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CARD15 in Hungarian patients with Crohn's disease
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Digestive and Liver Disease 39 (2007) 1064–1070
Alimentary Tract
NOD1 gene E266K polymorphism is associated with disease susceptibility but not with disease phenotype or NOD2/ CARD15 in Hungarian patients with Crohn’s disease T. Molnar a,∗ , P. Hofner a , F. Nagy a , P.L. Lakatos c , S. Fischer c , L. Lakatos d , A. Kovacs e , I. Altorjay f , M. Papp f , K. Palatka f , P. Demeter g , Z. Tulassay h , T. Nyari i , P. Miheller h , J. Papp c , Y. Mandi b , J. Lonovics a , the Hungarian IBD Study Group1 a
1st Department of Medicine, University of Szeged, Faculty of Medicine, Szeged, Hungary Department of Microbiology, University of Szeged, Faculty of Medicine, Szeged, Hungary c 1st Department of Medicine, Semmelweis University, Budapest, Hungary d 1st Department of Medicine, Csolnoky F. County Hospital, Veszprem, Hungary e 1st Department of Medicine, Erzsebet Hospital, Budapest, Hungary f 2nd Department of Medicine, University of Debrecen, Debrecen, Hungary g Department of Gastroenterology, St Margit Hospital, Budapest, Hungary h 2nd Department of Medicine, Semmelweis University, Budapest, Hungary Department of Medical Informatics, University of Szeged, Faculty of Medicine, Szeged, Hungary b
i
Received 8 March 2007; accepted 7 September 2007
Abstract Background. NOD1/CARD4, a member of the pattern-recognition receptor family, is a perfect candidate as a susceptibility gene for Crohn’s disease. Since only limited and conflicting data are available on G796A polymorphisms in inflammatory bowel disease patients, we set out to study the effect of this polymorphism on the susceptibility and course of Crohn’s disease in the Hungarian population. Methods. Four hundred thirty-four unrelated Crohn’s disease patients (age at presentation: 28.6 ± 9.6 years, female/male: 210/224, duration of Crohn’s disease: 8.2 ± 6.9 years) and 200 healthy subjects (blood donors) and 136 non-inflammatory bowel disease gastrointestinal controls with chronic gastritis were investigated. NOD1 G796A was detected by using polymerase chain reaction/restriction fragment length polymorphism. Detailed clinical phenotypes were determined by reviewing the medical charts. Results. The frequencies of the variant alleles of NOD1 G796A differed significantly between the Crohn’s disease patients and both healthy (GG 49.5% vs. 67%; AG 41.5% vs. 28%; and AA 9.0% vs. 5.2%; p < 0.0001) and non-inflammatory bowel disease controls with chronic gastritis. Carriage of the single nucleotide polymorphism of NOD1 G796A proved to be a highly significant risk factor for Crohn’s disease compared to both healthy (p < 0.0001, OR: 2.1, 95% CI: 1.5–2.9) and non-inflammatory bowel disease controls with chronic gastritis (p = 0.008). Significant associations were not found between the different genotypes and the demographic data on the patients or the clinical characteristics of Crohn’s disease. The different polymorphisms of pattern-recognition receptors (e.g. NOD2/CARD15 SNP8, SNP12 and SNP13 mutations, the TLR4 D299G polymorphism and NOD1 G796A) did not reveal a mutual basis. Conclusions. Our results suggest that carriage of the NOD1 G796A mutation increases susceptibility for Crohn’s disease in the Hungarian population. © 2007 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Crohn’s disease; E266K; Genetics; G796A; IBD; NOD1
∗
Corresponding author. Tel.: +36 62545186; fax: +36 62545185. Hungarian IBD Study Group: Peter Fuszek, Henrik Csaba Horvath, Ferenc Szalay (Budapest), Zsuzsanna Erdelyi, Tunde Pandur (Veszprem), Laszlo Bene (Budapest), Zsuzsanna Vitalis (Debrecen), Levente Balint, Ferenc Huoranszky, Istvan Dobo (Budapest), Laszlo Herszenyi, Annamaria Nemeth (Budapest). 1
1590-8658/$30 © 2007 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2007.09.003
T. Molnar et al. / Digestive and Liver Disease 39 (2007) 1064–1070
1. Introduction Inflammatory bowel disease (IBD) is a collective term for at least three heterogeneous gastrointestinal disorders (Crohn’s disease (CD), ulcerative colitis (UC) and indeterminate colitis), which affect the same age group, in very different manners, resulting in several distinct clinical phenotypes and subgroups. The epidemiological data suggest a genetic predisposition for IBD that does not follow Mendelian inheritance patterns or a simple segregation of the disease, the importance of which is more significant in CD. Although CD is manifested by almost identical clinical picture worldwide, there are significant differences in gene polymorphisms among different ethnicities. The discovery of mutations in the NOD2/CARD15 gene (the first susceptibility gene known for CD associated mainly with an ileal involvement) in Western European and North American countries as well as in Hungary was striking; however, patient–control studies of Japanese, Chinese, Korean and Turkish populations have not encountered NOD2/CARD15 polymorphisms in either patient or control groups [1–5]. The variation of mutations in different IBD populations necessitates repeated studies of the roles of candidate genes in various populations. The digestive mucosa has evolved various immune strategies to allow it to tolerate intimate contact with commensals and to prevent pathogenic bacteria from spreading into the host tissues. Innate immunity relies on the specific recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs). The nucleotidebinding and oligomerization domain (NBD and NOD) proteins are a family of intracellular PRRs that recognize PAMPs, which results in the activation of pro-inflammatory pathways [6–8]. NOD1 is a host cytosolic signalling PRR composed of a caspase-activating and recruitment domain (CARD), NBD, NOD and leucine-rich repeats. NOD1 (or CARD4) acts as a cytosolic receptor for the diaminopimelate (DAP)-containing GlcNAc-tripeptide muropeptide found mostly in Gram-negative bacterial peptidoglycans [9]. It plays a crucial role in innate immunity by activating the NFB pathway via its downstream effector, the kinase RICK (RIP2), and also by triggering interleukin-1 secretion by procaspase-1 activation, following the recognition of a specific bacterial ligand [10]. A British genome-wide scan for IBD linkage demonstrated a susceptibility locus on chromosome 7p14; the same locus within which is the NOD1 (CARD4) gene located [11]. Nevertheless, although both the detailed morphological research and the functional approach have emphasized the possible role of NOD1 mutations in the development of IBD, the latest studies have not resulted in any breakthroughs. The G796A variation encodes a non-conservative peptide change (E266K) in the NBD domain of NOD1. The corresponding glutamic acid residue appears to be conserved in CARD15, suggesting a potential functional effect of the polymorphism [12]. The earliest observations did not prove a significant association between this mutation and IBD
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in Western European populations or in one Turkish study [5,12,13]. We have, therefore, repeated such examinations in a different Central-East European (Hungarian) population with a known genetic background of two other PRR genes, NOD2 and TLR4.
