Advances in IBD genetics.

REVIEWS Advances in IBD genetics Johan Van Limbergen, Graham Radford-Smith and Jack Satsangi Abstract | IBD is a spectrum of chronic disorders that constitute an important health problem worldwide. The hunt for genetic determinants of disease onset and course has culminated in the Immunochip project, which has identified >160 loci containing IBD susceptibility genes. In this Review, we highlight how genetic association studies have informed our understanding of the pathogenesis of IBD by focusing research efforts on key pathways involved in innate immunity, autophagy, lymphocyte differentiation and chemotaxis. Several of these novel genetic markers and cellular pathways are promising candidates for patient stratification and therapeutic targeting. Van Limbergen, J. et al. Nat. Rev. Gastroenterol. Hepatol. advance online publication 11 March 2014; doi:10.1038/nrgastro.2014.27

Introduction IBD, comprising Crohn’s disease and ulcerative colitis, is emerging as a global health problem. 1 In spite of notable advances, IBD treatment remains challenging for a large number of patients, with repercussions on morbidity and mortality.2 The increasing incidence of IBD1 has prompted a sustained research interest over the past 30 years to understand the environmental triggers of these complex polygenic diseases, as well as help to predict the natural history and therapeutic response. In this Review, we highlight how genome-wide association studies (GWAS) have informed our understanding of the genetic architecture of IBD and which pathways are likely to be clinically useful for patient stratification and therapeutic targeting.

Familial genetic studies of IBD IBD Centre, Division of Gastroenterology and Nutrition, Department of Paediatrics, IWK Health Centre, Dalhousie University, 5850/5890 University Avenue, Halifax, NS B3K 6R8, Canada (J.V.L.). IBD Research Group, Queensland Institute of Medical Research and University of Queensland School of Medicine, Herston Campus, Brisbane, QLD 4029, Australia (G.R.-S.). Gastrointestinal Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XU, UK (J.S.). Correspondence to: J.V.L. johanvanlimbergen@ dal.ca

The genetic epidemiology of the family studies in patients with IBD laid the foundation for large-scale GWAS in IBD.3 Higher disease concordance in monozygotic twins with Crohn’s disease than those with ulcerative colitis, higher risk of IBD development in siblings of an affected individual than the general population and the high rate of a family history of IBD (especially in early-onset disease) have guided estimates of the genetic contribution to IBD susceptibility.4 However, whether these estimates of heritability obtained from family studies are reliable has been questioned.5

Missing heritability The conundrum of ‘missing heritability’ in many complex diseases has also troubled the IBD research commu nity as it tries to predict how much of the (patho)genetic map is uncharted (Figure 1).6 In the calculation of herita bility, observations in larger twin cohorts have suggested that the index studies might have overestimated the overall variance of disease due to inherited factors Competing interests The authors declare no competing interests.

(the denominator).7,8 The numerator, which consists of the additive effect of the genetic variants identified so far, is equally confounded by assumptions about how these genetic variants might contribute to disease susceptibility.8 Two examples of advances in our understanding of DNA biology and complex disease genetics illustrate why the heritability debate could focus research rather than cause distraction by chasing after elusive causative variants. The first example starts with the publication of the ENCODE project (Encyclopaedia of DNA elements) in 2012, which demonstrated how the proportion of the DNA sequence that encodes proteins (the exome) is small, compared with the regions of DNA regulating transcription and RNA biology.9 Although low-frequency variants (with a minor allelic frequency ranging 0.5–5.0%) do contribute to the allelic spectrum of immune-mediated diseases, a large number of single nucleotide polymorphisms (SNPs) discovered in the course of IBD GWAS published do not reside in the exome.10 However, conserved noncoding DNA sequence elements can be crucial in gene regulation as demonstrated by Zheng et al.11 for forkhead box protein P3 (FOXP3) and the differentiation of regulatory T (TREG) cells.

Very-early-onset IBD In IBD of very early onset (diagnosed before 2 years or even 6 years of age depending on the clinical context), it is important to perform an extensive diagnostic work-up for primary immunodeficiency syndromes, which are increasingly recognized by means of exome sequencing.12 Novel variants in the IL‑10 and IL‑10 receptor, X‑linked inhibitor of apoptosis deficiency, FOXP3 mutations associated with IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X‑linked) syndrome, TTC7A deficiency and mutations in the NADPH oxidase (for example, NCF2 and NCF4, leading to chronic granulo matous disease) have been identified.13–18 More than 40 monogenic diseases with associated intestinal inflam mation have been described to date, and are often

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REVIEWS Key points ■■ The Immunochip project has identified >160 loci containing IBD genes and 70% of these loci are shared with other immune-mediated inflammatory diseases ■■ Similar to other complex polygenic diseases, only a small fraction of heritability is explained by the genetic loci identified in IBD ■■ Hypothesis-free genetic association studies in IBD have identified key pathways involved in innate immunity, autophagy, lymphocyte differentiation and chemotaxis ■■ Genetic corroboration is now available for novel treatment strategies targeting IL-12–IL-23 signalling, JAK–STAT signalling and leukocyte chemotaxis ■■ The paradigm of personalized IBD care will probably only be achieved if we succeed in integrating the genetic and basic science advances with insights into the ecology of the gut microbiota

Gene × environment/microbiota ‘Missing heritability’

Gene × gene

Heritability

Gene × epigenetics Gene expression Protein × protein

Healthy

Rare variants

Common variants

IBD

Environment/microbiota Altered function

Gene regulation

Figure 1 | The complex polygenic pathogenesis of IBD. The complex polygenic pathogenesis of IBD and the concept of ‘missing heritability’ are two sides of the same coin. Rare and common variants contribute together with environmental triggers (for example, the gut microbiota) to the development of disease.

