Current Status of Genetics Research in Inflammatory Bowel Disease

REVIEW Current status of genetics research in inflammatory bowel disease

S Vermeire1 and P Rutgeerts1 1University Hospital Gasthuisberg Leuven, Division of Gastroenterology - Herestraat, Leuven, Belgium

The research on genetic susceptibility of inflammatory bowel diseases (IBD) has been tremendous and over 10 chromosomal regions have been identified by genome-wide scanning. Further fine mapping as well as candidate gene studies have already led to the identification of a number of susceptibility genes including CARD15, DLG5, OCTN1 and 2, NOD1, HLA, and TLR4. The CARD15 gene is undoubtedly replicated most widely and most understood at present. CARD15 is involved in the recognition of bacterial peptidoglycan-derived muramyl dipeptide (MDP) and will stimulate secretion of antimicrobial peptides including alpha-defensins (also called cryptdins) to protect the host from invasion. Genetic research in IBD has advanced our understanding of the clinical heterogeneity of the disease and has started to tackle the complex interactions between genetic risk factors and environmental risk factors in IBD. Genes also interfere with the metabolization of drugs and may influence the clinical response and the drug-related toxicity. Interesting pharmacogenetic data with respect to steroids, azathioprine, and infliximab have been generated in IBD. Overall, it is anticipated that genetic markers in the future will be implemented in an integrated molecular diagnostic and prognostic approach of our patients. Genes and Immunity (2005) 6, 637–645. doi:10.1038/sj.gene.6364257; published online 18 August 2005

Keywords: inflammatory bowel disease; Crohn’s disease; ulcerative colitis; genetics

