Inherited p40phox deficiency differs from classic chronic granulomatous disease

Abstract

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD.

Keywords: Genetics; Immunology; Inflammatory bowel disease; Macrophages; Neutrophils.

Figure 1. Identification of NCF4 mutations.

(A) Pedigrees of 12 families showing allele segregation. The index cases are indicated by an arrow, and “E?” indicates an unknown genotype. Black or white symbols represent individuals with or without clinical manifestations, respectively. Triangles represent pregnancies not carried to term. m, mutated. (B) Schematic diagram of the structure of the NCF4 gene and p40^phox protein, consisting of 9 exons and 3 protein domains, respectively. Newly discovered and previously reported mutations of the NCF4 gene are noted according to their localization. Red asterisks indicate mutations previously reported by Matute et al. (26).

Figure 2. p40^phox levels and NADPH oxidase activity of the NCF4-mutated alleles.

(A) Western blot of total protein extracts from HEK293T cells expressing NCF4 cDNAs. The upper-panel data were obtained with a polyclonal anti-p40^phox Ab, and the lower-panel data were obtained with an anti–DDK (Flag) Ab. An Ab against GAPDH was used as a loading control. (B) Western blot of total protein extracts from healthy control and NCF4^–/– EBV–B cells retrovirally transduced with NCF4 cDNAs. A polyclonal anti-p40^phox Ab was used, with an Ab against GAPDH as the loading control. NT, not transduced. (C) Production of O₂^– by NCF4^–/– EBV–B cells retrovirally transduced with NCF4 cDNAs upon PMA stimulation. The results are representative of 2 independent assays. LU, luminescence units. (D) DHR reaction of NB4 p40^phox-KO cells stimulated with PMA or (E) with E. coli. (F) Killing activity of NB4 p40^phox-KO cells infected with S. aureus (n = 2–3). Data represent the mean ± SEM (n ≥3) or the mean only (n <3) and were analyzed using a 2-tailed Mann-Whitney U test. EV, empty vector.

Figure 3. p40^phox and NADPH oxidase subunit expression in p40^phox-deficient cells.

(A) Western blot of total protein extracts from the neutrophils of healthy controls, patients, and heterozygous relatives. Abs against p40^phox protein were used. Actin or GAPDH protein was used as a loading control. (B) Western blot of total protein extracts from MDMs and MDDCs from a healthy control and p40^phox-deficient patients. Detection was done with a polyclonal anti-p40^phox Ab, and an Ab against GAPDH was used as a loading control. (C) Western blot of total extracts from EBV–B cells from a healthy control, p40^phox-deficient patients, and XR and AR CGD patients. Abs against p40^phox protein or gp91^phox, p22^phox, p67^phox, and p47^phox were used. Actin or GAPDH protein was used as a loading control. (D) Intracellular detection of gp91^phox, p22^phox, p67^phox, and p47^phox in EBV–B cells from a healthy control, p40^phox-deficient patients, and XR and AR CGD patients. The results shown are representative of 2 independent assays. p40^phox–/– indicates a patient reported in a previous study (26).

Figure 4. NADPH oxidase activity in p40^phox-deficient neutrophils.

(A) Intracellular ROS production by DHR in neutrophils from healthy controls (n = 37), p40^phox-deficient patients (n = 17), and CGD patients (n = 5) upon PMA stimulation (100 ng PMA/ml and 400 ng/μl). (B) Particle-induced DHR oxidation in neutrophils from healthy controls (n = 17 and n = 23), p40^phox-deficient patients (n = 12 and n = 14), and CGD patients (n = 3 and n = 6) upon stimulation with S. aureus (left) or E. coli (right). (C) DHR oxidation in neutrophils from healthy controls (n = 14 and n = 6), p40^phox-deficient patients (n = 6 and n = 5), and CGD patients (n = 4 and n = 3) upon addition of C. albicans, with (left) or without (right) opsonization. PMNs, polymorphonuclear neutrophils. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Mann-Whitney U test.

Figure 5. NADPH oxidase activity in p40^phox-deficient mononuclear cells.

(A) Extracellular H₂O₂ production by MDMs from healthy controls (n = 5), p40^phox-deficient patients (n = 3), and CGD patients (n = 1) upon PMA stimulation (with and without IFN-γ priming). (B) Extracellular H₂O₂ production by MDDCs from healthy controls (n = 5), p40^phox-deficient patients (n = 3), and CGD patients (n = 1) upon PMA stimulation (with and without LPS priming). (C) Production of O₂^– (left) and release of H₂O₂ (right) by EBV–B cells from healthy controls (n = 2), p40^phox-deficient patients (n = 6), and CGD patients (n = 1) upon PMA stimulation, assessed on the basis of luminol bioluminescence or Amplex Red test. The results are representative of 2 independent assays. Data represent the mean ± SEM. NS, no stimulation.

Figure 6. Pathogen killing activity by neutrophils from p40^phox-deficient patients.

(A) S. aureus viability was measured in infected neutrophils from healthy controls (n = 22), p40^phox-deficient patients (n = 13), and CGD patients (n = 3). (B) E. coli viability was measured in infected neutrophils from healthy controls (n = 20), p40^phox-deficient patients (n = 10), and CGD patients (n = 4). (C) A. fumigatus hypha viability was measured at different time points with infected neutrophils from healthy controls (n = 13), p40^phox-deficient patients (n = 13) and CGD patients (n = 3). (D) C. albicans conidial viability was measured with infected neutrophils from healthy controls (n = 15), p40^phox-deficient patients (n = 12), and CGD patients (n = 6). Data represent the mean ± SEM (n ≥3) or the mean only (n <3). (A–C) **P < 0.01 and ***P < 0.001, by 2-tailed Mann-Whitney U test. (D) ***P < 0.001, by 2-tailed Mann-Whitney U test, and P < 0.0063, by 2-tailed Mann-Whitney U test with Bonferroni’s correction for multiple comparisons.

Figure 7. Clinical manifestation and outcome of p40^phox-deficient patients.

(A) P20 images show cheilitis, episcleritis, severe chronic cutaneous lesions, and esophagitis. P21 images show esophagitis. P12 images show multifocal consolidations and infiltrates in both the lungs on x-ray and thorax on CT scan. P22 images show vulvar lichen planus before (top) and after (bottom) steroid treatment. (B) Distribution of age at diagnosis of CGD in the American (ref. 7) and European (ref. 9) cohorts and of the p40^phox-deficient patients in this study. (C) Kaplan-Meier survival curve compiling survival data on 363 CGD patients (n = 240 XR CGD and n = 123 AR CGD) from refs. and and on the p40^phox-deficient patients studied here.