Activation of the MAPK pathway.

<p><b>(</b>A<b>)</b> Western Blot of protein lysates from HEK293 cells transiently transfected with pCMV6-Myc-DDK (empty vector), pCMV6-BRAF-Myc-DDK (BRAF^WT) 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<b>)</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.</p

A new <i>GTF2I-BRAF</i> fusion mediating MAPK pathway activation in pilocytic astrocytoma

<div><p>Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. A recurrent feature of PA is deregulation of the mitogen activated protein kinase (MAPK) pathway most often through <i>KIAA1549-BRAF</i> fusion, but also by other <i>BRAF</i>- or <i>RAF1</i>-gene fusions and point mutations (<i>e</i>.<i>g</i>. <i>BRAF</i>V600E). These features may serve as diagnostic and prognostic markers, and also facilitate development of targeted therapy. The aims of this study were to characterize the genetic alterations underlying the development of PA in six tumor cases, and evaluate methods for fusion oncogene detection. Using a combined analysis of RNA sequencing and copy number variation data we identified a new <i>BRAF</i> fusion involving the 5’ gene fusion partner <i>GTF2I</i> (7q11.23), not previously described in PA. The new <i>GTF2I-BRAF</i> 19–10 fusion was found in one case, while the other five cases harbored the frequent <i>KIAA1549-BRAF</i> 16–9 fusion gene. Similar to other <i>BRAF</i> fusions, the <i>GTF2I-BRAF</i> fusion retains an intact <i>BRAF</i> kinase domain while the inhibitory N-terminal domain is lost. Functional studies on <i>GTF2I-BRAF</i> showed elevated MAPK pathway activation compared to <i>BRAF</i>^<i>WT</i>. Comparing fusion detection methods, we found Fluorescence in situ hybridization with <i>BRAF</i> break apart probe as the most sensitive method for detection of different <i>BRAF</i> rearrangements (<i>GTF2I-BRAF</i> and <i>KIAA1549-BRAF</i>). Our finding of a new <i>BRAF</i> fusion in PA further emphasis the important role of B-Raf in tumorigenesis of these tumor types. Moreover, the consistency and growing list of <i>BRAF/RAF</i> gene fusions suggests these rearrangements to be informative tumor markers in molecular diagnostics, which could guide future treatment strategies.</p></div

Expression analysis of fusion transcripts based on RNA-seq data.

<p>Log2 RNA-seq expression data for three fusion transcripts; <i>GTF2I-BRAF</i> 19–10, <i>DENND2A-GTF2IRD1</i> 14–2, <i>KIAA1549-BRAF</i> 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; <i>GTF2I-BRAF</i> 19–10 (exon 19- exon 10), <i>GTF2I</i> (exon 19- exon 20), <i>BRAF</i> e9-e10 (exon 9- exon 10), <i>DENND2A-GTF2IRD1</i> 14–2 (exon 14- exon 2), <i>KIAA1549-BRAF</i> 16–9 (exon 16- exon 9), <i>DENND2A</i> v1 (transcript version 1, exon 14- exon 15), <i>KIAA1549</i> (exon 16- exon 17), <i>BRAF</i> e8-e9 (exon 8- exon 9).</p

Summary of results from five different methods for BRAF alteraration detection.

<p>Summary of results from five different methods for BRAF alteraration detection.</p

Clinical data of the PA cases.

<p>Clinical data of the PA cases.</p

FISH analysis of <i>BRAF</i> fusions with <i>BRAF</i> break apart assay.

<p>Upper left: Schematic presentation of the <i>BRAF</i> 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 <i>BRAF</i> alleles, displayed as a merged (yellow) or two adjacent green (5’)/red (3’) signals. Lower panels: Fusion-positive tumor cells (<i>GTF2I-BRAF</i> in PA3 and <i>KIAA1549-BRAF</i> in PA4) showing the <i>BRAF</i> split pattern; two normal <i>BRAF</i> alleles green /red signals, as well as one additional split <i>BRAF</i> red signal representing the duplicated 3’ region in the fusion gene. The same split signal pattern is seen for different types of <i>BRAF</i> fusions; <i>GTF2I-BRAF</i> in case PA3 and <i>KIAA1549-BRAF</i> in cases PA1-2 and PA4-6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175638#pone.0175638.s003" target="_blank">S3 Fig</a>). Tissue sections were counterstained with DAPI (blue).</p

A new <i>GTF2I-BRAF</i> fusion mediating MAPK pathway activation in pilocytic astrocytoma - Fig 2

<p><b>(</b>A<b>)</b> 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 <i>KIAA1549-BRAF</i> duplication in 7q34. In case PA3 two novel duplications of approximately 250kb each were detected; one in 7q11.23 with breakpoint within <i>GTF2IRD1</i> and <i>GTF2I</i>, and one in 7q34 with breakpoint within <i>DENND2A</i> and <i>BRAF</i>. The two duplicated regions give rise to two fusion junctions; <i>DENND2A-GTF2IRD1</i> exons 14–2 and <i>GTF2I-BRAF</i> 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 (<a href="https://genome.ucsc.edu/" target="_blank">https://genome.ucsc.edu</a>). Positions marked with star (*) are approximate by manual inspection in the ChAS software. (B<b>)</b> Supporting reads for the <i>DENND2A-GTF2IRD1</i> 14–2 and <i>GTF2I-BRAF</i> 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 (<i>DENND2A</i>: NM_015689, <i>GTF2IRD1</i>: NM_016328, <i>GTF2I</i>: NM_032999), <i>BRAF</i>: 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 <i>DENND2A-GTF2IRD1</i> gene fusion and a total of 109 unique supporting read pairs/reads for the <i>GTF2I-BRAF</i> gene fusion. Only reads supporting the fusions are shown. Read pairs are in the same color. e = exon; i = intron.</p

Alessandro alle sedi degli antichi sovrani, dal Romanzo greco alle versioni persiane

L’articolo passa in rassegna le varianti di un episodio spesso riprodotto nella tradizione letteraria dedicata alla figura di Alessandro il Grande: l’arrivo del sovrano macedone ad una sede di re – città, palazzo o tomba. Vengono prese in considerazione le più antiche narrazioni greche appartenenti al ciclo del Romanzo di Alessandro dello Pseudo-Callistene, con le varianti latine, siriache, arabe, fino, soprattutto, a quelle persiane. Gli elementi simbolici che attraversano questa serie di brani (il trono meccanico, il letto mortuario, l’immagine del cinghiale, gli antichi obelischi, la coppa) vengono analizzati e messi in relazione testuale tra loro, fino a comporre un mosaico sostanzialmente unitario: quello di una riflessione – costante attraverso i secoli e le lingue – sul ruolo del re al centro del suo dominio, sui suoi compiti e i suoi limiti. Ciò che costituisce uno dei temi portanti della storia di Alessandro, ed una delle principali ragioni del suo successo editoriale

Additional file 5: Figure S2. of Targeted high-throughput sequencing for genetic diagnostics of hemophagocytic lymphohistiocytosis

Scatterplot of NK cell cytotoxic activity, displayed as lytic units at 25 % specific lysis, and frequency of NK cells in PBMCs. The patients are grouped by their molecular diagnosis. The individuals with very low NK cytotoxicity are also displayed in the zoomed plot for better resolution. Transport controls are included (triangles). (EPS 1171 kb