Introduction Genetic epidemiology The inflammatory bowel diseases (IBD), Crohn’s disease Several lines of evidence suggest a genetic contribution (CD) and ulcerative colitis (UC) are chronic relapsing to the pathogenesis of IBD. First, there are considerable inflammatory diseases of the gastrointestinal tract. The differences in the prevalence of IBD among ethnic precise aetiology of the disease is unknown, but an groups.1,2 The highest prevalence is seen among Cauca- interplay of environmental risk factors and immunologic sians. Afro-American and Asian individuals carry a changes will trigger the onset of the disease in a much lower risk, although the incidence of IBD is rising genetically susceptible host. Epidemiological studies in even in these ethnic groups. The prevalence in the Jewish the past suggested a genetic susceptibility that has been population is two to four times higher than any other confirmed by molecular data from total genome scans and ethnic group and is independent from geographic from candidate gene studies. Since the start of genetics location and the period of analysis. The highest risk research in IBD 10 years ago, important progress has been is seen among Ashkenazi Jews, in comparison with made and a number of susceptibility genes have been Sephardic Oriental Jews. identified. These have further advanced researchers in Family studies have reported an important familial unravelling the pathophysiology of IBD. In this respect, association of the disease.3–5 Around 5.5–22.5% of the identification of the caspase-recruitment domain patients with IBD report having another family member (CARD)15 gene has underscored the importance of the also affected with IBD. In fact, the most important risk bacterial flora and has focused attention on the role of factor for IBD is having a family member with the pattern recognition receptors (PRRs) and other pathways disease. The relative risk for a sibling of a CD patient to of the innate immune system. The clinical impact of the also become affected is 13–36, and for a sibling of a UC genetic findings so far is situated in understanding the patient this risk is 7–17. The greatest risk appears for heterogeneity of IBD in terms of location, age at onset and children of whom both parents have IBD (430% at the behaviour, and also in predicting answer to and side age of 28 years).6 effects of particular treatments. Patients with IBD and a familial history tend to get their disease at an earlier age than patients without a familial history and show an increased Correspondence: Dr S Vermeire, University Hospital Gasthuisberg concordance in disease type (CD or UC) and probably Leuven, Division of Gastroenterology – Herestraat, Leuven 49 - 3000, Belgium. E-mail: [email protected] also disease location. The severity of the disease, in Received 11 July 2005; accepted 11 July 2005; published online 18 contrast, does not differ between familial or sporadic August 2005 disease. Current status of genetics research in IBD S Vermeire and P Rutgeerts 638 Twin studies provide solid evidence for the contribu- Candidate gene analysis uses case–control cohorts or tion of genetic and/or environmental factors.7–10 The fact trios of affected offspring with both parents. Here, that monozygotic twins with CD have a much higher authors will investigate a specific gene, which has a disease concordance (37%) than dizygotic twins (7%) known or potential interest for the disease under study. indicates that genetic factors play an important role. For The allelic frequencies (in the case of case–control study) UC, the concordance rates are less (10 vs 3% for or the transmission of a single-nucleotide polymorphism monozygotic and dizygotic twins, respectively), suggest- (SNP) towards affected offspring (in the case of trios) will ing a less important genetic impact, in comparison with be studied and differences between patients and controls CD. or an overtransmission towards affected children might A number of subclinical markers have been identified point towards implication of this particular gene in the in unaffected family members of IBD patients. These pathogenesis of the disease under investigation. markers might indicate who is at risk of developing The human genome contains approximately 10 million the disease or in whom early disease is already present. SNPs and its genetic sequence is organized in haplotypes The markers studied most are intestinal permeability, or stretches of DNA. SNPs that lie closely together ( ¼ on and a number of anti-microbial antibodies: anti-Sacchar- the same haplotype block) tend to be inherited together, omyces cerevisiae antibodies (ASCA) and atypical pANCA whereas SNPs in different