Discussion 221 7 of course money. Because of this, we could include 5 control lines and 8 leukodystrophy lines, while maintaining feasible practical burden. Of course, there was variation and unfortunately, sample sizes were not large enough to address the cause of this variation. However, the experimental set-up, allowed for the comparison of findings between leukodystrophies. Interestingly, we identified two cell clusters, cycling radial glia and neural progenitors, being largely absent in all leukodystrophies. Additionally, cluster specific findings were identified, however, the comparison of these findings between leukodystrophies revealed that results need to be interpreted with caution. To illustrate, GSEA analysis revealed “ribosomal subunit” to be upregulated in 4H neuron mono-cultures compared to controls, although not p-adjusted significant. We utilized the scRNA sequencing data set to determine whether this was a true finding. Indeed, Ribosomal subunit related GO_CC terms were frequently significant in 4H, though they were not consistently up or down regulated. For example, certain clusters showed upregulation at D100 (proliferating OPC), but downregulation at D150 (maturing cells). Initially, we hypothesized this might reflect a 4H-specific translation dysfunction, which would be in line with 4H-related mutations in RNA Polymerase III. However, ribosomal-related GO_CC terms were also significant in datasets from GLD and CD, suggesting a possible leukodystrophyspecific phenomenon. This overlap between pathways suggests shared mechanisms or potential limitations in dataset resolution, complicating distinctions among these leukodystrophies. Upon closer inspection of genes within these pathways, we found an overexpression of ribosomal genes (e.g. RPL and RPS), raising the possibility of technical biases in the dataset that might also lead to their frequent significance. We need to continue our research on this, possibly, including pseudotime analysis using the D100 and D150 subsets we can shed light on which findings are relevant. Although findings such as rRNA processing gene sets being significant in 4H proliferating OPC, initially were linked to the POLR3 mutations in 4H, we showed that these pathways were also different in CD and GLD. The findings highlight the value of studying broader leukodystrophy cohorts to identify universal and subtype-specific mechanisms. Despite this progress, variability within small sample sizes prevented definitive conclusions about the causes of certain phenotypes. Future research should focus on further increasing sample sizes and standardizing experimental designs across collaborative studies.
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