Single nucleotide polymorphisms that increase expression of the guanosine triphosphatase RAC1 are associated with ulcerative colitis

Abstract

Background & aims: RAC1 is a guanosine triphosphatase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases are associated with dysregulation of immune defenses. We studied the role of RAC1 in inflammatory bowel diseases using human genetic and functional studies and animal models of colitis.

Methods: We used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 messenger RNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulfate sodium.

Results: We observed a genetic association between RAC1 with ulcerative colitis in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the single nucleotide polymorphisms rs10951982 (P(combined UC) = 3.3 × 10(-8), odds ratio = 1.43 [95% confidence interval: 1.26-1.63]) and rs4720672 (P(combined UC) = 4.7 × 10(-6), odds ratio = 1.36 [95% confidence interval: 1.19-1.58]). Patients with inflammatory bowel disease who had the rs10951982 risk allele had increased expression of RAC1 compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected against dextran sulfate sodium-induced colitis.

Conclusions: Human studies and knockout mice demonstrated a role for the guanosine triphosphatase RAC1 in the development of ulcerative colitis; increased expression of RAC1 was associated with susceptibility to colitis.

Figure 1. RAC1 Imputation Analysis

Regional blots plot of the negative decadic logarithm of the P-values of SNPs in the RAC1 region including both genotyped (diamond) and imputation (*) SNPs, based on the 2049 individuals from the discovery cohort. In IBD the strongest signal comes from 3 SNPs (rs836472, rs1880118, rs12536544) located at 6401429 to 6402426 of Chromosome 7 that are in strong LD (r² > 0.80) with rs10951982. RAC1 gene is depicted below LD plot with genotyed SNP in bold and r² SNP map.

Figure 2. RAC1 Expression Based on Genotype

(A) Plot of LOD score (y-axis) and SNP position of each chromosome (x-axis). The eQTL maximal LOD score on chromosome 7 is the RAC1 SNP (rs12536544) that is in strong disequilibrium to rs10951982 (r² of 0.82 to 1.0). (B) RAC1 expression from mRNA isolated from PBCs from 16 IBD patients with colonic disease based on rs10951982 genotype. `G' allele is the risk allele. Expression was normalized to β-actin expression. Wilcoxon exact test.

Figure 3. Impact of impairing Rac1 function in DSS induced colitis in mice

(A) Rac1-KO. Left Panel graphs changes in weight with DSS treatment. Middle Panel shows changes in rectal bleeding. # at risk indicates number of mice not bleeding at each time point. Right Panel shows Disease Activity Index. (Error bars represent SEM). (B) Colonic MPO Activity. Colonic neutrophil infiltration was measured by MPO activity. MPO activity was assessed in the distal colon of WT mice (WT + water), Rac1-KO mice (Rac1-KO + water), and Rac1-KO mice treated with 5% DSS (Rac1-KO + DSS). (Wilcoxon rank sum test; Error bars represent SEM). (C) Colonic Cytokine Assay. Cytokine assays performed using MESO scale discovery mouse TH1/TH2 9-plex assay kit. (Mann Whitney test Two Tailed; Error bars represent SEM).