haplotype blocks are less (perinuclear antineutrophil cytoplasmic antibodies).11–15 likely to be inherited together. Recently, an International Other microbial antibodies found in IBD patients as anti- Haplotype Mapping (HapMap) Consortium was estab- OmpC (directed against the outermembrane porin C of lished to develop a HapMap of the genome, with the Escherichia coli), I2 (antibodies directed against Pseudo- frequencies of common polymorphisms in the human monas fluorescens) and Cbir 1 (anti-flagellin antibodies) genome in various populations in the world and with the have not been studied in asymptomatic family mem- extent of linkage disequilibrium (LD) between these bers.16–18 However, the presence of these subclinical polymorphisms and the haplotype blocks in which they markers has no clinical implications yet and longitudinal fall.19 This information is freely available on the internet studies are needed first to evaluate their exact meaning. (http://www.hapmap.org/). Therefore, instead of genotyping all SNPs in the genome, genotyping haplotype- tagging SNPs (which are representative of a unique Identification of susceptibility genes haplotype) will undoubtedly facilitate the identification of causal variants in complex diseases. for IBD Also, technology has advanced greatly and the Segregation studies have not supported a simple introduction of the so-called chip-arrays, containing up Mendelian model of inheritance for IBD. Instead, CD to 100 000 SNPs (with even larger chips being developed and UC are considered to be complex polygenic as we speak), has allowed high-throughput and high- disorders. This means that several genes, together with resolution genotyping of the whole genome. However, environmental factors, contribute to the final clinical the high costs and also the informatics to analyse these phenotype, which inevitably will be heterogeneous. How huge amount of data are at this moment the bottle neck many susceptibility genes underlie IBD and how they for wide-spread use of these techniques. interact with each other and with environmental factors is not precisely known, but the list of genes and environmental factors is growing each month. Since both Results from linkage and candidate gene forms of IBD can coexist in single families (and maybe analyses in IBD also in the same individual?) with a frequency greater than expected by chance alone, CD and UC are likely to A total of 11 total genome-wide scans have been share some susceptibility genes. Orholm et al3 showed performed in IBD (Table 1), resulting in a number of that the crossdisease relative risks are 3.85 for UC, given susceptibility regions on chromosomes 1, 3, 4, 5, 6, 7, 10, a CD proband and 1.72 conversely, further suggesting 12, 14, 16, 19 and X. 20–30 the presence of shared genes among CD and UC. According to their initial date of reporting, and However, also disease-specific genes will exist since CD independent confirmations, the regions on chromosomes and UC are also very distinct in clinical features. 16, 12, 6, 14, 5, 19 and 1 have been renamed as IBD1–7, There are two main approaches to identify genes in respectively. It must be stressed that no single locus has complex multifactorial diseases: the positional cloning been reported in a constant manner through all genome approach based on linkage studies and the candidate– scans, suggesting important genetic heterogeneity. gene approach based on association studies. Linkage Nevertheless, variation in phenotypes and lack of power analysis studies the cosegregation of the disease under also explain some of the discrepancies. study with a marker within families. The most used To search for IBD regions that are common for all approach in the genetics of complex diseases is the populations, two meta-analyses have been per- method where allele sharing between affected sibling formed.27,31. The first by Williams et al included five pairs is used. A distortion of the observed ratio of genome scans22–24,26–27 for which the complete results sharing vs the expected is taken as evidence for linkage were published at that time. The highest evidence for with that particular marker. Linkage analysis allows linkage for IBD was obtained for the IBD3 locus, scanning of the whole genome and has already been followed by the IBD1 and IBD2 locus.27 Analysis of the successfully undertaken in many complex diseases such CD-only families gave strongest results for IBD3, as hypertension, schizophrenia, diabetes type I, asthma, followed by IBD5, IBD1, IBD2, IBD6 and IBD7. The rheumatoid arthritis, psoriasis, celiac disease, and also second meta-analysis by van Heel et al31 included data IBD. from 10 separate studies comprising 1952 IBD, 1068 CD