2. Patients and methods 2.1. Patients Four hundred thirty-four unrelated CD patients (age at presentation: 28.6 ± 9.6 years, female/male: 210/224, duration: 8.2 ± 6.9 years), 200 healthy subjects (blood donors) and 136 non-IBD patients with chronic gastritis were investigated. The clinical data of the CD patients are presented in Table 1. The diagnosis was on the basis of Lennard–Jones criteria [14]. The age at onset, duration of disease, presence of extraintestinal manifestations (arthritis: peripheral and axial; ocular manifestations: conjunctivitis, uveitis, iridocyclitis; skin lesions: erythema nodosum and pyoderma gangrenosum; and hepatic manifestations: primary sclerosing cholangitis), frequency of flare-ups (frequent flare-ups: Table 1 Clinical data on Crohn’s disease patients All (n = 434) Sex (female/male) Age at presentation (years) Duration (years) Familial IBD, n (%)
210/224 28.6 ± 9.6 8.2 ± 6.9 52 (12.0%)
Smoking habits, n (%) No Yes Previous
259 (59.7%) 143 (32.9%) 42 (9.7%)
Location, n (%) L1 L2 L3 L4
120 (27.6%) 105 (24.2%) 200 (46.1%) 9 (2.1%)
Behavior, n (%) B1 B2 B3
184 (42.4%) 103 (23.7%) 147 (33.8%)
Perianal disease, n (%) Frequent relapsea , n (%) EIMb , n (%)
100 (23.0%) 152 (35.0%) 216 (49.7%)
Steroid, n (%) Use Refractory
335 (77.2%) 23 (5.3%)
Azathioprine use, n (%) Surgery, n (%)
263 (60.6%) 173 (39.8%)
a
Frequent relapse: >1/year. EIM: extraintestinal manifestations (arthritis: peripheral and axial; ocular manifestations: conjunctivitis, uveitis and iridocyclitis; skin lesions: erythema nodosum and pyoderma gangrenosum; and hepatic manifestations: primary sclerosing cholangitis). b
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>1/year), effectiveness of therapy (e.g. steroid and/or immunosuppressive use, and steroid resistance), need for surgery (resections), occurrence of familial IBD, smoking habits and perianal involvement were investigated through a review of the medical charts and questionnaires. The disease phenotype (the age at onset, the duration, the location and the behaviour) was determined according to the Vienna classification [15]. The control group for the mutation analysis consisted of 200 age-matched and gender-matched healthy blood donors (male/female: 102/98, age: 38.05 ± 10.7 years). The control subjects, who had no gastrointestinal or liver diseases, were selected from consecutive blood donors in Szeged, Hungary. A second non-IBD gastrointestinal control group consisting of 136 chronic gastritis cases (male/female: 61/75, age: 41.03 ± 11.5 years). was also investigated. The study protocol was approved by the Ethical and Science Committee of the Ministry of Health and the University of Szeged Regional and Institutional Committee of Science and Research Ethics. Each patient was informed about the details of the study and signed the informed consent form.
The 796G-to-A transition (E266K) in exon 3 was analysed by PCR-RFLP. The 50 l reaction mixture contained 100 ng of genomic DNA, 20 pmol of each primer (sense: 5 TGA GAC CAT CTT CAT CCT GG 3 ; antisense: 5 CTT CCC ACT GAG CAG GTT G 3 ), 1.25 U Taq DNA polymerase (Fermantas, Vilnius, Lithuania), 1.5 mM MgCl2, 1×PCR Taq polymerase buffer + (NH4 )2 SO4 , and 25 mM of each dNTP (Fermentas). After denaturation (12 min at 95 ◦ C), the reaction consisted of 30 PCR cycles (30 sec at 94 ◦ C, 30 s at 58 ◦ C, and 30 s at 72 ◦ C) followed by a final extension by 7 min at 72 ◦ C. For RFLP analysis, the PCR products were digested with AvaI restriction enzyme (Fermantas) overnight at 37 ◦ C, electrophoresed on a 2% agarose gel (Sigma-Aldrich, St. Louis, MO, USA), visualized under UV illumination and stained with 0.4 mg/l ethidium bromide. The presence of an AvaI site (G allele) was indicated by cleavage of the 379 bp amplified PCR product to yield fragments of 209 bp and 170 bp.
2.2. DNA isolation
The χ2 -test and the Fischer exact test were used to evaluate differences between the IBD patients and the controls, as well as within subgroups of the IBD patients. Logistic regression was applied to compare genetic and clinical data; the results are expressed as odds ratios (OR) with 95% confidence intervals (95% CI). A p value <0.05 was considered significant. For the statistical analysis STATA (STATACOR LD, College Station, Texas, USA) was used.
Genomic DNA was isolated from whole blood according to the QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Germany). 2.3. Detection of NOD2/CARD15 SNP8, SNP12, SNP13 mutations and TLR4 D299G polymorphism The NOD2 SNP8, SNP12 and SNP13 and the TLR4 D299G genotypes were known for each patient. NOD2 variants were previously detected by means of denaturing high-performance liquid chromatography (dHPLC; Wave DNA fragment analysis system; Transgenomic Ltd., UK). Sequence variations observed in the dHPLC profile were sequenced on both strands to confirm the alteration. Sequencing reactions were performed with the ABI BigDye Terminator Cycle Sequencing Kit v1.1 (Applied Biosystems, Foster City, CA, USA) and samples were sequenced on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). The D299G polymorphism was detected using polymerase chain reaction/restriction fragment length polymorphism (PCRRFLP) and fragments were separated and visualized by gel electrophoresis (3% NuSieve GTG agarose gel; BMA, Rockland, ME, USA) [16]. 2.4. Detection of NOD1 polymorphisms The sequence variant of the studied single nucleotide polymorphism (SNP) in the NBD of NOD1 was as follows: CARD4 (NOD1) (Gene ID: 10392, localisation 7p15-p14, Genbank accession number NM 006092; c.709G>A, peptide change: E226K, protein domain: NBD, exon 3, SNP database ID: rs5743336).
3. Statistical methods
4. Results Fig. 1 shows representative results for NOD1 genotyping. To detect the nucleotid swap, RFPL was used. The homozygote mutant (AA), the heterozygote (GA) and the homozygote (GG) variants were well distinguishable. The frequencies of the variant alleles of NOD1 G796A among the CD patients and the healthy (p < 0.0001) and non-IBD GI controls (p = 0.008) differed significantly (Table 2). No significant departures were noted from the Hardy–Weinberg equilibrium. When the frequencies of the homo- and heterozygous mutations were examined together (50.5% vs. 33%), the carriage of the variant SNP of NOD1 G796A proved to be a highly significant risk factor for CD compared to both healthy (p = 0.0001, OR: 2.07, 95% CI: 1.46–2.93) and non-IBD GI controls. In addition, the A allele was significantly more frequent in CD compared to both control groups (p < 0.0001 and p = 0.019). There was no interaction between NOD1 G796A and the SNPs of NOD2 and TLR4 in Hungarian patients. The different polymorphisms of the PRRs (e.g. the NOD2/CARD15 SNP8, SNP12 and SNP13 mutations, the TLR4 D299G polymorphism and NOD1 G796A) did not reveal a mutual basis (Table 3).
