Page 128 - Molecular features of low-grade developmental brain tumours

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genes related to several pathways including ECM organization, but did not further study the role of the ECM in SEGA in detail 26. As MMPs and TIMPs may contribute to TSC pathology and cancer, understanding their role in SEGA development could provide new insights into the SEGA biological behaviour and potential new therapeutic targets. Therefore, in this study we investigated the expression of MMPs and TIMPs in SEGA. Furthermore, we identified differentially expressed miRNAs in SEGA compared to control tissue that could regulate the expression of MMPs and further investigated the role of these miRNAs in SEGA and their regulation of MMP expression in vitro.
Methods and Materials
Subjects
A total of 24 SEGA specimens (male/female: 16/8; age ranging from 1–28 years; ethnicity: Caucasian) were collected from the following hospitals: University Medical Centers Amsterdam (location Academic Medical Center, Amsterdam, The Netherlands), the University Medical Center Utrecht (Utrecht, The Netherlands), University Medical Center Groningen (Groningen, The Netherlands), Medical University of Vienna (Vienna, Austria), Children’s Memorial Health Institute (Warsaw, Poland), Anna Meyer Children’s Hospital (Florence, Italy) (Table 1). Twenty- two of the twenty-four SEGA samples included in this study were obtained from patients who met the clinical diagnostic criteria for TSC, whereas two samples were obtained from patients with no other clinical signs of TSC. Histological diagnosis was confirmed following the current WHO classification guidelines 51 by two independent neuropathologists. Additional TSC1/ TSC2 mutation analysis was performed as part of routine clinical care on blood or tumour sample DNA or was determined using massively parallel sequencing as described previously 23,52, if available (Table 1). Periventricular brain tissue along the lateral ventricle at the level of either the caudothalamic groove or hippocampus plus its associated cortex was obtained from autopsies of patients (post-mortem delay less than 24 hours) who did not have TSC, epilepsy or brain tumours and served as control tissue (frozen: n=8, male/female: 6/2; age ranging from 3-87 years; paraffin: n=4, male/female: 1/3; age ranging from 2 months-44 years). Specimens were obtained and used in accordance with the Declaration of Helsinki and this study was approved by the Medical Ethics Committee of each institution.
RNA-Sequencing
RNA-Sequencing (RNA-Seq) data sets produced by our laboratory, were retrieved from the European Genome-phenome Archive (EGA), which is hosted by the EBI and the CRG (accession number: EGAS00001003787) 26. Data was retrieved for 19 SEGA samples and 8 control and processed as previously described 26. Briefly, sequence reads were trimmed and filtered using FastQC v0.11.5 (Babraham Institute, Babraham, Cambridgeshire, UK) and Trimmomatic v0.36. Paired-end reads were aligned to the human reference genome (GRCh38) with TopHat2 v2.0.13 and default settings 53. For the small RNA-Seq dataset no mismatches were allowed between the trimmed reads and the reference genome and small RNA reads were allowed to align a maximum of ten times. Next, the number of reads that