associated with additional immunodeficiencies. In a comprehensive review, Uhlig 19 divided the spectrum of diseases into epithelial barrier and epithelial response defects, neutropenia and defects in phagocytic bacterial killing, hyperinflammatory and autoinflammatory dis orders, immune defects that include T‑cell and B‑cell selection and activation defects (including antibody defects), TREG-cell defects (including immune dys regulation) and intestinal innervation defects (for example, Hirschprung disease). Several proteins encoded by the genes involved in these monogenic conditions are positioned within established IBD protein networks.19 Clearly, appropriate genetic diagnostic investigations can profoundly alter the chosen therapy, ranging from immune suppression to allogeneic haematopoietic stem cell transplantation. A worldwide consortium (NEOPICS—the International Early Onset Paediatric

IBD Cohort Study) has brought together researchers and clinicians studying IBD in infants and young children and offers support for a structured approach to these challenging patients.20

Rare versus common variants in IBD In contrast with the approach in early-onset IBD, the first exome-sequencing studies in adult-onset IBD and the latest Immunochip project have suggested that only a limited number of rare and/or missense variants are likely to have a role in IBD (thus implying a larger number of variants involved in gene regulation).21–24 In addition to rare variants in NOD2 (also known as caspase recruitment domain-containing protein 11 or CARD15), these exome-sequencing studies also identified rare variants in IL23R, PTPN22, CARD9, PRDM1 and NDP52. 22,25,26 In turn, this finding has led to a new emphasis on how gene expression is regulated, which then provides a framework to understand how loci are shared between immune-mediated diseases. Parkes et al.10 demonstrated that for many of the shared loci of the Immunochip project (with associations at P 1 × 106 in the latest GWAS chipsets), it has been necessary to use strict thresholds of genome-wide significance to avoid excessive numbers of false-positive association signals. One of the goals of Immunochip was to provide deep replication for the top 2,000 independent association signals for each of the diseases studied.10 The Immunochip was custom designed by investigators of autoimmune and inflammatory diseases to include almost 200,000 SNPs and >700 small insertion–deletions. Previously identified GWAS loci for 12 immune-mediated diseases—Crohn’s disease, ulcerative colitis, coeliac disease, type 1 diabetes mellitus, psoriasis, ankylosing spondylitis, multiple sclerosis, rheumatoid arthritis, IgA deficiency, autoimmune thyroid disease, primary biliary cirrhosis and systemic lupus erythematosus—were sampled extensively to enable fine mapping of the GWAS association signal (a total of 186 loci were studied using SNP information from the 1,000 Genomes project, although genetic variation of individuals of non-European origin is under-represented, and previous resequencing studies of GWAS loci). For Immunochip, SNP selection was done without filtering based on physical genomic distance or linkage disequilibrium, as had been the case in earlier GWAS. Owing to the substantial cost-saving of this custom chip, it was possible to study many more individuals. The resultant increase in study power has meant additional new loci were identified—for IBD, the number of confirmed susceptibility loci increased from 100 to 163.10 Abbreviations: GWAS, genome‑wide association study; SNP, single nucleotide polymorphism.

as smoking cessation.37,38 Li et al.39 demonstrated the role of IBD phenotype and NOD2 variant carriage in shifts in overall microbial composition, whereas IBD phenotype, medication and smoking influenced the relative frequency of F. prausnitzii. Personalized IBD care will depend on the integration of genetic and basic science advances with insights into gut microbial ecology. Studies to incorporate the increasing number of IBD loci in diagnostic and/or severity of disease-prediction scores have shown a larger contribution of a limited number of genes and/or pathways (for example, NOD2, ATG16L1, IL-12–IL-23 signalling, IL-10–IL-19 signalling or C13orf31).27,40,41 To date, one of the most important achievements of IBD genetics has been to help provide these targets for further research, either directly (for example, TNF and IL-12–IL‑23 pathway modulation) or indirectly (for example, influence of NOD2 genetics on gut microbiota composition).

IBD genetic architecture Family-based studies identified a number of familial IBD loci using nonparametric linkage analyses. The loci IBD1 (NOD2), IBD3 (HLA-region) and IBD5 are the most replicated and confirmed in the GWAS and Immunochip project.42–44 In this project, the globalization of research efforts into the genetic susceptibility to 12 immune-mediated inflammatory disease groups (including Crohn’s disease, ulcerative colitis, coeliac disease, type 1 diabetes mellitus, psoriasis, ankylosing spondylitis, multiple sclerosis, rheumatoid arthritis, IgA deficiency, autoimmune thyroid disease, primary biliary cirrhosis and systemic lupus erythematosus) yielded a combined total of >75,000 cases and controls available for analysis (Box 1).23

