/l (ref < 400), indicative of a pituitary prolactinoma. (B) Hematoxylin and Eosin staining of case PA3, demonstrates biphasic pattern with compact (+) and loose (*) areas, including Rosenthal fibers (arrow) and eosinophilic granular bodies (arrow head).
Table 2.
Summary of results from five different methods for BRAF alteraration detection.
Fig 2.
(A) Copy number variation (CNV) genomic profiling with CytoScan HD SNP arrays. The weighted log2 ratio, smooth signal, and allele difference plot for chromosome 7 is shown for all six PA samples. Four out of five samples show the KIAA1549-BRAF duplication in 7q34. In case PA3 two novel duplications of approximately 250kb each were detected; one in 7q11.23 with breakpoint within GTF2IRD1 and GTF2I, and one in 7q34 with breakpoint within DENND2A and BRAF. The two duplicated regions give rise to two fusion junctions; DENND2A-GTF2IRD1 exons 14–2 and GTF2I-BRAF exon 19–10, probably through a circularization event followed by incorporation into the genome. The breakpoint positions are according GRCh37/Hg19 at UCSC Genome Browser (https://genome.ucsc.edu). Positions marked with star (*) are approximate by manual inspection in the ChAS software. (B) Supporting reads for the DENND2A-GTF2IRD1 14–2 and GTF2I-BRAF 19–10 fusion junctions in RNA sequencing data from case PA3. Spanning and split read pairs supporting the junction were extracted by BLAT and were aligned to 600bp of the predicted mRNA/cDNA sequence for each fusion. A schematic presentation of the mRNA junction is presented by the black box, showing exons (e), positions in cDNA, and GenBank accession numbers (DENND2A: NM_015689, GTF2IRD1: NM_016328, GTF2I: NM_032999), BRAF: NM_004333). Each row represent read pairs (or single reads) supporting a unique template. The RNA-seq data contained a total of 10 unique supporting read pairs/reads for DENND2A-GTF2IRD1 gene fusion and a total of 109 unique supporting read pairs/reads for the GTF2I-BRAF gene fusion. Only reads supporting the fusions are shown. Read pairs are in the same color. e = exon; i = intron.
Fig 3.
(A) RT-PCR verification of gene fusions in six PA cases. Gene fusion PCR products of a) DENND2A-GTF2IRD1 14–2 (222 bp), b) GTF2I-BRAF 19–10 (291 bp), c) KIAA1549-BRAF 16–9 (249 bp), and wild type (wt) positive control products of d) BRAF and e) DENND2A. e = exon; i = intron. (B) Sanger sequencing of RT-PCR fusion junction products. Translation of codons is shown below the electropherograms. Upper panel: The DENND2A-GTF2IRD1 14–2 junction result in an out-of-frame truncated protein generating 41 new amino acids of the C-terminal of DENND2A. The ATG translation starting site in GTF2IRD1 is underlined. Lower panel: The GTF2I-BRAF 19–10 in-frame junction results in a putative protein involving an integrated sequence of 16bp from BRAF intron 9. New amino acids produced by the junctions are marked in bold. (C) Schematic illustration of the GTF2I and BRAF protein domains and localization of the fusion breakpoints. The TFII-I GTF2I protein (998 amino acids (aa), NP_127492) consists of six helix-loop-helix–like domains (R1-R6); the DNA binding domain basic region (BR); the nuclear localization signal (NLS); the leucine zipper domain (LZ). The BRAF (766 aa, NP_004324) consists of the Ras binding domain (RBD), the Phorbol ester/diacylglycerol binding zink finger domain (C1) and the tyrosine kinase (TK) domain. The breakpoint locus in GTF2I (position 575 aa) and BRAF (position 394 aa) is marked by a dashed vertical line. The GTF2I-BRAF putative fusion protein is 955 aa long and contains the TFII-I DNA binding domains (R1-R3, BR and LZ) and nuclear localization (NLS) from GTF2I joined to the tyrosine kinase (TK) domain of BRAF. Domains and positions are according to NextProt (http://www.nextprot.org, accessing date 2016-04-25).
Fig 4.
Expression analysis of fusion transcripts based on RNA-seq data.
Log2 RNA-seq expression data for three fusion transcripts; GTF2I-BRAF 19–10, DENND2A-GTF2IRD1 14–2, KIAA1549-BRAF 16–9 compared to wild type fusion partner genes in six PA cases. Expression data was calculated as total number of supporting reads normalized to the total number of raw reads in each sample. Exon-exon junction in genes are as follows; GTF2I-BRAF 19–10 (exon 19- exon 10), GTF2I (exon 19- exon 20), BRAF e9-e10 (exon 9- exon 10), DENND2A-GTF2IRD1 14–2 (exon 14- exon 2), KIAA1549-BRAF 16–9 (exon 16- exon 9), DENND2A v1 (transcript version 1, exon 14- exon 15), KIAA1549 (exon 16- exon 17), BRAF e8-e9 (exon 8- exon 9).
Fig 5.
FISH analysis of BRAF fusions with BRAF break apart assay.
Upper left: Schematic presentation of the BRAF break apart assay, consisting of a 5’ 170 kb green probe and a 178 kb 3’ red probe in 7q34. Upper right (wt BRAF): Metaphase FISH of normal control and Interphase FISH of fusion-negative cell (right corner) showing two wild type BRAF alleles, displayed as a merged (yellow) or two adjacent green (5’)/red (3’) signals. Lower panels: Fusion-positive tumor cells (GTF2I-BRAF in PA3 and KIAA1549-BRAF in PA4) showing the BRAF split pattern; two normal BRAF alleles green /red signals, as well as one additional split BRAF red signal representing the duplicated 3’ region in the fusion gene. The same split signal pattern is seen for different types of BRAF fusions; GTF2I-BRAF in case PA3 and KIAA1549-BRAF in cases PA1-2 and PA4-6 (S3 Fig). Tissue sections were counterstained with DAPI (blue).
Fig 6.
Activation of the MAPK pathway.
(A) Western Blot of protein lysates from HEK293 cells transiently transfected with pCMV6-Myc-DDK (empty vector), pCMV6-BRAF-Myc-DDK (BRAFWT) or pCMV6-GTF2I-BRAF-Myc-DDK (GTF2I-BRAF) were probed with antibodies against FLAG-DDK, phosphorylated ERK-Thr202/Tyr204 (pERK), total ERK (tERK) and GAPDH. Bars show relative mean pERK/tERK protein expression for each construct performed in triplicates (mean±SEM) after normalization to GAPDH. (B) Activation of the MAPK pathway in PA tumor tissue. FFPE sections from the six primary PA cases were immunostained with phosphorylated-ERK-Thr202/Tyr204 (pERK) antibody. Tumor tissue (PA1-6) showing perinuclear (arrow), nuclear (arrow head) and to lesser extent cytoplasmic (*) pERK staining. Normal human brain cerebellum reference tissue section from an autopsy specimen showing negative staining for pERK. Negative control with omitted primary antibody showing negative staining for pERK. Some endothelial cells were also positive for pERK. Original magnification x400.