Next-generation profiling to identify the molecular etiology of Parkinson dementia

Abstract

Objective: We sought to determine the underlying cortical gene expression changes associated with Parkinson dementia using a next-generation RNA sequencing approach.

Methods: In this study, we used RNA sequencing to evaluate differential gene expression and alternative splicing in the posterior cingulate cortex from neurologically normal control patients, patients with Parkinson disease, and patients with Parkinson disease with dementia.

Results: Genes overexpressed in both disease states were involved with an immune response, whereas shared underexpressed genes functioned in signal transduction or as components of the cytoskeleton. Alternative splicing analysis produced a pattern of immune and RNA-processing disturbances.

Conclusions: Genes with the greatest degree of differential expression did not overlap with genes exhibiting significant alternative splicing activity. Such variation indicates the importance of broadening expression studies to include exon-level changes because there can be significant differential splicing activity with potential structural consequences, a subtlety that is not detected when examining differential gene expression alone, or is underrepresented with probe-limited array technology.

Figure 1. SpliceSeq “splice graphs” for the 7 qRT-PCR tested genes

RNA-seq transcript reads are aligned to composite gene graphs built from all possible isoforms existing in the Ensembl genome database. The graphs in this figure are partial splice graphs showing alternatively spliced regions. Values on graphs are splice observations per kilobase of transcript length per million aligned reads. Exon highlighting (determined by a log² exon expression ratio) indicates up- or downregulated exon expression or splicing as follows: green exons and splice arcs represent increased activity in the CON group and red exons and splice arcs represent either the PD or PD-D disease group. Gene splice graphs shown are specific for PD-D vs CON (ATXN2, DST, HSPH1, RELA, and SRRM1) or PD vs CON (LRRFIP1 and TRIM9). CON = control; PD = Parkinson disease; PD-D = PD with dementia; qRT = quantitative real-time.

Figure 2. dPSI values for SpliceSeq-predicted alternatively spliced exons within the labeled genes

Exon dPSI represents the difference in percent spliced in, disease vs CON (see table 2 for exon details). CON = control; dPSI = delta percent spliced in; PD = Parkinson disease; PD-D = PD with dementia.

Figure 3. qRT-PCR fold change comparison, disease vs CON, of splice vs no-splice primer sites

Exon regions with expected alternative splicing activity are compared with flanking exon regions without predicted alternative splicing, “no-splice.” The dotted line indicates 100% inclusion (of a queried exon). CON = control; PD = Parkinson disease; PD-D = PD with dementia; qRT = quantitative real-time.