Chapter 6 198 Further research is however required to elucidate the specific contribution of these findings to 4H phenotypes. Our study underscores the importance of context-specific and patientspecific research and how iPSC technology could contribute to personalized research approaches. Cell-specific POLR3 gene expression and implications One of the clearest findings is the upregulation of POLR3B gene expression in NES compared to iPSCs, and a similar trend in POLR3A. Possibly, there is an increased transcriptional demand during the early stages of neuronal differentiation. This upregulation could be part of the explanation why the neuronal lineage is affected by POLR3-variants found in 4H. However, further research is needed to validate this hypothesis and establish a direct link between POLR3 expression and neuronal vulnerability. Although we did not find an effect of 4H status on POLR3 gene expression levels, interestingly, one 4H patient with POLR3B variants was identified as influential outlier in the POLR3B gene expression analysis. This could possibly be 4H related, future studies with larger sample size are required to determine if gene expression levels are or are not affected. Disease-specific changes in Pol III protein We identified reduced Pol III protein levels in 4H cells. This is in line with previous case reports that describe reduced Pol III protein levels in 4H patients and in mutant cell lines (Choquet, Forget, et al., 2019; Perrier et al., 2020). Interestingly, the patients with POLR3B variants have the lowest levels of Pol IIIB expression. Additionally, we did observe one 4H patient with POLR3A variants to be an influential outlier in the nuclear localization of Pol IIIA. Again, due to small sample sizes we cannot rule out whether this is actually disease related. Since Pol III has its function in the nucleus, but assembles in the cytoplasm, reduced nuclear localization possibly has negative impact and contributes to the disease phenotype (Tian et al., 2023). In literature, impaired nuclear import is described for a 4H patient with biallelic variants in POLR1C (Thiffault et al., 2015). Additionally a number of other variants causing POLR3 related leukodystrophy are reported to impair proper assembly of Pol III (Choquet, Pinard, et al., 2019), often causing a retention of the unassembled subunits in the cytoplasm (Thiffault et al., 2015). Possibly, protein level and localization are early indicators of neuron cellular dysfunction, but this requires further exploration.
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