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Table 2 NOD1 E266K (G796A) genotypes in Hungarian patients with Crohn’s disease (CD) and controls NOD1 E266K (G796A) CD (n = 434)
Controls (n = 200)
Gentortype* Homozygous AA Heterozygous AG Wild-type GG
39 (9.0%) 180 (41.5%) 215 (49.5%)
10 (5%) 56 (28%) 134 (67%)
Allele frequency# A G
258 (29.7%) 610 (70.3%)
76 (19.0%) 324 (81.0%)
non-IBD GI controls (n = 136) 10 (7.5%) 41 (30%) 85 (62.5%) 61 (22.4%) 211 (77.6%)
(*) IBD vs. controls: χ2 = 17.907, p < 0.0001, ORhetero/homozygous vs. wild type : 2.07, 95% CI: 1.46–2.93. (*) IBD vs. non-IBD GI controls: χ2 = 6.977, p = 0.008, ORhetero/homozygous vs. wild type : 1.70, 95% CI: 1.14–2.52. (#) IBD vs. controls: χ2 = 16.229, p < 0.0001, ORA vs. G : 1.80, 95% CI: 1.35–2.41. (#) IBD vs. non-IBD GI controls: χ2 = 5.472, p = 0.019, ORA vs. G : 1.46, 95% CI: 1.06–2.01.
5. Discussion
Fig. 1. NOD1 E266K genotypes were deduced from the migration profile on a 2% agarose gel: wild-type DNA is visible as a double-band 209 bpand 170 bp (GG); the mutated DNA is visible as a single 379 bp band (AA); whereas heterozygotes give three bands (GA). Lane M is a molecular weight marker (Fermentas).
The results on the phenotype–genotype associations in the CD patients are shown in Table 4. No significant associations were found between the different genotypes and the demographic data on the patients or the clinical characteristics of CD.
This is the first report on the prevalence of the NOD1 (G796A) polymorphisms in patients with IBD from an Eastern European country and this is the first study when NOD1 G796A, NOD2/CARD15 SNP8, SNP12 and SNP13 mutations, as well as the TLR4 D299G polymorphism were examined simultaneously. The present study has demonstrated that the heterozygous/homozygous carriage of the variant SNP as well as the A allele of NOD1 G796A is a possible determinant of susceptibility in Hungarian patients with CD compared to both healthy and non-IBD GI controls. The different polymoprhism of PPRs did not revealed any connection. Why did we select this particular mutation of this gene? Linkage to the area that encompasses the NOD1/CARD4 gene, chromosome 7p14, was first found during a genomewide scan of the UK population in 1996 [11], and since that time this is a putative IBD susceptibility locus. This initial finding was replicated in two further genome-wide scans involving patients in North America (USA and Canada) [17,18]. In a multicenter Western European study [12] involving 381 IBD families (235 CD, 58 UC, 81 mixed and 7 indeterminate), no association was observed between the susceptibility to IBD and the E266K variant, but when 63 unrelated index patients (54 CD and 9 UC patients) were subjected to mutation screening, of the three frequently found polymorphisms that encode a mutated protein, altering a glutamic acid residue that is conserved in CARD1,
Table 3 Association between different NOD1 E266K (G796A) genotypes and the presence of TLR4 Asp299Gly SNP and SNP8, SNP12 and SNP13 mutations of NOD2 NOD1
TLR4
NOD2
E266K
D299G
SNP8
AA 39 (9.0%) AG 180 (41.5%) GG 215 (49.5%)
6 (13.6%) 13 (29.5%) 25 (56.8%)
7 (11.6%) 27 (45.0%) 26 (43.3%)
Patients with SNP (n)
44
60
p = NS, patient number = 434.
SNP12 2 (10.0%) 7 (35.0%) 11 (55%) 20
SNP13 8 (10.0%) 30 (37.5%) 42 (52.5%) 80
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Table 4 Clinical data on Crohn’s disease patients in the different NOD1 E266K (G796A) genotypes AA (n = 39)
AG (n = 180)
GG (n = 215)
Sex (female/male) Age at presentation (years) Duration (years) Familial, IBD n (%)
20/19 27.9 ± 11.3 8.4 ± 7.2 10.2
78/102 30.7 ± 9.0 8.0 ± 5.1 15.5
112/103 27.9 ± 8.8 7.4 ± 6.1 9.3
Smoking habits, n (%) No Yes Previous
58.9 33.3 7.7
57.7 33.9 13.9
61.4 32.1 6.5
Location, n (%) L1 L2 L3 L4
23.1 15.4 58.9 2.5
32.2 25.0 41.1 1.6
24.6 25.1 47.9 23.2
Behavior, n (%) B1 B2 B3
58.9 12.8 28.2
43.3 23.9 32.8
38.6 25.5 35.8
Perianal disease, n (%) Frequent relapsea , n (%) EIMb , n (%)
15.4 33.3 41.0
22.8 35.5 51.6
24.6 34.9 49.7
Steroid, n (%) Use Refractory
74.3 7.7
77.2 5.0
77.6 51.1
Azathioprine use, n (%) Surgery, n (%)
61.5 28.2
61.1 38.9
60.0 42.8
a
Frequent relapse: >1/year. EIM: extraintestinal manifestations (arthritis: peripheral and axial; ocular manifestations: conjunctivitis, uveitis and iridocyclitis; skin lesions: erythema nodosum and pyoderma gangrenosum; and hepatic manifestations: primary sclerosing cholangitis). b
the E266K variant was the only one detected, suggesting a potential functional effect of the mutation. There are no exact data that demonstrate how this polymorphism alter the function of NOD1. However, the computer data analysis had performed earlier by our group demonstrated that this single amino acid changing of glutamine to lysine results slight decreasing of the helix-formatting potential, parallel to the Kyte–Doolittle hydropathy scale and the antigenicity revealed drastic changes. This genetic polymorphism is associated with more sever disease manifestation in Helicobacter pylori positive duodenal ulcer, therefore the presence of the A allel might lead to altered activation of NFB [19]. The frequency of the K variant was also high (0.23) in familial IBD patients, though it was quite similar (0.25) in the panel of alleles not transmitted to the control IBD patients, without any significant difference. Comparison of IBD patients with healthy controls might have led to a different final result. Two other studies of the frequency of the SNP in rs5743336 have been published to date. Ozen et al. [5] examined seventy Turkish CD patients and found no significant polymorphisms, neither in the E266K region of CARD4, nor in R702W, G908R and 3020insC regions
