Gastroenterology
Volume 133, Issue 4 , Pages 1327-1339, October 2007

The Genetics of Inflammatory Bowel Disease

  • Judy H. Cho

      Affiliations

    • Inflammatory Bowel Disease Center, Section of Digestive Diseases, Yale University, New Haven, Connecticut
    • Corresponding Author InformationAddress requests for reprints to: Judy H. Cho, MD, Yale University, 333 Cedar Street, 1080 LMP, New Haven, Connecticut 06520-8019. fax: (203) 785-7273.
    • ,
  • Casey T. Weaver

      Affiliations

    • Department of Pathology, University of Alabama Birmingham, Birmingham, Alabama

Received 27 July 2007; accepted 1 August 2007.

Article Outline

Abbreviations used in this paper: GWA, genome-wide association, IL, interleukin, SNP, single nucleotide polymorphism

 

The inflammatory bowel diseases (IBDs) are subdivided into Crohn’s disease (CD) and ulcerative colitis (UC) based on well-established clinical and pathologic criteria. Recently, significant progress has occurred in our understanding of genetic variation associated with IBD, most notably through genome-wide association (GWA) studies. These GWA studies have shown encouraging consistency between cohorts, dramatically increasing the number of definitive, well-replicated genetic associations in IBD. These advances will improve understanding of pathophysiologic mechanisms required to develop new therapeutic interventions. Useful in this regard is integration of understanding between human studies and murine genetic models relevant to IBD. The role of the interleukin (IL)-23 pathway in multiple chronic inflammatory diseases in humans, including IBD, was foreshadowed by immunologic studies in murine models. This review outlines recent human genetic advances, with a particular focus on the association of IL-23 receptor (IL23R) polymorphisms and the IL-23 pathway with IBD.

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Value of Genetic Studies in IBD 

The primary goal of genetic studies in IBD is the identification and prioritization of mechanisms contributing to expression of clinical disease. The identification of key pathogenic mechanisms will serve to prioritize development of new therapeutic approaches in IBD. The plethora of murine genetic models of IBD1 (Table 1) shows the broad variety of very different mechanisms by which an IBD-like phenotype can result. The complex and delicate balance required to maintain intestinal homeostasis can be disrupted by increased or decreased expression of an ever-increasing catalog of genes.

Table 1. Representative Mouse Models of IBD
ModelAffected gene(s)/locusGenetic alterationPhenotypeReferences
MDR1α deficientAbcb1bTargeted deletionAltered epithelial function72, 130
Gαi2 deficientGnai2Targeted deletionDefective epithelial barrier131
EP4 deficientPtger4Targeted deletionAltered epithelial function125
TNF-αΔARETnfTargeted deletion: mRNA 3′UT AU repeatsIncreased tumor necrosis factor α production132
Macrophage-PMN STAT3 deficientStat3Targeted deletion in myeloid cellsImpaired IL-10R signaling133
A20 deficientTnfaip3Targeted deletionIncreased nuclear factor κB activity134
F59D-JAB transgenicSocs1Overexpressed DN mutantImpaired SOCS1/SOCS3 signaling; enhanced STAT3 activation135
IL-10 deficientIl10Targeted deletionImpaired regulatory T-cell function73, 136
IL-2 deficientIl2Targeted deletionImpaired regulatory T-cell maintenance/function137
SAMP1/YitPpargMultiple(?)Spontaneous ileitis7, 138

NOTE. Included here is a representative list of mouse models of IBD that highlight the range of epithelial, innate, and adaptive immune cell defects that can lead to IBD pathogenesis. Of note, although specific characterized defects are indicated for each of the models, there may be multiple levels of involvement among innate and immune cell populations for each of these pathways.

A second, related goal of IBD genetics studies is the development of predictive risk models, both in unaffected relatives of IBD probands and in predicting disease outcomes in patients with diagnosed IBD. The rational design of well-powered preventive interventions2 will require the identification of currently unaffected individuals at high risk for development of IBD. While numerous IBD genetic studies have tested for statistical interactions between established susceptibility genes, at present, definitive interactive effects have been established.

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Murine Genetic Models Relevant to IBD 

An extensive and growing number of animal studies have contributed to establishing the mechanistic bases for our current understanding of IBD pathogenesis. Mouse models with induced or spontaneous mutations in a diversity of genes have established the complex interrelationships that control the lifelong host response to the intestinal microbiota and have identified a number of pathways that can lead to IBD.1 With few exceptions, these models implicate a dysregulated dialogue between the intestinal microbiota and components of the innate and adaptive immune systems in disease development. The host response to the intestinal microbiota can be categorized into 3 basic compartments: the intestinal epithelium, innate immune cells of the myeloid lineages (eg, monocytes, dendritic cells, and granulocytes), and adaptive immune cells (B cells and T cells). Natural killer and natural killer T cells may also play important roles, although these are less well defined.3 Models with defects in each of these compartments have been associated with IBD pathogenesis in the mouse. Shown in Table 1 is an abbreviated listing of mouse models that highlights the diversity of genes and pathways associated with IBD in the mouse. In virtually all of the models characterized to date, a primary component of disease expression is unrestrained effector CD4 T-cell reactivity to enteric bacteria. Insofar as CD4 T-cell responses are induced and directed by the innate immune system, the interplay among the microbiota, the intestinal epithelial barrier, and innate immune cells are critical determinants of mucosal CD4 T-cell responses. Accordingly, dysregulation at any of these levels may lead to IBD. Furthermore, a substantial portion of the mucosal CD4 T-cell repertoire comprises regulatory T cells that control CD4 T-cell effector responses. To date, there are limited data from mouse models to suggest that B-cell responses play a central pathogenic role, and in some models, antibody responses to the intestinal flora appear to be protective.