The number of confirmed IBD loci has increased to 163 (with each locus containing an average of five genes).23 Two-thirds of these loci are shared between Crohn’s disease and ulcerative colitis. In addition, 30 Crohn’s-disease-specific loci and 23 ulcerative-colitisspecific loci have been identified. In Supplementary Table 1, the large number of prioritized candidate genes on these loci is shown. Most of these disease-specific loci have the same direction of effect in the nonassociated disease. Two notable exceptions are PTPN22 and NOD2, which show a protective effect in ulcerative colitis. The extent of shared genetic risk between Crohn’s disease and ulcerative colitis suggests that most of the pathways involved in one disease also contribute to the other. These findings open up tremendous avenues for targeted development of new therapies in IBD, but might also be relevant to understanding how treatment of one immune-mediated disease can trigger development of another (for example, the development of psoriasiform skin lesions during anti-TNF treatment).45,46 This new avenue of understanding ‘shared’ susceptibility can be particularly important as some of the most clinically relevant diseases to IBD clinicians are also showing the strongest enrichment of genetic risk overlap: ankylosing spondylitis, psoriasis, primary immunodeficiencies and susceptibility to infectious diseases (notably, mycobacterial infection).10 Only a minority of IBD-associated SNPs are in strong linkage disequilibrium with a missense variant in the 1,000 Genomes Project data, consistent with the hypothesis that a large fraction of the genetic risk of com plex diseases can be attributed to non-protein-coding DNA variation. 10,24,47 Twice as many IBD-associated variants (n = 64) are in linkage disequilibrium with variants already known to regulate gene expression.23 This number is expected to increase further as the findings of the ENCODE project are integrated with the disease‑associated GWAS data.9 Within each locus, the selection of candidate gene(s) for further study is crucial to appreciate the real contribution of the locus to disease susceptibility. To date, only a handful of loci have had their associated alleles subjected to functional studies, including NOD2, IL23R, ATG16L1, IRGM, PTPN22, IRF5, PRDM1 and NDP52. 23,26,28 However, for most loci, the strongest GWAS-associated allele does not explain the entire contribution of the locus indicating that either other variants across the gene are associated independently (for example, NOD2, IL23R, ATG16L1) or that other genes on the locus also contribute to disease susceptibility.21,48–52 Gene prioritization for each locus often relies on biological assumptions or strategies such as protein-network-based analyses (for example, GRAIL, Gene Relationships Among Implicated Loci), searching for enrichment across the IBD loci of established gene ontology terms and/or canonical pathways and enrichment of differentially expressed genes across immune-cell types.23,53 Although these analyses have been important to structure the IBD loci for further analyses, they can also shift the research focus away from novel pathways. Repeating such analyses regularly is necessary



REVIEWS Pro268Ser

CARD CARD

Arg702Trp

Gly908Arg

JW1

1007 (SNP13)

NBD LRR

Interaction with RIPK2

Oligomerization ATP-binding and hydrolysis Ligand binding?

Ligand sensing (MDP)

Figure 2 | NOD2 structure and position of the most common variants with Crohn’s disease.71 The 1007frameshift variant (SNP13) is most disruptive to MDP sensing. The JW1 variant (intervening sequence 8 + 158: a C>T mutation in the palindrome sequence in the intron 8 splicing region) and other, rare NOD2 variants (not shown), have been described.22,195 Abbreviations: CARD, caspase recruitment domain; LRR, leucine-rich repeat; MDP, muramyl dipeptide; NBD, nucleotide-binding domain; NOD, nucleotide-binding oligomerization domain-containing protein; RIPK2, receptor‑interacting serine/threonine‑protein kinase 2.

as our knowledge about gene–gene and protein–protein interactions expands, as illustrated below for NOD2 and ATG16L1.29,53–55

Functional implications NOD2, innate immunity and pathway analyses The fine mapping of the IBD1 region on chromosome 16 led to the identification of NOD2,48,56 which encodes an innate intracellular pattern-recognition receptor that recognizes microbial and viral components.57,58 More than a decade after its discovery, NOD2 variants remain the strongest determinants of susceptibility to Crohn’s disease, in genetic association studies and in clinical studies exploring patient risk stratification (discussed later).23,41,59,60 The discovery of NOD2 focused research interest on the innate immune response to the gut microbiota and priming of adaptive immunity. NOD2 directly binds ATP and muramyl dipeptide (MDP), a breakdown product of peptidoglycan derived from the cell wall of Gram-negative and Gram-positive bacteria.61 Coulombe et al.62 showed that TNF secretion by macrophages, infected with 12 different bacterial species, was NOD2-dependent only after infection by mycobacteria and Actinomycetes (Nocardia and Rhodococcus); these bacteria have N‑glycolylation of MDP in contrast with N‑acetylation in most bacteria. Three variants in the MDP-sensing domain of NOD2 account for >80% of the identified germline variants (encoding Arg702Trp [SNP8], Gly908Arg [SNP12] and 1007frameshift [SNP13], Figure 2).63 Studies have identified a number of additional NOD2 variants associated with Crohn’s disease (due to increased power of the study to detect statistical association in larger cohorts).21,22 In particular, the 1007frameshift NOD2 polymorphism disrupts sensing of MDP.57 Although the frequency of NOD2-variant carriage differs among high-incidence populations, a meta-analysis has confirmed that carriage of two NOD2 variants has a 98% specificity of having a complicated disease course in patients with Crohn’s disease (discussed later).59