of NOD2. The relatively small number of patients, the lack of NOD2 mutations and the specific, highly heterogeneous population might explain why their results differ from our own. McGovern et al. [13] examined 12 previously identified NOD1 polymorphisms in 556 IBD trios; the frequency of E266K SNP was less than 1% and was not tested further. However, it was found that the deletion allele of a complex functional NOD1 ins-del polymorphism (ND1 + 32656*1) was significantly associated with early onset IBD in unrelated cases and controls. ND1 + 32656*1 was also associated with extraintestinal manifestations of IBD. A similar positive result was ruled out in a British population by Tremmeling et al. [20], and in a Scottish and Swedish IBD population by van Limbergen [21] who examined 370 and 1791 unrelated IBD patients and 522 IBD-parents subjects and 760 and 1649 regionally matched controls. It seems clear that the results of the above studies are so conflicting that they do not allow for a decision concerning the possible role of G796A polymorphism, or of any mutation of the NOD1 gene at all, despite the encouraging data of the genome-wide screening. The importance of the commensal bacterial flora and its communication strategies with the host is illustrated by several transgenic mouse models and innate immunodeficiency syndromes, which present similar gastrointestinal disorders as in IBD. Innate immunity is much more complex than a mere mechanism is fully capable to distinguish self from non-self [22]. NOD1/CARD4 is a perfect candidate as a susceptibility gene for CD. Like NOD2, CARD 4 is a member of the nucleotide-binding site/leucine-rich repeat family (also known as CATERPILLERs). These proteins are located in the cytoplasm and are involved in the detection of bacterial PAMPs that enter the cell either with an invasive microbe or by translocation with certain pathogenic bacteria through various forms of specialized transport apparatus [23]. The cytoplasmic detection system mediated by NODs probably plays a key role in the host defence in those tissues where Toll-like receptors are absent or expressed only at low levels [24]. This occurs in epithelial cells that line mucosal surfaces, e.g. colonic epithelial cells. d-glutaminemesoDAP (iE-DAP) [25] is the NOD1 activation signal leading to NF-B induction. The two terminal amino acids are the minimal motif of the Gram-negative bacterial infection, as neither of them exists in mammals. In the context of in vivo infection, NOD1 appears to be a key molecule in the sensing of Gram-negative bacterial infection since isolated intestinal epithelial cells from mice deficient in NOD1 do not react to the intracellular presentation of Gram-negative bacterial products [26]. The latest several microbiological studies failed to find a potential pathogen micro-organism which lead to development of IBD, perhaps because there is no such a common microbe. As each bacterial sensors react with activation signal of different bacteria, theoretically the altered function of either of them can result a defective recognition of at least one part of the commensal flora. This theory might explain how IBD can develop
T. Molnar et al. / Digestive and Liver Disease 39 (2007) 1064–1070
in those patients who have not NOD2 polymorphism: other receptor(s) of PAMPs is responsible for the causative defect, and whoever this receptor can be the NOD1 in individual cases. We have not found any significant association between the different genotypes and the demographic data on the patients or the clinical characteristics of CD. Zouali et al. [12] reported similar data when a subgroup of 235 unrelated CD patients was randomly selected for more detailed analyses from families in which only CD segregated. These patients were subdivided into three groups on the basis of the number of K variants. No difference was observed between these groups with regards to: sex, age at onset, family history, disease location (at onset and at maximal severity), behaviour, granuloma formation, extraintestinal symptoms and therapeutic management. Furthermore, our results also demonstrated no relationship between CARD4 and CARD15 in CD patients to identify a putative gene interaction. The frequencies of the three CARD15 CDassociated variants (R702W, G908R and 1007fs) were similar in both studies among patients with the wild-type genotype or those carrying the K allele of the E266K CARD4. We know, that certain limitations must be considered when our data are interpreted. Expression analysis and transcription factor-binding studies are needed to clarify to functional role of this NOD1 polymorphism. Investigations of further SNPs of NOD1 may also be informative. In summary, this is the first report on the prevalence of the G796A (E266K) mutation in patients with IBD from an Eastern European country. Our results suggest that the carriage of this mutation is a significant susceptibility factor for CD in the Hungarian population.
Practice points • NOD1 G796A (E266K) mutation is a significant susceptibility factor for Crohn’s disease in the Hungarian population. • The carriage of the single nucleotide polymorphism of NOD1 G796A increases the risk for Crohn’s disease twofold in the Hungarian population. • A allele alone was significantly more frequent also in Crohn’s disease compared to healthy and non-IBD control groups. • There was no interaction between NOD1 G796A and the SNPs of NOD2 and TLR4 in Hungarian patients with Crohn’s disease. • NOD1 G796A (E266K) genotype do not demonstrated any connection with the demographic data on the patients or the clinical characteristics of Crohn’s disease.
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Research agenda • Expression analysis and transcription factorbinding studies are needed to clarify to functional role of this NOD1 polymorphism. • Investigations of further SNPs of NOD1 may also be informative. • It has to continue the research on the role of innate immunity in the pathogenesis of inflammatory bowel disease. • Further polymorphisms of patternrecognition receptors should be examined to determine the possible role of these mutations in the susceptibility of Crohn’s disease.
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TLR4 genes, are associated with Helicobacter pylori-induced duodenal ulcer and gastritis. Helicobacter 2007;12:127–31. Tremelling M, Hancock L, Bredin F, Sharpstone D, Bingham SA, Parkes M. Complex insertion/deletion polymorphism in NOD1 (CARD4) is not associated with inflammatory bowel disease susceptibility in east anglia panel. Inflamm Bowel Dis 2006;12:967–71. Van Limbergen J, Russell RK, Nimmo RR, Torkvist L, Lees CW, Drummond HE, et al. Contribution of the NOD1/CARD4 insertion/deletion polymorphism + 32656 to inflammatory bowel disease in Northern Europe. Inflamm Bowel Dis 2007;13:882–9. van Limbergen J, Russell RK, Nimmo RR, Gwo-Tzer Ho, Arnott ID, Wilson DC, et al. Genetics of the innate immune response in inflammatory bowel disease. Inflamm Bowel Dis 2007;13:338–55. Murray PJ. NOD proteins: an intracellular pathogen-recognition system or signal transduction modifiers? Curr Opin Immunol 2005;17: 352–8. Viala J, Sansonetti J, Philpott DJ. Nods and ‘intracellular’ innate immunity. CR Biol 2004;327:551–5. Chamaillard M, Hashimoto M, Horie Y, Masumoto J, Qiu S, Saab L, et al. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol 2003;4: 702–7. Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jehanno M, Viala J, et al. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 2003;6:1584–7.
