RNA-Seq and human complex diseases: recent accomplishments and future perspectives

Abstract

The availability of the human genome sequence has allowed identification of disease-causing mutations in many Mendelian disorders, and detection of significant associations of nucleotide polymorphisms to complex diseases and traits. Despite these progresses, finding the causative variations for most of the common diseases remains a complex task. Several studies have shown gene expression analyses provide a quite unbiased way to investigate complex traits and common disorders' pathogenesis. Therefore, whole-transcriptome analysis is increasingly acquiring a key role in the knowledge of mechanisms responsible for complex diseases. Hybridization- and tag-based technologies have elucidated the involvement of multiple genes and pathways in pathological conditions, providing insights into the expression of thousand of coding and noncoding RNAs, such as microRNAs. However, the introduction of Next-Generation Sequencing, particularly of RNA-Seq, has overcome some drawbacks of previously used technologies. Identifying, in a single experiment, potentially novel genes/exons and splice isoforms, RNA editing, fusion transcripts and allele-specific expression are some of its advantages. RNA-Seq has been fruitfully applied to study cancer and host-pathogens interactions, and it is taking first steps for studying neurodegenerative diseases (ND) as well as neuropsychiatric diseases. In addition, it is emerging as a very powerful tool to study quantitative trait loci associated with gene expression in complex diseases. This paper provides an overview on gene expression profiling of complex diseases, with emphasis on RNA-Seq, its advantages over conventional technologies for studying cancer and ND, and for linking nucleotide variations to gene expression changes, also discussing its limitations.

Figure 1

Nucleotide variations altering gene expression and splicing. (a) Graphical representation of nucleotide variations potentially affecting the binding of transcription factors (TFs) and/or RNA polymerase II, thus altering gene expression, detectable by integration of RNA-Seq and ChIP-Seq experiments. (b) SNP possibly occurring within the introns (black lines) affecting donor and acceptor splice sites (GU and AG) altering the splicing of the coding exons. In detail, in (1) a canonically spliced pre-mRNA following the GU-AG rule; (2) an example of nucleotide variation/s occurring within the introns and generating a novel acceptor ‘cryptic' splice site. In this case, two different mRNAs are produced, depending on the different used acceptor splice site; (3) SNPs within the donor site (GU to AU change), leading to intron retention.

Figure 2

Extended UTRs and epigenetics in gene expression regulation. (a) Graphical representation of mRNAs with putative extended untranslated regions (UTRs). RNA-Seq may reveal new unannotated extended 5' UTRs, potentially involved in the binding of previously unexplored stabilizing protein complexes, whereas in extended 3' UTRs there may be new putative binding sites for miRNAs. (b) Schematic representation of some epigenetic mechanisms, regulating gene expression, possibly investigated by combining RNA-Seq to other NGS applications (ie, ChIP-Seq). TF, transcription factor; miRNA, microRNA; DNMT, DNA methyltransferase; HDAC, histone deacetylase; CH_3, methyl groups; Ac, acetyl groups.