NOD2 is a key orchestrator of the homeostasis of the gut mucosal barrier through regulation of the gut microbiota, the innate and adaptive immune response to it and even intestinal angiogenesis. 64,65 One of the mechanisms by which NOD2 regulates the gut microbiota and mucosal immune response is through the production of antibacterial defensins.66 The relative contribution of NOD2 genotype and ileal inflammation to defensin production in Crohn’s disease is debated.67,68 Shanahan et al.69 have demonstrated the importance of experimental design in controlling for environmental influences in animal studies of NOD2-mediated production of defensins by Paneth cells, notably co-housing of wild-type and knockout mice. NOD2 signalling critically depends on its intracellular localization, which is disrupted by Crohn’s-disease-associated variants.70,71 In HEK293 cells, the 1007frameshift NOD2 variant results in a loss-of-function with regards to the ability of NOD2 to differentially regulate growth factors, chemokines and nuclear factor κB (NFκB) antagonists.72 As discussed later for other CARD proteins, differential effects of NOD2 in different cell types (for example, myeloid versus intestinal epithelial cells) also contribute to the complex biology of NOD-driven inflammation, as suggested by work using the spontaneous ileitis mouse model.73 NOD2 has been found to interact with several other IBD susceptibility pathways at the level of DNA (for example, TLE1 and ATG16L1), RNA (for example, IL-10, via nuclear ribonucleoprotein hnRNP‑A1) and IL-12– IL-23 (via microRNA29) and protein (for example, PTPN22, ATG16L1 and TGF‑β). 40,74–79 A novel role has been reported for ATG16L1 that is independent of autophagy: it is involved in downregulating NOD-driven inflammation by interfering with the polyubiquitination of the RIPK2 adaptor and recruitment of RIPK2 into large signalling complexes.55 The rs2241880 ATG16L1 allele (encoding Thr300Ala; associated with Crohn’s disease) was impaired in its ability to regulate these NOD-driven inflammatory responses.55 Additional risk alleles are being found in genes of which the protein product interacts with NOD2, notably RIPK2, RELA, TLE1, VIM and ATG16L1. 23,28,70,75,80,81 In the study by Nimmo et al.,75 SNPs within TLE1 were strongly associated with susceptibility to ileal Crohn’s disease and the TLE1 risk allele was even required for the increased susceptibility to Crohn’s disease in carriers of NOD2 mutations. An association was also found across the haplotype block containing VIM.80 Vimentin was required for NOD2-dependent NFκB activation and MDP-induced autophagy induction and, together with NOD2, regulated the invasion and survival of a Crohn’s-disease-associated adherent-invasive E. coli strain. Crohn’s-disease-associated NOD2 variants correlated with an inability to interact with vimentin.80 In turn, many of these proteins interacting with NOD2 are involved in other signalling pathways, which have been associated with Crohn’s disease susceptibility by GWAS or pathway-driven candidate gene analysis: for example, autophagy (ATG16L1); epigenetics (TLE1); effectortriggered immunity against pathogens (RIPK2); NFκB

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REVIEWS activation (RELA); lymphocyte differentiation (PTPN22); and innate immunity against fungi (CARD9).50,52,82–87

Lessons from CARDs In addition to shared susceptibility with several common immune-mediated diseases (such as coeliac disease and psoriasis), the Immunochip project highlighted the marked enrichment of genes involved in primary immunodeficiencies and response to infection, particularly mycobacterial disease (RIPK2, NOD2, TNFSF15, IL23R, HLA, IL12B, STAT1/3, TYK2, IFNGR1/2 and IL18RAP/R1).23,88–91 Rare variants within several of these genes predispose to immunodeficiency syndromes: Blau syndrome and/or early-onset sarcoidosis (NOD2), IL10– IL10R-mediated very-early-onset IBD, CD40 deficiency, hyper IgE syndrome (TYK2, STAT3), CARD11-mediated combined immunodeficiency and chronic mucocutaneous candidiasis and/or invasive fungal infection (CARD9).92–97 Although NOD2 variant carriage has a protective effect against development of ulcerative colitis, genetic variants in other CARDs (for example, CARD9 and CARD11) are associated with increased risk of ulcerative colitis. 23,85 CARD9 and CARD11 are particularly illustrative with regards to the intricate relationship between tissuespecific responses (for example, responses of myeloid versus lymphoid cell types to microorganisms), priming of the adaptive immune response (B-cell and T‑cell development) and susceptibility to IBD.98 Both CARD9 and CARD11 are intracellular signal transduction molecules in pathways activated by ITAM (immunoreceptor tyrosine-based activation motif) receptors. ITAMs are phosphorylation motifs found in a large number of receptors or adaptor proteins. Phosphorylated ITAMs recruit CARD9 in a complex with BLC10 and MALT1, which results in activation of NFκB or MAPK in myeloid-lineage cells, whereas CARD11 interacts with these same partners in lymphoid cells.98,99 Several ITAM receptors have been implicated in IBD pathogenesis, either functionally (T-cell receptor, B‑cell receptor and NK2GD receptor) or genetically (such as the fungal receptor CLEC7A (C-type lectin domain family 7 member A, also known as dectin‑1).100,101 Dectin‑1 is a C‑type lectin receptor that recognizes β‑1,3-glucans, which are present in the cell walls of nearly all fungi. A twomarker CLEC7A haplotype, rs2078178–rs16910631, was found to be associated with medically refractory ulcerative colitis.101 Many other ITAM-containing receptors, which are expressed on natural killer cells and subsets of CD8+ cells, CD4+ cells and B cells, reside in IBD loci (CD5, CD6, CD244, the FCγ receptor cluster on Chr.1q23.3, CRACC– SLAMF7, KIR2DL1).23 CARD9 and CARD11 can also interact with integrins, which also contain ITAMs and are promising therapeutic targets.102 Compared with the other established immuno deficiency syndromes mentioned earlier, studies in patients with CARD9 and CARD11 human deficiency syndromes and in corresponding mouse knockout models have identified key processes in which these innate signalling adaptor molecules are involved. CARD9 has a role in several antimicrobial responses, notably including

bacterial, viral, fungal and mycobacterial, linking innate immunity with T‑cell differentiation and type 17 T helper (TH17)-cell development.103–105 CARD11 controls a thymic checkpoint in Foxp3+ TREG-cell development and was also found to be necessary for acquisition of IL‑17A, IL‑17F, IL‑21, IL‑22, IL‑23R and CCR6 expression in T cells cultured under TH17 conditions.95, 106 In Card11-knockout T‑cells from knockout mice, chromatin loci of TH17 effector molecules failed to acquire an open conformation required for transcription.95

