Transcriptome sequencing reveals aberrant alternative splicing in Huntington's disease

Abstract

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG expansion in the gene-encoding Huntingtin (HTT). Transcriptome dysregulation is a major feature of HD pathogenesis, as revealed by a large body of work on gene expression profiling of tissues from human HD patients and mouse models. These studies were primarily focused on transcriptional changes affecting steady-state overall gene expression levels using microarray based approaches. A major missing component, however, has been the study of transcriptome changes at the post-transcriptional level, such as alternative splicing. Alternative splicing is a critical mechanism for expanding regulatory and functional diversity from a limited number of genes, and is particularly complex in the mammalian brain. Here we carried out a deep RNA-seq analysis of the BA4 (Brodmann area 4) motor cortex from seven human HD brains and seven controls to systematically discover aberrant alternative splicing events and characterize potential associated splicing factors in HD. We identified 593 differential alternative splicing events between HD and control brains. Using two expanded panels with a total of 108 BA4 tissues from patients and controls, we identified four splicing factors exhibiting significantly altered expression levels in HD patient brains. Moreover, follow-up molecular analyses of one splicing factor PTBP1 revealed its impact on disease-associated splicing patterns in HD. Collectively, our data provide genomic evidence for widespread splicing dysregulation in HD brains, and suggest the role of aberrant alternative splicing in the pathogenesis of HD.

Figure 1.

Gene expression analysis of HD and control motor cortex (BA4) samples using RNA-seq. (A) Heat map of 222 genes differentially expressed between HD and control motor cortex (BA4) samples. Color bars above sample IDs indicate the disease status: black, control; yellow, HD grade 2; orange, HD grade 3; red, HD grade 4. Each row of the heat map represents the Z-score transformed FPKM values of one differentially expressed gene across all samples. (B) Diagram depicting gene ontology (GO) terms that are significantly enriched in the differentially expressed genes. Categories in red and orange are enriched in up-regulated genes in HD. Categories in blue are enriched in down-regulated genes in HD. (C) Cell type composition analysis of human BA4 motor cortex samples. Geometric mean of the FPKM values of cell type marker genes of each cell type is used to indicate the change in specific cell type.

Figure 2.

Aberrant splicing program in Huntington's disease. (A) Differential alternative splicing events between HD and control motor cortex (BA4) samples. (B) Heat map of 316 differentially skipped exons in HD based on PSI (Percent Spliced In) values in HD and control motor cortex (BA4) samples. Color bars above sample IDs indicate the disease status: black, control; yellow, HD grade 2; orange, HD grade 3; red, HD grade 4. Each row of the heat map represents the Z-score transformed PSI values of one differentially spliced exon across all samples. (C) Examples of differential AS events in genes with neuronal functions. Box plots of PSI values of each alternative splicing event in 7 CTRL vs 7 HD samples. PSI values are represented by dots and dark horizontal lines represent the mean, with the box representing the 25th and 75th percentiles, the whiskers representing the 5th and 95th percentiles. (D) Significantly enriched binding sites of splicing factors and other RNA binding proteins in differentially skipped exons between HD and control samples. Gene symbols of splicing factors are followed by the consensus binding motifs and their Fisher’s exact test (one-sided) p-values (adjusted for multiple testing). IUPAC Ambiguity Codes were used to indicate motif patterns.

Figure 3.

qRT-PCR analysis of splicing factors and RNA binding proteins in two independent sets of control and HD motor cortex (BA4) samples: Set 1 (17 control and 37 HD) (A-C); Set 2 (27 control and 27 HD) (D-F). (A) and (D) Splicing factors and RNA binding proteins with significantly altered gene expression. (B) and (E) PTBP1 expression comparison between control and HD brains. (C) and (F) PTBP1 expression comparison among control and different HD grades. Lines indicate group means. Error bars indicate ±SEM. ∗ Nominal P < 0.05; ∗∗ P <0.01, ∗∗∗ P < 0.001. $: Genes with outlier data point detected and excluded from analysis; &: Genes with unequal variance and p-value calculated using unpaired t-test with Welch's correction.

Figure 4.

Splicing change of putative PTBP1 target exons in HD and the correlation between exon inclusion level and PTBP1 gene expression level (log2(PTBP1 fold change)). (A) ATP1B3 exon 2 inclusion level is negatively correlated with PTBP1 gene expression level. (B) DPF2 exon 7 inclusion level is negatively correlated with PTBP1 gene expression level. (C) KTN1 exon 40 inclusion level is positively correlated with PTBP1 gene expression level. Error bars indicate ±SEM. ∗∗ Nominal P < 0.01; ∗∗∗ P < 0.001.