Two CGD Families with a Hypomorphic Mutation in the Activation Domain of p67phox

Abstract

Study background: Chronic granulomatous Disease (CGD) is a rare immunodeficiency caused by a defect in the leukocyte NADPH oxidase. This enzyme generates superoxide, which is needed for the killing of bacteria and fungi by phagocytic leukocytes. Most CGD patients have mutations in CYBB, the X-linked gene that encodes gp91^phox, the catalytic subunit of the leukocyte NADPH oxidase. We report here three autosomal recessive CGD patients from two families with a homozygous mutation in NCF2, the gene that encodes p67^phox, the activator subunit of the NADPH oxidase.

Methods: Leukocyte NADPH oxidase activity, expression of oxidase components and gene sequences were measured with standard methods. The mutation found in the patients' NCF2 gene was expressed as Ala202Val-p67^phox in K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67^phox from the cytosol of the patients' neutrophils to the plasma membrane was measured by confocal microscopy and by Western blotting after membrane purification.

Results: The exceptional feature of the A67 CGD patients reported here is that the p.Ala202Val mutation in the activation domain of p67^phox was clearly hypomorphic: substantial expression of p67^phox protein was noted and the NADPH oxidase activity in the neutrophils of the patients was 20-70% of normal, dependent on the stimulus used to activate the cells. The extent of Ala202Val-p67^phox translocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67^phox in K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67^phox.

Conclusion: The mutation found in NCF2 is the cause of the decreased NADPH oxidase activity and the (mild) clinical problems of the patients. We propose that the p.Ala202Val mutation has changed the conformation of the activation domain of p67^phox, resulting in reduced activation of gp91^phox.

Keywords: Chronic granulomatous disease; NADPH oxidase; NCF2; hypomorphic mutation; p67phox; p67phox activation domain; p67phox translocation.

Figure 1. Characteristics of patients’ neutrophils

(A) NADPH-oxidase activity. Neutrophils were incubated with serum-treated zymosan (STZ, 1 mg/ml, 70 particles per neutrophil) or PMA (100 ng/ml), and the oxygen consumption was measured with an oxygen electrode (10). The maximal rate of oxygen consumption of patient cells is displayed in nmoles/min/10⁶ cells. Alternatively, the release of hydrogen peroxide from the cells was measured with the Amplex Red assay (14) after stimulation with zymosan (1 mg/ml), STZ (1 mg/ml), PMA (100 ng/ml or PAF (1 μM) followed by fMLP (1 μM). The maximal rate of H₂O₂ release is displayed as nmoles/min/10⁶ cells. Open bars, control neutrophils; closed bars, patient neutrophils (patients A1, A2, and B). Mean ± SEM of 3 (oxygen consumption) or 4 (H₂O₂ release) independent experiments (in the H₂O₂ release assay, one patient was tested twice). Significance of differences was calculated with the paired, two-tailed t-test. (B) Western blot of p47^phox and p67^phox. Neutrophils from a control donor (lane 1), patient B (lane 2), patient A1 (lane 3), patient A2 (lane 4) and a CGD patient with a p.Trp137Arg mutation in p67^phox (lane 5) were lysed and subjected to SDS-PAGE as described under Methods. The proteins were blotted onto nitrocellulose, treated with antibodies to p47^phox and to p67^phox and visualized by fluorescence. The lower (green) band indicates the presence of p47^phox, the upper (red) band the presence of p67^phox.

Figure 2. DNA and RNA sequencing of NCF2 (end exon 6)

Figure 2A shows the sequence obtained from genomic DNA of family A, with patients A1 and A2 being homozygous for the c.605C>T mutation and all other family members heterozygous for this mutation. Figure 2B shows the sequence obtained with cDNA of patient B and her mother. The c.605C peak and the c.605T peak in the mother have a similar height, indicating that the mutated c.605T mRNA is as stable as the wild-type c.605C mRNA.

Figure 3. Expression and function of p67^phox in K562 cells

K562 cells containing all NADPH oxidase components except p67^phox were transfected with cDNA encoding p67^phoxAla202Val (p67A202), p67^phoxVal204Ala with a myc-tag (p67V204myc) or p67^phoxAla202 (p67wt). A representative Western blot in Figure 3A shows that all proteins were expressed in similar amounts in the cells. Figure 3B shows that upon activation with PMA, the mutated proteins did not support the oxidase activity in K562 cells, in contrast to the wild-type protein.

Figure 4. Translocation of p67phox to the cell membrane in intact neutrophils

(A) Quantification of p67phox translocation in stimulated human neutrophils. Human neutrophils (5×10⁶/ml) were stimulated with PMA (100 ng/ml) for 10 min or with STZ (1 mg/ml) for 20 min. Separation of cytosol and the rest of the cells, followed by Western blot analysis of p67^phox was performed as described under Materials and Methods. The amount of p67^phox was quantified by means of fluorescently labeled conjugates, and detected by scanning with the Odyssey Infrared Imagine System and Odyssey Application Software V3.0. Black bars, control neutrophils; Red bars, patient neutrophils (patient A1, A2, and B). Mean ± SEM of 3 independent experiments for PMA. With STZ only the cells of patient B were tested. Significance of differences was calculated with the paired, two-tailed t-test. (B) Visualization of p67^phox translocation in stimulated human neutrophils. Neutrophils from a control donor and from patient B were incubated with PMA (100 ng/ml) or left untreated for 10 minutes at 37°C in suspension. The cells were then allowed to adhere on fibronectin-coated glass covers, followed by a 10-minute incubation with STZ (1 mg/ml) or left untreated. Thereafter, the cells were fixed with formaldehyde and permeabilized with Triton X-100. To visualize p67^phox protein, the cells were incubated with rabbit-anti-human-p67^phox, followed by incubation with a secondary goat-anti-rabbit-Ig ALEXA-568-labeled. Coverslips were mounted with Vectashield on microscope slides and imaged with a confocal microscope through a 63× oil-objective. Note that with PMA, p67^phox translocates to the plasma membrane of control neutrophils (arrows), but much less so to the plasma membrane of patient neutrophils. In contrast, translocation of p67^phox to the phagosomal membrane surrounding internalized STZ (arrowheads) is similar in control and patient neutrophils.