Clinical and demographic characteristics of the IBD cohort are given in Table 1. The SNPs selected for analysis were: c.156C>G (rs2075818; synonymous SNP), g.27606T>C (rs2075822; intronic SNP), and g.45343G>A (rs2907748; intronic SNP) (Table 2; 6).

Carrying the NOD1 c.156C>G variant led to statistically significant changes in CD or overall IBD risk, but not UC risk. Table 3 shows a significant difference in the major (C) allele frequencies between CD patients (81.1%) and controls (73.9%) (p = 0.004), such that those individuals carrying the C allele have an increased probability of developing CD in this population (OR = 1.51, 95% CI = 1.13–2.02, p = 0.004). CG heterozygotes have a reduced risk of CD (OR = 0.53, 95% CI = 0.37–0.75, p = 0.002), and of developing IBD as compared with CC homozygotes (OR = 0.64, 95% CI = 0.46–0.88, p = 0.01).

Carrying the NOD1 g.27606C>T variant led to statistically significant changes in the probability of developing UC, but not CD (Table 3). The CT heterozygote had a 2.65-fold (OR = 2.65, 95% CI = 1.29–5.41) greater risk, while TT homozygotes had a 2.45-fold (OR = 2.45, 95% CI = 1.23–4.85) greater risk of developing the disease, as compared with the risk for CC homozygotes (p = 0.02). For g.45343G>A SNP, it is the major allele that carries the increased risk. AG heterozygotes (OR = 2.32, 95% CI = 1.18–4.53) and GG homozygotes (OR = 2.19, 95% CI = 1.14–4.22) have an increased risk of developing the disease, as compared with AA homozygotes (p = 0.04). There was no significant difference in the allele frequencies between control subjects and CD, UC, or IBD patients (Table 3).

We also report results from extensive subgroup analysis using sets of cases defined by gender, age of diagnosis, disease location, disease behaviour, need for surgery, smoking history, use of immunomodulators, and presence of family history of IBD (Table 4). While several of these categories showed substantially increased risks associated with different SNPs, none explained an undue proportion of the CD cases.

Table 5 summarises haplotype analysis. A positive hap-score implies that the haplotype occurs more frequently in the CD or UC case group. A global p-value tests the overall association between haplotypes and the response.

The gastrointestinal colon cell line Caco2 was infected with LF82 for 6 h and 9 h, respectively. Infection with this bacterial strain leads to increased Nod1 m RNA levels for the 6 h time point (4.3 fold increase as measured using quantitative real time PCR), whereas after 9 h infection the Nod1 expression levels were similar to the uninfected state (Figure 1). As Nod1 signalling leads to the activation of NF-κB, we also tested the mRNA expression for this gene. Infection of Caco2 cells with the E. coli strain LF82 results in an upregulation for NF-κB for 6 h (3.2 fold increase) and 9 h (2.4 fold increase).

Cases for this study were provided through the Canterbury Inflammatory Bowel Disease Project, and controls randomly selected from a population-based cohort, both of which are described more fully elsewhere 2021. Both CD and UC were defined using standard diagnostic criteria, and cases phenotyped according to the Montreal Classification systems 22.

This study had ethical approval from the Canterbury Ethics Committee and the Southern Regional Health Authority's Ethics Committee in Canterbury, New Zealand, and all participants gave their written informed consent.

Genotyping was performed using the Sequenom MassArray^® platform (Sequenom, San Diego, CA, USA) 23 using a chip-based matrix-assisted laser desorption ionisation-time-of-flight (MALDI-TOF) mass spectrometer 24, as previously described 1213. Multiplex SNP assays were designed using SpectroDesigner software and realSNP.com online tools (Sequenom, San Diego, CA, USA). Primers, sequences and assay conditions are shown in Table 6.

To estimate genotype accuracy, approximately 12% of the samples were genotyped in duplicate or triplicate. We estimated the error rate to be <1%.

E. coli strain LF82, isolated from a chronic ileal lesion of a French patient with CD, was a kind gift of A. Darfeuille-Michaud, Université Clermont I, Pathogénie Bactérienne Intestinale, Clermont-Ferrand, France. Bacteria were grown routinely in Luria-Bertani (LB) broth or on LB agar plates overnight at 37°C.

The human colorectal cancer cell line Caco-2 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle medium (DMEM) with L-glutamine supplemented with 10% heat-inactivated fetal calf serum (Moregate, Hamilton, New Zealand), 1 mM sodium pyruvate and 1 mM non essential amino acids. All reagents were supplied from Invitrogen, Auckland, New Zealand. Cultures were maintained at 37°C in a humidified 5% CO₂ atmosphere. Monolayers were seeded in 24-well tissue culture plates (BD, Bioscience, Auckland, New Zealand) with 2 × 10⁵ cells/well and incubated for 14 days. During this time the cell medium was constantly replenished.

For infection experiments, bacteria were grown overnight in LB broth at 37°C. Before starting the infection the cell monolayers were washed twice with phosphate-buffered saline (PBS, pH 7.2). Each monolayer was infected in 1 ml of the cell culture medium without FCS at a multiplicity of infection (MOI) of 10 bacteria per epithelial cell. After a 6-h and 9-h incubation period at 37°C with 10% CO₂, infected monolayers were washed three times with PBS and the cells were trypsinated with 10% Trypsin-EDTA (Invitrogen, Auckland, New Zealand). To remove the trypsin cells were centrifuged for 1 min at 1500 rpm and the cell pellets were stored at -80°C.

Total RNA was extracted from non-infected and infected Caco2 cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following manufacturer's protocols. The RNA was quantified spectrophotometrically and then stored at -80°C. Reverse transcription of 1.5 μg of total RNA to complementary DNA (cDNA) was carried out using the SuperScript™III First-strand Synthesis – Super Mix Kit (Invitrogen, Auckland, New Zealand) and oligo(dT)₂₀ primers, according to the standard protocol. The primers used for PCR had the following sequences: NF-κB, 5'-ACAAATGGGCTACACCGAAG-3', 5'-GGACAACGCAGTGGAATTTT-3'; Nod1, 5'-AGCTGAAGATGAATTTGGGAAA-3', 5'-GCCGAGAAGTAGTCATTCTTCAG-3' and Actin 5'-CATTGCCGACAGGATGCA-3', 5'-CCGATCCACACGGAGTACTTG-3'. These sets of primers yield to PCR products that were 198 bp (Nod1), 341 bp (NF-κB) and 102 bp (Actin) long, respectively. Results are expressed as the relative mRNA accumulation corrected using Actin mRNA as an internal standard.

The allelic trend test 25 and Fisher's exact genotypic test were used to compare case and control allele frequencies. An exact test was used to test for departures from Hardy-Weinberg equilibrium (HWE) in the case and the control samples 26. Allelic odds ratios were calculated and confidence intervals for the allelic odds ratio were also calculated under the assumption of HWE in the cases and the control groups. Logistic regression analysis was used for subgroup analysis. Other analyses were carried out using R 27, SHEsis 28 (available from http://analysis.bio-x.cn/myAnalysis.php), and SAS (V9.1 SAS Institute., Cary, NC, USA).