Genome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region

Ulcerative colitis (UC) is a common form of inflammatory bowel disease with a complex aetiology. As part of the Wellcome Trust Case Control Consortium 2, we performed a genomewide association scan for UC in 2361 cases and 5417 controls. Loci showing evidence of association at P < 1 × 10−5 were followed up by genotyping in an independent set of 2321 cases and 4818 controls. We find genome-wide significant evidence of association at three new loci, each containing at least one biologically relevant candidate gene, on chromosomes 20q13 (HNF4A; P = 3.2 × 10−17), 16q22 (CDH1 and CDH3; P = 2.8 × 10−8) and 7q31 (LAMB1; 3.0 × 10−8). Of note, CDH1 has recently been associated with susceptibility to colorectal cancer, which is an established complication of longstanding UC. The new associations suggest that changes in the integrity of the intestinal epithelial barrier may contribute to the pathogenesis of UC. Genetic epidemiological data clearly implicate inherited susceptibility in the pathogenesis of UC and Crohn's disease (CD), which represent the two common forms of inflammatory bowel disease (IBD) and together affect at least 1 in 250 of the Northern European population.1 Notwithstanding recent therapeutic advances, disease-related morbidity in ulcerative colitis continues to be high. Recognized complications of severe disease refractory to medical therapy include colectomy, often as an emergency, in 15-20% of patients, as well as colorectal cancer2. Substantial progress has been made in understanding IBD pathogenesis in recent years. In genetically susceptible individuals it appears that a dysregulated mucosal immune response to commensal enteric bacteria predisposes to chronic, relapsing intestinal inflammation which is the hallmark of IBD.3 Clinical features combined with epidemiological evidence have long suggested that CD and UC are related polygenic diseases. This has recently been corroborated by the results of genetic association studies, which have highlighted both disease-specific loci and others which are shared between UC and CD. For example, while genetically-determined defects in the handling of intracellular bacteria (NOD2 and the autophagy genes ATG16L1 and IRGM) are specific to CD, multiple components in the Th17 pathway (IL23R, IL12B, JAK2, STAT3) are associated with both CD and UC.4-12 Until recently most attention had focused on CD, with genome-wide association (GWA) studies and subsequent meta-analysis yielding more than 30 confirmed CD susceptibility loci.4, 6, 7, 10-12 In addition to the longstanding known association in the MHC,13 the first Correspondence to: JCB ([email protected]), CCAS ([email protected]). Author contributions JL, CL, NP, AP, EW, KP, HZ, HD, ERN, DM, KB, TE, LC were involved in establishing DNA collections, and/or assembling phenotypic data; CL, AP, EW, DM, HD, AL, CM, JeS, DPJ, CE, TA, JCM, JS, MP recruited patients; WN, CE, TA, JCM, JS, MP, CGM supervised clinical and laboratory work; WTCCC2 DNA, Genotyping, Data QC and Informatics group executed GWAS sample handling, genotyping and QC; WTCCC2 Data and Analysis group, JCB, CAA performed statistical analyses; JCB, JL, CL, CCAS, CAA, TA, PD, JS, MP, CGM contributed to writing the manuscript. WTCCC2 Management Committee conceived and oversaw the design and execution of the GWAS. WTCCC2 group memberships are specified in the full author list. Europe PMC Funders Group Author Manuscript Nat Genet. Author manuscript; available in PMC 2010 June 01. Published in final edited form as: Nat Genet. 2009 December ; 41(12): 1330–1334. doi:10.1038/ng.483. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts GWA scans in UC reported associations at IL23R, IL10 and loci on chromosomes 1p36 and 12q15 which meet accepted genome-wide significance thresholds.14, 15 As part of the Wellcome Trust Case Control Consortium 2 (WTCCC2) study of 15 complex disorders and traits, we report here the results of the largest GWA scan in UC to date. All study subjects were UK residents of white, European ancestry; clinical data are presented in Table 1. Cases and controls were genotyped on the Affymetrix 6.0 array. After application of quality control filters (see Methods), we analysed GWA data from 2361 individuals with UC and 5417 controls (Figure 1). An initial analysis revealed 24 distinct loci (comprising 156 SNPs) which showed evidence of association at P < 1×10−5. Sixteen of these had not been previously reported, and were followed up by genotyping the most strongly associated SNP from each locus using the Sequenom iPlex platform in an independent panel of 2321 UC cases and 4818 controls. Three new loci showed evidence for association at P < 5 × 10−8 in the combined panel, with three further new loci showing nominal (P < 0.05) replication (Table 2 and Figure 2). We describe these loci below and highlight the most plausible candidate gene for each, recognizing that fine mapping and functional studies are required to define causal variants and identify the gene from which each signal arises. A list of all loci for which replication was attempted is shown in Supplementary Table 1. The most significant new association was seen at rs6017342 (GWA scan P = 3.2 × 10−13; combined GWA and replication P = 8.5 × 10−17), which maps within a recombination hotspot on chromosome 20q13 containing the 3′ untranslated region (UTR) of just one gene, HNF4A. The SNP rs6017342 itself maps 5kb distal to the 3′UTR. Although