Chapter 6 190 ABSTRACT Induced pluripotent stem cell (iPSC) technology offered new tools for studying disease mechanisms by modeling patient-specific genetics in disease-relevant cell types. Here, we focus on 4H leukodystrophy, a genetic brain white matter disorder linked to POLR3 variants with distinct clinical characteristics, which manifests with considerable clinical variability. Although 4H leukodystrophy primarily features white matter abnormalities, emerging research highlights the involvement of neuronal pathology. To address this, we analyzed POLR3 gene and protein expression throughout neuronal lineage differentiation, from the iPSC state to neuroepithelial stem cells (NES) and mature neurons. We identified elevated POLR3 gene expression in NES. However, Pol III protein levels were notably reduced in 4H patient cells. Despite these protein-level alterations, overall Pol III-transcribed transcript levels, including tRNAs and BC200 RNA were unchanged in 4H cells. Notably, patient-specific genetic backgrounds were found to have a significant impact on POLR3A expression. These results underscore the necessity of considering individual genetic backgrounds and specific developmental cell states when investigating the pathology of 4H leukodystrophy. Furthermore, our work demonstrates the utility of iPSC-based models in unraveling patientspecific disease mechanisms, thereby facilitating the development of more tailored therapeutic strategies. Highlights: - cell type dependent POLR3 gene expression, increased in neuronal lineage cells - 4H patient-specific reductions in Pol III protein - iPSC models reveal (possible) patient-specific molecular changes in 4H Keywords iPSC, 4H Leukodystrophy, RNA polymerase III, neuronal cell lineages, gene expression
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