Autophagy, ER stress and Paneth cells One of the key advantages of performing hypothesisfree research such as GWAS is the potential to discover novel disease-associated pathways. Autophagy was first implicated in the pathogenesis of IBD by the discovery of a strong signal of the Thr300Ala variant in ATG16L1 in a nonsynonymous SNP association study and later confirmed in several GWAS and meta-analyses.50,52,107 Autophagy comprises a number of cellular processes involving the delivery of portions of the cytoplasm to the lysosome for degradation, in response to metabolic (cellular starvation) and/or infectious (bacterial, viral or mycobacterial) triggers, with several autophagy-modulating drugs awaiting further study of their efficacy in IBD.82,108–111 ATG16L1 function has been studied in mice, healthy individuals and patients with Crohn’s disease. 112–116 In Atg16l1-deficient and hypomorphic mice, canonical and bacteria-induced autophagy, Paneth-cell homeostasis and Il‑1β secretion were dependent on ATG16L1; interestingly, changes in the morphology of Paneth cells were only seen in the presence of mouse norovirus.52,112,114 By contrast, studies focusing on Thr300Ala (rs2241880) have shown conflicting results.117,118 Further fine mapping of the ATG16L1 gene has confirmed that the genetic susceptibility association signal extends across ATG16L1 (that is, a number of SNPs in ATG16L1 are associated with Crohn’s disease), involving domains of inter action with another autophagy protein (the N-terminal domain interacts with ATG5) and with the WD domain, which interacts with NOD1 and NOD2.52,82,119 In agreement with the work by Sorbara et al.55 demonstrating a novel autophagy-independent role for ATG16L1 in regulating NOD-driven inflammation (as mentioned earlier), Marchiando and colleagues120 have shown that Atg16l1-hypomorphic mice have a hyperimmune phenotype that protects them from Citrobacter infection and that this protection is lost in mice deficient for both Nod2 (knockout) and Atg16l1 (hypomorphic). In contrast with the effect of ATG16L1 specific to Crohn’s disease, other autophagy genes are associated with risk of both Crohn’s disease and ulcerative colitis (IRGM, LRRK2 and SMURF1).23,111 The investigation of the association signal of IRGM has highlighted several new insights into what we consider ‘risk alleles’.121 Exonsequencing studies of IRGM have not found novel nonsynonymous variants.21,22,122–125 McCarroll et al.123 identified a common, 20 kb deletion polymorphism, upstream of IRGM, which altered IRGM expression (at the RNA level) and therefore modulated cellular



REVIEWS

Healthy gut

Microbial sensing Autophagy ER-stress

Defensins

Ileitis ∆ Defensins

Environment (e.g. Norovirus) Microbiota NOD2 ATG16L1

Paneth Stem cell cell

XBP1 ATG16L1 Microbiota

Dendritic Neutrophil Macrophage cell Lymphocyte

Figure 3 | Paneth cells as the site of origin of intestinal crypt inflammation as well as crypt homeostasis. In the healthy intestinal crypt, Paneth cells maintain the stem cell niche and secretion of defensins into the lumen by integrating microbial and nutrient signalling.67,68,211–213 Inherited variants and/or altered expression of several genes associated with susceptibility to Crohn’s disease (for example, NOD2, ATG16L1 and XBP1) contribute to ileitis through defects in microbial sensing, autophagy, handling of ER-stress and regulation of inflammation.55,128 Abbreviations: ATG16L1, autophagyrelated protein 16‑1; ER, endoplasmic reticulum; NOD2, nucleotide‑binding oligomerization domain-containing protein 2; XBP1, X‑box-binding protein 1.

autophagy of internalized bacteria. Moreover, Brest and colleagues125 demonstrated that a synonymous variant within the IRGM coding region (rs10065172, 313C>T, encoding Leu105Leu), in perfect linkage disequilibrium with the 20 kb deletion upstream of IRGM, constitutes a microRNA196 (miR‑196) binding site. miR‑196 expression correlated with downregulated IRGM expression (and autophagy) in human epithelial cells (except in Paneth cells). A GWAS in a Korean cohort of patients with Crohn’s disease identified a novel association with ATG16L2 (11q13), in addition to two other new susceptibility loci (4p14 and 10q25) confirming loci containing TNFSF15, IL23R, MHC region and CCR6 (but importantly not several previously identified others genes such as NOD2 and ATG16L1).126 ATG16L2 is a novel isoform of mammalian ATG16L that is not essential for canonical autophagy (yet still forms a complex with other autophagy proteins, such as ATG5 and ATG12).127 The adaptor protein CALCOCO2 (also known as NDP52), involved in selective autophagy of intracellular bacteria, was implicated in Crohn’s disease susceptibility by means of a whole-exome sequencing study followed by an association study of candidate SNPs.26 A common missense variant rs2303015 (encoding Val248Ala) was found to impair the regulatory function of NDP52, resulting in inhibition of NFκB-mediated activation of genes that regulate inflammation and also affecting the stability of proteins in Toll-like receptor pathways.26 Autophagy, endoplasmic reticulum (ER) stress (owing to the unfolded protein response) and bacterial sensing all interact in the Paneth cell to orchestrate intestinal inflammation (Figure 3).128 In mice, deletion of X‑box binding protein 1 (Xbp1) in intestinal epithelial cells results in ER stress, Paneth-cell impairment and severe

spontaneous transmural ileitis (similar to Crohn’s disease) if the autophagy process is also compromised.128 Rare variants in XBP1 have previously been associated with IBD.129 Activation of autophagy has been reported in Paneth cells of untreated patients with Crohn’s disease, independent of ATG16L1 or IRGM genotype, which was associated with a marked decrease in the number of secretory granules and features of crinophagy, which might account for the disorganization of secretory granules previously reported in Paneth cells from patients with Crohn’s disease.112,113,118 Deuring and colleagues130 demonstrated a higher expression of markers of ER stress in Paneth cells from patients with Crohn’s disease carrying an ATG16L1 (rs2241880, encoding Thr300Ala) risk allele than patients not carrying the allele, even during quiescent disease. In turn, patients with ER‑stressed Paneth cells had elevated numbers of adherent-invasive E. coli in ileal biopsy samples and tended to have more ileal disease, fistulizing disease and need for intestinal surgery.130 Furthermore, the proportion of abnormal Paneth cells was found to be associated with the cumulative number of NOD2 and ATG16L1 risk alleles.131

