A08 Early transcriptional changes in human HD-IPS cell lines revealed by RNASEQ

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Abstract

HD is mainly a late-onset disorder, however, subtle symptoms in patients may occur years or even decades prior to diagnosis. Such changes at a molecular level may begin much earlier, even in stem cells. Here, we present a study defining the transcriptional profiles and early HD alterations in human HD-iPS cell lines. For the RNA sequencing analysis, we used three clonal HD lines with 71 CAG repeats, three juvenile HD clonal lines with 109 CAG repeats and control lines. HD-iPSC lines (71Q and 109Q) were compared with control lines where 82 significantly deregulated mRNAs were identified (30 downregulated and 52 upregulated). In addition 71Q lines were compared with control lines yielding 113 significantly deregulated mRNAs (33 downregulated and 80 upregulated). In the last group, in which 109Q lines were compared with control lines, 169 significantly deregulated mRNAs were identified (90 downregulated and 79 upregulated). The analysis revealed mRNAs which occurred in both HD lines (ex. OTOGL, TRIM69) but also many unique mRNAs, deregulated in 71Q (ex. PIWIL2, HIST1H3C) or HD109Q-iPSC lines (ex. TP53, CDKN1A). RNA sequencing was also focused on circular RNA (circRNA) profiling. Nearly 100 significantly deregulated circRNAs were identified, also showing many transcriptomic differences between 71Q and 109Q lines. The high-throughput RNA screening was followed by bioinformatics analyses, such as differential expression analysis and also over-representation and enrichment analyses which demonstrated several affected biological processes in iPSC. These processes were related with central nervous system development, disruption of the apoptosis pathway, enhanced DNA methylation and negative regulation of Wnt signaling pathway. RNA-seq and in silico analyses were then followed by experimental validation of the most deregulated and the most interesting mRNAs and digital droplet PCR for chosen circRNAs.

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