Association of NOD1, CXCL16, STAT6 and TLR4 gene polymorphisms with Malaysian patients with Crohn’s disease

Introduction

Both Crohn’s disease (CD) and ulcerative colitis (UC), the major sub-types of inflammatory bowel disease (IBD), are regarded as important global health issue, especially in the Western countries (Hilmi, Tan & Goh, 2006). Despite the similarities of some of the clinical symptoms shared by CD and UC, they can be differentiated by the affected locations. CD patients often suffer from multiple inflammation along the entire gastrointestinal tract, including mouth and anus. Occasionally, CD also inflicts complications that involve other organs, such as eyes, joints, blood, skin, and endocrine system. On the other hand, UC displays relatively restricted clinical manifestations, which are always localised at colon and rectum. To date, there is yet a cure for both CD and UC. Treatments and medical procedures are given to patients mainly for symptomatic relief and maintaining remission, which may also come with the cost of side effects, such as nausea and skin rashes (Sandborn et al., 2007).

Despite numerous extensive research studies that were conducted to find the etiology of CD in the past decades, the exact cause of CD remains inconclusive. However, it is generally accepted that, like other autoimmune diseases, the onset of CD could be triggered by the interaction of two major factors, i.e., genetic and environment (Baumgart & Carding, 2007). The increased disease concordance rate in monozygotic twins and higher risk of disease development in siblings of affected individuals highlight the importance of genetic predisposition in the disease development (Orholm et al., 2000). A number of genes have been identified through large scale Genome Wide Association Studies (GWAS) and meta-analysis to have significant linkage with the development of CD in the past (Duerr et al., 2006; Hampe et al., 2007). However, none of these genes was found as the sole contributor to the disease, suggesting polygenic traits in the disease development and progression.

CD are commonly observed in the Western countries, e.g. northern Europe, United Kingdom, and North America (Hilmi, Tan & Goh, 2006). The prevalence rate of CD has been relatively low in developing countries in Asia. Until recently, several studies had reported a rising trend of CD in Sri Lanka, Hong Kong, Japan, and Singapore (Morita et al., 1995; Lee et al., 2000; Niriella et al., 2010). Scientists postulate that this phenomenon might result from the adoption of modern lifestyles from Western counterparts (Thia et al., 2008). The overall prevalence rates of CD in Malaysia are 26 per 100,000 person (Tan & Goh, 2005; Hilmi, Tan & Goh, 2006). The prevalence rate differs among the three main ethnic groups that make up the multiracial society of Malaysia, i.e., Malay, Chinese, and Indian. CD appears most commonly in Indians, followed by Chinese and Malays, with prevalence rates of 52.6, 26.9, and 9.2 per 100,000 person respectively (Tan & Goh, 2005; Hilmi, Tan & Goh, 2006). Some gene and CD association studies have also been conducted previously (Chua et al., 2011; Chua et al., 2012; Chua et al., 2015); however, the actual genetics architecture of CD in the Malaysian populations still remains unclear.

Nucleotide-binding Oligomerization Domain-containing 1 (NOD1) protein is a member of NOD-like receptor (NLR) family. It is involved in the activation of NF-κB and caspase-9 that are important to cellular apoptosis and immune response, respectively (Inohara et al., 1999). NOD1 recognises the invasion of bacteria by reacting with a unique dipeptide, γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), that is present in the bacterial peptidoglycan (Chamaillard et al., 2003). The gene that encodes NOD1 protein is located on chromosome 7p14. Genetic variations in this gene have been associated to asthma and IBD (Hysi et al., 2005; McGovern et al., 2005). The NOD1 rs2075820 involves the replacement of glutamic acid (E) by lysine (K) at amino acid position-266. This SNP has been shown to increase the susceptibility of gastric mucosal inflammation following Helicobacter pylori infection, possibly due to altered plasma level of NF-κB (Kara et al., 2010).