Cytokines aplenty: to block or not to block Among gene ontology terms and pathways demonstrating notable enrichment in genes contained within IBD loci of the Immunochip project, the regulation of cytokine production (specifically IFN‑γ, IL-12, TNF and IL‑10), lymphocyte activation (including T cells, B cells and natural killer cells), response to molecules of bacterial origin and the JAK–STAT (Janus kinase– signal transducer and activator of transcription) signalling pathway were most represented.23 In both Crohn’s disease and ulcerative colitis, strong, but distinct, associations with the HLA locus on chromsome 6p have been identified.23,132 The new pathway associations expand previous work that had identified the central role of the IL‑12–IL‑23 pathway.27 The biological complexity of IBD is illustrated by the finding that so many cytokines or their receptors play a part in genetic susceptibility to the disease: IL‑1 to IL-6, IL‑10, IL‑12 to IL-13, IL‑15, IL‑17 to IL-24, IL‑26 and IL‑27 ligands and/or receptors; several TNF superfamily ligands and receptors; interferon and interferon-regulatory factors. This complexity also explains how challenging the selective blockade or activation of any of these pathways can be in terms of expected therapeutic efficacy or treatment safety. The therapeutic efficacy of any treatment that incorporates targeted blockade will be greatly enhanced if we can better identify patients who are likely to benefit from this therapy, based on clinical criteria but also on genotypic data.133 That the regulation of all these cytokines will be genetically altered in all patients with IBD is clearly not the case. Wang et al.40 showed that the difference in average cumulative risk allele scores (of a total of 71 Crohn’s-disease-associated SNPs in their study) between Crohn’s diseases cases and healthy individuals was relatively small (although statistically significant): individuals with Crohn’s disease (49.1 ± 3.3; range, 37–60) and healthy individuals as controls (46.9 ± 3.4; range, 36–56).



REVIEWS One of the key challenges of the next generation of genotype–phenotype studies will be to establish a phenotype linked with a set of genetic markers (probably also incorporating novel gut microbiota data and serology) rather than trying to match our current disease classifications to genotypic markers in regression analyses that have so far yielded disappointing receiver operating characteristic (ROC) curves 75%) have ileal involvement, hence the issue of statistical power with pure colonic Crohn’s disease. This feature is further complicated and confounded by fairly high inter-observer variation in terms of what constitutes colonic Crohn’s disease, as distinct from ulcerative colitis or IBD-undetermined. A strong association between the HLA-DRB1 locus and ‘colonic IBD’ has been reported.168,169 Studies of predictors of natural history A number of clinical features have been identified as predictors of a complicated disease course in Crohn’s disease, including disease onset before age 40 years, smoking and presence of IBD in the small bowel (jejunal and ileal Crohn’s disease).170,171 Studies have demonstrated that carriage of a NOD2 variant, and specifically, the frameshift mutation (which causes a premature stop codon downstream and because of this stop codon, a truncated protein), is associated with an increased risk of complications (fibrostenotic disease and resection).59 NOD2 frameshift status in conjunction with serology has been used to develop a predictive model of Crohn’s disease course, which shows good correlation between modelled and observed data (r = 0.973) with an AUC of 0.81 (0.757–0.846).134 A potential link between NOD2,

tissue damage and treatment has been demonstrated.60 By measuring bacterial DNA in peripheral blood samples, together with a range of other factors including TNF and infliximab levels, the study authors identified NOD2 variants as a risk factor for bacterial translocation, leading to a heightened TNF response and a greater requirement for anti-TNF therapy.60 What the exact sequence of events is and whether increased TNF levels leads to greater tissue damage and hence increased translocation, or whether increased TNF levels is the result of increased bacterial translocation is not currently clear. Impressive tissue healing rates with anti-TNF agents would support the former. The need for rigorously collected longitudinal data on large numbers of IBD cases has been underlined by attempts to interrogate genotype–phenotype relationships at an international level.41,172 In the IBD chip European project, NOD2 variant carriage was also associated with ileal Crohn’s disease, stenosing and/or penetrating disease and need for surgery.41 The association with ileal Crohn’s disease was confirmed in the genotype–phenotypeanaly sis of the Immunochip project.169 Henckaerts et al.172 studied 875 patients with Crohn’s disease who had adequate longitudinal follow-up (median disease duration of 14 years; interquartile range, 7–22 years). Several novel genetic associations were identified with penetrating complications, but these findings are yet to be replicated. However, as with the majority of these studies, the role of medication in reducing the risk of complications was not addressed. In 2013, the group of Charles Bernstein showed that in a predictive model including serology (notably anti-Sachharomyces cerevisiae antibody levels), genotypes of Crohn’s-disease-associated variants of NOD2, ATG16L1 and IL23R did not associate with complicated disease or surgery.173 By contrast, Dubinsky et al.174 showed a combination of genetic and clinical risk factors was most predictive of time to surgery. Three Crohn’s disease susceptibility loci were independently associated with surgery within 5 years (IL12B, IL23R and C11orf30).174 Using an ‘extremes of phenotype’ approach to identify genetic markers of prognosis in Crohn’s disease, Lee et al.175 compared patients requiring at least two immunomodulators and/or two intestinal resections with those running an indolent disease course (disease duration of at least 4 years with no requirement of immunomodulators or surgery). The study identified an association between an intronic SNP (rs12212067, T>G change) within FOXO3A and prognosis, which was replicated in three independent cohorts. However, this SNP did not contribute to disease risk. Functional studies implicated a downregulation of inflammatory responses in monocytes mediated, at least in part, by TGF‑β. The same minor allele (G) within FOXO3A was associated with a milder course in rheumatoid arthritis, and greater disease severity in malaria. Studies in ulcerative colitis are less common and have focused on potential predictors of complications, including extent of disease, colectomy for refractory disease and predictors of chronic pouchitis. In the Immunochip dataset, the HLA locus was the strongest determinant



