a total of 2877 individuals with ibd ( 1902 with cd and 975 with uc ) were recruited in 5 centers across england and scotland . cd phenotype was classified by age at diagnosis , location , and behavior of disease . control allele frequencies were obtained from 1345 individuals recruited across britain as part of the 1958 british birth cohort . cases and controls were categorized into 12 broad geographical regions within great britain to minimize confounding due to variation in allele frequencies across the country . genotyping of cases was undertaken with iplex chemistry on a matrix - assisted laser desorption / ionization time - of - flight massarray platform ( sequenom , san diego , ca ) . cases were genotyped for 8 il23r markers reported in the index study , including the nonsynonymous single nucleotide polymorphism ( snp ) rs11209026 encoding amino acid change arg381gln ( primer sequences in supplementary table 1 ; see supplemental material online at www.gastrojournal.org ) . two of the north american markers ( rs7517847 , rs2201841 ) were omitted due to their location within a sequence of interspersed low - complexity repeats . genotyping of controls was undertaken at the wellcome trust sanger institute using the illumina 550k chip ( illumina , san diego , ca ) . concordance of genotype calls between the different platforms was confirmed by genotyping 87 control dnas for all 8 markers using the massarray platform with strong concordance of calls between technologies98.99% for the 8 markers overall . there was 100% concordance for 3 markers , including the coding variant arg381gln ( supplementary table 2 ; see supplemental material online at www.gastrojournal.org ) . the data for 1594 cases of cd genotyped for card15 mutations in earlier studies were used to undertake analysis for evidence of interaction between card15 and il23r . allele frequencies were compared between cases and controls and between phenotypic subgroups using  tests of 2  2 tables . odds ratios were calculated for the minor allele at each snp ; confidence intervals ( cis ) were calculated using woolf s method . haenszel test for association conditioning on geographical region was implemented using plink ( http://pngu.mgh.harvard.edu/purcell/plink/ ) . median age at disease diagnosis between groups was compared using the wilcoxon rank sum test . unless specified otherwise , all analyses were performed using r version 2.2 for windows ( http://www.r-project.org ) . all genotypes were in hardy  weinberg equilibrium in both cases and controls ( p > .05 ) . a highly significant association with cd was observed across the region ( table 2 ) . the strongest association was observed at the nonsynonymous snp arg381gln , where the frequency of the a allele was 2.5% in cd compared with 6.2% in controls ( p = 1.1 10 ) . the odds ratio for this protective allele was 0.38 ( 95% ci , 0.290.50 ) . alternatively , the common wild - type homozygous gg genotype can be considered as the risk genotype with an odds ratio of 2.70 . to minimize potential confounding from regional differences in allele frequencies , a mantel  haenszel test was performed across 12 regional strata . haenszel odds ratios was very similar to those obtained from pooled data for all snps . the strongest signal was observed with common snps rs1004819 ( p = .0071 ) and rs10889677 ( p = .0042 ) . the frequency of arg381gln was only marginally different between cases and controls ( uc , 0.046 ; controls , 0.062 ; p = .029 ) , with an odds ratio of 0.73 ( 95% ci , 0.550.96 ) . the nonsynonymous snp arg381gln was in tight linkage disequilibrium with one other snp ( rs11465804 , r = 0.85 ) but weak linkage disequilibrium with all 6 other snps ( r = 0.030.1 ) . a separate test for cd association was performed for each snp conditioning on arg381gln by conditional regression modeling . this showed a significant association at all snps ( p < .001 ) except rs11465804 , with the strongest residual association detected at rs10889677 ( p = 4.6  10 ) . hence , the nonsynonymous snp does not account for all the association signal at this locus . data were then analyzed for evidence of significant genotype - phenotype correlations based on age at onset of cd , disease location , and disease behavior ( table 3 ) . no significant subgroup association was observed . in particular , the subgroup of subjects with cd affecting the colon only without small bowel disease ( n = 539 ) appeared to be as strongly associated as those with exclusively ileal / small bowel involvement ( n = 668 ) ( minor allele frequencies , 2.3% and 2.0% , respectively ) . the age at disease onset ranged from 12 to 67 years in patients with cd who carried the a allele of arg381gln and from 0 to 80 years in wild - type gg cases . there was no difference in the median age of onset between these 2 groups ( aa / ag : median , 28 years [ n = 85 ] ; gg : median , 26 years [ n = 1650 ] ; p = .26 ) . stratification of cases by age at diagnosis according to the montreal classification revealed similar genotype frequencies in all groups ( table 3 ) . for uc , subgroup analysis by disease extent , smoking history , and sex also revealed no significant subgroup association . age at onset of uc ranged from 14 to 79 years in cases who carried the a allele of arg381gln and from 2 to 81 years in wild - type gg cases , with no difference in the median age of onset between the 2 groups ( aa / ag : median , 34 years [ n = 72 ] ; gg : median , 33 years [ n = 708 ] ; p = .14 ) ( table 4 ) . a total of 1540 subjects with cd were fully genotyped for the 3 card15 mutations ( g908r , l1007fs , r702w ) ( table 3 ) . the frequency of arg381gln in 460 cases carrying at least one card15 mutation ( 2.2% ) was not significantly different from that in 1081 cases who carried none ( 2.7% ; p = .47 ) . none of the 3 cases who were homozygous for the rare a allele also carried a card15 mutation . this