Demographic and clinical characteristics of the CD (cohorts 1 and 2) and UC patients are shown in tables 1 and 2. There were more women than men in CD cohort 1, with a younger age at diagnosis and a higher prevalence of a familial history. More patients suffered from penetrating diseases in CD cohort 2 (tables 1, 2).
We first examined the prevalence of the TLR4 Asp299Gly genotype in CD and UC patients. There were significantly more CD patients in cohort 1 carrying the TLR4 Asp299Gly variant compared with the control population (20.7% v
0.012). This was also confirmed in CD cohort 2 (22.1% v
0.017) (table 3). Mutant allele frequency was significantly higher in CD patients from cohorts 1 (11% v
5%, OR 2.31 (95% CI 1.28–4.17); p
0.004) and 2 (12% v
5%, OR 2.45 (95% CI 1.248–4.816); p
0.007) compared with the control population (combined cohorts 1 and 2: 11% v
5%, OR 2.35 (95% CI 1.32–4.18); p
0.001) (fig 1A). Similarly, for UC patients, the allele frequency of the TLR4 polymorphism was significantly higher than in controls (10% v
5%, OR 2.05 (95% CI 1.072–3.930); p
0.027) (fig 1A). TLR4 allele frequencies were in Hardy-Weinberg equilibrium in all cohorts and controls. In order to further assess the relevance of the association of the TLR4 Asp299Gly polymorphism with CD, TDT testing was performed on 318 IBD trios (table 5). Overtransmission of the TLR4 Asp299Gly polymorphism from heterozygous parents to affected children was observed in the CD trios, with 52 transmissions versus 29 non-transmissions (χ2
0.01). When the CD, UC, and IC trios were combined, this overtransmission was still observed (64 transmissions versus 38 non-transmissions; χ2
0.01). We then examined whether this TLR4 polymorphism could be related to particular CD or UC phenotypes. Detailed analysis did not show any association of the TLR4 polymorphism with either CD or UC patient subgroups (data not shown). Thus the TLR4 Asp299Gly polymorphism was significantly associated with CD and UC without influencing disease phenotype.
Table 3 Toll-like receptor 4 (TLR4) genotypes in the study populations
Figure 1 (A) Allele frequencies for the NOD2 variants (Arg702Trp, Gly908Arg, and Leu1007fsinsC) and the toll-like receptor 4 (TLR4) Asp299Gly polymorphism in the Crohn’s disease (CD) (more ...)
Table 5 Transmission disequilibrium test shows overtransmission of the Toll-like receptor 4 (TLR4) polymorphism to CD patients
TLR4 and NOD2 are both PRRs involved in bacterial recognition.8
Three NOD2 variants (Arg702Trp, Gly908Arg, and Leu1007fsinsC) have been reported previously to be associated with CD.11,12
We then sought to explore the influence of the interaction between the TLR4 Asp299Gly polymorphism and the three NOD2 variants on the relative risk (RR) of CD and on CD phenotype. Consistent with previous studies, all three NOD2 polymorphisms were significantly associated with CD, as observed in cohort 1 (44.9% v
22.2%, OR 4.4 (95% CI 2.99–6.48); p<0.001) and cohort 2 (41.2% v
22.2%, OR 2.19 (95% CI 1.36–3.51); p
0.004) but were not associated with UC (OR 0.76 (95% CI 0.45–1.27); p
0.296) (table 4). When examining the interaction between the TLR4 Asp299Gly polymorphism and NOD2 genotype (table 6), no interaction between TLR4 and NOD2 was observed as the allele frequency of the TLR4 Asp299Glyc risk allele did not differ whether NOD2 variants were absent (10.9%) or present (10.2%). The RR of carrying one variant in TLR4 or NOD2 was 2.652 and 2.528, respectively. Table 7 shows the RR of carriers heterozygous or homozygous for TLR4 or NOD2 variants. The RR of carriers homozygous for both TLR4 and NOD2 variants could not be measured as there were no patients with this genotype combination. The RR of each of the NOD2 polymorphisms stratified by TLR4 genotype could not be calculated because of the limited sample size. Defining the RR of CD when carrying no variants in TLR4 or NOD2 as 1, the RR in carriers of either TLR4 or NOD2 variant was 3.071, and the RR in carriers of both TLR4 and NOD2 variants was 4.73. Thus the TLR4 Asp299Gly polymorphism increases the RR of developing CD either alone or when combined with the NOD2 polymorphisms.
Table 4 NOD2 genotypes in the study populations
Table 6 Stratification of Toll-like receptor 4 (TLR4) allele frequency by NOD2 genotype
Table 7 Relative risks (expressed as odds ratios) for the Toll-like receptor 4 (TLR4) and NOD2 genotypes
Phenotypic analysis demonstrated an association of the NOD2 variants with small bowel involvement and fibrostenotic disease, and a negative association with colonic involvement.15
In our CD population (cohorts 1 and 2), multiple logistic regression showed a positive and negative association with ASCA (OR 1.94 (95% CI 1.29–2.90); p
0.001) and colonic involvement (OR 0.45 (95% CI 0.24–0.84); p
0.012), respectively. We then examined whether CD clinical characteristics were associated with any of the four genotypes (TLR4−/NOD2+, TLR4−/NOD2−, TLR4+/NOD2+, and TLR4+/NOD2−). The NOD2+/TLR4− genotype was the only combination with a significant genotype-phenotype association. Indeed, the NOD2+/TLR4− genotype was significantly associated with ASCA (p
0.003; OR 1.92 (95% CI 1.242–2.953)) and negatively associated with colitis (p
0.015; OR 0.43 (95% CI 0.211–0.848)).
Analysing SNP interactions influencing the level of expression and/or function of signalling molecules will allow an understanding of the phenotypic variation in LPS transduction. LPS may first interact with soluble or membrane bound CD14 before binding to TLR4.8
The recently described C−260T promoter polymorphism is associated with increased expression and levels of soluble CD14, which may further amplify LPS signalling.29
While reported to be associated with CD or UC,30
both the genotype and allele frequencies of this polymorphism were not higher in our CD (cohort 1 and 2) and UC patients compared with controls (fig 1B). Furthermore, this polymorphism, either alone or in combination with the Asp299Gly TLR4 polymorphism, was not associated with any CD or UC clinical characteristics.