Digestive and Liver Disease 39 (2007) 1064–1070
Alimentary Tract
NOD1 gene E266K polymorphism is associated with disease susceptibility but not with disease phenotype or NOD2/ CARD15 in Hungarian patients with Crohn’s disease T. Molnar a,∗ , P. Hofner a , F. Nagy a , P.L. Lakatos c , S. Fischer c , L. Lakatos d , A. Kovacs e , I. Altorjay f , M. Papp f , K. Palatka f , P. Demeter g , Z. Tulassay h , T. Nyari i , P. Miheller h , J. Papp c , Y. Mandi b , J. Lonovics a , the Hungarian IBD Study Group1 a
1st Department of Medicine, University of Szeged, Faculty of Medicine, Szeged, Hungary Department of Microbiology, University of Szeged, Faculty of Medicine, Szeged, Hungary c 1st Department of Medicine, Semmelweis University, Budapest, Hungary d 1st Department of Medicine, Csolnoky F. County Hospital, Veszprem, Hungary e 1st Department of Medicine, Erzsebet Hospital, Budapest, Hungary f 2nd Department of Medicine, University of Debrecen, Debrecen, Hungary g Department of Gastroenterology, St Margit Hospital, Budapest, Hungary h 2nd Department of Medicine, Semmelweis University, Budapest, Hungary Department of Medical Informatics, University of Szeged, Faculty of Medicine, Szeged, Hungary b
i
Received 8 March 2007; accepted 7 September 2007
Abstract Background. NOD1/CARD4, a member of the pattern-recognition receptor family, is a perfect candidate as a susceptibility gene for Crohn’s disease. Since only limited and conflicting data are available on G796A polymorphisms in inflammatory bowel disease patients, we set out to study the effect of this polymorphism on the susceptibility and course of Crohn’s disease in the Hungarian population. Methods. Four hundred thirty-four unrelated Crohn’s disease patients (age at presentation: 28.6 ± 9.6 years, female/male: 210/224, duration of Crohn’s disease: 8.2 ± 6.9 years) and 200 healthy subjects (blood donors) and 136 non-inflammatory bowel disease gastrointestinal controls with chronic gastritis were investigated. NOD1 G796A was detected by using polymerase chain reaction/restriction fragment length polymorphism. Detailed clinical phenotypes were determined by reviewing the medical charts. Results. The frequencies of the variant alleles of NOD1 G796A differed significantly between the Crohn’s disease patients and both healthy (GG 49.5% vs. 67%; AG 41.5% vs. 28%; and AA 9.0% vs. 5.2%; p < 0.0001) and non-inflammatory bowel disease controls with chronic gastritis. Carriage of the single nucleotide polymorphism of NOD1 G796A proved to be a highly significant risk factor for Crohn’s disease compared to both healthy (p < 0.0001, OR: 2.1, 95% CI: 1.5–2.9) and non-inflammatory bowel disease controls with chronic gastritis (p = 0.008). Significant associations were not found between the different genotypes and the demographic data on the patients or the clinical characteristics of Crohn’s disease. The different polymorphisms of pattern-recognition receptors (e.g. NOD2/CARD15 SNP8, SNP12 and SNP13 mutations, the TLR4 D299G polymorphism and NOD1 G796A) did not reveal a mutual basis. Conclusions. Our results suggest that carriage of the NOD1 G796A mutation increases susceptibility for Crohn’s disease in the Hungarian population. © 2007 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Crohn’s disease; E266K; Genetics; G796A; IBD; NOD1
∗
Corresponding author. Tel.: +36 62545186; fax: +36 62545185. Hungarian IBD Study Group: Peter Fuszek, Henrik Csaba Horvath, Ferenc Szalay (Budapest), Zsuzsanna Erdelyi, Tunde Pandur (Veszprem), Laszlo Bene (Budapest), Zsuzsanna Vitalis (Debrecen), Levente Balint, Ferenc Huoranszky, Istvan Dobo (Budapest), Laszlo Herszenyi, Annamaria Nemeth (Budapest). 1
1590-8658/$30 © 2007 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2007.09.003
T. Molnar et al. / Digestive and Liver Disease 39 (2007) 1064–1070
1. Introduction Inflammatory bowel disease (IBD) is a collective term for at least three heterogeneous gastrointestinal disorders (Crohn’s disease (CD), ulcerative colitis (UC) and indeterminate colitis), which affect the same age group, in very different manners, resulting in several distinct clinical phenotypes and subgroups. The epidemiological data suggest a genetic predisposition for IBD that does not follow Mendelian inheritance patterns or a simple segregation of the disease, the importance of which is more significant in CD. Although CD is manifested by almost identical clinical picture worldwide, there are significant differences in gene polymorphisms among different ethnicities. The discovery of mutations in the NOD2/CARD15 gene (the first susceptibility gene known for CD associated mainly with an ileal involvement) in Western European and North American countries as well as in Hungary was striking; however, patient–control studies of Japanese, Chinese, Korean and Turkish populations have not encountered NOD2/CARD15 polymorphisms in either patient or control groups [1–5]. The variation of mutations in different IBD populations necessitates repeated studies of the roles of candidate genes in various populations. The digestive mucosa has evolved various immune strategies to allow it to tolerate intimate contact with commensals and to prevent pathogenic bacteria from spreading into the host tissues. Innate immunity relies on the specific recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs). The nucleotidebinding and oligomerization domain (NBD and NOD) proteins are a family of intracellular PRRs that recognize PAMPs, which results in the activation of pro-inflammatory pathways [6–8]. NOD1 is a host cytosolic signalling PRR composed of a caspase-activating and recruitment domain (CARD), NBD, NOD and leucine-rich repeats. NOD1 (or CARD4) acts as a cytosolic receptor for the diaminopimelate (DAP)-containing GlcNAc-tripeptide muropeptide found mostly in Gram-negative bacterial peptidoglycans [9]. It plays a crucial role in innate immunity by activating the NFB pathway via its downstream effector, the kinase RICK (RIP2), and also by triggering interleukin-1 secretion by procaspase-1 activation, following the recognition of a specific bacterial ligand [10]. A British genome-wide scan for IBD linkage demonstrated a susceptibility locus on chromosome 7p14; the same locus within which is the NOD1 (CARD4) gene located [11]. Nevertheless, although both the detailed morphological research and the functional approach have emphasized the possible role of NOD1 mutations in the development of IBD, the latest studies have not resulted in any breakthroughs. The G796A variation encodes a non-conservative peptide change (E266K) in the NBD domain of NOD1. The corresponding glutamic acid residue appears to be conserved in CARD15, suggesting a potential functional effect of the polymorphism [12]. The earliest observations did not prove a significant association between this mutation and IBD
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in Western European populations or in one Turkish study [5,12,13]. We have, therefore, repeated such examinations in a different Central-East European (Hungarian) population with a known genetic background of two other PRR genes, NOD2 and TLR4.