For most human IBD susceptibility genes, functional polymorphisms will not result in gross defects in genes of the innate or adaptive immune system; therefore, many of the mouse models appear to be more important in identifying pathways of susceptibility rather than establishing strict pathogenetic associations. Illustrative of the complexity of genetic factors contributing to human and murine IBD has been the use of induced or spontaneous mutant mice to map IBD modifier genes. In several cases, it has been shown that strain background can have profound effects on expression of disease in experimental models. For example, identification of the C3H/HeJ and C57BL/6 strains as highly susceptible and resistant genotypes, respectively, was used as a basis for QTL analyses in IL-10–deficient mice to map a colitis susceptibility gene locus on murine chromosome 3, termed Cdcs1, which appears to regulate the magnitude of innate responses to TLR ligands.4, 5 Similarly, genome-wide linkage analysis performed on crosses of the spontaneously ileitic SAMP1/YitFc mouse with the resistant C57BL/6 strain identified a susceptibility locus on chromosome 9 that encompasses candidate genes encoding IL-18 and the IL-10 receptor α chain.6 More recent analyses of this phenocopy of human CD in backcrosses to the parental AKR strain have identified an additional susceptibility locus that includes the inflammation-modulating transcription factor peroxisome proliferator-activated receptor γ, for which susceptibility alleles have been reported in humans.7 As the number and power of GWA studies progresses, it is likely that an increasing number of correlates for human IBD will be identified for these and other genes.

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Naturally Occurring Human Genetic Variation 

The comprehensive sequencing and characterization of genetic variation in humans and other species has provided an enormous wealth of biologic information.8 Recent studies estimate the presence of 25,000 genes contained within 3 billion DNA nucleotides.9 Naturally occurring interindividual variations in DNA sequences include large-scale copy number polymorphisms (variations in the number of copies of a given DNA sequence), repetitive sequences of varying length, a variety of insertions and deletions, and, most commonly, single nucleotide polymorphisms (SNPs). Salient features of SNPs (single base pair substitutions at given DNA sequences) include the following: (1) their relative frequencies within populations, (2) whether or not functional alterations can be attributed to the substitution, and (3) their organization within the human genome relative to other genetic variants.

Common Versus Rare SNPs 

Most SNPs within the human genome are diallelic, and genotyping platforms directly measure genotypes (homozygous wild-type, heterozygous, and homozygous mutant). Within populations, genotype frequencies can then be utilized to estimate allele frequencies (wild-type or major allele vs mutant or minor alleles). It is estimated that there are 10 million SNPs within the human genome with a minor allele frequency of greater than 5% in at least one population, representing an enormous source of “common” human genetic variation. As a general rule, commoner alleles are more ancient, having had a greater time to propagate within the population. SNPs having a minor allele frequency greater than 5%–10% will more likely be observed commonly in European, Asian, and African populations, with rare variants more likely to be uniquely observed in one population or another. Accordingly, each of the NOD2 CD-associated susceptibility alleles has a frequency of less than 5% in European ancestry controls10, 11 and is not observed in Asian12, 13, 14 or African15 populations. Both common and uncommon human genetic variants contribute to complex multigenic disorders such as IBD (Figure 1); a variety of approaches will be required for the comprehensive identification of genetic variation contributing to IBD phenotypes.

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  • Figure 1. 

    Spectrum of CD susceptibility alleles. Minor allele frequencies are plotted as a function of allelic odds ratios for the most well-established CD susceptibility alleles. With the exception of the NOD2 risk alleles, functional studies establishing a direct pathogenic role have not been performed, and the plotted odds ratios represent estimates of representative, associated markers. The NOD2 risk alleles are estimated from a population-based cohort from Manitoba.32

Functional Polymorphisms 

A number of forces contribute to the history and frequency of SNPs, including, most notably, evolutionary selection resulting from the functional effects of genetic polymorphisms. It should be recognized that only a fraction of human SNPs will have direct functional consequences. The precedent from single-gene, Mendelian disorders shows a preponderance of variants directly affecting protein structure (eg, frameshift mutations, amino acid polymorphisms, variation in consensus messenger RNA [mRNA] splice site sequences) and function.16 However, not all amino acid polymorphisms will have significant functional effects. Bioinformatic analyses showing amino acid conservation between species enrich for more likely functional polymorphisms. Ultimately, however, proof of a direct role in disease pathogenesis for putative functional polymorphisms requires experimental support using mutagenesis approaches and/or primary human cells stratified on the polymorphism of interest. More challenging to characterize is the vast array of noncoding polymorphisms, a fraction of which will more subtly affect protein expression through tissue- and context-specific mechanisms. A large variety of approaches are being used to address these challenges.17 High throughput application of chromatin immunoprecipitation identifies DNA-protein interaction sites more comprehensively.18 GWA mapping of mRNA expression levels with genetic polymorphisms has identified successfully highly regulated genes.19

