POLR3 gene and protein expression dynamics in 4H 199 6 Absence of POLR3 Transcript Product Alterations Despite reduced protein expression in 4H cells, there is no significant alteration in level of Pol III transcribed transcript, raising questions about the downstream functional effects of POLR3 variants. No effect of disease status was found on expression levels of tRNAs, which is contradicting literature that describes altered tRNA profiles in a mouse model of 4H (Moir et al., 2024). Additionally, we did not identify a cell type effect on tRNA expression, while it is known that human tRNA expression vary widely in human tissue (Dittmar et al., 2006). Together, this made us believe that we did not have either sensitive enough assays or large enough sample sizes to detect the altered tRNA levels. The structural complexity of tRNAs poses additional challenges for accurate quantification via qPCR, as their secondary and tertiary structures can interfere with detection. Additionally, we also found no effect of disease status on BC200 expression, despite reports of BC200 downregulation in POLR3A mutants (Choquet, Forget, et al., 2019). Given that we analyzed POLR3A and POLR3B patients together, it is conceivable that BC200 downregulation may be specific to certain subgroups. Further studies are needed to clarify these inconsistencies and determine the functional relevance of Pol III transcript expression. Impact of individual variability on POLR3 expression Our study highlighted considerable inter-individual variability in POLR3 gene expression, independent of disease status and cell type. However, this variability was not mirrored at the protein level, suggesting that post-transcriptional regulatory mechanisms may buffer these differences. The substantial individual differences underscore the complexity of POLR3 gene regulation and could have implications for personalized treatment approaches in 4H leukodystrophy. The need for tailored therapies becomes particularly apparent when considering the genetic and phenotypic heterogeneity observed among patients. Conclusions Overall, our findings support the idea that genetic and molecular alterations in 4H are highly context-dependent, reinforcing the importance of identifying the specific cell populations and developmental stages most affected by these variants. The inter-individual variability we observed in POLR3 gene expression across individuals also points to a need for personalized approaches in both research and therapy. By using patient-derived iPSCs, we can better capture this complexity and work towards refining targeted interventions. Future studies should delve deeper into splicing variations and protein assembly disruptions, as these may hold the key to understanding and potentially mitigating the cellular dysfunction observed in 4H leukodystrophy.
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