Aberrant Splicings Due to Microsatellite Instability in Colorectal Cancer : Physiopathological and Clinical Impact (MICROSPLICOTHER)

WP1 - To identify exon/intron junctions that are specifically affected by aberrant splicing events in MSI CRC. Sequences will first be evaluated at CNG using Illumina's Pipeline CASAVA (Consensus Assessment of Sequence And Variation) software. This program converts intensity scores into base calls, quality scored alignments and additional formats for downstream analysis, thus rapidly transforming data into biologically relevant information. The filtered data will then be transferred to the CIT platform (Carte d'Identité des Tumeurs; http://cit.ligue-cancer.net, dir: A. de Reynies) to be analysed by our bio-informatics expert. The Cufflinks workflow will be used to quantify transcript expression levels in MSI tumours. This allows transcript assembly, discovery and differential expression measures at transcript-level resolution. Because the standard Cufflinks workflow does not support gene fusion discovery or quantification, several new features will be incorporated into it. Firstly, SoapFuse will be used to detect fusion break points and to predict fusion junction sequences. These will be integrated into the human reference genome and gene annotation from the Gencode project to provide a comprehensive, integrated annotation of gene features for mapping of splicing reads. The integration INCa - PRTK 2014 17/51 process will abide by several rules to minimise the potential to disturb the quantification of expression level in forthcoming analyses. With the help of our customised reference genome and annotation, TopHat, an aligner that supports splice junction and gene fusion mapping will be used for RNASeq mapping. Cufflinks will then be used to find new splice variants, including new exon skipping isoforms.

These will be integrated into the annotation using Cuffmerge. Depending on the alignment results obtained with TopHat, several filters will be applied to remove low quality candidates for splice variation and gene fusion, as well as candidates that are incompatible with existing annotated transcripts. Where necessary, reads will be assembled by AbySS and then aligned onto the reference genome by BLAT to provide more information for refining the annotation. This will produce a transcriptome assembly containing high confidence gene fusion and exon skipping events. Identification of these events will then be performed in individual samples. Cuffdiff will be used to analyse the mapping result, also based on this transcriptome assembly, for calling differentially expressed genes and transcripts and for detecting differential splicing changes. Finally, CummeRbund will be used to interpret and visualise the results.

The reliability of RNA-seq analysis will be verified by searching for aberrantly spliced transcripts already reported in MSI cancers (eg. MRE11 and HSP110) and for point mutations in the coding sequences of target genes for MSI (eg.TGFBR2, IGF2R, TCF7L2, AXIN2, PTEN, RIZ) and in other cancer-related genes that serve as internal positive controls (eg. KRAS, BRAF, TP53, PIK3CA). It is worth noting this project is part of several others developed jointly with CIT-Ligue and that are aimed at characterizing MSI CRC using Omics technologies. Importantly, this data is already available for a significant number of samples and could therefore be exploited if required.

Data from the RNASeq cohort of patients will be comprehensively analysed to identify recurrent splicing aberrations (expected to be mostly exon skipping) that occur specifically in MSI colon tumours compared to MSS CRCs and matching normal colonic mucosa. Amongst these, the study will focus on splicing aberrations that are due to MSI and that affect exons with a flanking upstream intron containing a ≥ 15 bp LNCR that is located ≤ 6 bp from the intron-exon junction (splice acceptor site) (RNASeqMSI-exon pre-list). As stated above, about 2,000 human genes contain at least one intron with a LNCR very close to the AG splice acceptor site at the intron-exon junction. Approximately 100 human genes could be affected by recurrent and specific aberrant splicing events due to MSI in CRC (mostly exon skipping; deduced from experiments performed in a limited series of CRC cell lines and primary tumours using exon arrays; preliminary and unpublished results).

WP2 - To investigate for functional links between MSI and aberrant splicing events.