study provides unequivocal confirmation of association between variants in the il23r gene and ibd , suggesting a major effect on overall susceptibility to cd and a more modest effect on uc . importantly , this study also shows the association at il23r for the first time in a non - american population . the strength of this association at il23r and the fact that it reaches such a magnitude in 2 independent data sets leaves no doubt that it is a true finding . in addition , this is one of the first instances of highly significant , independent replication of data derived from a genome - wide association scan and provides important validation of this technique as a hypothesis - free method for the identification of complex disease genes . as with the north american genome - wide scan , the strongest evidence for association was seen at the nonsynonymous snp arg381gln , where the frequency of the a allele was 2.5% in cd compared with 6.2% in controls ( p = 1.1  10 ) . there was no evidence that il23r variants associate with any particular subphenotype of cd based on disease behavior or location . hence , there was no difference in minor allele frequency even between the extremes of pure ileal / small bowel cd and pure colonic cd ( 2.7% and 2.3% , respectively ) . likewise , analysis based on disease behavior did not show any specific subgroup associations ( table 3 ) . this negative result is interesting because it contrasts with the other confirmed cd susceptibility locus card15 , which seems to have definite associations with ileal disease . these findings are extended by the observation of association with uc overall but not with any known uc subphenotype group , suggesting that il23r variants may exert a rather generic effect on chronic intestinal inflammation , although the effect size in uc does appear to be smaller than in cd . it is noteworthy that the odds ratio confidence interval at arg381gln for uc ( 0.73 [ 95% ci , 0.550.96 ] ) does not overlap with that for cd ( 0.38 [ 95% ci , 0.290.50 ] ) , suggesting a significantly less marked protective effect of the rare allele for uc compared with cd . based on data from our large , independent panel of cd cases , it is possible to provide an accurate estimate of the size of the effect conferred by il23r variants with regard to the risk of cd . we estimated an odds ratio of 0.38 ( 95% ci , 0.290.49 ) for arg381gln . this is likely to be a more accurate estimate than that provided in the index report from the north american study ( odds ratio , 0.26 ; 95% ci , 0.150.43 ) due to the well - recognized bias of the so - called winner s curse ,  which leads to overestimation of effect size in discovery panels . characterizing the exact effect size is important to permit sample size calculation for any further attempts at replication . where the effect size is overestimated , there is a risk that apparently appropriately powered studies will fail to observe the effect and erroneously conclude that it is a false positive . in some previous reports of genetic association , effect sizes have been quantitated as a population - attributable risk in addition to the odds ratio . this figure is intended to estimate the proportion of disease incidence attributable to a specific variant . however , it can not be calculated for a protective minor allele as in the case of arg381gln . further , while it is possible to think of the effect at arg381gln as an increased risk conferred by the common g allele , calculations of population - attributable risk based on this assumption lead to an implausibly high figure due to the very high carriage rate of the g allele in the control population . one important question is whether the nonsynonymous variant accounts for all of the association signal at il23r . this was tested by conditional regression modeling , looking for evidence of association while controlling for the effect at arg381gln . from this analysis , it is clear that there is a strong residual signal , maximal at rs10889677 ( p = 4.6  10 ) , and hence that variation at loci in addition to arg381gln , either within or adjacent to il23r , exert an influence on ibd susceptibility . whether this reflects a functional impact of the noncoding variants themselves or the fact that they are in linkage disequilibrium with other functionally significant or coding variants is yet to be established . analysis within haploview ( http://www.broad.mit.edu/mpg/haploview/ ) of data available from the international hapmap project ( http://www.hapmap.org ) shows that this selection of 8 tag snps captures only 18 out of 83 informative snps , within il23r and the 3 intergenic region covered by these markers , genotyped in the ceph ( utah residents with ancestry from northern and western europe ) panel at r > 0.8 . any additional coding variation is likely to be rare because interrogation of ensembl database release 41 ( october 2006 ) ( http://www.ensembl.org/ ) revealed the presence of only 2 additional nonsynonymous coding variants ( rs1884444 and rs7530511 ) in il23r with minor allele frequency > 1% in healthy european populations , both of which were investigated in the north american study with neither showing evidence of association . however , it is known that different splice isoforms of il23r exist and it is possible that their expression is determined by some of the documented noncoding variation . to clarify these issues , future studies will need to include resequencing of il23r in a cd panel and fine mapping across the gene using markers identified as a result , as well as studies to assess the potential functional impact of variants identified . the data with regard to arg381gln provide evidence of a very common variant being a disease risk allele , or conversely protection from cd being conferred by the rare allele . the explanations are likely to be complex but for immune - mediated conditions may include the fact