Crohn’s disease is a chronic, nonspecific, idiopathic gastrointestinal inflammatory disease that often leads to fibrosis and obstructive symptoms and can affect any part of the gastrointestinal tract from the mouth to the anus. The etiology of Crohn’s disease has not been clearly identified. Many factors have been suggested, but none are proven. Possible risk factors include immunologic factors, infectious agents (such as bacteria, viruses or amoebae), and dietary factors (including chemicals and drugs) [1].

Over the last decade, major advances have occurred that contribute to the understanding of the genetics of Crohn’s disease (CD), including studies based on single nucleotide polymorphism (SNP) and candidate gene approaches and studies in mouse experimental colitis using transgenic and deletion (knockout) techniques. Genome-wide linkage screens have identified more than 31 genetic loci containing putative inflammatory bowel disease (IBD) risk factors that could be associated with CD; however, estimates suggest that these 31 loci only explain approximately 20% of the heritability of Crohn’s disease [2].

The discovery of CARD15/NOD2 has focused interest on the regulation of innate immune responses and apoptosis. This gene, with important polymorphisms, encodes the intracellular bacterial sensor. The CARD15/NOD2 gene is located on chromosome 16 and is expressed mainly in monocytes, dendritic cells, intestinal epithelium and Paneth cells [3]. The CARD15/NOD2 protein recognizes components of the bacterial cell wall [4], is involved in the innate immune response and leads to the mobilization of the intracellular nuclear factor κB (NF-κB) [5].

The three major variants of CARD15/NOD2, Gly908Arg, Arg702Trp, and Leu1007fsinsC, are associated with a deficit in NF-κB activation in response to bacterial components, providing a unifying mechanism for the major CD-associated CARD15/NOD2 variants [6]. Lesage et al. suggested that patients carrying two variant copies of the CARD15/NOD2 gene are at increased risk of early age and fibrostenotic behavior of CD [2].

The contribution of the CARD15/NOD2 gene to CD has been studied in different ethnic populations with positive associations in up to 50% of CD patients [2]. However, data from Asian countries such as Japan and China failed to show any significant association, and no data from the Arab world could support or deny such association [7,8]. Although it remains uncertain how or whether the CARD15/NOD2 protein interacts with other protein families involved in the innate immune response, the C-terminal domain of CARD15/NOD2 comprises a leucine-rich repeat (LRR) region, which has sequence homology with a number of plant disease resistance genes in the toll and toll-like receptor (TLR) gene superfamilies.

Toll-like receptor 4 (TLR4) was found to be strongly up-regulated in patients with CD, and the allele 299Gly has been associated with the risk of developing IBD and particularly CD [9–11]. The TLR4 gene is expressed mainly in macrophages, dendritic cells and endothelial cells and to a lesser extent in the intestinal epithelium. The TLR family is involved in the recognition of pathogen-associated molecular patterns by the immune system. The TLR4 protein recognizes lipopolysaccharide (LPS) from gram-negative bacteria and binds to LPS using the extracellular LRR domain, which leads to intracellular activation of NF-κB [12].

Klein et al. demonstrated an association of CD with a functionally relevant single nucleotide polymorphism in the promoter of the CD14 gene (T/C at position −159) and suggested that the interaction of the CARD15/NOD2 and CD14 polymorphisms increases the risk for developing CD [13]. The human CD14 gene lies on the long arm of chromosome 5 (5q31.1), a region (IBD5) that has been reported to be associated with CD [14]. Membrane-bound CD14 is expressed on monocytes and macrophages [15].

To date, there has been no study performed in Asian Arabia, in particular Saudi Arabia, to evaluate genetic risk factors for CD among the Arab ethnic populations despite the increasing prevalence of the disease. Therefore, we have investigated the contribution of the three common CARD15/NOD2 mutations, as well as TLR4 and the CD14 −159C/T gene promoter polymorphism, to the predisposition to CD among the Saudi population.

The demographic and clinical characteristics of the CD patients are shown in Table 1. The demographic characteristics of the control population were similar to those of the patients. Among the CD patients, 31 males and 15 females, the mean age was 30.43 ± 10.20; 50% had a history of surgery; and 66% had been treated with biological therapy, 28 (62%) with infliximab and 3 (6%) with Adalimumab.

Blood samples from 46 patients with CD and 50 age- and sex-matched healthy individuals were collected at the IBD Outpatient Clinic at King Khalid University Hospital (KKUH) between May 2010 and February 2011. The diagnosis of CD was based on standard clinical, endoscopic, radiological, and histological criteria [1]. Clinical and demographic characteristics were recorded, including age at diagnosis, gender, family history, smoking habits, disease behavior, disease location, and need for surgery.