Genomic Organization: Linkage Disequilibrium Patterns 

The organization of SNPs within the genome represents an additional feature of interest. Because nearby SNPs are often highly correlated with each other (linkage disequilibrium), it is not necessary to directly type millions of SNPs to comprehensively compare common genetic variation between IBD cases and controls. Through the human HapMap Project,20 the correlative patterns of common SNPs have been characterized; this information has been utilized to develop and interpret genotyping platforms testing several hundred thousand SNPs that successfully capture a large fraction of common human variation.21 Early results from GWA studies have significantly increased the number of definitive IBD associations in a very short time. While advantageous in developing genome-wide genotyping platforms, linkage disequilibrium patterns may complicate efforts to precisely identify directly causative functional polymorphisms. This will be discussed with respect to the IBD5 association on chromosome 5q31.22

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IBD Genetics Before the GWA Era 

Efforts to identify IBD susceptibility alleles before the advent of GWA studies involved focused candidate gene studies combined with fine-mapping efforts in regions of genetic linkage (demonstrations of increased genomic sharing between multiple, close relatives sharing a disease).23 The candidate gene approach resulted in well-replicated associations of IBD with various major histocompatibility complex class II associations.24 The NOD2 association with CD10, 11 was preceded by the identification of well-replicated linkage in the pericentromeric region of chromosome 16.25, 26 The interpretative challenges of genetics studies in complex disorders result from a number of sources, including the following: (1) the presence of genuine population differences, (2) inconsistent genotyping approaches between studies (eg, changing typing technologies for class II HLA associations has complicated interpretation of existing studies), (3) underpowered studies resulting in type I error (this is particularly an issue with respect to associations reported solely in patient subsets for which no compelling prestudy hypothesis existed), and (4) publication bias with respect to the reporting of positive versus negative association studies. Definitive IBD associations are established by the reporting of associations in large cohorts that are consistently replicated by independent studies (Table 2). Before GWA studies, the NOD2, IBD5, and HLA class II associations represent the most consistently replicated associations; in addition, we briefly summarize other associations for which additional studies will be required to establish a definitive role in IBD.

Table 2. Definitive IBD Associations
Gene or LocusGenomic LocationFunction and/or Genes in the RegionAssociationContributing AllelesReferences
NOD216q12Intracellular sensor of peptidoglycanCD onlyArg702Trp, Gly908Arg, Leu1007fsinsC10, 11
IL23R1p31IL-23 receptorCD and UCArg381Gln, additional undefined alleles85
ATG16L12q37Autophagy geneCD onlyAla197Thr120, 121
Intergenic region5p13?PTGER4; CARD6Reported in CD onlyUndefined91
IBD55q31?SLC22A4, ?SLC22A5, ?IRF1CD and UCUndefined22, 53, 66
MHC region6p21Major histocompatibility complexCD and UC; distinct association signalsUndefined24
PTPN218p11T-cell protein tyrosine phosphataseReported in CD onlyUndefined92, 93
IL12B5q33Interleukin-12/23 p40 subunitReported in CD onlyUndefined92, 93
NKX2-3 region10q24Gut immune developmentReported in CD onlyUndefined92, 93
Intergenic region10q21?ZNF365; ?EGR2Reported in CD onlyUndefined121
Gene-rich region3p21ManyReported in CD onlyUndefined92, 93
IRGM gene region5q33Autophagy geneReported in CD onlyUndefined92, 93

CD-Associated NOD2 Polymorphisms 

Genetic epidemiology 

The NOD2 gene is located on chromosome 16q12, and the encoded intracellular protein activates nuclear factor κB and mitogen-activated protein kinase pathways in response to stimulation by components of peptidoglycan (minimal bioactive component, muramyl dipeptide), present in the cell wall of gram-positive and gram-negative bacteria.27, 28, 29 Three variants, Arg702Trp, Gly908Arg, and Leu1007fsinsC, each independently increase risk for ileal-only and ileocolonic CD, but not colonic-only CD or UC.10, 11, 30 A recent meta-analysis of all previously reported association studies in European ancestry cohorts estimated that homozygous or compound heterozygous carriage of NOD2 risk alleles confers a 17.1-fold (95% confidence interval, 10.7–27.2) increased risk of CD, whereas heterozygous carriage of NOD2 increases the risk of CD 2.4-fold (95% confidence interval, 2.0–2.9).31 These odds ratios represent some of the most significant risk alleles among complex, multigenic disorders. However, it is estimated that among NOD2 homozygote/compound heterozygote carriers, significantly less than 10% of individuals will manifest CD, an estimate of the genetic penetrance of the NOD2 mutations.32, 33 In addition to the 3 major CD-associated mutations, a number of very rare amino acid polymorphisms either within or near the C-terminus leucine rich repeat peptidoglycan-sensing domain have been reported that likely contribute to CD susceptibility.30, 34 The 3 major CD mutations are not observed in Asian patients with CD12, 14, 35 and very uncommonly in black patients with CD.15

