GSE124664
Identification of altered developmental pathways in human juvenile HD iPSC with 71Q and 109Q using transcriptome profiling
Purpose: In Huntington disease (HD) subtle symptoms in patients may occur years or even decades prior to diagnosis. HD changes at a molecular level may begin as early as in cells that are non-lineage committed such as stem cells or HD patients induced pluripotent stem cells (iPSCs) offering opportunity to enhance the understanding of the HD pathogenesis. In addition, juvenile HD non-linage committed cells were previously not directly investigated in detail by RNA-seq. Methods: Strand-specific RNA-seq of the whole transcriptome was performed using 3 clonal lines from each of 2 HD patients  (6 HD iPSC lines) with 71 or 109 CAG repeats in exon 1 of the HTT gene, and 2 healthy individuals, with 17/18 (2 clonal iPSC lines) and 21 CAG repeats (one iPSC line), to comprehensively identify mRNAs related to HD. A DESeq2 pipeline for transcripts of HD was developed to identify significantly dysregulated mRNAs. During the RNA-seq data analysis, we compared 6 HD iPSC lines vs control lines and also compared separately each set of 3 HD lines from one patient vs 3 control lines. As a result of such an approach, we generated statistical values for three comparison groups, HD vs WT, HD71Q vs WT and HD109Q vs WT Methods: 9 pM-indexed libraries were sequenced with the use of Illumina HiSeq2000, rapid run with paired-end 75 bp reads. On average, 65 mln reads were collected per library Results: We identified 107 (6 HD lines), 198 (3 HD71Q lines) and 217 (3 HD109Q lines) significantly dysregulated mRNAs in each comparison group. Conclusion: The analyses showed that many of dysregulated transcripts in HD109Q iPSC lines are involved in DNA damage response and apoptosis, such as CCND1, CDKN1A, TP53, BAX, TNFRSF10B, TNFRSF10C, TNFRSF10D, DDB2, PLCB1, PRKCQ, HSH2D, ZMAT3, PLK2, and RPS27L. Most of them were identified as downregulated and their proteins are direct interactors with TP53. HTT probably alters the level of several TP53 interactors influencing apoptosis.This may lead to accumulation of an excessive number of progenitor cells and potential disruption of cell differentiation and production of mature neurons. In addition, HTT effects on cell polarization also demonstrated in the analysis may result in a generation of incorrect progenitors. Overall design: Total RNA from 9 samples were isolated for RNA-seq analysis, including 3 from control iPSC lines, 3 from clonal iPSC lines with 71 CAG repeats and 3 from clonal iPSC lines with 109 CAG repeats.