REVIEWS of disease extent (extensive versus left-sided colitis).169 Haritunians et al.176 used strict definitions for refractory ulcerative colitis (n = 324) requiring colectomy, non refractory cases (n = 537) and a large cohort of healthy individuals as controls, to develop a risk score based upon 46 SNPs identified by GWAS. This score accounted for 48% of the variance across colectomy risk. Using the Immunochip dataset, 45 of 46 SNPs were retested in a cohort of 111 patients with acute, severe colitis who had failed medical therapy (early colectomy cases), 820 patients with mild ulcerative colitis (disease duration ≥10 years, no intravenous steroid use, no immuno modulators), and 8,393 healthy controls.177 One SNP of 45 (rs2403456, on chromosome 11) replicated the association with severe disease (OR 2.15, 1.57–2.95; P = 1.22 × 10–6). NOD2 variants have been identified as potential markers for recurrent pouchitis.178,179 Tyler et al.15 confirmed these initial observations and combined NOD2 frameshift genotype with clinical parameters (smoking), additional loci (NOX3, DAGLB) and serology (antiCBir1 antibody) to generate a weighted risk score. This score performed best when differentiating between patients with acute, intermittent pouchitis (no history of chronic pouchitis) and those with a phenotype similar to Crohn’s disease in the pouch, with 80% sensitivity and 70% specificity.15 Additional subphenotypes comprising ‘hard end points’ such as colorectal cancer complicating colonic IBD and extraintestinal manifestations of IBD are being actively pursued using similar GWAS strategies.

Pharmacogenetic studies To cover pharmacogenetics in IBD in depth is beyond the scope of this Review. However, in terms of maximizing cost-effectiveness and minimizing harm, pharma cogenetic studies offer the potential of personalized therapy, as illustrated by advances in the treatment of hepatitis C.180 Given the costs associated with biologic therapy and the published rates of both primary nonresponse and secondary loss of response with anti-TNF therapies, a number of studies have investigated the association between genetic polymorphisms and response to infliximab using a candidate gene approach.181 The numbers of patients (range, 40–444) and definitions of response (Crohn’s Disease Activity Index, Harvey– Bradshaw Index, C‑reactive protein level) vary between studies, but the majority focus on pathways involved in mononuclear cell apoptosis and TNF biology. Four genes have been investigated in two or more cohorts and, thus far, only the association between the synonymous SNP TNFRSF1A (36A>G, rs767455) and infliximab response has been independently replicated by some, but not all, other studies.182–184 Studies such as these are essential to better inform the field of IBD, but at this stage do not seem to attract the peer-reviewed funding needed to support an adequately powered and appropriately designed protocol. The number of genetic susceptibility loci containing genes of the TNF and TNF receptor superfamily (in addition to the established TNFSF15 association) has now increased substantially, which might enhance research interest in this area.23 Clinical

trials could be an alternative pathway of investigation, but are limited by several confounders, including the crosssectional nature of the case population, heterogeneity in phenotyping and sponsorship.

Future directions Studies across ethnic groups Although much of the current research effort continues to focus on patients with IBD of European ancestry, it is important to note that landmark observations were made in the Japanese population: the early and striking association of ulcerative colitis with HLA-DR2 and the discovery of TNFSF15 by genome-wide analysis.185,186 Association studies in East Asian populations have co nfirmed several IBD loci with an equivalent or greater contribution (notably polymorphisms in TNFSF15, CTLA4 and MICA) but not all, with no effect found for ATG16L1 or indeed the three most important NOD2 variants in Western populations.126,186,187 However, before discussing studies including a number of ethnic groups in further detail, it would be reasonable to consider first the changing epidemiology of IBD in Asia. Although the incidence is still much lower than in Western populations, multiple studies demonstrate notable increases in disease incidence across Asian cohorts, including individuals from Japan, Hong Kong and South Korea.188,189 Similarly, IBD in highly developed economies has become more multi ethnic and less familial.190 In the presence of a stable human genome, this alteration in disease is probably driven by changes in the environment and a positive family history of IBD is indeed less common.189 Top of the list of suspects is the gut microenvironment, specifically the gut microbiome, which, in turn, is affected by modifications in diet and lifestyle including hygiene.191,192 At this early stage in determining the epidemiology of IBD in Asian populations, as was determined in white individuals with IBD 80 years ago, ulcerative colitis seems to have a markedly higher prevalence in these populations than Crohn’s disease. Migrant studies support some of these observations, and underline the importance of the complex relationship between multiple commensal microorganisms and the epithelial surface of the human gut. On this background, it is perhaps not surprising then that IBD leads the field in susceptibility loci identified. Initial studies of the NOD2 gene in non-white patients with Crohn’s disease focused on the three variants (as discussed earlier) associated with Western populations. Strong associations were found between these NOD2 variants and Crohn’s disease in Middle Eastern populations (Turkey and Iran), but not with a range of Asian populations.187 Subsequent studies have identified novel associations with the NOD2 SNP5 and JW1 variants in Malaysian, Han Chinese and Indian Crohn’s disease populations.193–195 Importantly, these mutations demonstrated similar relationships with disease phenotype as seen in studies of NOD2 in white populations, including individuals with early age of disease onset, ileal location and stricturing behaviour. Asano et al.196 previously showed the strong contribution of the MHC to Japanese ulcerative colitis. Moreover, the HLA-Cw*1202‑B*5,201-DRB1*1502



