Cortical interneuron development is affected in 4H leukodystrophy 43 2 Transcriptome analysis revealed 4H-associated changes in gene expression RNA sequencing analysis showed several DEGs between 4H patients and controls. In fibroblasts, 29 genes showed a significantly altered expression (Supplementary Table 4), while in iPSCs 20 genes were differentially regulated (Fig. 2A, Supplementary Table 5). Notable DEGs that were downregulated in 4H iPSCs include several genes involved in neural development (OTX2, NPTX1, SLITRK6, SEMA6A) and a downregulation of TMEM64 that plays a role in osteoclast differentiation (Boles et al., 2014; Kim et al., 2013; Mitsogiannis et al., 2017; Mortensen et al., 2015; Sandberg et al., 2014). In 4H cerebellar cells, 16 genes showed a significant differential expression (Fig. 2B, Supplementary Table 6). 4H cerebellar cells showed an increased expression of PDPR, which is associated with intellectual disability (Bruno et al., 2021) and an increased expression of CBLN1, a cerebellum-specific precursor protein (Hirai et al., 2005). The downregulation of ARX appears to be a potentially significant discovery. ARX mutations are associated with several brain disorders, and ARX loss of function is associated with abnormal cortical interneuron development and migration (Gecz et al., 2006; Kitamura et al., 2002; Marsh et al., 2016; Sherr, 2003). Cortical interneuron changes in 4H We studied whether the decreased ARX expression point to cortical interneuron involvement in 4H. iPSCs were differentiated into cortical neurons, i.e. a mixture of GABAergic interneurons and glutamatergic cortical projection neurons, according to previously established protocols (Nadadhur et al., 2017). At this stage, we were able to include 3 additional iPSC lines of POLR3A patients (P4-P6, Supplementary Table 1). At a neuronal precursor cell state, cells were harvested for RNA analysis. A selected number of DEGs from the cerebellar cells were tested on immature (day 18) neurons of 4H and controls. No differences in expression levels of PDPR, ITGA11 or MACIR (not shown) were observed (Fig. 3A-B). Consistent with the cerebellar cells, ARX expression was decreased in neurons of 4H patients (control 1.08 ± 0.31, 4H 0.26 ± 0.10, t(6) = 12.073, P < 0.001, Fig. 3C), confirming that ARX dysregulation may be a common feature in 4H.
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