Page 70 - Molecular features of low-grade developmental brain tumours

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CHAPTER 3
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on request.
Results
The methylation profile of SEGAs
To characterize the methylation profile of SEGAs, DNA was extracted from SEGA samples and control brain samples and analyzed using the 450k methylation array. In total 42 SEGA samples were included from 39 TSC patients and 3 patients with no other manifestations of TSC (surgical specimens) and 8 location-matched periventricular controls (autopsy specimens; see materials and methods and Table 1). After filtering for probes with a detection p-values of more than 0.01, located on the sex chromosomes, or on SNPs as well as cross-hybridization probes a total of 421,352 CpGs were analyzed with a principal component analysis (PCA) indicating that the major source of variability was the diagnosis (SEGA or control; Figure 1a), which was confirmed with a spearman’s correlation matrix on the top 5% most variable CpGs (Figure 1b). Furthermore, no specific clustering was seen based on the TSC mutation (Figure 1b). To assess other potential confounders on the methylation profile PVCA was performed, showing that the major contributor to the variance between the samples was the diagnosis (Supplementary Figure 1a). The majority of the filtered CpGs were located outside of the promoter region of genes either on the body region (36.67%), the IGR (23.18%) or the 3’UTR (3.93%; Figure 1c). Within the promoter region 13.55% was located on the TSS200 region of the gene, 17.71% on the TSS1500 region, 8.4% on exon 1 and 13.94% on the 5’UTR. After selecting for an adjusted p-value<0.01 and a β-value difference of >0.2 this distribution shifted towards the promoter region (20.93% TSS200, 42.55% TSS1500, 13.78% exon 1 and 18.85% 5’UTR), whereas 41.21% was located on the body region, 1.74% on the 3’UTR and no CpGs were located within the IGR (Figure 1c).
Since the promoter region is important for the regulation of RNA expression, we narrowed our data set to CpGs located at the promoter region (TSS200, TSS1500, exon 1 and 5’UTR) and found 4616 CpGs hypomethylated and 2526 hypermethylated in SEGA compared to control (adjusted p-value 0.01, β-value difference of >0.2, promoter region; Figure 1d). The 7142 differentially methylated CpGs were located on 3875 genes. We identified 227 enriched GO terms (adjusted p-value<0.05) for these genes (Figure 1e; Table 2) including adaptive immune system, T cell activation, leukocyte mediated immunity, extracellular structure organization and the ERK1 & ERK2 cascade. The majority of the enriched pathways contained more hypomethylated genes then hypermethylated genes (Figure 1f; Table 2). Methylation of the mTOR pathway in SEGAs
In order to compare the TSC1 mutated SEGA samples with the TSC2 mutated SEGA samples, three differential analyses were carried out: TSC1 mutated SEGAs compared to control (TSC1-control) and TSC2 mutated SEGAs compared to control (TSC2-control) and TSC1 mutated SEGAs compared to TSC2 mutated SEGAs (TSC1-TSC2). We identified 4008 hypomethylated and 2111 hypermethylated CpGs in TSC1-control (Figure 2a), 4721