S1 Data -

(XLSX)</p

Gene expression levels of PAI-1 in A. platelets compared with reference genes and B. in subcutaneous adipose tissue compared with reference genes.

There were no significant differences in gene expression levels of PAI-1 in platelets between participants with T2D, obese and lean control subjects (T2D n = 6, obese n = 7 and lean control subjects n = 7)(A.). There was a significant difference in gene expression levels of PAI-1 in subcutaneous adipose tissue between lean control subjects and T2D or obese non-diabetic control subjects (T2D n = 7, obese n = 6 and lean control subjects n = 6) * P = 0.003, ** P = 0.038 (B.). Abb. PAI-1: Plasminogen Activator Inhibitor -1; T2D: type 2 diabetes.</p

Platelets in T2D patients compared with obese and lean control subjects.

Platelets in T2D patients compared with obese and lean control subjects.</p

Gene expression of PAI-1 in subcutaneous adipose tissue of lean, obese and T2D normalized against LRP10.

ANOVA with Bonferroni’s multiple comparisons test. N = 6 for all groups. Abb. PAI-1: Plasminogen Activator Inhibitor -1; T2D: type 2 diabetes.</p

Pool of PAI-1 in A. plasma, B. serum and C. platelets in T2D patients, lean and control participants.

There was a significant increase of plasma PAI-1 in participants with T2D and obese non-diabetic control subjects compared with lean control subjects (T2D n = 6, obese n = 7 and lean control subjects n = 8). * P˂0.001, ** P˂0.001. B. No significant difference in serum PAI-1 between the three groups (T2D n = 6, obese n = 7 and lean control subjects n = 8). C. There was no significant difference in platelet PAI-1 between the three groups (T2D n = 4, obese n = 5 and lean control subjects n = 8). Abb. PAI-1 = plasminogen activator inhibitor -1; T2D = type 2 diabetes.</p

Primers and probes for qPCR.

Primers and probes for qPCR.</p

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

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

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