Genes and Immunity Current status of genetics research in IBD S Vermeire and P Rutgeerts 639 and 457 UC affected relative pairs. Again, the IBD3 locus (also called cryptdins) to protect the host from invasion.42 on chromosome 6p (containing the MHC region) met A reduced expression of these defensins has been found genome-wide significance for IBD. Loci meeting sugges- in CD patients and this reduction is even more tive significance for IBD were 2q, 3q, 5q, 7q and 16 pronounced in patients with CARD15 mutation.42,43 (IBD1). For CD-only families, chromosomes 2q, 3q, IBD3, However, the exact pathways through which signalling IBD1, 17q and 19p (IBD6) gave the strongest evidence for occurs are still unclear. The frameshift mutation linkage. 1007fsinsC, which leads to a truncated protein lacking Fine mapping of the IBD1 locus led Hugot et al32 to the 33 distal amino acids, was associated with impaired identify the underlying gene on chromosome 16 as the activation of the transcription factor NF-kappa B (NF-kB) CARD15 (previously NOD2) gene. Parallel to this after stimulation in the initial paper by Ogura et al.34 discovery, Ogura et al33 and Hampe et al34 at the same However, this is contradictory to the increased NF-kB time also identified the CARD15 gene by means of activity observed in CD. In vivo studies using CARD15 the candidate gene approach. In all, 30 nonconservative À/À KO animals have also shown conflicting results, polymorphisms have been identified within the gene whereas Watanabe et al44 showed that intact CARD15 that are associated with CD, but only three are common signalling inhibits Toll-like receptor (TLR) 2-driven (Arg702Trp, Gly908Arg and Leu1007insC). The three activation of NF-kB, and that the presence of a CARD15 common variants, however, account for approximately mutation increased TLR 2-mediated activation of 82% of the mutated alleles.35 NF-kB; the study by Maeda et al45 showed that CARD15 CARD15 is associated with CD only, and not with UC. mutant mice had increased NF-kB activation directly in CARD15 codes for a protein expressed in monocytes, response to MDP. macrophages, dendritic cells, epithelial cells and Paneth cells.36–40 The protein consists of two N-terminal CARD, a central nucleotide-binding domain and a C-terminal leucin-rich-repeat region (LRR) (Figure 1). CARD15 is Other susceptibility genes involved in the recognition of bacterial peptidoglycan- derived muramyl dipeptide (MDP) through the LRR In the last year, more genes have been reported using region.38,41 Recognition of MDP will stimulate secretion positional cloning techniques and fine mapping of of anti-microbial peptides including alpha-defensins identified susceptibility regions from total genome scans.

