Introduction Inflammatory Bowel Disease (IBD) is a complex genetic disease characterised by chronic inflammation of the gastro-intestinal tract. Genome-wide association study (GWAS) meta-analysis and replication has identified over 200 genomic regions involved in IBD susceptibility although only a few of the associated single nucleotide polymorphisms (SNPs) directly alter the coding sequence of a gene. In addition, >92 of the IBD-susceptibility loci are known to co-localise with active regulatory elements, suggesting the majority of IBD associated SNPs alter gene expression in some way. Here we employ whole transcriptome sequencing to investigate the role of altered gene expression in intestinal tissue.
Methods Patients and non-IBD control individuals were recruited after informed consent through endoscopy clinics at Guy’s and St Thomas’ Hospital, London. Whole RNA was extracted from 2–4 colonic biopsies from 104 IBD patients (76 CD, 28 UC) and 24 non-IBD controls and ribo-depleted (Ribo-zero, Illumina). We performed whole RNA sequencing using Illumina TruSeq and HiSeq2500 on pools of 4 indexed RNA libraries across 1–2 lanes. Quality control of sequence reads was performed via FastQC. Subsequent alignment and assembly, quantification and differential expression (DE) analysis of each transcript was performed using Tophat2, Cufflinks, Cuffmerge and EdgeR.
Results So far a detailed bioinformatics analysis has been carried out on the first 32 whole RNA-seq libraries. We have observed expression above background (FPKM > 1) for approximately 46% of all known transcripts located within known IBD susceptibility loci. DE analysis identified 249 transcripts with significant difference in expression (q < 0.06) between IBD cases and controls, 23 of which were located within 500 Kbp of a known IBD susceptibility locus including HLA-DRB5, MUC19, LRRC7 and 4 long non-coding RNAs (lncRNAs). Gene set enrichment analysis (GSEA) on the differentially expressed genes identified 19 functional sets with significant enrichment (FDR < 0.25), of which, 13 were immune response related. Most notably, enrichment was seen in gene sets related to Th1, Th2 immune response pathways, known to be relevant in IBD.
Conclusion We have generated high coverage whole RNAseq data on intestinal biopsy samples from 128 individuals. We have shown <50% of genes mapping to IBD-associated loci are expressed in these intestinal samples and DE analysis have identified genes of potential interest in IBD pathogenesis. Further bioinformatics analysis of the remaining 96 colonic RNA libraries is ongoing.
Disclosure of Interest None Declared
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