Abstract
BackgroundThe vast majority of human genes (>70%) are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes.Methodology/Principal Findings We combined GO term to parent threshold-based and threshold-independent ad-hoc functional statistics with in-depth analysis of key modified transcripts to highlight various exon-specific changes. These denote alternative splicing in MG-thymoma tumors compared to healthy human thymus and to in-house and Affymetrix datasets from colon cancer and healthy tissues. By using both global and specific, term-to-parent Gene Ontology (GO) statistical comparisons, our functional integrative ad-hoc method allowed the detection of disease-relevant splicing events.Conclusions/SignificanceHyper-spliced transcripts spanned several categories, including the tumorogenic ERBB4 tyrosine kinase receptor and the connective tissue growth factor CTGF, as well as the immune function-related histocompatability gene HLA-DRB1 and interleukin (IL)19, two muscle-specific collagens and one myosin heavy chain gene; intriguingly, a putative new exon was discovered in the MG-involved acetylcholinesterase ACHE gene. Corresponding changes in spliceosome composition were indicated by co-decreases in the splicing factors ASF/SF2 and SC35. Parallel tumor-associated changes occurred in colon cancer as well, but the majority of the apparent hyper-splicing events were particular to MG-thymoma and could be validated by Fluorescent In-Situ Hybridization (FISH), Reverse Transcription–Polymerase Chain Reaction (RT-PCR) and mass spectrometry (MS) followed by peptide sequencing. Our findings demonstrate a particular alternative hyper-splicing signature for transcripts over-expressed in MG-thymoma, supporting the hypothesis that alternative hyper-splicing contributes to shaping the biological functions of these and other specialized tumors and opening new venues for the development of diagnosis and treatment approaches.
Highlights
Changes in gene expression, and in alternative splicing patterns are often disease-associated [1], and aberrant alternative splicing is one of the characteristics of cancer cells [2], as well as of inflammation and autoimmune muscle diseases[3]
Our findings demonstrate a particular alternative hyper-splicing signature for transcripts over-expressed in myasthenia gravis (MG)-thymoma, supporting the hypothesis that alternative hyper-splicing contributes to shaping the biological functions of these and other specialized tumors and opening new venues for the development of diagnosis and treatment approaches
Each term was compared to the global array population and to all its direct parent Gene Ontology (GO) terms as well as to the root of the tree: Molecular Function (MF) or Biological Process (BP) (Figure 1)
Summary
In alternative splicing patterns are often disease-associated [1], and aberrant alternative splicing (hyper-splicing) is one of the characteristics of cancer cells [2], as well as of inflammation and autoimmune muscle diseases[3]. The vast majority of human genes (.70%) are alternatively spliced [4,5], and 75% of alternative splicing events affect coding regions, yielding subtle amino acid substitutions, removal of protein motifs or protein truncations [6]. This can alter protein structures, yield cell-specific protein patterns [7] and enlarge protein versatility in a tissue-specific manner [6]. We report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes
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