that genetic variation at a particular locus confers a spectrum of risk , being protective against some diseases , such as infections , while increasing the risk of others , such as autoimmunity or inflammatory conditions . these variations will have been subject to differing selection pressures in diverse populations as a result of different environmental exposures . it is also noteworthy that for some of the markers showing evidence of association , it is the rarer allele that is associated with increased risk of cd . this further supports the argument for more than one variant in il23r with different effects on gene function . recent studies have identified il-23 , the cognate ligand of il23r , as a key player in both innate and adaptive immune systems . most il-23 is secreted by activated dendritic cells , monocytes , and macrophages following their exposure to pathogen - derived molecules that bind at toll - like receptors . il-23 stimulates a unique cd4 helper t - cell population characterized by the production of il-17 , tumor necrosis factor , and il-6 and known as th17 cells . these cells play a central role in driving autoimmune inflammation in a number of animal models . il-17 stimulates monocytes and endothelial cells to produce proinflammatory mediators , which in turn promote rapid neutrophil recruitment . the effect of il-23 has recently been distinguished from that of the related heterodimer il-12 , with which it shares a common p40 subunit . importantly in this regard , 2 studies in knockout mice lacking the p19 subunit of il-23 showed marked attenuation of t cell  mediated colitis , while knockout of the p35 subunit of il-12 produces no such attenuation , suggesting that il-23 but not il-12 is essential for the development of colitis . the identification of different roles for il-12 and il-23 in control of immune pathways together with the current genetic data suggest that targeting il-23 ( and components of its downstream effector pathway ) may be a useful and specific strategy to inhibit ibd while sparing systemic host protective immunity . as well as focusing attention on the il-23 pathway in the pathogenesis of ibd , the current study also provides key validation of genome - wide association scanning as a means of identifying complex disease susceptibility genes . the north american study group applied an appropriate , genome - wide significance level , and use of such a stringent threshold has immediately led to replication in our independent panel with a level of significance that makes the association indisputable . to date , complex disease genetic studies have been beset by poor study design , particularly use of nonconservative thresholds for significance , resulting in publication of many unreplicated false - positive results across the spectrum of common disease , hence the importance of the current study in providing unequivocal early replication in an independent panel of the principal findings from one of the first reported genome - wide association scans . there are recent reports in another complex disease ( age - related macular degeneration ) that also provide grounds for optimism that this technique produces replicable genetic association data . the clear message is that genome - wide association scanning works and that this study design , which is being so vigorously applied across a number of common diseases , is likely to be highly productive . the hope is that with use of appropriately stringent statistical thresholds and appropriately powered data sets , the success seen here in cd will be generally applicable without the plethora of false - positive results that have vexed the field of complex disease genetics to date . supplementary table 1primer sequences for all 8 snps using iplex chemistry on a massarray platformsupplementary table 2results of genotyping to confirm concordance between illumina and massarray technologies primer sequences for all 8 snps using iplex chemistry on a massarray platform results of genotyping to confirm concordance between illumina and massarray technologies
background & aims : identification of inflammatory bowel disease ( ibd ) susceptibility genes is key to understanding pathogenic mechanisms . 
 recently , the north american ibd genetics consortium provided compelling evidence for an association between ileal crohn s disease ( cd ) and the il23r gene using genome - wide association scanning . external replication is a priority , both to confirm this finding in other populations and to validate this new technique . 
 we tested for association between il23r and ibd in a large independent uk panel to determine the size of the effect and explore subphenotype correlation and interaction with card15 . 
 methods : eight single nucleotide polymorphism markers in il23r tested in the north american study were genotyped in 1902 cases of crohn s disease ( cd ) , 975 cases of ulcerative colitis ( uc ) , and 1345 controls using massarray . 
 data were analyzed using 2 statistics , and subgroup association was sought . 
 results : a highly significant association with cd was observed , with the strongest signal at coding variant arg381gln ( allele frequency , 2.5% in cd vs 6.2% in controls [ p = 1.1  1012 ] ; odds ratio , 0.38 ; 95% confidence interval , 0.290.50 ) . 
 a weaker effect was seen in uc ( allele frequency , 4.6% ; odds ratio , 0.73 ; 95% confidence interval , 0.550.96 ) . 
 analysis accounting for arg381gln suggested that other loci within il23r also influence ibd susceptibility . within cd 
 , there were no subphenotype associations or evidence of interaction with card15 . 
 conclusions : this study shows an association between il23r and all subphenotypes of cd with a smaller effect on uc . 
 this extends the findings of the north american study , providing clear evidence that genome - wide association scanning can successfully identify true complex disease genes .