Functional NOD2 studies 

Both transfection studies of mutant and wild-type NOD2 as well as studies in primary human cells stratified on NOD2 genotype11, 17, 29, 34, 36, 37, 38, 39 demonstrate decreased response with acute, short-term muramyl dipeptide stimulation. By contrast, the murine knock-in of the frameshift mutation demonstrated increased nuclear factor κB activation40; additional studies will be required to reconcile differences between the human and murine findings. If the CD-associated NOD2 mutations represent loss-of-function mutations, the NOD2 knockout represents an important model for assessing the functional consequences of NOD2 polymorphisms. The NOD2 knockout does not spontaneously develop ileitis,41, 42 demonstrating the requirement for other genetic variants and environmental and developmental factors for disease expression. Potential mechanisms of disease pathogenesis include altered cytokine regulation,43, 44 dysregulated killing of intracellular bacteria,45 and decreased cryptdin expression observed in NOD2-deficient mice.41 The latter observation corresponds with decreased α-defensin expression observed in human CD generally,46 especially pronounced in individuals with NOD2 mutations. A number of NOD2-positive and -negative signaling partners have been identified, including RIP2,47 erbin,48 centaurin β1,49 GRIM-19,50 and TAK1.51 The NOD2 discovery provides specific support for the long-held hypothesis that CD results from a genetically dysregulated host immune reponse to luminal bacteria.52

Association of IBD5 on Chromosome 5q31 With CD and IBD 

An association with CD has been consistently observed for a 250-kilobase genomic region on chromosome 5q31.22 A relatively common haplotype (Figure 1) in the region has been reported, conferring a modestly increased risk of CD, with allelic odds ratios reported typically less than 1.3. The region of association includes a relatively gene-rich area containing several candidate genes. It has been reported that at the Leu503Phe variant in SLC22A4, the less common, CD-associated phenylalanine allele, is associated with decreased transporter activity53; however, this finding has not been replicated in independent studies. Due to linkage disequilibrium patterns, Leu503Phe demonstrates statistically equivalent association with a number of variants throughout the region, and therefore additional studies will be required to definitively determine contributing functional variants in the region. Despite a high frequency of the risk haplotype in Ashkenazi Jewish cohorts, no evidence for an association with IBD is observed, in contrast to IBD in patients of non-Jewish European ancestry.54 This could reflect either the presence of distinct risk alleles in various population cohorts or that the actual causative variant(s) has not yet been identified. Similarly, no association at IBD5 has been observed in multiple Japanese CD cohorts,55, 56, 57 with the risk haplotype being at a significantly lower frequency in the Japanese population. Phenotypic correlates for the IBD5 CD association have been reported for perianal disease,58 colonic location,59 disease complications and progression,60, 61 extensive disease in UC,62 and decreased height and weight at diagnosis in pediatric cohorts.63 In contrast, some studies have not observed clear genotype-phenotype correlations.54, 64 Both positive61, 65, 66 and nonsignificant55, 58, 59, 60, 62, 67, 68 studies of IBD5 associations with UC have been reported, and meta-analyses of all existing data sets will be required to assess the overall contribution of this region to risk of UC.

HLA Class II Associations 

Both genetic linkage and association studies support the contribution of genetic variants within the major histocompatibility complex region to IBD. The major histocompatibility complex region is characterized by enormous genetic diversity driven by significant selection forces, with complex patterns of linkage disequilibrium. A plethora of IBD association studies in this region has been reported. Interpretation of such studies has been hampered by the use of different typing platforms, small study cohorts, and comparisons between different population groups. However, several broad conclusions have been forwarded. First, the strongest associations have been observed in the class II as opposed to the class I region. The HLA class II genes encode α and β receptors that form heterodimeric receptors on antigen-presenting cells. Secondly, the associations between UC and CD are distinct, with the evidence for class II associations particularly strong for colonic disease. Replicated HLA class II associations in IBD include HLA-DRB1*1502 (serologic marker HLA-DR2)69 association with UC and HLA-DRB1*0103 association with UC and colonic CD.70, 71 HLA-DRB1*0103 is noteworthy in that it is a risk factor for both UC and colonic CD, suggesting it may play a role in chronic inflammation of the colon independent of major IBD phenotype (ie, CD or UC).71 As is the case with IBD5, the DRB1*0103 and DRB1*1502 class II variants are in strong linkage disequilibrium with SNPs on multiple immunologically active candidate genes. Whether the association signal is driven by class II genes themselves, in nearby genes, or by a combination of variants has not been fully defined. The advent of new genotyping platforms, including dense typing in this region, will result in a more complete understanding of IBD associations in this region.

