Elevation in cell cycle and protein metabolism gene transcription in inactive colonic tissue from Icelandic patients with ulcerative colitis

BACKGROUND & AIMS: Ulcerative colitis (UC) is a chronic relapsing inflammatory disorder that affects the colonic epithelium. A combination of genetic and environmental factors is thought to be involved in the pathogenesis, resulting in an abnormal immune response and destruction of the colonic epithelium. In Iceland, the incidence of UC is one of the highest in the world and is three times more common than Crohn’s disease. Previous studies have suggested a genetic component may have an influence on the increased incidence. The aim of this study was to characterise a cohort of patients with UC and identify potential germ line mutations and pathways which could be associated with UC in this population. METHODS: We performed exome sequencing of genomic DNA and genome-wide microarray analysis on macroscopically non-inflamed colonic mucosa from Icelandic patients with UC and age and sexed matched controls. Gene-ontology analysis was used to identify common processes and pathways differentially expressed in tissue from UC cases. Exome sequence data were examined for very rare or novel mutations which might be over represented in the UC cohort. Combined matching of variant analysis and downstream influence on transcriptomic expression in the rectum was also analysed. RESULTS: Non-inflamed colonic tissue from patients with UC demonstrated a significant alteration in the transcriptomic profile compared to controls. Over 2,000 genes were differentially expressed in rectal tissue from UC patients and gene ontology analysis identified an up-regulation in genes associated with cell cycle control and protein processing in the endoplasmic reticulum. Exome sequencing identified two missense mutations in thiopurine S-methyltransferase (TPMT) with a minor allele frequency of 0.22 in the UC patients compared to a reported 0.062 in the Icelandic population and 0.03 in the non-Finnish European populations. A predicted damaging mutation in the inflammatory bowel disease associated gene SLC26A3 was identified which was associated with increased expression of DUOX2 and DUOXA2 in UC rectal tissue. CONCLUSIONS: In patients with UC, the colonic mucosa demonstrates a clear alteration in gene expression compared to control subjects. There is evidence of an elevation in genes involved with cell proliferation and the processing of proteins within the endoplasmic reticulum. Exome sequencing identified an increased prevalence of two damaging TPMT variants within the Icelandic UC population, which would suggest screening the UC population prior to initiation of therapy is warranted in order to avoid the serious toxicity associated with these mutations and azathioprine treatment. The original expression data contained 47,198 probes and 57 samples: 27 ascending colon and 30 rectal taken from the 35 individuals. For ascending colon samples, there were 12 cases and 15 controls, and for rectal samples, 15 cases and 15 controls. Probes with a minimum detection P-value of < 0.01 in at least 2 biopsies were retained (n = 22,716). Two case samples (1 ascending colon HCA12, 1 rectal HCR10) with significantly lower numbers of detected probes were excluded. Retained probes were mapped to gene IDs by the illuminaHumanv4.db package. Adjustment of the expression data for chip-level batch effects was done by the empirical Bayes method ComBat24 (sva R package25). The recorded bowel sites were verified by examining expression of genes known to significantly differ between the ascending colon and rectum and through a PCA on all probes: 1 ascending colon (UCA20) and 2 rectal samples (UCR11, UCR18) demonstrated discordant expression from expected and were removed. Post-QC, there were 52 samples (24 cases, 28 controls), of which 25 were ascending colon (11 cases, 14 controls), and 27 were rectal (13 cases, 14 controls). These data were log2 transformed, quantile normalised and analysed in R using various Bioconductor packages.