within an expressed sequence tag DB076868, this has been detected in just a single testis cDNA library and does not encode a significant open reading frame. The region contains two small blocks of sequence that are conserved in mammals and may include regulatory sequences affecting the expression of surrounding genes. Since rs6017342 is located within a recombination hotspot, there are few known SNPs in strong linkage disequilibrium (r2 > 0.5) with it; there are none on the Affymetrix chip used in this study or on the Illumina chips used in previous studies. As the evidence for this association rests on this single SNP, we subjected these data to careful scrutiny; genotype cluster plots for this SNP showed clear resolution of the 3 genotype classes (Supplementary Figure 1), with 99.3% completeness of genotypes within this dataset. Rare HNF4A mutations account for approximately 4% of UK cases of maturity-onset diabetes of the young (MODY),16 a monogenic form of diabetes mellitus characterized by autosomal dominant inheritance, young age of onset, pancreatic b-cell dysfunction and sensitivity to sulphonylureas. Common variants of HNF4A influence predisposition to Type II diabetes (rs2144908)17 and dyslipidaemia (rs1800961).18 The UC associated SNP, rs6017342 is not in LD with either of these 2 common variants, nor did it not show association in our study of CD (P=0.92).3 HNF4A encodes the transcription factor hepatocyte nuclear factor 4 α which regulates the expression of multiple components within all three key compartments of the cell-cell junction, namely the adherens junction, the tight junction and the desmosome.19 Such cellcell junctions are fundamental to epithelial organization and barrier function. HNF4α also plays a key role in the development of the embryonic mammalian gastrointestinal tract. Previous studies demonstrated that mice with targeted deletion of HNF4α in epithelial cells of the foetal colon die perinatally. Histological analysis of colonic tissue recovered during late development (E18.5) demonstrated absent crypt formation, reduced epithelial cell proliferation and defective goblet cell maturation.20 In order to explore the role of HNF4α in murine intestinal inflammation, Ahn and colleagues circumnavigated the embryonic lethality of Hnf4α−/− mice by generating a conditional model of intestinal Hnf4α deletion. and Page 2 Nat Genet. Author manuscript; available in PMC 2010 June 01. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts 21 These Hnf4αΔIEpC mice (floxed Hnf4α driven by the villin promoter) developed increased epithelial permeability and a markedly more severe colitis following dextran sodium sulphate (DSS) challenge, than their wild-type littermates.21 The same investigators provided preliminary evidence for dysregulated HNF4A gene expression in the intestinal epithelium in Crohn's disease and in ulcerative colitis,21 a finding which now merits detailed re-exploration. Significant association was also seen for a locus on chromosome 16q22, with the strongest signal at rs1728785 (GWA scan P = 1.8 × 10−5; combined GWA and replication P = 2.8 × 10−8). The interval bounded by recombination hotspots spans 411 kb and encodes several genes. Among the strongest candidates for UC susceptibility is CDH1 which encodes Ecadherin. This transmembrane glycoprotein is one of the main components of the adherens junction and a key mediator of intercellular adhesion in the intestinal epithelium. It also plays a key role in epithelial restitution and repair following mucosal damage and expression of CDH1 is known to be significantly reduced in areas of active UC.22 Given the well-recognised association between UC and colorectal cancer,2 the observation of correlated association signals at the CDH1 locus in both diseases is striking. Thus variants in LD (r2 = 0.5) with the most strongly UC associated SNP in our study were recently identified in a GWA scan meta-analysis to be associated with colorectal cancer susceptibility23; conversely, we find that a perfect proxy for the most associated SNP in the colorectal cancer study is also associated with UC (P = 8 × 10−4). This locus did not show association with CD in a large international GWA meta-analysis of CD (P = 0.549)6 (Supplementary Table 2). However, evidence for association of CDH1 with CD was reported recently in the Canadian population using a candidate gene approach,24 and the CD associated SNPs resulted in a truncated E-cadherin protein in vitro which accumulated in the cytoplasm and led to disorganized epithelial architecture.24 Of great potential relevance is the evidence that HNF4A and E-cadherin co-operate to maintain epithelial barrier integrity in the intestine. In experiments focused on the liver, HNF4α knockout mice failed to express E-cadherin,19 while in the gut E-cadherin dependent cell-cell contact was found to be critical in determining the amount and binding activity of nuclear HNF4α. This in turn affected the expression of several genes including ApoA-IV,25 an anti-inflammatory protein known to inhibit experimental colitis.26 The third newly confirmed UC susceptibility locus was a region on chromosome 7q31, previously suggested by a recent North American GWA scan.14 In the current study the peak association was seen at rs886774 (GWA scan P = 4.8 × 10−7; combined GWA and replication P = 3.0 × 10−8). A strong positional candidate gene at this locus is LAMB1, encoding the laminin beta 1 subunit. Laminins are heterotrimers; the beta-1 light chain is