Fig. 1.1
Associations of the IL12/IL23- and IL27-regulating genes with IBD in keeping with the TH1 and TH2/TH17 theory (Wang et al. 2010). Only the main proteins in these pathways are shown. For each gene, the most significant P value among SNPs closest to the gene was annotated (figure originally published in Wang et al. Analysing biological pathways in genome-wide association studies. Nature Review Genetics 2010;11:843–854)
Meta-Analysis
The associated common variants identified by single GWAS usually have modest individual effects, often with odds ratios of smaller than 1.2 for binary traits, or with explained variance of less than 1% for quantitative traits [65]. To discover common variants with even smaller effects, a sample size larger than that of single studies is required. Meta-analysis combines large datasets and is an economical way to improve sample size. An early meta-analysis of three genome-wide CD scans identified 21 new CD susceptibility loci. It increased the number of independent loci conclusively associated with CD to 32, explaining approximately 20% of Crohn’s disease heritability [66]. Including three additional GWAS scans, the most recent meta-analysis added 39 new confirmed Crohn’s disease susceptibility loci [67]. These 39 new loci increase the proportion of explained heritability to only 23.2%, indicating their rather modest effects. While some of these newly identified loci contain a single gene, others contain multiple genes or none at all. Some functionally interesting candidate genes in the implicated regions including STAT3, JAK2, ICOSLG, ITLN1, and SMAD3 are briefly described below.
STAT3 (signal transducer and activator of transcription 3) and JAK2 (Janus kinase 2) both come from the JAK-STAT pathway. This major signaling pathway transmits information from cell surface receptors stimulated by cytokine and growth factors to the nucleus to regulate transcription of various genes. STAT plays a central role in Th17 differentiation [68] while both contribute to IL23R signaling [43]. ICOSLG (inducible T-cell co-stimulator ligand) is a co-stimulatory molecule expressed on intestinal (and other) epithelial cells. It has been suggested that ICOS may have a key role in controlling the effector functions of regulatory T cells [69]. There is direct evidence showing that maturing plasmacytoid dendritic cells express different sets of molecules including ICOSLG for T cell priming [70]. ITLN1 (intelectin-1) is known to be expressed in human small bowel and colon. It is found that the lactoferrin receptor (LFR), which is structurally identical to human ITLN1, seems critical in membrane stabilization, preventing loss of digestive enzymes and protecting the glycolipid microdomains from pathogens [71]. SMAD3 (SMAD family member 3) binds the TRE element in the promoter region of many genes that are regulated by TGF-beta and, on formation of the SMAD3/SMAD4 complex, activates transcription. It has been demonstrated that SMAD3 deficiency will enhance Th17 during the TGF-β–mediated induction of Foxp3+ regulatory T cells [72].
Genetic Studies in Ulcerative Colitis
The search for genetic variants has yielded fewer candidate genes associated with ulcerative colitis than with Crohn disease. One potential candidate, the interleukin-1 receptor antagonist (IL1RA) gene, was first identified in 1994 and remains controversial. Data from the primary investigators reported an association between IL1RA and increased susceptibility to ulcerative colitis with an odds ratio (OR) of 1.3. Subgroup analysis showed that this relationship was strongest for individuals with extensive disease (OR 1.5), those undergoing colectomy (OR 1.5), and patients who developed pouchitis [73, 74]. However, other researchers studying cohorts from the same region failed to replicate an association between IL1RA and Crohn disease or ulcerative colitis, suggesting the connection may be weak [75].
The multidrug resistance gene, MDR1, which resides in a region of linkage on chromosome 7 has also been postulated to play a role in colitis. Encoding a set of membrane transporters, variants of the MDR1 gene modulate epithelial transport and barrier function. Animal studies have shown that murine MDR1 knockouts develop spontaneous colitis reminiscent of inflammatory bowel disease [76]. In humans, diminished mRNA expression of MDR1 from intestinal tissue of patients with IBD implies this pathway may be involved in disease pathogenesis [77]. Genetic analyses in humans, however, have provided contradictory results regarding susceptibility to UC and SNP C3435T. Taken together, these data suggest that variants of the MDR1 gene may warrant further study in relation to ulcerative colitis [78].