2. Patients and methods 2.1. Patients Four hundred thirty-four unrelated CD patients (age at presentation: 28.6 ± 9.6 years, female/male: 210/224, duration: 8.2 ± 6.9 years), 200 healthy subjects (blood donors) and 136 non-IBD patients with chronic gastritis were investigated. The clinical data of the CD patients are presented in Table 1. The diagnosis was on the basis of Lennard–Jones criteria [14]. The age at onset, duration of disease, presence of extraintestinal manifestations (arthritis: peripheral and axial; ocular manifestations: conjunctivitis, uveitis, iridocyclitis; skin lesions: erythema nodosum and pyoderma gangrenosum; and hepatic manifestations: primary sclerosing cholangitis), frequency of flare-ups (frequent flare-ups: Table 1 Clinical data on Crohn’s disease patients All (n = 434) Sex (female/male) Age at presentation (years) Duration (years) Familial IBD, n (%)
210/224 28.6 ± 9.6 8.2 ± 6.9 52 (12.0%)
Smoking habits, n (%) No Yes Previous
259 (59.7%) 143 (32.9%) 42 (9.7%)
Location, n (%) L1 L2 L3 L4
120 (27.6%) 105 (24.2%) 200 (46.1%) 9 (2.1%)
Behavior, n (%) B1 B2 B3
184 (42.4%) 103 (23.7%) 147 (33.8%)
Perianal disease, n (%) Frequent relapsea , n (%) EIMb , n (%)
100 (23.0%) 152 (35.0%) 216 (49.7%)
Steroid, n (%) Use Refractory
335 (77.2%) 23 (5.3%)
Azathioprine use, n (%) Surgery, n (%)
263 (60.6%) 173 (39.8%)
a
Frequent relapse: >1/year. EIM: extraintestinal manifestations (arthritis: peripheral and axial; ocular manifestations: conjunctivitis, uveitis and iridocyclitis; skin lesions: erythema nodosum and pyoderma gangrenosum; and hepatic manifestations: primary sclerosing cholangitis). b
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>1/year), effectiveness of therapy (e.g. steroid and/or immunosuppressive use, and steroid resistance), need for surgery (resections), occurrence of familial IBD, smoking habits and perianal involvement were investigated through a review of the medical charts and questionnaires. The disease phenotype (the age at onset, the duration, the location and the behaviour) was determined according to the Vienna classification [15]. The control group for the mutation analysis consisted of 200 age-matched and gender-matched healthy blood donors (male/female: 102/98, age: 38.05 ± 10.7 years). The control subjects, who had no gastrointestinal or liver diseases, were selected from consecutive blood donors in Szeged, Hungary. A second non-IBD gastrointestinal control group consisting of 136 chronic gastritis cases (male/female: 61/75, age: 41.03 ± 11.5 years). was also investigated. The study protocol was approved by the Ethical and Science Committee of the Ministry of Health and the University of Szeged Regional and Institutional Committee of Science and Research Ethics. Each patient was informed about the details of the study and signed the informed consent form.
The 796G-to-A transition (E266K) in exon 3 was analysed by PCR-RFLP. The 50 l reaction mixture contained 100 ng of genomic DNA, 20 pmol of each primer (sense: 5 TGA GAC CAT CTT CAT CCT GG 3 ; antisense: 5 CTT CCC ACT GAG CAG GTT G 3 ), 1.25 U Taq DNA polymerase (Fermantas, Vilnius, Lithuania), 1.5 mM MgCl2, 1×PCR Taq polymerase buffer + (NH4 )2 SO4 , and 25 mM of each dNTP (Fermentas). After denaturation (12 min at 95 ◦ C), the reaction consisted of 30 PCR cycles (30 sec at 94 ◦ C, 30 s at 58 ◦ C, and 30 s at 72 ◦ C) followed by a final extension by 7 min at 72 ◦ C. For RFLP analysis, the PCR products were digested with AvaI restriction enzyme (Fermantas) overnight at 37 ◦ C, electrophoresed on a 2% agarose gel (Sigma-Aldrich, St. Louis, MO, USA), visualized under UV illumination and stained with 0.4 mg/l ethidium bromide. The presence of an AvaI site (G allele) was indicated by cleavage of the 379 bp amplified PCR product to yield fragments of 209 bp and 170 bp.
2.2. DNA isolation
The χ2 -test and the Fischer exact test were used to evaluate differences between the IBD patients and the controls, as well as within subgroups of the IBD patients. Logistic regression was applied to compare genetic and clinical data; the results are expressed as odds ratios (OR) with 95% confidence intervals (95% CI). A p value <0.05 was considered significant. For the statistical analysis STATA (STATACOR LD, College Station, Texas, USA) was used.
Genomic DNA was isolated from whole blood according to the QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Germany). 2.3. Detection of NOD2/CARD15 SNP8, SNP12, SNP13 mutations and TLR4 D299G polymorphism The NOD2 SNP8, SNP12 and SNP13 and the TLR4 D299G genotypes were known for each patient. NOD2 variants were previously detected by means of denaturing high-performance liquid chromatography (dHPLC; Wave DNA fragment analysis system; Transgenomic Ltd., UK). Sequence variations observed in the dHPLC profile were sequenced on both strands to confirm the alteration. Sequencing reactions were performed with the ABI BigDye Terminator Cycle Sequencing Kit v1.1 (Applied Biosystems, Foster City, CA, USA) and samples were sequenced on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). The D299G polymorphism was detected using polymerase chain reaction/restriction fragment length polymorphism (PCRRFLP) and fragments were separated and visualized by gel electrophoresis (3% NuSieve GTG agarose gel; BMA, Rockland, ME, USA) [16]. 2.4. Detection of NOD1 polymorphisms The sequence variant of the studied single nucleotide polymorphism (SNP) in the NBD of NOD1 was as follows: CARD4 (NOD1) (Gene ID: 10392, localisation 7p15-p14, Genbank accession number NM 006092; c.709G>A, peptide change: E226K, protein domain: NBD, exon 3, SNP database ID: rs5743336).
3. Statistical methods
4. Results Fig. 1 shows representative results for NOD1 genotyping. To detect the nucleotid swap, RFPL was used. The homozygote mutant (AA), the heterozygote (GA) and the homozygote (GG) variants were well distinguishable. The frequencies of the variant alleles of NOD1 G796A among the CD patients and the healthy (p < 0.0001) and non-IBD GI controls (p = 0.008) differed significantly (Table 2). No significant departures were noted from the Hardy–Weinberg equilibrium. When the frequencies of the homo- and heterozygous mutations were examined together (50.5% vs. 33%), the carriage of the variant SNP of NOD1 G796A proved to be a highly significant risk factor for CD compared to both healthy (p = 0.0001, OR: 2.07, 95% CI: 1.46–2.93) and non-IBD GI controls. In addition, the A allele was significantly more frequent in CD compared to both control groups (p < 0.0001 and p = 0.019). There was no interaction between NOD1 G796A and the SNPs of NOD2 and TLR4 in Hungarian patients. The different polymorphisms of the PRRs (e.g. the NOD2/CARD15 SNP8, SNP12 and SNP13 mutations, the TLR4 D299G polymorphism and NOD1 G796A) did not reveal a mutual basis (Table 3).