Additional Reported IBD Associations 

Over the years, a number of IBD associations have been reported that have demonstrated varying levels of replication by independent studies. For many of these candidate genes, either inadequate or conflicting replicative studies have been reported, and therefore, definitive conclusions regarding association with IBD cannot be made. Rationales for testing these genes have included functional plausibility (murine models of IBD, homologues of established genes), genes in regions of increased linkage, and association of genes in related disorders. Specifically, a number of candidate genes have been tested based on prior observations that targeted disruption of the gene in murine models results in an IBD-like phenotype. For example, because MDR1 (multidrug resistant)72 and IL-10–deficient mice develop intestinal inflammation,73 multiple human genetics studies74 have been reported. However, because of the typing of different SNPs between studies, as well as the presence of both positive and negative studies, definitive support for association in humans is presently lacking. Similarly, given the close homology of NOD1 (CARD4) and NOD2, reports of IBD association with NOD1 are of great interest75 but require additional study.76, 77 The preponderance of studies thus far have reported positive association of the uncommon glycine allele of Arg299Gly within the TLR4 (Toll-like receptor 4) with CD78; however, this uncommon variant has not been well sampled in GWA studies. In a large case-control study, association was observed in UC and CD for Ala1011Ser within the Myo IXb (MYO9B)79 gene that had been previously implicated in celiac disease.80 If replicated, this would indicate shared susceptibility pathways between multiple types of intestinal inflammation.

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GWA Studies in IBD 

Progress from the Human Genome Project and HapMap Project, combined with markedly decreasing genotyping costs, has made possible the performance of adequately powered GWA studies in complex genetic disorders such as IBD. The capacity to detect association to an (untyped) susceptibility variant by genotyping a neighboring marker will be proportionate to the r2 value (measure of linkage disequilibrium, or correlation) between the susceptibility allele and genotyped marker. In European ancestry populations, presently available genotyping platforms, which include 300,000–500,000 SNPs throughout the genome, will sample approximately 80% of the common variation with an r2 value of greater than 0.8.21 This would suggest that present GWA approaches will successfully test a significant fraction of common human variation throughout the genome. Present GWA approaches provide a broad, unbiased landscape of contributing disease variants. When performing such a large number of statistical tests, the capacity to dissect signal from noise is dependent both on the magnitude of the underlying signal (eg, as assessed by the odds ratio) and the size of the study population. In CD, early GWA studies have identified a number of already well-replicated disease associations, dramatically advancing our understanding of IBD genetic associations.

Limitations of present GWA studies in IBD should be recognized, however. The extent to which rare functional variants contribute to overall disease burden is unestablished at present. However, the GWA genotyping platforms were developed to sample common variation having minor allele frequencies of greater than 5% in the population of interest, with poor coverage of rare polymorphisms achieved. A second limitation of present GWA studies is that they have largely examined European ancestry cohorts. Importantly, recently reported associations in European ancestry CD cohorts identified through GWA studies have thus far not been replicated in Asian cohorts.81 Greater comparative analyses of Asian and African IBD populations may provide important insight into alternative pathophysiologic mechanisms in IBD.

GWA Study of Japanese Patients With CD Implicates Polymorphisms in TNFSF15 

The first GWA in CD was performed using a 2-stage approach initially testing nearly 80,000 SNPs in 94 Japanese patients with CD.82 Follow-up studies for those markers demonstrating P values less than .01 performed in a second stage involving 484 patients with CD identified 22 markers with P values less than 10−4. Several of these significant markers were contained within a 280-kilobase region on chromosome 9q32, with the most significant markers clustered within the TNFSF15 (tumor necrosis superfamily) gene (odds ratio, 2.17; P = 1.71 × 10−14). Replicative evidence for association was observed in European family and case-control cohorts.82 Protein and mRNA levels of TNFSF15 are up-regulated in macrophages and CD4+/CD8+ lymphocytes of the intestinal lamina propria of patients with CD.83 TNFSF15 functions as a ligand for TNFRSF25 (tumor necrosis factor superfamily receptor) and is induced by FcγR stimulation of monocytes and dendritic cells.84 No replicative evidence in this region has been reported in independent European ancestry GWA studies; this may reflect the different markers present on the different GWA genotyping platforms and/or increased contribution of TNFSF15 in Asian IBD cohorts.

IL23R as an IBD Susceptibility Gene 

IBD association 

In a GWA study involving a North American ileal CD case-control cohort typing more than 300,000 SNPs, the most significant associations were observed in the previously identified NOD2 gene as well as in the IL23R gene region on chromosome 1p31.85 Within the IL23R gene, the most significant association was observed for the amino acid polymorphism, Arg381Gln, with the less common glutamine allele conferring significant protection (non-Jewish CD: odds ratio, 0.26; 95% confidence interval, 0.15–0.43) against developing CD. The allele frequency of the glutamine allele was 1.9% and 7.0% in non-Jewish CD cases and controls, respectively; therefore, approximately 1 in 7 European ancestry controls are heterozygous carriers for the protective glutamine allele. In addition to Arg381Gln, a number of additional SNPs demonstrated independent evidence for association, suggesting the presence of at least 2 independent risk alleles in the region. The gene immediately centromeric to IL23R is its closely related homologue, IL12RB2. Most of the association signals in this region are contained within a haplotype block encompassing the centromeric portion of IL23R (containing exons 5–11), which extends into the intergenic region between IL23R and IL12RB2.85 Multiple mRNA splicing variants have been reported for IL23R, most commonly involving exons 7 and/or 10.86 The haplotype block containing the IL12RB2 gene itself did not show evidence for CD association. The IL23R associations are observed in non-Jewish and Jewish CD case-control and family cohorts, as well as in UC.85, 87 Specifically, similar patterns of protection against UC are observed for the glutamine allele at Arg381Gln; however, the patterns of association may be distinct between CD and UC for the noncoding susceptibility alleles.85 Since the initial report, this association has been replicated in pediatric88, 89, 90 and adult87, 91, 92, 93, 94 European ancestry CD cohorts, confirming the role of IL23R in IBD susceptibility. Importantly, no replicative evidence was observed in a well-powered Japanese CD cohort,81 indicating alternative disease mechanisms in different population groups.

