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P018. Linking risk conferring mutations in NCF4 to functional consequences in Crohn's disease

R. Somasundaram1, J. Deuring1, C.J. van der Woude2, M.P. Peppelenbosch3, G. Fuhler4

1Erasmus University Medical Center Rotterdam, Gastroenterology and Hepatology, Rotterdam, Netherlands; 2Erasmus Medical Center, Department of Gastroenterology & Hepatology, Rotterdam, Netherlands; 3Erasmus Medical Center, Department of Gastroenterology and Hepatology, Rotterdam, Netherlands; 4Erasmus Medical Center, Gastroenterology and Hepatology, Rotterdam, Netherlands

Background: Genome wide association studies (GWAS) are a promising tool to identify genetic variants of genes linked to an increased risk of developing Crohn's disease (CD). Although much is made of the importance of GWAS-identified single nucleotide polymorphisms (SNPs), it remains as yet unclear how these SNPs affect disease pathology, as cell functional studies are scarce. Amongst the SNPs identified so far is a T to C substitution in intron 1 of NCF4, a gene coding for the p40phox NADPH protein. The NADPH oxidase complex drives the production of reactive oxygen species (ROS) during the bactericidal response of innate immune cells. Disturbed granulocytic ROS output as a result of defective oxidase genes has been shown in a number of diseases, including chronic granulomatous disease. These diseases have been linked to development of a colitis resembling CD, suggesting a potential role for impaired ROS production in the pathogenesis of CD.

Methods: To address the potential of SNP variants in NADPH oxidase genes to confer a functional consequence in CD, we investigated granulocyte ROS production in CD patients carrying either the NCF4 rs4821544 risk allele (C) (n = 8), or patients homozygous for the non-risk allele (T) (n = 10).

Results: There were no significant differences in demographics and disease characteristics between patient groups. No differences in fMLP-triggered intracellular ROS production between carriers and non-carriers of the risk allele was observed. However, fMLP-induced ROS production was significantly lower in GM-CSF primed neutrophils from CD patients with an NCF4 mutation. No differences in granulocyte respiratory burst were found when patients were stratified according to ATG16L1 (rs10210302, rs2241880), IRGM (rs13361189) or NOD2 (rs2066844, rs2066845, rs2066847) SNP variants.

Conclusions: We demonstrate for the first time that GWAS-identified risk-conferring SNPs within the NADPH oxidase machinery lead to functional alterations in granulocyte ROS production in CD patients. Impaired bacterial clearance in patients carrying the NCF4 risk allele may contribute to the risk of getting CD, and this SNP may thus allow stratification of patient groups. These data further show that although many of the SNPs found to be linked to CD, including rs4821544, are synonymous, they may nevertheless convey functional consequences.