Recent studies have also identified associations between ulcerative colitis and polymorphisms of the NFκB1 promoter and IBD2 susceptibility locus. Genes in the NFκB pathway merit special interest based upon the integral role the signal transduction pathway plays in inflammatory processes. Murine models of colitis have demonstrated strong linkage with a locus on chromosome 3 that contains the NFκB1 gene. Translated to humans, a Dutch study found that allele frequency for the 94ins/delATTG variant was significantly higher in UC patients compared to controls or those with Crohn. Individuals homozygous for the deletion also appeared to have younger age at onset of disease [79]. The IBD2 locus located on chromosome 12 has also been implicated in the pathogenesis of ulcerative colitis. In a linkage study of 904 affected relative pairs, the IBD2 locus was associated with a subset of patients manifesting extensive ulcerative colitis [80].
Genome-Wide Meta-Analysis
Recent GWAS and candidate gene association studies have identified 18 susceptibility loci for UC, which explain approximately 11% of the heritability for this disease. A recent meta-analysis combining data from six GWAS identified 29 additional UC risk loci, increasing the number of confirmed associations to 47 [81]. Examination of the gene content of the 47 associated regions shows that three regions each contain a single gene, most (35 out of 47) contain multiple genes and nine contain no genes. Some noteworthy candidate genes including PRDM1, TNFRSF14, TNFRSF9, IL1R2, IL8RA, and IL8RB are briefly described below.
PRDM1 (PR domain containing 1, with ZNF domain) is the master transcriptional regulator of plasma cells and acts as a transcriptional repressor of the IFN-β promoter by binding specifically to the PRDI element. It drives the maturation of B-lymphocytes into Ig secreting cells. TNFRSF14 (tumor necrosis factor receptor superfamily, member 14) has an important role in preventing intestinal inflammation in a T-cell transfer model of colitis [82]. TNFRSF9 (tumor necrosis factor receptor superfamily, member 9) is a co-stimulator in the regulation of peripheral T-cell activation, with enhanced proliferation and IL-2 secretion. This factor is expressed by dendritic cells, granulocytes, and endothelial cells at inflammation sites. IL1R2 (Interleukin 1 receptor, type II) can reduce IL1B activities by competitive binding to IL1B, preventing its binding to IL1R1. It is found that IL1B production by lamina propria macrophages is increased in patients with ulcerative colitis [83]. IL8RA and IL8RB (chemokine (C-X-C motif) receptor 1/2) are two receptors for interleukin-8, which is a powerful neutrophil chemotactic factor. Binding of IL-8 to the receptor also causes activation of neutrophils. IL8RA, but not IL8RB, expression is found to be increased in macrophages, lymphocytes, and epithelium in ulcerative colitis. It has been suggested that IL8RA may help IL-8 to play a role beyond neutrophil recruitment in mediating the immune response in UC [84].
Risk Prediction in IBD
Encouraged by the notable success of GWAS in Crohn disease (CD) and ulcerative colitis (UC), it is tempting to ask if these advances can deliver sufficiently accurate predictions to make targeted intervention realistic. Several efforts have been made, but most results are generally modest or even negative. For example, in a recent study Kang and colleagues reported the best AUC (area under the receiver operating characteristic curve) score of 0.72 in predicting CD risk using GWAS genotype data [85]. This best AUC is obtained assuming the optimal number of predictors is given. The practical AUC may be even lower because the optimal number of predictors is usually unknown and has to be inferred from data itself. However, it is noted that these early efforts usually use small or modest sample sizes. As in meta-analysis, when we are able to compile a large sample size by combining as many cohorts as possible, we may expect a boost of prediction performance. Finally, based on our successful experience in Type 1 Diabetes risk prediction [86], we may further optimize prediction performance by employing sophisticated analytical skills from the machine learning field.
Genotype–Phenotype Correlations in Pediatric IBD
Disease Type and Location
The discovery of genetic polymorphisms in IBD has afforded investigators with the opportunity to identify predictive correlations between specific variants and phenotypic disease characteristics. Analyses of adult populations have demonstrated that carriage of NOD2/CARD15 risk alleles predicts disease onset at an earlier age and ileal disease location in a dose-dependent manner. Subsequently, a meta-analysis of 16 case–control studies confirmed the association of NOD2/CARD15 carriage with ileal disease location, and also identified a correlation with fibrostenosing behavior and family history of IBD [16].