Table 1 Total genome scans in IBD

Author Year N asp CD/UC/mixed Chromosomal regions of linkage

Hugot et al20 1996 112 110/0/0 16cen Satsangi et al21 1996 186 81/64/41 12, 7q22, 3p21 Cho et al22 1998 151 (297 relative pairs) 97/18/36 (175/26/96) 16cen, 1p, 3q, 4q Hampe et al23 1999 268 129/90/49 16cen, 10q, 1q, 6p, 12, Â , 22, 4q Ma et al24 1999 65 65/0/0 14q11, 17q21, 5q33 Duerr et al25 2000 94 94/0/0 14q11 Rioux et al26 2000 183 116/20/47 19p13, 5q31, 3p, 6p Williams et al27 2002 70 (187 relative pairs) 40/13/17 (105/29/53) 16cen, 11p, 6p21 Paavola-Sakki et al28 2003 138 72/19/47 11p12, 2p11, 12p13, 12q23, 19q13 Vermeire et al29 2004 149 129/0/20 14q11, Xq, 1q, 6q, 20p, 4q, 10q Barmada et al30 2004 260 108/72/0 12, 6p, 6q, 8q, 15q, 22, 2q

The number of affected sibling pairs (asp) studied and the regions of linkage identified in each genome scan are indicated.

CARD 1 CARD 2 NBD LRR

128 124 127 220 273 577 744 1020 1040

Arg702Trp Gly908Arg SNP8 SNP12

Leu1007fsinsC 3020insC SNP13 Figure 1 Structure of the CARD15 (previously NOD2) protein on chromosome 16: the CARD15 protein consists of two N-terminal caspase- recruitment domains (CARD), a central nucleotide-binding domain (NBD) and a C-terminal leucin-rich-repeat region (LRR). The arrows show the three main associated variants associated with CD.