Chemokine (C-X-C motif) Ligand 16 (CXCL16) protein is produced by dendritic cells and splenic red pulp cells (Wilbanks et al., 2001). It is also expressed in macrophages. It interacts with CXC chemokine receptors 6 (CXCR6) during inflammation and recruits the CXCR6 expressing cells, such as naive CD8 cells, intraepithelial lymphocytes, natural killer T cells, activated CD8 and CD4 T cells, to the affected site (Matloubian et al., 2000). CXCL16 is constantly expressed in human cells in two forms, i.e., membrane-bound and soluble forms. The membrane-bound CXCL16 aids the adhesion of bacteria for phagocytosis, whereas the soluble form induces the migration of activated T-cells (Abel et al., 2004; Gough et al., 2004; Nakase et al., 2012). The gene for human CXCL16 is on chromosome 17p13. Study has found that the serum level of soluble form of CXCL16 was increased more than 10 times in CD patients compared to controls (Lehrke et al., 2008). Previous study has demonstrated the association of CXCL16 Ala181Val (rs2277680) variant with severe disease phenotype in young CD patients (Seiderer et al., 2008).

Signal Transducer and Activator of Transcription 6 (STAT6) is a transcription factor in STAT family (Hamlin et al., 1999). Unlike other members in the family, STAT6 is activated by interleukin-4 (IL-4) through tyrosine phosphorylation. The phosphorylated STAT6 dimerizes and translocates to cell nucleus. It binds to specific DNA and activates the transcription of certain IL-4-induced genes, which leads to the initiation of humoral immunity respond by T-helper 2 (Th2) cell. Th2 cells promote the production of IgE and the proliferation of mast cells/eosinophils, leading to inflammation (Romagnani, 1999). The STAT6 gene is located on chromosome 12q13. Studies had shown that the G2964A variation (rs324015) within its 3′ untranslated region (UTR) was related to familial CD (Klein et al., 2005).

Toll-Like Receptor 4 (TLR4) gene is located on chromosome 9q33. TLR4 protein is highly conserved and functions as a mediator for production of cytokines in innate immune system. It also plays a vital role in pathogen recognition. TLR4 senses the lipopolysaccharides (LPS) from Gram-negative bacteria. Binding of the bacterial LPS to TLR4, together with other accessory proteins such as LPS-binding protein, MD2, and CD14, activates the production of NF-κ B and other cytokines (Philpott & Girardin, 2004; Lavelle et al., 2010). Alteration of the protein structure, due to mutations or SNP, results in differential responsiveness to LPS and may induce massive inflammatory reaction. TLR4 T399I (rs4986791) was related to weakened LPS response and other diseases, i.e., diabetes, rheumatoid arthritis, Alzheimer’s disease, renal and cardiovascular disease (Arbour et al., 2000; O’Neill, Bryant & Doyle, 2009). It was also found to associate with the development of CD and UC (Torok et al., 2004; Zouiten-Mekki et al., 2009).

In the present study, we aim to investigate the distribution of four SNPs in NOD1 (rs2075820), CXCL16 (rs2277680), STAT6 (rs324015) and TLR4 (rs4986791) genes in Malaysian patients with CD. We reported the association of alleles and genotypes of these SNPs with the onset of CD in the Malaysian population.

Materials and Methods

Results

Discussion

The investigation of NOD1 rs2075820 in Malaysian CD patients showed that there was no significant association between the SNP with the onset of CD. Our result is in contrast to a genetic study conducted on Hungarian population, where the A allele was significantly observed in CD patients (Molnar et al., 2007). The rs2075820 presents in an evolutionary conserved region that consists of 171 amino acids. It interacts with a number of other domains such as CARD, WD40 repeat and LRR. It functions to hydrolyze ATP or GTP (Koonin & Aravind, 2000). The A allele was suggested to decrease the helix-formatting potential and affect the binding affinity of ATP or GTP to the domain (Molnar et al., 2007). NOD1 polymorphisms were also often linked to inflammatory reaction following Helicobacter pylori infection, such as duodenal ulcer and gastritis (Hofner et al., 2007; Kara et al., 2010). Therefore, NOD1 polymorphisms may not be related directly to the onset of CD, but through a complicated mechanism that is triggered by infection.

