AIM: To evaluate the role of genetic factors in the pathogenesis of Crohn’s disease (CD) and ulcerative colitis (UC), we investigated the single nucleotide polymorphisms (SNPs) of NOD2/CARD15 (R702W, G908R and L1007finsC), and Toll-like receptor 4 (TLR4) genes (D299G and T399I) in a selected inflammatory bowel disease (IBD) population coming from Southern Italy.

METHODS: Allele and genotype frequencies of NOD2/CARD15 (R702W, G908R and L1007finsC) and TLR4 (D299G and T399I) SNPs were examined in 133 CD patients, in 45 UC patients, and in 103 healthy controls. A genotype-phenotype correlation was performed.

RESULTS: NOD2/CARD15 R702W mutation was significantly more frequent in CD (9.8%) than in controls (2.4%, P = 0.001) and in UC (2.3%, P = 0.03). No significant difference was found between UC patients and control group (P > 0.05). In CD and UC patients, no significant association with G908R variant was found. L1007finsC SNP showed an association with CD (9.8%) compared with controls (2.9%, P = 0.002) and UC patients (2.3%, P = 0.01). Moreover, in CD patients, G908R and L1007finsC mutations were significantly associated with different phenotypes compared to CD wild-type patients. No association of IBD with the TLR4 SNPs was found in either cohort (allele frequencies: D299G-controls 3.9%, CD 3.7%, UC 3.4%, P > 0.05; T399I-controls 2.9%, CD 3.0%, UC 3.4%, P > 0.05).

CONCLUSION: These findings confirm that, in our IBD patients selected from Southern Italy, the NOD2/CARD15, but not TLR4 SNPs, are associated with increased risk of CD.

Crohn’s disease (CD) and ulcerative colitis (UC) are idiopathic chronic inflammatory bowel disease (IBD). The molecular basis of their pathogenesis is not completely clear, but contributing factors may include persistent bacterial infection, a defective mucosal barrier, and an imbalance in the regulation of the intestinal immune response[1].

Animal models of IBD support the concept that genetic factors, environmental triggers, and immune dysregulation may have a potential role in developing uncontrolled intestinal inflammation that determines the typical endoscopic manifestations and mucosal lesions compatible with CD or UC[1–4].

Over the last decade, multiple genome-wide linkage searches have delineated numerous genomic regions containing putative IBD risk factors. In studies performed on unselected populations, an average of 8%-10% of CD patients and 6%-8% of UC patients have at least one relative affected by some type of IBD. However, these values vary from study to study and percentages of CD familial aggregation of less than 4% and more than 20% have been reported[2].

Moreover, studies on twins demonstrate a greater genetic influence for CD compared with UC; combined study concordance rates for monozygotic twins are 36% for CD and 16% for UC[5].

Recently, an association between CD and mutations in the NOD2/CARD15 gene located on chromosome 16q12 (IBD1) has been reported. NOD2/CARD15 acts as an intracellular receptor in monocytes for bacterial components, triggering activation of NFκB and thus leading to subsequent activation of the inflammatory response. Within the NOD2/CARD15 gene, three mutations have been identified as being associated with CD: two missense mutations (Arg702Trp in exon 4 and Gly908Arg in exon 8) and an insertion mutation of a C in exon 11 (1007finsC), the latter resulting in a truncated NOD2/CARD15 protein. These NOD2/CARD15 variants alter the structure of either the leucine-rich repeat (LRR) domain of the protein or the adjacent region. The activating function of nuclear factor NFκB is regulated by the carboxy-terminal LRR domain, which has an inhibitory role and also acts as an intracellular receptor for components of microbial pathogens. These observations suggest that the NOD2\CARD15 gene can confer susceptibility to CD by altering the recognition of these components and/or by over-activating NFκB in monocytes[6–9]. The question arises as to how NOD2/CARD15 mutations and impaired NFκB activation can confer susceptibility to CD. It has been suggested that the answer most likely lies within the leucine-rich repeats (LRR) of the NOD2/CARD15 gene and the family of Toll-like receptors (TLRs). These receptors, a family composed of at least 10 mammalian homologs of Drosophila Toll, serve as pattern recognition receptors for various microbial products and can mediate production of proinflammatory cytokines[10]. Toll-like receptor 4 (TLR4) functions as the main receptor for lipopolysaccharide (LPS) of Gram-negative bacteria[11]. After the recognition of pathogen-associated molecular patterns, the TLRs activate signal transduction pathways of the innate immune response genes including inflammatory cytokines and the NFκB signalling pathway[12]. TLR4 is expressed in macrophages, dendritic cells, endothelial cells, and, less abundantly, in intestinal epithelial cells, which are partly tolerant to LPS, thereby preventing an exaggerated immune response caused by the large number of bacteria in the intestinal lumen[13].

Mutations of the TLR4 gene are known to abolish responses to endotoxin in mice, as shown for the mice strains C3H/HeJ and C57BL/10SeCr[14]. Therefore, the ability to recognize bacterial wall products and to activate proinflammatory mechanisms by TLRs may be of great importance for immune reactions in the intestinal mucosa. The recently characterised D299G and T399I single nucleotide polymorphisms (SNPs) of TLR4 gene are probably associated with impaired LPS signalling and increased susceptibility to Gram negative infections[15].

In this study, we investigated the frequencies of the three NOD2/CARD15 gene mutations (Arg702Trp, Gly908Arg and 1007finsC) and of the TLR4 gene D299G and T399I SNPs in a group of 178 Italian adult patients affected by IBD: 133 patients with CD and 45 with UC. The allele frequencies of the NOD2/CARD15 and TLR4 genes were evaluated, and a detailed genotype-phenotype correlation was performed.