Genes and Immunity Current status of genetics research in IBD S Vermeire and P Rutgeerts 640 So has the gene underlying the chromosome 10 linkage produces weight loss and bloody diarrhoea in rats with region, originally identified by Hampe et al,23 been histological signs of ulcers, mucus cell depletion, vessel identified as DLG5 (for its homology with Drosophila dilatation and increase of acute inflammatory cells.61 Discs Large Homolog 5). In the study by Stoll et al,46 two Peltekova et al51 showed that fibroblasts expressing the haplotypes were associated with IBD. One haplotype, 503F variant had a 2.7 times lower carnitine uptake and characterized by the haplotype-tagging SNP G113A an increased transport of xenobiotics compared to the (leading to a change from arginine to glutamine at cells expressing the conservative 503L. The OCTN2 amino-acid position 30 (R30Q)), was overtransmitted to promoter variant disrupts a heat-shock element (HSE) affected offspring with CD and UC. This SNP is located 207 bp upstream of the start codon. in the DUF622 domain and influences binding to Rab- Recently, a strong association between a complex GTPase, which is important in inflammatory signalling. functional NOD1 (CARD4) insertion/deletion poly- In an independent case–control sample, 25% of IBD morphism (ND(1) þ 32 656*1) and IBD was found by patients carried at least one 113A risk allele compared to investigators at the University of Oxford.62 NOD1 has 17% of healthy controls (P ¼ 0.001). The overall risk for analogy to CARD15 and is located in the region of IBD associated with the 113A variant was however linkage on 7p14, identified in the original genome scan moderate (odds ratio (OR) ¼ 1.6). The second haplotype, from the same group.20 Intriguingly, this gene was characterized by the presence of an insertion of 13 bp in examined previously by the French group lead by Hugot exon 26 (DLG5_e26), was undertransmitted. DLG5 is a et al, but no association with this gene in CD patients was widely expressed protein, found in placenta, small observed.63 Again, independent studies are necessary bowel, colon, heart, skeletal muscle, liver and pancreas. before firm conclusions can be made. It is a member of the MAGUK family (membrane- From a candidate gene approach, the MHC region is associated guanylate kinase) of scaffolding proteins the region studied most extensively. HLA class II important in signal transduction and epithelial cell molecules present partially digested antigen to the T-cell integrity. The authors also examined gene–gene inter- receptor and play a central role in the immune response. actions between DLG5 and CARD15, and showed In contrast with other immune-mediated complex dis- interaction between the 113A variant on haplotype D eases such as rheumatoid arthritis, multiple sclerosis and and the presence of CARD15 variants. Meanwhile, IDDM, results on the role of the MHC complex in IBD are replication studies have emerged, but results are con- inconsistent, heterogeneous and often very weak.64–67 In flicting. Whereas Daly et al47 was able to show Japanese studies, HLA DR2 (HLA DRB1*1502) has been independent replication in two cohorts from Quebec/ implicated in UC, whereas HLA DR3 (HLA DRB1*0103) Italy and Quebec/UK, other recent studies were not able has been implicated in European studies. In CD, HLA to show association.48–50 More studies are inevitably associations are less convincing. HLA DR1 has been needed to understand more about this gene. associated with the CD phenotype. At the same time last year and published in the same After the identification and knowledge of the function issue of Nature Genetics, Peltekova et al51 identified the of CARD15, researchers have expressed major interest in gene underlying the IBD5 locus as the SCL22A4 and the PRRs to which CARD15, and also the membrane- SLC22A5 genes, coding for, respectively, the OCTN1 and expressed TLRs belong. A Belgian collaborative study 2 (novel organic cation transporter) proteins. It was described an association between the TLR4 Asp299Gly Rioux et al26 who first reported linkage for CD on 5q31 in polymorphism and IBD in two independent cohorts of the Canadian population. IBD5 is very attractive as a patients.68 This polymorphism is associated with im- candidate region for IBD, since it harbours the cytokine paired LPS signalling and increased susceptibility to gene cluster. Fine mapping of this locus refined Gram-negative infections. In the Belgian study, the allele the region to a 250 kb risk haplotype (surrounding the frequency of the TLR4 Asp299Gly polymorphism was OCTNs), but precise identification of the underlying significantly higher in CD (11 vs 5%, OR 2.31 (95% causal genetic variants was impossible due to strong LD confidence interval (CI) 1.28–4.17), P ¼ 0.004) and UC across the region.52 At least three independent groups patients (10 vs 5%, OR 2.05 (95% CI 1.07–3.93), P ¼ 0.027) subsequently confirmed association of the IBD5-risk compared with the control population. A TDT on 318 IBD haplotype with CD53–56 and also with UC.53,55 Peltekova trios demonstrated preferential transmission of the TLR4 et al resequenced the five known genes in the IBD5 Asp299Gly polymorphism from heterozygous parents to region and identified 10 new SNPs, including two novel affected children (T/U: 68/34, P ¼ 0.01). Replication SNPs in SLC22A4 and SLC22A5 that were predicted studies performed in UC as well as CD have shown to have functional effects (a missense substitution in conflicting results so far.69–72 SLC22A4 (L503F) and a G-C transversion in the SLC22A5 promoter) and that were associated with susceptibility to CD. The OCTN family is a family of Translation of IBD-genetic research into transporter proteins for organic cations, and may also transport carnitine, an essential cofactor of the metabo- the clinic lism of lipids.57–60 Carnitine is involved in the transport CARD15 variants are found in the majority of the of long-chain fatty acids into the mitochondria, where Caucasian CD patients and vary between 35 and fatty acids will undergo b-oxidation. Hence, carnitine is 45%,73–81 with the exception of Scandinavian,81 Irish82 important in the energy production of the cell. There is and Scottish83 CD patients where the prevalence is much evidence that inhibition of fatty acid oxidation in the lower. In the Japanese84,85 and Chinese,86 as well as in epithelium of the colonic mucosa is associated with the African-American populations,87 no CARD15 variants development of UC. Rectal instillation of sodium 2- could be identified. CARD15 explains around 20% of bromo-octanoate, a specific inhibitor of b-oxidation, the genetic predisposition to CD. The relative risk of