This study is the first to determine the prevalence of CARD15/NOD2, TLR4 and CD14 −159C/T gene mutations in Saudi patients with CD. This genetic study provides evidence that the three major CARD15/NOD2 variant alleles and the CD14 −159C/T polymorphism are associated with Crohn’s disease susceptibility in the Saudi; however, there is no evidence that the TLR4 (Thr399Ile) polymorphism is associated with Saudi CD.

Comparison of the frequencies of the mutations (Table 4) shows that TLR4 Thr399Ile is the least significant contributor to CD disease compared with the other mutations. The three main CARD15/NOD2 mutations are well-known CD-susceptibility alleles, but the risk associated with the mutations at the individual and population levels remains largely indeterminate. The data presented here and the reports from the literature [16–19] demonstrate that in different populations, large variations are observed for the frequencies of the common mutations. In Europe, CARD15/NOD2 mutations (especially the Gly908Arg and Leu1007fsinsC mutations) are relatively rare (2.4–7%) in northern countries including Scotland, Finland, Norway, and Sweden. The frequencies of the mutations, especially the Leu1007fsinsC mutation, are relatively high (7–11.5%) in Central Europe including Ireland, United Kingdom, Belgium, France, Germany, Croatia, and Hungary. Finally, in Southern countries, including Spain, Portugal, and Italy, the mutation frequency is intermediate (6–7.5%) with a lower frequency for the Leu1007fsinsC mutation [20]. In our study, the frequencies of the CARD15/NOD2 mutations in the control group were 0–4% (Table 2), with the lowest frequency for the Leu1007fsinsC mutation.

All these data (with the exception of those for Greece, where a very high rate of Leu1007fsinsC and a very low rate of R702W mutations are observed) are in accord with the more general observations of North-South gradients for genetic polymorphisms in Europe that are compatible with a spread of Neolithic farmers from the Levant 10,000 years ago [20]. The low penetrance observed in at-risk genotypes demonstrates that CARD15/NOD2 does not delineate a subgroup of simple Mendelian diseases.

Our data confirmed that CARD15/NOD2 mutations are more common in CD patients than in a the control Saudi population, although our data showed that these mutations were not the only CD risk factor and that additional genetic and/or environmental cofactors not discovered yet are required for disease development at the individual and population levels.

The polymorphism in the CD14 gene has been shown to increase TNF-α production in response to bacterial LPS and to amplify mucosal immune responses, suggesting that CD14 may play a role in the etiology of CD. Previous studies showed that the polymorphism in the promoter of the human CD14 gene was associated with CD and ulcerative colitis (UC), either alone or through interaction with polymorphisms in the CARD15/NOD2 gene [21]. We have shown that the CD14 −159C/T polymorphism in the CD14 gene was more common in CD patients than in a control Saudi population; however, an association of the CD14 −159C/T polymorphism with CD in other ethnic populations was heterogeneous and scanty.

The importance of TLR4 polymorphisms in CD is less clear than the importance of the CARD15/NOD2 mutations. Franchimont et al. have reported a two-fold elevation in allele frequency of TLR4 Asp299Gly in Belgian CD patients [22]. The TL4 Thr399Ile variant was associated with UC in a German study [23]; however, Japanese [24] and Hungarian studies [25] failed to detect any individuals displaying the mutant alleles of TLR4.

The three candidate genes of CD14, CARD15/NOD2 and TLR4, studied in a Tunisian population [26], were not associated with CD, contrary to the results obtained with European and American populations [10].

These findings support our results because in our study of TLR4 polymorphisms no significant differences in the allele or genotype frequencies between the patient and control groups were identified. The small size of our samples may have contributed to these negative results.

The coexistence of the TLR4 and CD14 mutated alleles was higher in CD patients than in healthy subjects, and this association with increased risk of CD in the Saudi population was significant.

Analyzing the SNP interactions will contribute to an understanding of the phenotypic variation of the disease, but currently, the results with CD are controversial. In fact, some studies show that the coexistence of mutations of the CARD15/NOD2 and TLR4 genes increases the risk of developing CD [27,28] or is associated with the phenotype of the disease, but these results have not been confirmed.

The coexistence of the TLR4, CARD15/NOD2 and CD14 mutated alleles was not the same in CD patients and controls (Table 5). It seems that the coexistence of the three alleles could be a major risk of developing CD and that they may play a role in phenotype determination.

Haplotype frequency analysis of the CARD15/NOD2 gene showed that the haplotype with the Arg702Trp confers the single highest genetic risk for disease. This result is in agreement with previous studies in a Spanish population [29]. However, this mutation was not associated to CD in Galician, Finnish or Scottish populations [30]. The risk for CD was much greater in the presence of more than one of the studied CARD15/NOD2 variants.

The presented data suggest a need for further work and larger studies to determine other genetic and environmental factors influencing CD susceptibility and behavior in the Saudi population.