Common variants in the NLRP3 region contribute to Crohn's disease susceptibility

Abstract

We used a candidate gene approach to identify a set of SNPs, located in a predicted regulatory region on chromosome 1q44 downstream of NLRP3 (previously known as CIAS1 and NALP3) that are associated with Crohn's disease. The associations were consistently replicated in four sample sets from individuals of European descent. In the combined analysis of all samples (710 father-mother-child trios, 239 cases and 107 controls), these SNPs were strongly associated with risk of Crohn's disease (P(combined) = 3.49 x 10(-9), odds ratio = 1.78, confidence interval = 1.47-2.16 for rs10733113), reaching a level consistent with the stringent significance thresholds imposed by whole-genome association studies. In addition, we observed significant associations between SNPs in the associated regions and NLRP3 expression and IL-1beta production. Mutations in NLRP3 are known to be responsible for three rare autoinflammatory disorders. These results suggest that the NLRP3 region is also implicated in the susceptibility of more common inflammatory diseases such as Crohn's disease.

Figure 1. Association results for the five Crohn's disease sample sets

Top panel shows SNPs, their positions in the genes and the linkage disequilibrium structure between them. SNP names in red were genotyped in the second phase of the study, subsequent to the sequencing experiment. Middle panel shows D′ in the upper left and r² in the lower right. (a–e) Lower panels show results from association analysis of Leuven trios (a), Liège trios (b), Liège case-control cohort (c), Québec trios (d) and Toronto trios (e). P values for individual alleles are reported in a logarithmic scale on the y axis. Color spectrum represents strength of linkage disequilibrium and frequency of the associated alleles.

Figure 2. NLRP3 functional study results

(a,b) Linear regression analysis of NLRP3 mRNA level versus rs4353135 genotype in DNA-RNA matched freshly isolated PBCs (a; n = 30) and monocytes (b; n = 31) obtained from healthy individuals. Genotypes of the six Crohn's disease (CD)-associated SNPs (Table 1) were obtained by sequencing. Mean threshold cycle (C_t) was calculated for each sample from three replicates and then used to calculate relative expression level (ΔC_t), which is the difference between NLRP3 C_t and endogenous control 18S RNA C_t. Fold change in NLRP3 expression was calculated using comparative C_t method (see Methods), using as a reference the average ΔC_t of homozygosity for the risk allele. (c,d) Linear regression analysis of IL-1β production (pg/ml) versus rs6672995 genotype for unstimulated (c) and LPS-stimulated (d; 1.0 µg/ml) conditions after 3 h of incubation. ΔC_t (a,b) and IL-1β level (c,d) for each individual are shown in red; regression lines are shown as dashed lines (a–d). (e) Quantitative real-time PCR analysis of Nlrp3 expression in colons of healthy mice (n = 6), mice with acute TNBS-induced colitis (n = 12) and mice with chronic TNBS-induced colitis (n = 6). (f) Quantitative real-time PCR analysis of NLRP3 expression in colon specimens from healthy individuals (n = 35) and individuals with Crohn's disease (n = 25). Expression was normalized to 18S RNA expression; each bar represents mean fold change in NLRP3 expression ± s.e.m. normalized to that of healthy colon specimens (e,f).