Chapter 4 124 Disease specific differential gene expression in oligodendrocyte lineage For the cell cluster specific investigation, we used the contrasts HD x GLD, HD x 4H and HD x CD. First, the oligodendrocyte lineage was investigated at D150, as oligodendrocyte clusters were more abundant at this timepoint. For all leukodystrophies we identified multiple differentially expressed genes (DEGs). To note, for GLD in all three clusters of the oligodendrocyte lineage the genes C5orf17, CSMD1, DPP6 and CHCHD2 were significantly downregulated (Fig. 7A-C, note: gene labels are not always attributed in the graphs, but raw data was consulted to draw conclusions). C5orf17 is also down regulated in the oligodendrocyte lineage of 4H cells. Interestingly, CHCHD2 is upregulated in 4H OPC, and not significantly different in proliferating OPC and oligodendrocytes. For 4H there is upregulation of MEG3 and MEG8 in all cells of the oligo lineage (Fig. 7D-F). In addition, there is down regulation of MAG (Myelin Associated Glycoprotein) in 4H specifically (Fig. 7F). For CD oligo lineage, it stands out that many mitochondrial related genes are upregulated at the OPC stage (Fig. 7G). RPS4Y1 is downregulated in all cells of the oligodendrocyte lineage (Fig. 7G-I). MEG3, which was also identified for 4H is also up in all clusters of the oligodendrocyte lineage in CD. The gene AX119673.2 is upregulated in the oligodendrocyte cluster of all leukodystrophies. Since the investigation of DEG is very limited and often biased, we also applied gene set enrichment analysis (GSEA). Although the current data set, shows ample pathways to be significantly different in LDs, their significance and direction are context dependent. Considering the size of the data set this makes it hard to draw conclusions. To illustrate, GSEA on GLD oligodendrocyte lineages using the pathways database shows “Disease of signal transduction by growth factor receptors and second messengers” to be down regulated in OPC and proliferating OPC at D100 while upregulated in D150 (Supplemental Fig. 4A-D). Initially, this could be identified as a disease specific alteration. However, if we look at significant pathways like “translation” and “rRNA processing”, which are consistently upregulated in the oligodendrocyte lineage clusters of GLD (Supplemental Fig. 4A-F), we show that those pathways are also often significant in other leukodystrophies 4H and CD (Supplemental Fig. 5 & 6). This dysregulation of translation could be leukodystrophy specific, however when looking at 4H, the pathways are inconsistent. Sometimes they are upregulated other times downregulated (Supplemental Fig. 5). Similarly, the pathway of oxidative phosphorylation was highly significant when comparing HDs to any of the LDs. Also this pathway, was context dependent (Supplemental Fig. 7).
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