T. Molnar et al. / Digestive and Liver Disease 39 (2007) 1064–1070
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Table 2 NOD1 E266K (G796A) genotypes in Hungarian patients with Crohn’s disease (CD) and controls NOD1 E266K (G796A) CD (n = 434)
Controls (n = 200)
Gentortype* Homozygous AA Heterozygous AG Wild-type GG
39 (9.0%) 180 (41.5%) 215 (49.5%)
10 (5%) 56 (28%) 134 (67%)
Allele frequency# A G
258 (29.7%) 610 (70.3%)
76 (19.0%) 324 (81.0%)
non-IBD GI controls (n = 136) 10 (7.5%) 41 (30%) 85 (62.5%) 61 (22.4%) 211 (77.6%)
(*) IBD vs. controls: χ2 = 17.907, p < 0.0001, ORhetero/homozygous vs. wild type : 2.07, 95% CI: 1.46–2.93. (*) IBD vs. non-IBD GI controls: χ2 = 6.977, p = 0.008, ORhetero/homozygous vs. wild type : 1.70, 95% CI: 1.14–2.52. (#) IBD vs. controls: χ2 = 16.229, p < 0.0001, ORA vs. G : 1.80, 95% CI: 1.35–2.41. (#) IBD vs. non-IBD GI controls: χ2 = 5.472, p = 0.019, ORA vs. G : 1.46, 95% CI: 1.06–2.01.
5. Discussion
Fig. 1. NOD1 E266K genotypes were deduced from the migration profile on a 2% agarose gel: wild-type DNA is visible as a double-band 209 bpand 170 bp (GG); the mutated DNA is visible as a single 379 bp band (AA); whereas heterozygotes give three bands (GA). Lane M is a molecular weight marker (Fermentas).
The results on the phenotype–genotype associations in the CD patients are shown in Table 4. No significant associations were found between the different genotypes and the demographic data on the patients or the clinical characteristics of CD.
This is the first report on the prevalence of the NOD1 (G796A) polymorphisms in patients with IBD from an Eastern European country and this is the first study when NOD1 G796A, NOD2/CARD15 SNP8, SNP12 and SNP13 mutations, as well as the TLR4 D299G polymorphism were examined simultaneously. The present study has demonstrated that the heterozygous/homozygous carriage of the variant SNP as well as the A allele of NOD1 G796A is a possible determinant of susceptibility in Hungarian patients with CD compared to both healthy and non-IBD GI controls. The different polymoprhism of PPRs did not revealed any connection. Why did we select this particular mutation of this gene? Linkage to the area that encompasses the NOD1/CARD4 gene, chromosome 7p14, was first found during a genomewide scan of the UK population in 1996 [11], and since that time this is a putative IBD susceptibility locus. This initial finding was replicated in two further genome-wide scans involving patients in North America (USA and Canada) [17,18]. In a multicenter Western European study [12] involving 381 IBD families (235 CD, 58 UC, 81 mixed and 7 indeterminate), no association was observed between the susceptibility to IBD and the E266K variant, but when 63 unrelated index patients (54 CD and 9 UC patients) were subjected to mutation screening, of the three frequently found polymorphisms that encode a mutated protein, altering a glutamic acid residue that is conserved in CARD1,
Table 3 Association between different NOD1 E266K (G796A) genotypes and the presence of TLR4 Asp299Gly SNP and SNP8, SNP12 and SNP13 mutations of NOD2 NOD1
TLR4
NOD2
E266K
D299G
SNP8
AA 39 (9.0%) AG 180 (41.5%) GG 215 (49.5%)
6 (13.6%) 13 (29.5%) 25 (56.8%)
7 (11.6%) 27 (45.0%) 26 (43.3%)
Patients with SNP (n)
44
60
p = NS, patient number = 434.
SNP12 2 (10.0%) 7 (35.0%) 11 (55%) 20
SNP13 8 (10.0%) 30 (37.5%) 42 (52.5%) 80
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Table 4 Clinical data on Crohn’s disease patients in the different NOD1 E266K (G796A) genotypes AA (n = 39)
AG (n = 180)
GG (n = 215)
Sex (female/male) Age at presentation (years) Duration (years) Familial, IBD n (%)
20/19 27.9 ± 11.3 8.4 ± 7.2 10.2
78/102 30.7 ± 9.0 8.0 ± 5.1 15.5
112/103 27.9 ± 8.8 7.4 ± 6.1 9.3
Smoking habits, n (%) No Yes Previous
58.9 33.3 7.7
57.7 33.9 13.9
61.4 32.1 6.5
Location, n (%) L1 L2 L3 L4
23.1 15.4 58.9 2.5
32.2 25.0 41.1 1.6
24.6 25.1 47.9 23.2
Behavior, n (%) B1 B2 B3
58.9 12.8 28.2
43.3 23.9 32.8
38.6 25.5 35.8
Perianal disease, n (%) Frequent relapsea , n (%) EIMb , n (%)
15.4 33.3 41.0
22.8 35.5 51.6
24.6 34.9 49.7
Steroid, n (%) Use Refractory
74.3 7.7
77.2 5.0
77.6 51.1
Azathioprine use, n (%) Surgery, n (%)
61.5 28.2
61.1 38.9
60.0 42.8
a
Frequent relapse: >1/year. EIM: extraintestinal manifestations (arthritis: peripheral and axial; ocular manifestations: conjunctivitis, uveitis and iridocyclitis; skin lesions: erythema nodosum and pyoderma gangrenosum; and hepatic manifestations: primary sclerosing cholangitis). b
the E266K variant was the only one detected, suggesting a potential functional effect of the mutation. There are no exact data that demonstrate how this polymorphism alter the function of NOD1. However, the computer data analysis had performed earlier by our group demonstrated that this single amino acid changing of glutamine to lysine results slight decreasing of the helix-formatting potential, parallel to the Kyte–Doolittle hydropathy scale and the antigenicity revealed drastic changes. This genetic polymorphism is associated with more sever disease manifestation in Helicobacter pylori positive duodenal ulcer, therefore the presence of the A allel might lead to altered activation of NFB [19]. The frequency of the K variant was also high (0.23) in familial IBD patients, though it was quite similar (0.25) in the panel of alleles not transmitted to the control IBD patients, without any significant difference. Comparison of IBD patients with healthy controls might have led to a different final result. Two other studies of the frequency of the SNP in rs5743336 have been published to date. Ozen et al. [5] examined seventy Turkish CD patients and found no significant polymorphisms, neither in the E266K region of CARD4, nor in R702W, G908R and 3020insC regions
of NOD2. The relatively small number of patients, the lack of NOD2 mutations and the specific, highly heterogeneous population might explain why their results differ from our own. McGovern et al. [13] examined 12 previously identified NOD1 polymorphisms in 556 IBD trios; the frequency of E266K SNP was less than 1% and was not tested further. However, it was found that the deletion allele of a complex functional NOD1 ins-del polymorphism (ND1 + 32656*1) was significantly associated with early onset IBD in unrelated cases and controls. ND1 + 32656*1 was also associated with extraintestinal manifestations of IBD. A similar positive result was ruled out in a British population by Tremmeling et al. [20], and in a Scottish and Swedish IBD population by van Limbergen [21] who examined 370 and 1791 unrelated IBD patients and 522 IBD-parents subjects and 760 and 1649 regionally matched controls. It seems clear that the results of the above studies are so conflicting that they do not allow for a decision concerning the possible role of G796A polymorphism, or of any mutation of the NOD1 gene at all, despite the encouraging data of the genome-wide screening. The importance of the commensal bacterial flora and its communication