The majority of pediatric studies have concurred with findings from adult counterparts that carriage of NOD2/CARD15 variants is associated with ileal disease. Estimates suggest that 20–65% of children with ileal Crohn disease possess at least one NOD2/CARD15 mutation; consistent phenotypic associations have not been seen for other regions of the gastrointestinal tract [55, 87–91]. In contradistinction to the adult literature, correlates of NOD2/CARD15 variants with fibrostenosing disease have demonstrated conflicting results [19, 87, 88, 90, 91]. Two large studies from the US and Scotland found that 34–45% of Crohn patients possessing NOD2/CARD15 polymorphisms had evidence of fibrostenosing disease, especially the 1,007 fs and R720W variants [88, 91]. Three other pediatric studies, however, found no correlation of NOD2/CARD15 with fibrostenosing disease [19, 87, 90].
Disease Age of Onset
Individuals with a family history of IBD are more likely to have early onset disease, suggesting there is greater influence of genetic factors in those diagnosed with IBD at an earlier age. Analysis of age of disease onset with respect to NOD2 carriage, however, has not shown a clear association between age and these allelic variants [19, 88, 91]. Russell et al. reported a median age at diagnosis of 11 years for carriers of NOD2 variants and 11.3 years for noncarriers [91]. Similar results were published by Kugathasan et al. who found no difference in the mean age of disease onset among Caucasian children with Crohn disease possessing NOD2 mutations vs. those who did not (11.98 years vs. 12.76 years, respectively) [88]. Only one study of 55 German pediatric Crohn patients showed a higher frequency of two NOD2 mutations among children than adults (35% vs. 17%) [89]. Thus, while genetic factors may predispose individuals to developing IBD at a young age, there is insufficient evidence at this time to conclude that NOD2 variants are associated with earlier onset disease in pediatric IBD.
Growth Parameters
As growth failure is an important feature of pediatric IBD, several groups have investigated the relationship between anthropometric parameters and NOD2/CARD15 status. A study of 101 Crohn patients demonstrated that 44% of participants possessing a NOD2/CARD15 polymorphism were <5% for weight at the time of diagnosis, while only 15% of those without a genetic variant were <5% for weight at the time of diagnosis [87]. Although similar trends were seen for height, these results did not reach statistical significance. Another study of 93 Crohn patients, however, did not show any correlation between NOD2/CARD15 status and height or weight Z scores at disease onset or for the lowest Z score during childhood [90]. Rather, disease severity was the strongest predictor for impaired growth, and ileal involvement was associated with height retardation at disease onset and the lowest Z-score during follow-up. Finally, a German group did not find any statistically significant difference in mean body mass index (BMI) or mean height percentiles at diagnosis between patients with and without NOD2/CARD15 variants [92]. The authors did note a nonsignificant trend of greater numbers of patients possessing NOD2/CARD15 polymorphisms being below the 3% for BMI. These data imply that while NOD2/CARD15 variants may be associated with poor growth, this effect may be more a reflection of malnutrition secondary to ileal location and disease severity as opposed to an inherent genetic effect.
Association with Risk of Surgery
Results of pediatric studies correlating NOD2/CARD15 status with the need for small bowel surgical resection have consistently delineated a positive association. Russell et al. estimated an odds ratio for risk of surgery among children with Crohn possessing any NOD2 mutation to be 4.45 [91]. Pediatric Crohn patients with the 1007fsInsC variant appeared to have a greater likelihood of requiring surgery with an odds ratio of 4.8. Among US Caucasian Crohn patients, hazard ratios for surgery indicate that children possessing the 3020insC variant are at sixfold greater risk of requiring surgical intervention [88]. Furthermore, these children also showed a trend toward a need for earlier surgery at median of 14 vs. 23 months after diagnosis. Finally, it has been suggested that there is a positive correlation between the number of mutations possessed by an individual and increased risk of surgery [89].
Summary
Both family and twin-based studies lend strong support for a genetic basis of IBD. This is further supported by observations of racial/ethnic variations in disease prevalence. The recent advent of GWAS has markedly advanced the identification of well-replicated IBD association and has substantiated this concept at a molecular level and catapulted the field of IBD genetics into a new realm of discovery. Future sequencing studies are likely to identify more rare variants that confer greater risks at the individual level and may help uncovering new gene interactions and networks that contribute to the pathogenesis of IBD allowing for stratification of IBD patients into different therapeutic pathways and interventions in the future.
Acknowledgement
We are most grateful to Dr. Judy H. Cho and Dr. Nancy McGreal who wrote the earlier version of this chapter and authorized us to use it as a template for the updated studies presented.
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