Chapter 6 192 as neuroepithelial stem cells (NES). To address this, we investigated how patient-specific POLR3 variants influence gene expression, protein levels, and subcellular localization in different neuronal lineage cell types, including iPSCs, NES, and neurons. Additionally, we examined the impact of individual genetic variability on these molecular characteristics. Our findings demonstrate that POLR3 gene expression is different in cell subtype- and between individuals. In contrast, Pol III protein levels were affected by disease status but showed no significant variation across different individuals. Interestingly, the localization of Pol IIIA was determined to be an influential outlier, warranting further investigation to determine if this is disease related. Importantly, we did not find significant differences in Pol III transcript products related to disease status and cell type. These insights provide a deeper understanding of the molecular alterations contributing to neuronal dysfunction in 4H leukodystrophy. Furthermore, our study highlights the potential of iPSC-based models to uncover patient-specific genetic effects, offering a foundation for future therapeutic development. RESULTS POLR3 gene expression varies by cell type To investigate how POLR3 gene expression contributes to neuronal vulnerability in 4H leukodystrophy, we analyzed POLR3A and POLR3B gene expression in iPSCs, NES, and neurons derived from a 4H patient cohort with POLR3A and POLR3B variants, along with controls (Supplementary Table I and Supplementary Fig. 1A-C). A linear mixed-effects model (LMEM) indicated some evidence of a global significant effect of cell type on POLR3A expression (χ2 (2) = 6.180, p = 0.046). However, when focusing on specific level contrasts for cell type (via post-hoc pairwise comparisons) such statistical significance was not revealed. From visual inspection of Fig. 1A, some trends of increased expression were observed between iPSCs and neurons (p = 0.0723) and between iPSCs and NES (p = 0.0723). (Fig. 1A1). There was no difference between controls and 4H patients (Fig. 1A2; χ2(1) = 2.541, p = 0.111). POLR3B expression also varied significantly across cell types (χ2(2) = 21.340, p <0.001), with significant differences identified between iPSCs and NES (p = 0.0024) and between NES and neurons (p = 0.0021) (Fig. 1 B1). There was no significant effect of disease status on POLR3B expression (Fig. 1B2; χ2(1) = 0.077, p=0.782). In conclusion, both POLR3A and POLR3B gene expressions are influenced by cell type, with higher expression levels observed in NES compared to iPSCs, suggesting a cell-type-specific regulation of POLR3 genes.
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