In our study, we investigated the prevalence of NOD2/CARD15 and TLR4 genetic variants in CD and UC patients. Moreover, we compared the results with clinical phenotype characteristics of IBD of our patients to identify a possible genotype-phenotype association.

There are several controversial data about the role of the SNPs of the NOD2/CARD15 (R702W, G908R and L1007finsC), and TLR4 genes (D299G and T399I) in the pathogenesis of CD. Indeed, there are significant phenotypic differences that exist among populations.

The NOD2/CARD15 mutations are absent in Asian CD populations and controls[19–21]. In this case, the findings indicate that the NOD2/CARD15 is not a major contributor to CD susceptibility in the Japanese population. Similar data have been found in Turkish patients with IBD[22].

The highest recorded frequencies are reported in a small study of 55 paediatric patients in Europe with two thirds of the patients having at least one NOD2/CARD15 mutation[23]. Within Europe, there is evidence of a north-south gradient with lower allele frequencies in the Celtic and Scandinavian countries compared to Southern Europe[23].

To our knowledge, this is the first study in a large series of sporadic IBD patients coming from Eastern Sicily and Calabria. Indeed, previous reports regarded a Sicilian, small town population in which a familial study was performed[24]. Other studies have examined a group of sporadic Sicilian IBD patients, but the number of cases was smaller than in our study[25].

In our study, the reported rates of 48.8 % of patients carrying at least one NOD2/CARD15 mutation in CD and 19.4% in controls are consistent with previously reported rates of 30%-50% in CD and 7%-20% in controls from other European regions[26–31]. Moreover, 36.1% had two mutations (compound heterozygotes). Recently, Renda et al examined a group of 182 CD patients coming from Western Sicily and they found that 56 patients (30%) had at least one mutation of the NOD2/CARD15 gene[32].This percentage was lower in respect to our data (48.8%). This difference may be ascribed to a different ethnic background. Indeed, the patients of our study coming both Eastern Sicily and Calabria. Today, populations genetically similar to that of the Northern Italy (as well as of the Northern Africa) are present in the Eastern Sicily. This heterogeneous population, during the middle age, might explain the genetic differences in the patient CD samples of the Eastern in respect to Western Sicily[33].

The allele frequencies of the R702W (9.8%) and L1007finsC (9.8%) mutations were significantly higher in CD patients compared to UC patients and controls, whereas the frequency of the G908R (4.5%) mutation was similar in CD and UC patients, and in the control group. Collectively, our study confirmed previous studies, which reported increased mutation carrier frequencies of one of the three variant alleles in CD patients compared to UC patients or healthy controls[2283234–36].

We also found that different risk alleles might be associated with different clinical features: in particular, the G908R allele seems to be associated with stenosing phenotype and need for surgery. The L1007finsC seems to correlate with ileal localization and resective surgery. These data suggest a more aggressive course of the disease in carriers of risk alleles. The strongest observed effect for ileal location is consistent with the proposed involvement of ileal Paneth cells in the pathophysiology of NOD2/CARD15-mediated disease susceptibility[303637]. NOD2/CARD15 mutations may, thus, abrogate normal Paneth cell behaviour, explaining preferential involvement of the terminal ileum[26].

Moreover, in our study, the risk of developing CD with a more aggressive course was increased in compound heterozygotes. In other populations, stronger associations have been reported for homozygotes and compound heterozygotes than for simple heterozygotes. One copy of the risk alleles confers a 2-4-fold risk for developing CD, whereas double-dose carriage increases the risk by 20-40-fold[23]. Our study is in agreement with such a gene-dosage effect, although at lower levels.

In our IBD patients, we also examined the allele frequencies of the TLR4 D299G and T399I SNPs and the possible genotype-phenotype correlation.

With regard to the role of the TLR4 gene in the pathogenesis of IBD, several studies have been conducted leading to divergent results[23]. The allele frequency of the D299G mutation ranges between 8%-13% in CD, 0%-10% in UC and 3%-15% in healthy controls[38]. This TLR4 SNP was associated with CD and UC in a Belgian study[39]. This association was replicated in Dutch, German, Australian and Greek populations with CD, and an association with colonic disease has been described[8343540]. In one German cohort, an association was demonstrated between UC and the TLR4 T399I SNP[41]. However, there is substantial heterogeneity between populations, and no association was noted in Scottish CD patients[30].

In our study, we found no difference in the prevalence of these mutations in our CD and UC patients, and controls. Recently, other studies have failed to find the association of the D299G and T399I SNPs of TLR4 gene[2442–45]. In a retrospective German and Hungarian cohort study, patients with CD and UC were genotyped for the presence of the CD14 c.1-260C>T promoter variant and the TLR4 D299G variant. In this study, in German and Hungarian populations, IBD appears to be associated with the CD14 c.1-260C>T promoter variant, but not with the TLR4 D299G variant[45]. Recent data suggest that neither of these 2 variants is causal, but they may be in linkage disequilibrium with, as yet unidentified, causal variants[46–48].

We examined also whether the TLR4 D299G and T399I SNPs could be related to particular CD or UC phenotypes. Detailed analysis did not show any association of the examined TLR4 gene variants with either CD or UC patient subgroups. In other studies, in CD patients, an association has been reported between D299G SNP and ileal localization and structuring phenotype[49]. Our data are similar to those previously reported[28]. These contrasting results can be ascribed to the different ethnic background of the various IBD populations studied.

Although several studies have been performed, further research is warranted to clarify the role of the genetic variants of NOD2/CARD15 and TLR4, and to investigate whether these genetic risk factors might be confirmed and considered clinically relevant. Indeed, an eventual goal in the genomic study of IBD is to identify these biologically relevant genotype-phenotype associations and to apply them to clinical practice.