Residual NADPH oxidase and survival in chronic granulomatous disease

Abstract

Background: Failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs) is the major defect in chronic granulomatous disease, causing recurrent infections and granulomatous complications. Chronic granulomatous disease is caused by missense, nonsense, frameshift, splice, or deletion mutations in the genes for p22(phox), p40(phox), p47(phox), p67(phox) (autosomal chronic granulomatous disease), or gp91(phox) (X-linked chronic granulomatous disease), which result in variable production of neutrophil-derived ROIs. We hypothesized that residual ROI production might be linked to survival in patients with chronic granulomatous disease.

Methods: We assessed the risks of illness and death among 287 patients with chronic granulomatous disease from 244 kindreds. Residual ROI production was measured with the use of superoxide-dependent ferricytochrome c reduction and flow cytometry with dihydrorhodamine oxidation assays. Expression of NADPH oxidase component protein was detected by means of immunoblotting, and the affected genes were sequenced to identify causal mutations.

Results: Survival of patients with chronic granulomatous disease was strongly associated with residual ROI production as a continuous variable, independently of the specific gene affected. Patients with mutations in p47(phox) and most missense mutations in gp91(phox) (with the exception of missense mutations in the nucleotide-binding and heme-binding domains) had more residual ROI production than patients with nonsense, frameshift, splice, or deletion mutations in gp91(phox). After adolescence, mortality curves diverged according to the extent of residual ROI production.

Conclusions: Patients with chronic granulomatous disease and modest residual production of ROI have significantly less severe illness and a greater likelihood of long-term survival than patients with little residual ROI production. The production of residual ROI is predicted by the specific NADPH oxidase mutation, regardless of the specific gene affected, and it is a predictor of survival in patients with chronic granulomatous disease. (Funded by the National Institutes of Health.).

Figures

Figure 1. Production of Reactive Oxygen Intermediates and Survival in Chronic Granulomatous Disease

A scatter plot (Panel A) shows the relationship between the production of superoxide (O2·̄) and the mean fluorescence intensity (MFI) of dihydrorhodamine in polymorphonuclear neutrophils from patients with chronic granulomatous disease. The empirical discriminator for the x axis was set at 2.3 nmol of O2·̄ per 106 cells per hour and that for the y axis at an MFI of 225 arbitrary units (AU). Kaplan–Meier survival curves (Panel B) were calculated on the basis of the empirical separation of patients into two distinct populations according to the neutrophil production of reactive oxygen intermediates (determined on the basis of separations depicted in Panel A without consideration of the specific genotype). A Kaplan–Meier survival plot (Panel C, upper left) separates the 227 study patients into quartiles according to residual O2·̄ production, without consideration of genotype. The bar graph (Panel C, lower right) shows the increases in hazard ratios when quartile 1 (Q1) is compared with the other quartiles (Q2, Q3, and Q4). The ranges of O2·̄ production for Q1, Q2, Q3, and Q4 are 0.26 to 0.94, 0.95 to 1.67, 1.70 to 2.71, and 2.72 to 60.5, respectively. The data shown in Panel D represent the specific subunit mutation of each patient in Panel A; data from normal subjects were added. The black rectangles enclose 2 SD for O2·̄ production and the MFI of dihydrorhodamine in patients with nonsense, frameshift, splice, or deletion mutations in gp91phox (lower left) and in patients with mutations in p47phox (upper right), respectively.

Figure 2. Defining Characteristics and Subtype Composition of the Study Quartiles for Patients with Chronic Granulomatous Disease

Characteristics of the study quartiles are listed in Panel A. The subtype composition of each quartile is shown in Panel B. Differences in the proportion of the subtypes in the quartiles were determined with the use of a chi-square test for trend. Orange blocks with an upward slope indicate significant increases in subtype composition, and blue blocks with a downward slope significant decreases in subtype composition. Patients with gp91phox chronic granulomatous disease were divided into two subgroups: one for patients with nonsense, frameshift, splice, or deletion mutations and another for patients with missense mutations. Patients with missense mutations were further divided into two additional subgroups: one for patients with missense mutations in amino acids 1 through 309 (except His222) and another for patients with missense mutations in amino acids 310 through 570 (plus His222). The numbers in parentheses in the row for gp91phox, columns for Q2, Q3, and Q4, refer to patients whose mutations in gp91phox could not be determined with the use of standard approaches.

Figure 3 (facing page). Distribution of Mutations in gp91phox, p22phox, p47phox, and p67phox and the Consequences of Mutations in gp91phox

Panel A shows the positions of all identified mutations in our cohort within the protein subunits of NADPH oxidase. Changes in amino acids caused by missense mutations and 3-nucleotide in-frame deletions are indicated according to the mutation. The yellow highlight identifies amino acids 310 to 570 of gp91phox, a region in which missense mutations result in a loss of NADPH oxidase activity. For clarity, missense mutations in transmembrane regions (TM I, TM II, and TM IV) are indicated on the left side of the panel. Panel B shows the superoxide O2˙̄ production and expression of gp91phox as a function of the nucleotide position of the mutation in patients with missense mutations in gp91phox as compared with patients with all other mutations. Normal O2˙̄ production is 226±3 nmol per 10 cells per hour. The domain structure of gp91phox is depicted with transmembrane domains (TM I through TM VI), extracellular domains (EC), intervening cytosolic domains, and flavin adenine dinucleotide (FAD) and NADPH binding domains. The level of expression of gp91phox was scored on a scale from 0 (undetectable) to 3 (normal expression) (Fig. 1 in the Supplementary Appendix). In families with multiple patients, the mean value for the family members is presented.