The IL-23 pathway and chronic inflammation 

The functional IL-23 receptor is heterodimeric, comprised of the IL23R subunit on chromosome 1p31, as well as IL12RB1, located on chromosome 19p13. The IL-23 cytokine is also heterodimeric and is composed of p19 (chromosome 12q13) and p40 (chromosome 5q33) subunits. The IL12RB1 (receptor subunit) and p40 (cytokine subunit) are common to the IL-12 signaling pathway, with IL12RB2 (chromosome 1p31) and p35 (chromosome 3q25) representing unique components of the functional IL-12 receptor and cytokine, respectively. Of interest, GWA analyses in a large UK cohort showed modest evidence for association near the IL12B (p40) subunit of the IL-23 cytokine.92, 93 Interestingly, psoriasis cohorts show similar protective effects for Arg381Gln in IL23R, as is observed in IBD.95, 96 This finding may account in part for the previously observed cosegregation of psoriasis and CD cases within individuals. In contrast to the IBD findings, a strong association with psoriasis was observed for SNPs within the p40 gene region as well, potentially demonstrating overlapping and unique genetic features of chronic inflammatory disorders.

The human genetic associations with the IL-23 pathway coincide with significant advances in understanding of its role in mediating chronic inflammation. IL-23 has been recently associated with a new lineage of CD4 T cells, the Th17 lineage, that has now been linked to the pathogenesis of IBD.97, 98, 99 Th17 cells are distinct from classic Th1 and Th2 cells, both in the cytokines that induce their differentiation and the cytokines they produce (Figure 2A). Th17 cell development is initiated from naive CD4 T cells by the sequential actions of the inductive cytokines, transforming growth factor β and IL-6, and IL-23, which appears to act downstream of transforming growth factor β/IL-6–induced lineage commitment to amplify mature Th17 differentiation and the production of effector cytokines such as IL-17A, IL-17F, IL-21, and IL-22, among others.100, 101, 102, 103 IL-21 was identified recently as an important intermediate in Th17 differentiation that is induced by IL-6 and can act in an autocrine fashion to drive Th17 differentiation downstream of IL-6.104, 105, 106

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  • Figure 2. 

    (A) CD4 T-cell effector development. This schematic highlights key inductive and effector cytokines associated with the development and function of the Th1, Th2, and Th17 lineages in mice. TN, naive CD4 T cell. (B) Developmental divergence of the Th1 and Th17 lineages. This schematic highlights key cytokines, signaling components, and transcription factors that distinguish Th1 and Th17 development (see text for details). TN, naive CD4 T cell. (C) Shown are the key receptors and ligands expressed by naïve T, Th17, and Th1 cells. Structural homology between the IL-6, IL-12, IL-23, and IL-27 receptors is evident, including the shared component of the IL-6 and IL-27 receptors, gp130. The IL-6 and IL-27 receptors are expressed by naive T cells, as is the IL-12Rβ1 chain that is common to the IL-12 and IL-23 receptors. The inducible chain of the IL-23 receptor, IL-23R, is expressed downstream of Th17 lineage commitment. Activation of the principal STATs, believed to be important in Th17 and Th1 differentiation, is indicated in black; other STATs recruited by these receptors are indicated in gray. (D) Positive and negative regulators of Th17 development. This schematic highlights key cytokines that promote or inhibit Th17 development.

While the shared component of the IL-12 and IL-23 receptors, IL-12Rβ1, is expressed by naive CD4 T cells, induction of the IL-23–specific component of the IL-23 receptor complex, IL-23R, is limited to developing Th17 cells downstream of IL-6 signaling via STAT3105, 107, 108 (Figure 2B). In comparison, developing Th1 cells up-regulate IL-12Rβ2 downstream of interferon signaling via STAT1 or via TCR signaling, rendering them sensitive to IL-12–induced production of Th1 cytokines such as interferon gamma.109 Expression of IL-12Rβ2 or IL-23R by mature Th1 or Th17 effector cells appears to be stable, such that these cells retain IL-12 or IL-23 responsiveness for their lifetime. In this way, differential expression of the inducible components of the IL-23 or IL-12 receptor complexes stabilizes and reinforces lineage specification for Th17 or Th1 and may play an important role in maintenance of these differentiated cell populations. Importantly, IL-23 can act independently of T-cell receptor stimulation in mature Th17 cells to elicit cytokine production via coordinate signaling with IL-1,110, 111 analogous to similar effects of IL-12 and IL-18 on mature Th1 cells. In this way, mature Th17 cells can act to amplify tissue inflammation without a requirement for antigen recognition. Accordingly, IL-23 acts to potently amplify the inflammatory cascade driven by the Th17 adaptive immune response at several levels.