Genes and Immunity Current status of genetics research in IBD S Vermeire and P Rutgeerts 641 developing CD in the presence of one mutation is 2–4, altered Pgp expression and function in humans.97 but increases dramatically to 20–40 in the case of two Overexpression of MDR-1 leads to an increased efflux mutations (compound heterozygous or homozygous). of drugs and to decreased cytoplasmic drug concentra- Several studies have investigated the phenotypic expres- tions. Several drugs, including glucocorticoids, are sion of CARD15 variants and the most widely replicated known Pgp-170 substrates. Farrell et al98 showed that, association is seen with small bowel disease.35,73–74,76–81 compared to controls, peripheral blood lymphocyte Patients with double-dose mutations in a French study (PBL) MDR was significantly elevated in CD patients were characterized by a more frequent structuring who required bowel resection for failed medical therapy phenotype (53 vs 28%; P ¼ 0.00003 OR 2.92),35 which (mean7s.e.m. 26.772.8 vs 11.971.0; Po0.0001). Also, has also been confirmed by others.73,78,81,88 patients with UC who required proctocolectomy Besides the implication of CARD15 mutations in CD, for failed medical therapy had elevated PBL MDR this gene seems implicated in graft-versus-host disease compared to controls (mean7s.e.m. 20.372.5 vs (GvHD) and complications following allogeneic stem cell 11.971.0; Po0.0001). A second mechanism responsible transplantation.89 In patients receiving stem cell trans- for the glucocorticoid resistance lies at the level of plantation, the transplant-related mortality in donor/ the glucocorticoid receptor (GR) and polymorphisms in recipient pairs with mutations was much higher (49%) in the gene encoding this receptor (GRb) influence the contrast to the mortality in donor/recipient pairs with- response to corticosteroid treatment in IBD. Overexpres- out mutated CARD15 (20%). The mortality was even sion of GRb is associated with steroid-refractory UC, in higher (83%) in pairs with mutated alleles in both donor contrast to steroid-responsive UC.99 and recipients (Po0.001). Similar associations have been Of all drugs used in IBD, azathioprine (AZA) is the observed for severe overall and severe gastrointestinal only drug where pharmacogenetics have shown clinical GvHD. relevance to date. AZA is one of the best examples of Very little is known so far about the clinical expression genetically influenced heterogeneity in drug response. of the novel reported genes DLG5, OTCN, NOD1 or The pathway of AZA metabolism is illustrated in TLR4. For IBD5 and OCTN1 and 2, results have not been Figure 2. In a first step, AZA is converted by a consistent so far and associations with perianal dis- nonenzymatic reaction into 6-mercaptopurin (6-MP). ease,50,90,91 with ileal disease92 as well as with colonic Subsequently, 6-MP is metabolized by competing catabo- disease, with an earlier age of disease onset and reduced lic and anabolic enzymatic pathways: xanthine oxidase need for surgery49 have all been reported. Large studies converts 6-MP into the inactive 6-thiouric acid (6-TU). with phenotypically well-characterized patients are Alternatively, 6-MP may be methylated to 6-methyl MP therefore greatly needed. (6-MMP) by the enzyme thiopurine methyl transferase Also, the HLA system influences the disease pheno- (TPMT). A third pathway is metabolization towards type. Genetic markers in the MHC, especially the class II active 6 thioguanine (6-TG) nucleotides, which are HLA DRB1*0103 allele, have been associated with severe incorporated as false bases into the DNA and would cortico-dependent and refractory UC as well as severe explain the cytotoxicity of the drug. The activity of TPMT UC necessitating colectomy.66,93 It is well possible that is genetically regulated.100,101 In the Caucasian popula- the HLA gene plays a disease-modifying role, rather than tion, TPMT*3A and TPMT*3C are the most common being an important gene in the susceptibility for variant alleles. Approximately 11% of the population is developing IBD: a UK study found that certain HLA heterozygous for the common TPMT polymorphisms alleles are associated with different disease behaviour and around 1/300 is homozygous mutant. Heterozygous and extraintestinal manifestations such as IBD-arthro- patients show decreased activity of TPMT and patients pathy, as well as eye and skin manifestations.94,95 The homozygous have almost absent TPMT activity. A HLA-B27 and -B35 were associated in this study with the deficiency of TPMT results in a preferential production large joint type of IBD-arthropathy, whereas HLA-B44 of 6-TG nucleotides, which in its turn explains the risk of was increased in the small joint type of IBD-arthro- bone marrow suppression and neutropenia.102,103 How- pathy.94–96 ever, Colombel et al104 genotyped 41 patients who previously demonstrated signs of bone marrow toxicity (leucopenia or thrombocytopenia) upon AZA treatment The emerging field of pharmacogenetics and showed only 65.8% (27/41) of TPMT mutations in these patients (10 were homozygous and 17 hetero- in IBD zygous). The duration of onset of the bone marrow Genes interfere with the metabolizing pathways of drugs suppression was significantly shorter in the homozygous and may influence the clinical response and the drug- patients (o1.5 months) compared to the wild-type related toxicity. With increasing knowledge of the patients (0.5–87 months) and the heterozygous patients variations within the human genome sequence, research- had an onset in between that of wild-type and of ers and clinicians aim at personalizing medicine and at homozygous patients (1–18 months). However, these optimizing a patient’s treatment based on his/her results suggest that also other factors are responsible for genotype. bone marrow suppression. Besides bone marrow sup- Interesting results have been generated to predict the pression, hepatotoxicity is a second adverse event of answer to steroid treatment in IBD. One mechanism of AZA metabolism and would be related to 6-MMP. action is related to the multidrug resistance gene MDR-1. Dubinsky et al105 showed that the 16/173 CD patients MDR-1 encodes the drug efflux pump P-glycoprotein 170 with hepatotoxicity had high MMP levels (median 5463) (Pgp-170). Various polymorphisms have been identified compared to patients without those symptoms (median within MDR-1: a mutation C3435T in exon 26 and a MMP 2213; Po0.05). A cutoff of MMP45700 pmol/ mutation G2677T in exon 21 have been correlated with 8 Â 108 RBC was associated with hepatotoxicity. Remark-