strategies with the host is illustrated by several transgenic mouse models and innate immunodeficiency syndromes, which present similar gastrointestinal disorders as in IBD. Innate immunity is much more complex than a mere mechanism is fully capable to distinguish self from non-self [22]. NOD1/CARD4 is a perfect candidate as a susceptibility gene for CD. Like NOD2, CARD 4 is a member of the nucleotide-binding site/leucine-rich repeat family (also known as CATERPILLERs). These proteins are located in the cytoplasm and are involved in the detection of bacterial PAMPs that enter the cell either with an invasive microbe or by translocation with certain pathogenic bacteria through various forms of specialized transport apparatus [23]. The cytoplasmic detection system mediated by NODs probably plays a key role in the host defence in those tissues where Toll-like receptors are absent or expressed only at low levels [24]. This occurs in epithelial cells that line mucosal surfaces, e.g. colonic epithelial cells. d-glutaminemesoDAP (iE-DAP) [25] is the NOD1 activation signal leading to NF-B induction. The two terminal amino acids are the minimal motif of the Gram-negative bacterial infection, as neither of them exists in mammals. In the context of in vivo infection, NOD1 appears to be a key molecule in the sensing of Gram-negative bacterial infection since isolated intestinal epithelial cells from mice deficient in NOD1 do not react to the intracellular presentation of Gram-negative bacterial products [26]. The latest several microbiological studies failed to find a potential pathogen micro-organism which lead to development of IBD, perhaps because there is no such a common microbe. As each bacterial sensors react with activation signal of different bacteria, theoretically the altered function of either of them can result a defective recognition of at least one part of the commensal flora. This theory might explain how IBD can develop
T. Molnar et al. / Digestive and Liver Disease 39 (2007) 1064–1070
in those patients who have not NOD2 polymorphism: other receptor(s) of PAMPs is responsible for the causative defect, and whoever this receptor can be the NOD1 in individual cases. We have not found any significant association between the different genotypes and the demographic data on the patients or the clinical characteristics of CD. Zouali et al. [12] reported similar data when a subgroup of 235 unrelated CD patients was randomly selected for more detailed analyses from families in which only CD segregated. These patients were subdivided into three groups on the basis of the number of K variants. No difference was observed between these groups with regards to: sex, age at onset, family history, disease location (at onset and at maximal severity), behaviour, granuloma formation, extraintestinal symptoms and therapeutic management. Furthermore, our results also demonstrated no relationship between CARD4 and CARD15 in CD patients to identify a putative gene interaction. The frequencies of the three CARD15 CDassociated variants (R702W, G908R and 1007fs) were similar in both studies among patients with the wild-type genotype or those carrying the K allele of the E266K CARD4. We know, that certain limitations must be considered when our data are interpreted. Expression analysis and transcription factor-binding studies are needed to clarify to functional role of this NOD1 polymorphism. Investigations of further SNPs of NOD1 may also be informative. In summary, this is the first report on the prevalence of the G796A (E266K) mutation in patients with IBD from an Eastern European country. Our results suggest that the carriage of this mutation is a significant susceptibility factor for CD in the Hungarian population.
Practice points • NOD1 G796A (E266K) mutation is a significant susceptibility factor for Crohn’s disease in the Hungarian population. • The carriage of the single nucleotide polymorphism of NOD1 G796A increases the risk for Crohn’s disease twofold in the Hungarian population. • A allele alone was significantly more frequent also in Crohn’s disease compared to healthy and non-IBD control groups. • There was no interaction between NOD1 G796A and the SNPs of NOD2 and TLR4 in Hungarian patients with Crohn’s disease. • NOD1 G796A (E266K) genotype do not demonstrated any connection with the demographic data on the patients or the clinical characteristics of Crohn’s disease.
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Research agenda • Expression analysis and transcription factorbinding studies are needed to clarify to functional role of this NOD1 polymorphism. • Investigations of further SNPs of NOD1 may also be informative. • It has to continue the research on the role of innate immunity in the pathogenesis of inflammatory bowel disease. • Further polymorphisms of patternrecognition receptors should be examined to determine the possible role of these mutations in the susceptibility of Crohn’s disease.
Conflict of interest statement None declared. References [1] Lakatos PL, Lakatos L. Current concepts on the genetics and pharmacogenetics of inflammatory bowel disease: do they help in clinical management? Curr Genom 2006;7:149–62. [2] Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 2001;411:599–603. [3] Yamazaki K, Takazoe M, Tanaka T, Kazumori T, Nakamura Y. Absence of mutation in the NOD2/CARD15 gene among 483 Japanese patients with Crohn’s disease. J Hum Genet 2002;47:469–72. [4] Leong RWL, Armuzzi A, Ahmad T, Wong ML, Tse P, Jewel DP, et al. NOD2/CARD15 gene polymorphisms and Crohn’s disease in the Chinese population. Aliment Pharmacol Ther 2003;17:1465–70. [5] Ozen SC, Dagli U, Kilic MY, Toruner M, Celik Y, Ozkan M, et al. NOD2/CARD15, NOD1/CARD4 and ICAM-1 gene polymorphisms in Turkish patients with inflammatory bowel disease. J Gastroenterol 2006;41:304–10. [6] Inohara N, Nunez G. NODs. intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol 2003;3:371–82. [7] Chamaillard M, Girardin SE, Viala J, Philpott DJ. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol 2003;5:581–92. [8] Kufer TA, Fritz JH, Philpott DJ. NACHT-LRR proteins (NLRs) in bacterial infection and immunity. Trends Microbiol 2005;13:381–8. [9] Girardin SE, Boneca IG, Carneiro LAM, Antignac AM, Jehanno M, Viala J, et al. Nod1 detects a unique muropeptide from Gram-negative bacterial peptidoglycan. Science 2003;300:1584–7. [10] Manon F, Favier A, Nunez G, Simorre JP, Cusack S. Solution structure of NOD1 CARD and Mutational analysis of its interaction with the CARD of downstream kinase RICK. J Mol Biol 2007;365:160–74. [11] Satsangi J, Parkes M, Louis E, Hashimoto L, Kato N, Welsh K, et al. Two stage genome-wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes 3, 7 and 12. Nat Genet 1996;14:199–202. [12] Zouali H, Lesage S, Merlin F, Cezard JP, Colombel JF, Belaiche J, et al. CARD4/NOD1 is not involved in inflammatory bowel disease. Gut 2003;52:71–4.
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