Evidence of Extensive Alternative Splicing in Post Mortem Human Brain HTT Transcription by mRNA Sequencing

Abstract

Despite 20 years since its discovery, the gene responsible for Huntington's Disease, HTT, has still not had its function or transcriptional profile completely characterized. In response to a recent report by Ruzo et al. of several novel splice forms of HTT in human embryonic stem cell lines, we have analyzed a set of mRNA sequencing datasets from post mortem human brain from Huntington's disease, Parkinson's disease, and neurologically normal control subjects to evaluate support for previously observed and to identify novel splice patterns. A custom analysis pipeline produced supporting evidence for some of the results reported by two previous studies of alternative isoforms as well as identifying previously unreported splice patterns. All of the alternative splice patterns were of relatively low abundance compared to the canonical splice form.

Fig 1

A) Read pileup of the superset reads showing coverage in exons and specific introns. Canonical HTT gene model is in blue. B) Relative contribution of reads from each disease dataset to the superset binned into exon (top) and intron (bottom) features. Each bar is the average number of covered bases per base position in the given feature divided by the number of samples in the corresponding condition. The canonical gene model lies between the bar charts. High intronic coverage is apparent in introns 9, 10, 12, 41, 49, and 58, highlighted in grey. None of the features are obviously biased toward any of the conditions. The rightward skew of counts is indicative of the poly-A selection method used in library prep.

Fig 2. Splicing events detected in reads of both novel and previously reported splice forms.

Grey areas indicate overall aligned read coverage in the region, black areas are the spliced reads that contribute to the splicing events. Blue lines indicate detected splicing events with a minimum of 10 supporting reads. The blue track across the top of all plots is the canonical HTT splice form, with red boxes indicating the position in the gene region displayed. Splicing patterns shown in B, E, and F support the spliceforms HTT-d13, HTT-41b, and HTT-d46 reported in Ruzo et al. In C, the skipped exon 28 is consistent with an isoform identified in mouse and human [Hughes JMB 2014] but, in these data, is only seen in a splice pattern where exon 27 is also skipped.