In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 2 ( 2012-01-10)
Abstract:
The results presented here demonstrate that a single identified mutation in NCF2 leads to decreased ROS production and plays a role in predisposition to SLE. This finding also provides support for an emerging paradigm shift that ROS are not solely proinflammatory byproducts of cellular responses to infectious or inflammatory stimuli. They may instead have a more nuanced function in regulating immune responses and inflammation-limiting processes, with notable consequences for autoimmune disease development. Our analysis demonstrated that the association of NCF2 with SLE can be attributed to a single mutation, which alters one amino acid at the H389 position from histidine (H) to glutamine (Q) in the PB1 domain of the NCF2 protein, with Q conveying an increased risk for acquiring SLE. We then investigated possible effects of this mutation on NCF2's protein-protein interactions. We used a computational method developed by one of us (M.E.), which has previously been shown to be effective ( 5 ). The resulting model supported all available information and predicted a mode of interaction between NCF2 and Vav1 that involves their respective PB1 and ZF domains ( Fig. P1 ). Our computational modeling further predicted that H389 residue of NCF2 resides in a pocket on the surface of Vav1 ZF domain and that the NCF2 H389Q mutation reduces the binding efficiency of NCF2 with Vav1. To test this prediction, we examined the effect of the NCF2 H389Q mutation on NADPH oxidase function, using site-specific mutations at the 389 position of NCF2. Results showed that the H389Q mutation indeed causes a twofold decrease in Vav1-dependent ROS production but has no effect on Vav1-independent activity. We identified a causal mutation in the NCF2 gene of patients who have lupus that causes a reduction in leukocyte NADPH oxidase function, reflected by a specific change in the NCF2 protein affecting its binding to Vav1. We obtained strong evidence for the association of SLE with NCF2 in two independent populations, a childhood-onset SLE cohort and a large adult-onset SLE cohort. The distinctive first step of using a childhood-onset cohort was based on our hypothesis that an earlier disease onset, more severe disease course, and more commonly seen family history of disease would all translate to a higher frequency of the disease-causing mutations and less masking attributable to environmental factors, facilitating gene discovery. In our current study, we build on this work by applying a computational method to search for genes associated with SLE. The first step in our method draws on a family-based study to identify “noteworthy” genes from a larger panel selected on the basis of increased likelihood of association because of their known function or genomic location. The second step follows up these noteworthy genes in a targeted investigation. Another unique aspect of our approach is extensive use of computer modeling to generate hypotheses pointing out likely functional consequences of mutations identified in the association study. These hypotheses are then tested in appropriate cellular systems. The NADPH oxidase complex is composed of multiple subunits [neutrophil cytosolic factor (NCF) 1, NCF2, and NCF4] ( 3 ). The formation of the completed, active complex requires the participation of many other proteins, including Vav1. Vav1 can facilitate the transition of Rac, another protein, into its active state, a necessary step in the completion of the NADPH complex. A pivotal recent study demonstrated a direct interaction of Vav1 with the C-terminal end of the NADPH subunit, NCF2, that enhances the activity of Vav1 leading to a positive feedback loop, which amplifies Rac activation ( 4 ). The white blood cell (i.e., leukocyte) NADPH oxidase is an enzyme complex that contributes to the formation of reactive oxygen species (ROS), which are used to kill invading microorganisms. Mutations that interfere with the formation of ROS may cause chronic granulomatous disease, which is associated with life-threatening bacterial and fungal infections ( 2 ). In addition, ROS have long been thought to lead to collateral tissue damage in inflammatory diseases. Genome-wide association studies are widely used to search organisms’ genomes for common alterations at single-nucleotide positions that are associated with complex diseases. Although such an approach has some advantages, it typically ignores valuable known information about the mechanisms of development and genetic basis of diseases, which inevitably requires researchers to analyze many regions of the genome that are unlikely to be associated with a disease. This process increases the number of tests needed, and thereby reduces the statistical power of the analysis. We developed an alternative bioinformatics-driven method to avoid this reduction of power by drawing on previously discovered information ( 1 ). We have applied this methodology to discover genes associated with systemic lupus erythematosus (SLE, or lupus), the prototypical autoimmune disease.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1113251108
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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