Genes and Immunity Current status of genetics research in IBD S Vermeire and P Rutgeerts 642 Circulation Intracellular: 6-MP taken up by erythrocytes and organ tissues

6-MMP 6-MMP Purine inactive ribonucleotides synthesis

TPMT TPMT

AZA 6-MP 6-TIMP 6-TGN DNA/RNA HGPRT IMPDH Cytotoxicity and XO immunosuppression

6-TU inactive

Figure 2 Azathioprine metabolism. In a first step, azathioprine is rapidly converted by a nonenzymatic reaction into 6-mercaptopurin (6- MP). Subsequently, 6-MP is metabolized by competing catabolic and anabolic enzymatic pathways: xanthine oxidase (XO) converts 6-MP into the inactive 6-thiouric acid (6-TU). Alternatively, 6-MP may be methylated to 6-methyl MP (6-MMP) by the enzyme thiopurine methyl transferase (TPMT). The active 6 thioguanine nucleotides (6-TGNs) are generated in a multistep pathway, which is initiated by hypoxanthine guanine phosphoribosyl transferase (HGPRT).

ably also was the fact that none of the patients with significant association between the Fas ligand CÀ843T hepatotoxicity carried genetic variants in the TPMT gene. polymorphism and the caspase-9-C93T polymorphism. These data suggested that certain patients preferentially Patients carrying the Fas ligand À843 CC/CT genotype metabolize through TPMT, resulting not only in higher (n ¼ 135) responded in 74.7% in contrast to 38% response 6-MMP levels and more risk of hepatotoxicity but also in the patients carrying the TT genotype (n ¼ 21) in lower 6-TG levels. (OR ¼ 0.11; 95% CI ¼ 0.08–0.56; P ¼ 0.002). Patients with Other side effects of AZA as pancreatitis, fever, the caspase-9 93 TT (n ¼ 9) genotype all responded, in myalgias and arthralgias are type I hypersensitivity contrast with 66.7% (n ¼ 147) with the CC and CT reactions and should not lead to genetic testing. In these genotype (P ¼ 0.04; OR ¼ 1.50, 95% CI ¼ 1.34–1.68). Inter- patients, AZA should be stopped and rechallenge is not esting was that concomitant 6-MP/AZA therapy was recommended. able to overcome the low response observed with these Also for the novel biological treatment infliximab, a genotypes. chimeric monoclonal antibody to TNFa approved for the Taken together, the first results on pharmacogenetics in treatment of active luminal and fistulizing CD, genetic IBD are promising and urge more research in this field. factors have been investigated to explain the lack of response seen in 25% of patients. Various polymorphisms in the TNFa and b genes and in their receptors have been Conclusion examined but failed to show clear associations.106–108 Following the discovery of NOD2/CARD15, it was The recent advances in the research on genetic suscept- postulated that mutations in NOD2/CARD15 might ibility of IBD have been tremendous. Total genome scans predict therapy outcome based on changes in NF-kB have generated over 10 regions of linkage, which are in activation and subsequent TNF production. However, the process of fine mapping. These efforts have already three independent cohorts of patients treated with led to the identification of a number of susceptibility infliximab (one Belgian n ¼ 245; one German n ¼ 90 and genes (CARD15, DLG5, OCTN1 and 2, NOD1, HLA, the ACCENT I Cohort n ¼ 444) failed to show an TLR4), of which the CARD15 gene is undoubtedly association.109,110 In a Belgian collaborative study, an replicated most widely and most understood at present. association with FCGR3A-158 was described.111 The FcG However, even for the CARD15 gene, a number of recptor 3a is expressed on macrophages and NK cells questions remain especially concerning the mechanisms and is involved in ADCC. In the study by Louis et al in of signalling. Answers to these questions will further 200 patients treated with infliximab, all patients with the improve our knowledge on the pathogenesis of the V/V genotype showed biological response compared disease in the common years. Genetic research in IBD has to 69.8% of patients carrying the F allele (P ¼ 0.0002; RR advanced our understanding of the clinical heterogeneity 1.43, 95% CI 1.27–1.61). Interestingly, the FCGR3A of the disease and has started to tackle the complex polymorphism has also been associated with a positive interactions between genetic risk factors and environ- response to rituximab, a recombinant IgG1 antibody mental risk factors in IBD. It is anticipated that these used in non-Hodgkin’s lymphoma.112,113 The effect of discoveries in the future will be translated back into infliximab is ascribed to induction of apoptosis. In a clinical practise where genetic markers will find their cohort of 204 consecutive patients treated with infliximab place in an integrated molecular diagnostic and prog- for refractory luminal CD, Hlavaty et al114 found a nostic approach of our patients.

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