Production of IL-23 appears limited to cells of the innate immune system, particularly dendritic cells and macrophages. The IL-23–specific component of IL-23, p19, is induced by stimulation of pattern recognition receptors, particularly TLRs and dectin receptors, which are activated by conserved structures of bacteria or fungi.112, 113 Recent studies indicate that IL-23 production is synergistically enhanced by coordinate stimulation of NOD family members.114 Thus, bacterial or fungal components of the intestinal microbiota would be expected to activate IL-23 production by innate immune cells in the setting of an epithelial barrier breach and may continually stimulate low-level IL-23 production in dendritic cells that sample the intestinal flora in the resting state.115

Although dysregulated Th1 responses to the microbiota have been traditionally associated with IBD, the recent shift in focus to the Th17 lineage in mouse models has been reinforced by genetic association data linking IL-23R to human CD. In its role as a participant in Th17 differentiation and potent amplifier of Th17 responses, both through antigen-dependent and -independent pathways, IL-23 interactions with its receptor appear to play a central role in the pathogenesis of IBD. IL-23R is also expressed by mature dendritic cells and natural killer cells, where its function is less clearly defined but could also be important in disease pathogenesis. Accordingly, genetic variations that might enhance or blunt production of IL-23 by innate immune cells or IL-23 signaling in adaptive T cells, natural killer cells, or dendritic cells might be expected to be significant genetic markers or risk factors for IBD. Similarly, additional factors associated with either the induction or function of the Th17 pathway might be anticipated as genetic factors in susceptibility or protection for IBD. For example, IL-6 appears to play a nonredundant role in the initiation of Th17 development through its induction of IL-21, which then induces IL-23R (Figure 2C). STAT3, which is a critical signaling component of IL-6R, IL-21R, and IL-23R in Th17 development and function, might also be anticipated as a central player in genetic associations, as might the orphan nuclear receptor, retinoic acid receptor–associated receptor (ROR)γt, which has been identified as a critical transcription factor involved in Th17 differentiation.116 By extension, factors that normally down-regulate induction and function of the Th17 pathway might also play a role and are yet to be defined (Figure 2D). Thus, interferon gamma and IL-27, both of which can block Th17 development via STAT1 signaling,107, 117, 118 may be implicated as more extensive genetic analyses are performed. Suffice it to say that there are multiple genes and pathways associated with the development and function of Th17 cells that are likely to be elucidated as significant targets for analyses.

Therapeutic implications 

The genetic association and the proinflammatory role of IL-23 strongly prioritize this pathway as a therapeutic target in IBD. Anti-p40 administration, which blocks both IL-23 and IL-12 activities, has proved promising119 in the treatment of CD. The contribution of the IL23R pathway to IBD will likely involve more than simple gain or loss of function IL23R variants, and ongoing studies of this pathway may reveal new therapeutic options. Future studies should examine mechanisms of the strong protective effect of the Arg381Gln allele, which could be potentially exploited for clinical benefit.

ATG16L Association With CD 

In a genome-wide survey of nearly 20,000 nonsynonymous SNPs, an amino acid polymorphism, Ala197Thr, within the ATG16L1 gene was found to be highly associated with CD, with the less common threonine allele conferring protection against CD.120 The ATG16L1 protein is comprised of N-terminal APG16 domain consisting of coiled coils and 8 C-terminal WD repeats. ATG16L1 is expressed in intestinal epithelial cells, as well as in CD4+, CD8+, and CD19+ primary human lymphocytes. The Ala197Thr variant is located at the N-terminus of the WD-repeat domain in ATG16L1. The ATG16L1 gene is part of the autophagosome pathway and has been implicated in the processing of intracellular bacteria. Knockdown of ATG16L1 decreased autophagy of Salmonella typhimurium in HeLa cells.121 Of interest, no association was observed in UC and a statistical interaction was reported with the NOD2 CD associations.120 Since the initial report, this association has been replicated broadly.91, 92, 93, 121, 122, 123, 124 However, in subsequent studies, no evidence for interaction with the NOD2 risk alleles was identified. In these studies, similar to the initial report, no association was observed in UC. Taken together, the ATG16L1 association represents a well-replicated, CD-specific association and suggests that autophage and host cell responses to intracellular microbes are involved in the pathogenesis of CD.

Association of a Gene Desert on Chromosome 5p13.1 That Modulates Expression of the Prostaglandin Receptor EP4 (PTGER4) 

In a GWA study in a Belgian CD cohort,91 in addition to the NOD2, IL23R, and ATG16L1 associations, association was observed in a region on chromosome 5p13.1. The most significant association was observed in a gene desert region flanked by a number of potential candidate genes, including CARD6, complement factors C6, C7, and C9, and the prostaglandin receptor, EP4 (PTGER4). No association was observed in UC for markers in this region. PTGER4 is a compelling candidate, in part because PTGER4-deficient mice develop a more severe colitis with dextran sodium sulfate treatment.125 In elegant analyses, the investigators compared SNP data in this genomic region with mRNA expression levels of flanking genes from the corresponding, individual lymphoblastoid cell lines. Throughout the region of association, a number of SNPs were significantly associated with mRNA expression levels of PTGER4, including at least one SNP demonstrating both CD association as well as correlation with PTGER4 expression. However, the susceptibility allele at this marker corresponds with increased PTGER4 expression, which is not consistent with the increased colitic susceptibility observed in PTGER4-deficient mice.125 However, taken together, these findings suggest that genetic variation in this region is associated with IBD and significantly regulates PTGER4 expression.

