Analysis of Germline GLI1 Variation Implicates Hedgehog Signalling in the Regulation of Intestinal Inflammatory Pathways

Abstract

BackgroundUlcerative colitis (UC) and Crohn's disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis.Methods and FindingsUsing a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C→G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09–1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1+/lacZ mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1+/lacZ mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model.ConclusionsHH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.

Evidence that glioma-associated oncogene homolog 1 is not a universal risk gene for inflammatory bowel disease in Caucasians

“…A quality threshold of 0.90 was set for imputation of these data sets. 2 reported no association of rs2228226 with IBD in the smaller Swedish data set 2 and proposed that GLI1 was predominantly a risk factor for UC. This is despite an equally strong association of rs2228226 with CD in the Scottish data set and a subsequent meta-analysis that produced the same effect size (OR ¼ 1.2) regardless of whether controls were compared with UC patients only or all IBD patients.…”

“…1 A recent study used a tagged single-nucleotide polymorphism (SNP) approach to characterize the variation in glioma-associated oncogene homolog 1 (GLI1) and tested for association with IBD in Scottish Caucasians. 2 Analysis of two tSNPs (rs2228224 and rs2228226) in this cohort demonstrated a significant association of GLI1 with both IBD and UC and a borderline association of GLI1 with CD, largely due to the non-synonymous rs22282260 (Q1100E), with the minor allele conferring susceptibility. The association of rs2228226 with UC, but not CD, was replicated in an IBD case-control data set of English Caucasians, but the minor allele frequency of this SNP did not differ significantly between Swedish IBD patients and controls.…”

“…This is despite an equally strong association of rs2228226 with CD in the Scottish data set and a subsequent meta-analysis that produced the same effect size (OR ¼ 1.2) regardless of whether controls were compared with UC patients only or all IBD patients. 2 To test for possible association with CD, we performed separate meta-analyses for CD and UC. In contrast to the combined analysis of Lees et al, 2 which showed association of rs2228226 with overall IBD (P ¼ 0.0002, OR ¼ 1.19, 95% CI ¼ 1.09-1.31) and UC (P ¼ 0.0008, OR ¼ 1.20, 95% CI ¼ 1.08-1.33), our UC-specific metaanalysis showed no significant association with UC (P ¼ 0.089, OR ¼ 1.07, 95% CI ¼ 0.92-1.24) (Figure 1a).…”

“…The association of rs2228226 with UC, but not CD, was replicated in an IBD case-control data set of English Caucasians, but the minor allele frequency of this SNP did not differ significantly between Swedish IBD patients and controls. 2 This as yet unconfirmed IBD gene is located within the IBD2 linkage region 12q13. 3.…”

“…This pathway may also be important for inflammatory responses as Hedgehog signalling is critical for the development of T lymphocytes, 5 CD4 þ T-cell activation, 6 myeloid cell maturation 7 and a direct transcriptional target of nuclear factor-kB. 8 The aim of our study was to genotype rs2228224 and rs2228226 in two additional Caucasian case-control data sets, combine this data with the Lees et al 2 and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) CD data sets, and to conduct a meta-analysis to assess the cumulative evidence for association of GLI1 with IBD.…”