The CXCL16 rs2277680 was significantly associated to CD patients in the Malaysian cohort. Overall, the G allele was found to have a weak predisposing effect on CD. When the data was analyzed according to ethnic group, significant association was only observed in the Malays. The G allele and homozygous G genotype carriers exerted two- and four-fold higher risk to develop CD in the Malay cohort. The statistical power of the sample size and corrected P values however, did not support the association. Therefore, more Malay CD patients could be recruited in future study to confirm this observation. There was no significant association established between the G allele with Chinese and Indian CD patients. This could be due to the difference in genetic background of these ethnic groups. Increased serum level of CXCL16 has been reported in CD patients, suggesting a pro-inflammatory effect of CXCL16 through the stimulation of C-reactive protein (Lehrke et al., 2008). The CXCL16 rs2277680 involves the substitution of Alanine by Valine, which may enhance the efficiency of CXCL16 protein and lead to the development of CD. The SNP was also reported to have no association to the susceptibility of CD in Caucasian population (Seiderer et al., 2008). However, it was shown to influence the clinical development of the disease, such as early age of onset.

The STAT6 gene is situated well within IBD2 susceptibility locus on chromosome 12. It is also one of the components in the pathway that triggers T-cell differentiation in inflammatory responses. Therefore, it may be a potential predisposing gene to the development of CD. In the present study, we did not observe significant association of rs324015 polymorphism, located in the 3′ UTR region of STAT6, with the establishment of CD in Malaysian cohort. Similar findings were also reported in Caucasian patients with CD from the Netherlands (De Jong et al., 2003; Xia et al., 2003). Interestingly, the G allele and homozygous G genotype were found significantly increased in CD patients without any variation in CARD15 gene (Klein et al., 2005). The relationship of STAT6 rs324015 with Asthma was also studied extensively but thus far, no significant association has been reported (Li et al., 2013; Qian et al., 2014). Based on these genetic studies, it is plausible to suggest that STAT6 may not have a leading role in the development of inflammatory diseases.

In the overall Malaysian cohort, the TLR4 rs4986791 polymorphism was shown to have very strong association to CD. The homozygous C genotype confers protection against the disease, whereas the heterozygous C/T carriers were seen to have three-fold higher chance to contract CD. Stratification analysis revealed that these associations were actually attributed to the Malay ethnic group, which showed more stringent levels of significance (P < 0.0003). Computation via Quanto software indicates that the sample size is sufficient to express high statistical power for the association analysis. In addition, the corrected P values for all associations remain significant. These further strengthen the association of the TLR4 polymorphisms with the onset of CD in the Malay population. However, there was no significant association observed in the Chinese and Indian cohorts. It is interesting to note that the distribution of TLR4 rs4986791 varied greatly in our study. It was monomorphic in the Chinese, where only homozygous C genotype was detected. Only two genotypes, homozygous C and heterozygous C/T, were seen in the Malays, and all three genotypes were observed in Indians. This may due to the discrepancy in genetic background of different ethnic groups and provide a likely explanation to many controversial case-control studies of TLR4 rs4986791 on diverse populations. Many genetic studies could not establish the link between TLR4 rs4986791 polymorphism and IBD patients from various Asian and Western countries (Browning et al., 2007). Several meta-analysis studies however, concluded that the T allele increases the risk of CD and/or UC, especially in Caucasian populations (Shen et al., 2010; Senhaji et al., 2014; Cheng et al., 2015). The T allele was suggested to impact the TLR4 protein by affecting the transcription and expression of the gene (Cheng et al., 2015).

In conclusion, we reported significant associations of two loci (CXCL16 rs2277680 and TLR4 rs4986791) with Malay CD patients in Malaysia. However, there was no significant association observed for Chinese and Indian patients. A limitation of the current study was the small sample size of the CD patient cohort, particularly for the Malay ethnic group. Another limitation was the unknowing sub-ethnicity stratification within each of the three studied ethnic groups that may lead to ambiguous associations.

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