The potential of GWA studies to discover modest genetic risk factors in complex disease has been widely debated for several years. The simultaneous emergence of resources, such as the data from the International HapMap Project and dramatic leaps in genotyping technology (both clearly motivated by this potential), has now enabled the actual test of this hypothesis. In the case of CD, years of research effort by a substantial international community of researchers had identified only two uniformly replicated, genuinely associated, genetic risk factors for this disease (CARD15 and IBD5). Encouragingly, after only the first, moderately-sized GWA study and modest replication attempts, compelling evidence that this list has now grown substantially has been provided, as unequivocally strong evidence of replication has emerged from this study for variation at IL23R, ATG16L1 and an intergenic region on 10q. Moreover, the excess of additional low p-values in this study – in particular three additional top-ranking SNPs (in PHOX2B, NCF4, and FAM92B) which replicated at p<.01 - along with the fact that other, large GWA attempts are ongoing, offers the potential that the list of replicated loci will grow even further in a short period of time. In fact, since the submission of the current study, reports describing a genome-wide association studies of 735 German patients with CD and of 547 Belgian patients with CD have been published 23,24
. In the German study, after association analyses of 7,159 nonsynonymous SNPs, the Ala197Thr in ATG16L1 was found to be associated with CD, confirming this gene's involvement in CD susceptibility. In contrast to our findings, however, this group reports an epistatic interaction with CARD15. It should be noted, however, that rather than an aberrant joint frequency estimates in cases (as would be expected in the case of epistasis promoting a rare disease), the interaction signal in that study was mostly due to deviation from expectation in the control genotypes. Although this is possible for some genetic models, it requires a careful investigation. Furthermore, since different IBD genes show different degrees of phenotypic specificity, genes which promote the same specific sub-phenotype may be perceived as interacting if that specificity is not accounted for in the analyses. In the Belgian study, using the Illumina HumanHap300 Genotyping BeadChip, the authors identified an associated SNP (rs1373692) that implicated the prostaglandin receptor EP4 (PTGER4) gene in CD pathogenesis. In the current study, we also found evidence of association of the rs1373692 SNP with ileal CD (p=5.37E-04), as well as to a SNP (rs4613763; p=1.21E-04) less than 40kb away, providing the first replication of this finding. Full release and integration of results from these and other ongoing studies seems likely to add further to this burgeoning list of confirmed associations.
It is without doubt, however, that there are limitations to the GWA approach, just like any other genetic approach, and therefore complementary strategies for gene discovery will continue to be necessary25
. One limitation is related to our ability to type large enough cohorts to have sufficient statistical power to detect loci of very modest genetic effect25
. One example from our current study is the lack of genome-wide significant association of the IBD5 haplotype, despite the fact that this is a confirmed CD risk haplotype26
and despite the fact that in a targeted replication study performed with the Replication Cohort #1 presented in the current study we find significant evidence of replication27
. The strength of the association of the IBD5 in the current GWA study, it should be noted however, is consistent with modest risk conferred by this locus28
. While recognizing the limitations inherent to the GWA study design, these current and future association findings also have the potential for dramatically changing our understanding of the biological processes that are crucial to disease pathogenesis, given the fact that genome-wide searches are not biased by our current understanding of pathogenic mechanisms. A prime example of this is our discovery that the ATG16L1 gene is associated with IBD, given that this gene, or the biological pathway of autophagy to which it belongs, was not previously implicated in IBD pathogenesis before the current generation of genome scans.
Autophagy is a constitutive process required for proper cellular homeostasis and organelle turnover. However, recent data has revealed a role for autophagy in innate and adaptive immune responses to pathogens29
. This places autophagy as a gate-keeper for innate immune ligands and antigen presentation from intracellular compartments. So far the role of autophagy in innate immune recognition has been little studied, but several systems have been identified which show a potential role in host-defense30-32
. We have exploited a model system using the invasive bacterium S
. Typhimurium in cultured human epithelial cells22
to demonstrate that in addition to classical autophagic stimuli, such as serum starvation and rapamycin, ATG16L1 is required for autophagic targeting of a subset of intracellular Salmonella exposed to the cytoplasm. While we do not propose that this represents the physiological stimulus involved in CD, we do believe it provides initial evidence that ATG16L1 variants with altered autophagic efficiency/efficacy might alter either bacterial replication and immune control, or delivery of antigens to adaptive immune pathways. For example, interactions between innate immunity, the inflammasome and autophagy have been proposed for Legionella pneumophila 33
. The proposed mechanism relies upon a NOD-LRR family member (Naip5) to detect invading L. pneumophila and subsequently activate autophagy, or if Naip5 signalling is sustained, proinflammatory cell death via caspase 1. Associations between the response to microbial products and chronic intestinal inflammation have been noted for flagellin, with both colitic mice and CD patients exhibiting elevated anti-flagellin serum IgG34
. Since it is now known that TLR7- mediated detection of viral replication is autophagy-dependant35
, it is tempting to speculate that differential autophagic effectiveness might enhance defects in NOD-LRR family based signaling from intracellular pathogens.
Adaptive responses are also influenced by autophagic processing, since HLA class II molecule loading occurs in lysosomes and autophagy delivers cytoplasmic components into this compartment. Antigen loading via this pathway has been observed in professional antigen presenting cells as well as epithelial cells36
. In cells with low levels of endocytosis, such as epithelial cells, this is likely to be an important route for antigen presentation and immune surveillance37
. It is possible that differential rates or substrate specificities induced by variation within the autophagic apparatus will result in differential antigen presentation, as has been observed for B cells in starvation stress37
. In many models of CD and other inflammatory disorders, T-cells are the primary effectors and autophagy is a vital process for T cell maintenance and homeostasis, as demonstrated by the increased cell death seen in ATG5−/−
T cells and their inability to proliferate following stimulation38
. T cells may also be stimulated directly by bacterial products in the gut microenvironment, demonstrated by the discovery of the I2 superantigen39
. Given the role of autophagy in controlling T-cell death and proliferation, ATG16L1-mediated alterations in the proliferative and survival abilities of T cells provide a possible explanation for some of the T-cell driven pathology observed in CD. The relationships between T cell survival and regulation and the ATG16L1 variants discussed here will be an important area of further study.
Although our observation of potential association of the NCF4 gene to ileal CD is not unequivocally confirmed, the known functions of this gene warrant further examination. In particular, previous studies of the p40phox protein encoded by the NCF4 gene have demonstrated that it plays an important role in NADPH oxidase activity and the generation of reactive oxygen species (ROS) production upon phagocytosis17,18
; both important for mounting an effective anti-microbial response. An alteration in gene expression could potentially have an effect on phagosome function, less effectively kill phagocytosed microbes, and could result in prolonged immune activation, incomplete pathogen clearance or might influence TLR activation and antigen presentation due to inappropriate ROS generation40,41
. The constant exposure to bacterial challenge and high antigen load in the gut may pose special difficulties for even slightly altered innate immune responses, resulting in gut-restricted phenotypes. Such gut restriction has been previously observed for much more severe phox deficiencies in a number of chronic granulomatous disease (CGD) patients, whose initial symptoms resulted in a diagnosis of CD. Thus we propose that mutations in NCF4 might interfere with the stochiometry of interaction with p67 and other p47phox interacting partners resulting in relatively moderate phagosome dysfunction phenotypes sufficient to trigger the onset of IBD.
Although additional functional studies are required to place all of these novel loci into a biological context that will enable a deeper understanding of their roles in normal homeostasis and disease pathogenesis, there can be little question that the opportunity to make advances in 12 our understanding and eventual treatment of CD is being offered by the successful application of robust GWA studies.