Leukodystrophy -affected patients harboring mutations in Pol III gene present a profound transcriptional dysregulation

Leukodystrophies (LDs) are genetic disorders which impact the white matter in the central nervous system, leading to a deficiency in myelin development. Mutations in RNA Polymerase III (PolIII) subunits cause POLR3-related LDs, such as those present in POLR3A and POLR3B genes. PolIII is involved in the transcription of small ribosomal units, tRNAs, 7SL RNA, U6 spliceosomal RNA, and more. tRNAs are implicated in translation, whilst small RNAs can perform regulatory functions on mRNA transcripts. The work hereby presented aimed to identify the transcriptional dysregulations present in primary fibroblasts of POLR3-mutated patients and to also assess alterations in the translation process. Fibroblasts were obtained from skin biopsies of 2 POLR3A and 1 POLR3B mutated patients and matched controls. RNA was extracted with Trizol reagent and Total RNA sequencing was performed with the CORALL Total RNA-Seq Library Prep Kit using Illumina NextSeq 500 Sequencing. Differential expression analysis was performed with DESeq.2 package and enrichment analyses were performed on differentially expressed genes (DEGs). Click-iT Protein Labeling approach was used to analyze newly synthesized proteins. Trascriptomic profiling highlighted a strong dysregulation in POLR3 patients, identifying 418 DEGs when comparing LDs patients to controls. Moreover, when comparing each patient to its matched control, a personalized DEGs signature was also observed, suggesting caution when grouping patients harboring different mutations in the same gene. Moreover, nascent protein synthesis analysis highlighted a down-regulation in nascent protein synthesis, suggesting an impaired translation possibly due to alterations in tRNA metabolism. In conclusion, our results highlight a profound impact on transcription and translation processes, in patients’ specific primary cells used as pre-clinical experimental model of the disease.