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Glioblastoma is a molecularly heterogeneous and highly aggressive brain tumor with the worst 5-year survival of all cancer types. It’s the most common brain tumor in adults and many efforts are being made to understand the complexity that comes with the intra- and intertumoral heterogeneity and their fast-evolving pace. Coding mutations have explained a lot of this, but since the coding parts only make up about 1 % of the genome there’s valid reason to include non-coding regions as well in mutational studies. There lie many important regulatory elements that could alter cellular functions if disrupted. To narrow down the extensive mutational burden of the entire genome, and filter out most of the silent passenger mutations, the host lab chose to only focus on evolutionary conserved regions in flanking regions of a gene. A mutation fulfilling these criteria was then called a “non-coding constraint mutation” (NCCM). 

Here, to further study the function of these NCCMs, RNA-seq from 78 patient derived glioma stemlike cell lines were used together with data on the NCCMs found through whole genome sequencing of matched tumour and blood samples from the same patients. This was done through a series of analyses using R, such as comparing the expression of a gene in multiple cell lines with and without NCCMs linked to that gene, and investigating the regions binding affinity and how it might be altered by a NCCM to find out more about the region’s functional properties. 

From the analysis it was found that out of the highly enriched NCCM genes, EBF1 and IGF1 was significantly downregulated in the presence of a NCCM. This is interesting findings for different reasons as IGF1 is a previously known GBM related gene as it affects the tumour phenotype, where EBF1 is novel to GBM but linked to other types of cancers. And while potentially important, neither would have been found if only looking at coding mutations. 

These NCCMs also seem to have a local effect as only the closest related gene expression is altered, not any other gene in a spatial proximity. Glioma stemlike cell lines were cultured and prepared to be used as in vitro models to further study the phenotypic effects of these NCCMs, although many experiments weren’t followed through with due to time constraints.