present in laminins-1 -2 and -10. Laminins are expressed in the intestinal basement membrane, and play a key role in anchoring the single-layered epithelium; expression is known to be down-regulated in UC.27 rs886774 was not associated with CD in the metaanalysis.5 (Supplementary Table 2) Two other loci previously implicated in UC-related phenotypes showed strong (but not genome-wide significant) association with UC. These comprise a SNP previously associated with osteoporosis28 (rs7524102 on chromosome 1p36, combined GWA and replication P = 3.1 × 10−7) and a SNP nearby (though not in LD with) a marker known to be associated with psoriasis29 (rs9548988 on 13q.13, combined GWA and replication P = 2.7 × 10−7). In addition to the novel loci described above, our GWAS detected strong association at established UC loci such as the MHC, IL23R, 3p21/MST1 and NKX2-3 (one tailed P values in the direction of the previously reported association in Table 3). We also provide robust and Page 3 Nat Genet. Author manuscript; available in PMC 2010 June 01. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts confirmation of two UC loci reported recently in genome-wide scans, the IL10 locus11 and the OTUD3/PLA2G2E locus12 on chromosome 1q31 and 1p36 respectively. Also of interest is our finding that the PSMG1 locus on chromosome 21, which has previously been associated with pediatric-onset IBD,30 is likely to contribute specifically to disease susceptibility in UC. Variable degrees of support were obtained for some previously reported UC loci, including ECM1, CARD9,31 KIF21B/chromosome 1q32, and JAK2/ chromosome 9p24, but weaker support for other loci such as IL2/IL21,32 IL12B and 12q15 (Table 3). Some of the UC loci are clearly associated with CD, while others are not, or have not been tested (Supplementary Table 2). We also tested for epistatic interaction among all pairwise combinations of these loci (both previously described and new) but found none. This is the first report of a new series of GWA scans undertaken by the WTCCC2 consortium. We have identified three new susceptibility loci for UC, and provide the first genetic link between UC and colorectal cancer. Each of the strongest new association intervals that we have identified contains respectively HNF4A, CDH1 and LAMB1 as the most plausible positional candidate genes, thus providing further evidence for the reemerging concept that altered epithelial barrier function may be a key factor in UC pathogenesis.8 Indeed, this is the first time that variants within genetic loci encoding such epithelial barrier genes have shown association with IBD at stringent genome-wide significant thresholds. Fine mapping and functional studies are clearly required to investigate this connection further, but our study provides strong scientific justification for the exploration of new therapeutic targets relevant to epithelial barrier function. METHODS Subjects Cases—A total of 5319 unrelated patients of white, European, non-Jewish ancestry with a diagnosis of ulcerative colitis established using standard endoscopic, radiological and histological criteria, were recruited from ten centres within the United Kingdom (Cambridge, Oxford, London, Newcastle, Sheffield, Edinburgh, Dundee, Manchester, Torbay and Exeter, Supplementary Table 4). All patients provided written consent and either a sample of blood or saliva, from which DNA was extracted according to standard protocols. Research Ethics Committee approval was obtained prior to sample collection (Cambridge, Oxford, London, Newcastle, Sheffield, Edinburgh, Dundee, Manchester, Torbay and Exeter Local Research Ethics Committees). After QC (see below), we analyzed a total of 4682 samples, which were divided between the discovery panel (2361 samples) and replication panel (2321 samples). Controls—A total of 10,235 control DNA samples from 3 sources passed our QC filters (see below). 5417 samples of the WTCCC2 common control set were used for the GWA experiment. This comprised 2675 healthy blood donors recruited from the United Kingdom Blood Service (UKBS), and 2742 samples from the 1958 Birth Cohort (1958BC) obtained from EBV-transformed cell lines from individuals born in England, Wales and Scotland during one week in 1958. The 4818 samples used as controls for the replication cohort were recruited from the Wellcome Trust-funded People of the British Isles (PoBI) DNA collection, obtained from rural populations throughout the British Isles, and from a further independent set of DNA samples obtained from 1958BC. All of the control samples used were from individuals with self-reported Caucasian ethnicity. A summary of patients and controls is shown in Table 1 and Supplementary Table 4. DNA sample preparation: Genomic DNA for all cases was shipped to the Sanger Institute, Cambridge. DNA quality plus subject identity were validated using the Sequenom iPLEX and Page 4 Nat Genet. Author manuscript; available in PMC 2010 June 01. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts assay designed to genotype 4 gender SNPs and 26 SNPs present on the Affymetrix array. DNA concentrations were quantified using a PicoGreen assay (Invitrogen) and an aliquot assayed by agarose gel electrophoresis. A DNA sample was considered to pass quality control if the original DNA concentration was ≥50ng/ul, the DNA was not degraded, the gender assignment from the iPLEX assay matched that provided in the patient data manifest and genotypes were obtained for over 65% of the SNPs on the iPLEX.