POLR3 gene and protein expression dynamics in 4H 191 6 INTRODUCTION Induced pluripotent stem cell (iPSC) technology has revolutionized the study of patientspecific genetics, enabling researchers to investigate how genetic variations influence cellular phenotypes. This is especially important for disorders with complex and variable clinical presentations, where treatment targets remain ambiguous (Nicholson et al., 2022; Shi et al., 2017). By modeling patient-derived cells, iPSCs offer a unique platform to explore the genetic and molecular underpinnings of such diseases and identify how these differences manifest in relevant cell types. However, despite advances in identifying disease-causing genes, translating these findings into effective therapies has been challenging. This is largely due to the context-dependent nature of gene expression and protein function, which vary across cell types and developmental stages. Therefore, it is critical to determine the specific cell populations and developmental contexts that are most affected by disease variants to design targeted therapeutic strategies. In this study, we focus on the genetic brain white matter disorder known as 4H leukodystrophy, or POLR3-related leukodystrophy. This disorder, caused by biallelic variants in either POLR3A, POLR3B, POLR3K, POLR3D or POLR1C genes, is characterized by a clinical triad: hypomyelination, hypogonadotropic hypogonadism, and hypodontia (Bernard et al., 2011; Dorboz et al., 2018; Macintosh et al., 2023; Tetreault et al., 2011; Thiffault et al., 2015). Despite these hallmark features, 4H leukodystrophy exhibits considerable clinical variability. Some patients present with significant neurological involvement despite minimal hypomyelination (Harting et al., 2020; La Piana et al., 2016), while others may experience isolated endocrine abnormalities such as hypogonadotropic hypogonadism (Richards et al., 2017). The causative genes encode subunits of RNA Polymerase III, which synthesizes various essential small non-coding RNAs, including transfer (t), ribosomal (r), small nuclear (sn), small nucleolar (sno), and micro (mi) RNAs. Given the progressive and debilitating nature of 4H leukodystrophy, there is an urgent need for therapeutic development. Current treatments are limited to symptomatic management (Adang et al., 2017). Identifying the specific cell types vulnerable to POLR3 variants is crucial for developing effective, targeted therapies. Previous studies have suggested a neuronal component to 4H pathology, at least in some patient subsets (Dooves et al., 2023; Harting et al., 2020; La Piana et al., 2016). Yet, it remains unclear which molecular mechanisms underlie these neuronal phenotypes and whether these alterations are exclusive to neurons or present earlier in neural precursor cells, such
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