REVIEWS haplotype increases susceptibility to ulcerative colitis, but reduces risk of Crohn’s disease, based on a GWAS in a Japanese population.197 Yamazaki et al.198 reported associations of Crohn’s disease with variants in MHC, TNFSF15 and STAT3, in addition to two new susceptibility loci (4p14 and 13q14). Other major IBD susceptibility loci in white individuals, including IL23R but not ATG16L1, have shown association with Crohn’s disease in Japanese, South Korean and Han Chinese populations, whereas data for IRGM are conflicting. 199–203 GWAS studies for both ulcerative colitis and Crohn’s disease have been completed in the South Korean population;126,204 further evidence for shared genetic susceptibility across ethnic groups includes 21 Crohn’s disease loci from a meta-analysis in white populations, that demonstrated consistent association in the Korean population, four of which reached genome-wide significance.126,204 In a study of African American patients with Crohn’s disease, the admixture between West African (80%) and European (20%) ancestry, was proposed as an explanation for the low allelic frequency of the NOD2 1007 frameshift variant: no known NOD2 risk alleles were seen in either a cohort of West African origin or in individuals of African Ancestry in the 1,000 Genomes project.205 Wang et al.206 demonstrated that European admixture is similar between IBD cases and controls of African American descent. The International IBD genetics consortium is in advanced stages of a so-called trans ethnic meta-analysis of association studies including African American, Puerto Rican and several Asian populations, which will have substantially increased power to detect variants in non-European ancestry populations.

Exploring the epigenome Technological and analytical advances over the past few years have enabled scientific exploration of the extent of epigenetic alterations associated with IBD, and indeed other complex immune-mediated diseases.207 Alterations in DNA methylation, and the role of microRNAs in regulating gene transcription have received most attention, and a series of reports provide optimism that these studies might either highlight new aspects of disease pathogenesis, or identify clinically useful biomarkers or indeed therapeutic targets. Of particular excitement are

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a series of highly influential papers providing substantial evidence for interaction between epigenetic alterations, germline variation and relevant functional effects, notably TH17 cellular activation and xenophagy.79,208,209 To ascribe causality to observed epigenetic alterations in gut or blood with many confounding factors—for example, cell type, inflammation, drug therapy, germ line variation—is of course not straightforward, and the challenges inherent to study design are substantial. Nonetheless, consistent data are emerging both with respect to microRNA production and methylation that will be taken forward in ongoing international studies. Parallel studies applying other emerging technologies are highly pertinent in terms of biomarker discovery (such as IBDBIOM, IBD biomarkers programme, or IBD character from the Seventh Framework programme of the European Union).

Conclusions The advances in our understanding of the genetic architecture of IBD have uncovered a large number of bio logical processes involved in IBD pathogenesis. The major challenges ahead will be to integrate exome and whole-genome sequencing data (focused on the regions identified by the Immunochip project), with new insights in regulation of transcription of key genes, cell-type sp ecific experiments (as heralded by the ENCODE project and breakthroughs in epigenetics) and data from the complex ecology of our gut microbiota (probably moving beyond diversity mapping towards metagenomic approaches).9,210 International efforts are underway to assess the contribution of the known IBD susceptibility loci in populations of non-European ancestry. In years to come, these advances will hopefully increasingly affect patient care by offering the prospect of prediction of disease course and early intervention with genotype and microbiota-type targets. Review criteria Full-text articles were selected from PubMed with the following search terms: “inflammatory bowel disease”, “genetics”, “Crohn’s disease” and “ulcerative colitis”. References from 2013–2011 were prioritized in addition to landmark papers from before 2011.

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Retinitis pigmentosa--new advances in ophthalmic genetics.

Recent advances in the genetics of dystonia.

Myotonic dystrophy: advances in molecular genetics.

Recent advances in the genetics of diabetes.

Recent advances in primary ciliary dyskinesia genetics.

Advances in Faba Bean Genetics and Genomics.

NAFLD in 2014: Genetics, diagnostics and therapeutic advances in NAFLD.

hyperactivity disorder.

Clinical neurogenetics: recent advances in the genetics of epilepsy.

Advances in the Molecular Genetics of Non-syndromic Syndactyly.

Recent advances in the molecular genetics of frontotemporal lobar degeneration.

Some legal implications of advances in human genetics.

Recent advances in the genetics and management of harlequin ichthyosis.

Recent advances in the genetics of emotion regulation: a review.

Dystonia--new advances in classification, genetics, pathophysiology and treatment.

Recent advances in hemophilia. Part II. Genetics. Discussion paper: Prospects.

Trichoderma-plant-pathogen interactions: advances in genetics of biological control.

Recent advances in the genetics of testicular failure.

Genetics advances in autosomal dominant focal epilepsies: focus on DEPDC5.

Advances in genetics: widening our understanding of prostate cancer.