Additional Definitive CD Associations 

Definitive CD associations of more modest statistical effect have been identified, largely through a recent, well-powered UK genome-wide screen of 2000 CD cases.92, 93 While statistically less significant, these definitive associations shed crucial insight into the roles of intracellular processing of bacteria as well as the IL-23 pathway in CD pathogenesis. In none of these instances are clearly causal alleles identified. The most likely genes driving the association signal may or may not be identifiable, depending on the location of the observed associations and the local patterns of linkage disequilibrium.

A clear example of where the association signal is strongly localized to a single gene is for the PTPN2 gene, a protein tyrosine phosphatase expressed in T cells.126 One study implicates the PTPN2 protein regulating IL-6 signaling through STAT3 dephosphorylation.127 Given the role of IL-6 in Th17 differentiation, combined with the role of STAT3 activation and phosphorylation in response to IL-23, it may be speculated that the PTPN2 association may contribute to altered IL-23 pathway function. Similar associations with PTPN2 have been reported in type 1 diabetes.93 Modest but significant association signals have been observed in the IL12B (p40) gene region, highlighting another member of the IL-23 pathway in CD pathogenesis.92, 93 The patterns of CD association at IL12B appear to be similar to those reported for psoriasis.96 Finally, CD association is observed in a region on chromosome 10q24 containing the compelling candidate gene NKX2-3 (NK2 transcription factor related, locus 3).92, 93 NKX2-3–deficient mice manifest alterations in tissue architecture within intestinal and mesenteric lymphoid tissues potentially mediated by NKX2-3 transcriptional regulation of MAdCAM-1 (mucosal addressin cell adhesion molecule-1) expression.128

In several cases, the association signals are confined to intergenic regions, similar to the chromosome 5p13 region, or alternatively span several candidate genes. For example, replicated associations in multiple cohorts have been observed in an intergenic region on chromosome 10q21.92, 93, 121 Flanking genes include ZNF365, a zinc finger protein, and EGR2, an early growth response gene. In contrast, the reported association signal on chromosome 3p21 and 5q33 spans multiple genes and includes compelling candidates such as MST1 (macrophage stimulating 1) and IRGM (immunity-related GTPase protein type M), respectively.92, 93 In particular, the IRGM gene is a compelling candidate, given its role in autophagy and regulation of intracellular bacteria.129

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Emerging Themes in IBD Genetics and Future Directions 

Significant recent progress in the genetics of IBD has fundamentally advanced understanding of disease pathogenesis. A unique feature of the intestinal immune system is its close apposition to high concentrations of luminal bacteria. The initial association of the NOD2 gene clearly established that altered host responses in intracellular bacterial processing contribute to CD pathogenesis. This theme has been advanced with the report of CD associations with the ATG16L1 gene and IRGM gene region, genes both involved in intracellular bacterial processing and autophagy. For both NOD2 and ATG16L1, the association appears to be unique to CD, not being observed in UC. A second major theme revolves around the importance of the IL-23 pathway in IBD pathogenesis, notably the presence of multiple associations within the IL23R gene to CD and UC. More modest associations of the IL12B and PTPN2 genes implicate multiple members of the IL-23 pathway in IBD pathogenesis. The importance of the IL-23 pathway in mediating peripheral tissue inflammation generally is underscored by similar IL23R and IL12B associations observed in psoriasis,95, 96 as well as the association of PTPN2 with type 1 diabetes.93 Finally, the presence of CD and UC associations with potentially different patterns of associations in the IL23R and major histocompatibility complex regions may highlight important disease-modifying regions. The patterns of association in these 2 regions may provide important comparative insight between CD and UC.

Future genetic studies will be directed toward combining large GWA data sets to identify common variation of more modest effects contributing to CD pathogenesis. Additional genetic, bioinformatic, and laboratory assessments of association signals will be required to define more clearly susceptibility genes and contributing susceptibility alleles, especially where the association signals span several candidate genes. Similar GWA studies in UC will provide important comparative insight to the CD GWA studies. The capacity of GWA studies to sample common variation should be supplemented by deep resequencing efforts to identify rare variants contributing to IBD pathophysiology. Finally, successful translation of genetic advances to clinical practice will require improved understanding of intermediate phenotypes, or biomarkers, that can be objectively applied to measure disease activity, pathophysiologic mechanisms, and/or therapeutic response.

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 Supported by grants R01DK072373, U01 DK62429, and U01 DK062422 and Burroughs Wellcome Translational Research Award (all to J. H. C.), as well as grants U19 AI056542, PO1 DK071167, R24 DK064400, RO1 AI057956, and RO1 AI035783 (all to C. T. W.) and the Crohn’s and Colitis Foundation of America (C. T. W.).

PII: S0016-5085(07)01488-6

doi:10.1053/j.gastro.2007.08.032

Gastroenterology
Volume 133, Issue 4 , Pages 1327-1339, October 2007

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