NADPH oxidase complex and IBD candidate gene studies: identification of a rare variant in NCF2 that results in reduced binding to RAC2

Abstract

Objective: The NOX2 NADPH oxidase complex produces reactive oxygen species and plays a critical role in the killing of microbes by phagocytes. Genetic mutations in genes encoding components of the complex result in both X-linked and autosomal recessive forms of chronic granulomatous disease (CGD). Patients with CGD often develop intestinal inflammation that is histologically similar to Crohn's colitis, suggesting a common aetiology for both diseases. The aim of this study is to determine if polymorphisms in NOX2 NADPH oxidase complex genes that do not cause CGD are associated with the development of inflammatory bowel disease (IBD).

Methods: Direct sequencing and candidate gene approaches were used to identify susceptibility loci in NADPH oxidase complex genes. Functional studies were carried out on identified variants. Novel findings were replicated in independent cohorts.

Results: Sequence analysis identified a novel missense variant in the neutrophil cytosolic factor 2 (NCF2) gene that is associated with very early onset IBD (VEO-IBD) and subsequently found in 4% of patients with VEO-IBD compared with 0.2% of controls (p=1.3×10(-5), OR 23.8 (95% CI 3.9 to 142.5); Fisher exact test). This variant reduced binding of the NCF2 gene product p67(phox) to RAC2. This study found a novel genetic association of RAC2 with Crohn's disease (CD) and replicated the previously reported association of NCF4 with ileal CD.

Conclusion: These studies suggest that the rare novel p67(phox) variant results in partial inhibition of oxidase function and are associated with CD in a subgroup of patients with VEO-IBD; and suggest that components of the NADPH oxidase complex are associated with CD.

Figure 1

Flow diagram of NADPH oxidase genetic experiments. VEO-IBD, very early onset inflammatory bowel disease.

Figure 2

Functional studies of the p67^phox R38Q polymorphism. (A) p67^phox R38Q variant reduces binding to RAC. Interaction between RAC and wild-type p67^phox and p67^phox R38Q. GFP-tagged RAC2 and MYC-tagged p67^phoxand p67^phox R38Q were co-transfected into T293 cells. After 20 h p67^phox was immunoprecipitated using anti-MYC antibody and blotted for MYC and GFP. RAC2 showed a 51% reduction of binding to p67^phox R38Q in comparison with RAC2 binding to wild-type p67^phox. Representative blot of three independent experiments. IP, immunoprecipitation; WB, western blot. (B) p67^phox R38Q variant results in a conformational change. The model shows a close-up view of the RAC binding cavity of the wild-type p67^phox and p67^phox R38Q as adapted from the RAC/p67^phox complex crystal structure (PDB 1E96). The proteins are shown in ribbon representation, coloured according to secondary structure, with RAC residues labelled in blue and p67^phox residues labelled in red. Residues within 7×10^-8 cm of p67^phox R38 are represented by stick models with nitrogen and oxygen atoms indicated in blue and red, respectively. Water molecules found within the crystal structure are denoted by red spheres. Dashed lines denote putative hydrogen bond interactions between p67^phox wild-type and p67^phox L112 as well as water molecule 2013. In the p67^phox R38Q variant, bond distances between p67^phox R38Q and p67^phox L112 are reduced, the interaction with water molecule 2013 is lost, and a new putative interaction between p67^phox R38Q and water molecule 2025 forms. In addition, owing to the reduced carbon chain length between the arginine and glutamine side chains, a hydrophilic cavity is present perpendicular to the RAC/p67^phox R38Q interface that is visible through electron density mapping (not shown). All distances labelled are ×10^-8 cm. Images were generated with PyMOL (PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC.)