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Technical Briefs |
1 Institut für Laboratoriumsmedizin und Pathobiochemie, Charité, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany
2 Klinik für Kinderheilkunde, Universitätsklinikum Carl Gustav Carus, TU, D-01307 Dresden, Germany
3 Abteilung Klinische Chemie und Molekulare Diagnostik, Philipps-Universität, D-35033 Marburg, Germany
aauthor for correspondence: fax 004930-450569598, e-mail andreas.lun{at}charite.de
The multisubunit enzyme NADPH oxidase [phagocyte oxidase (phox)] of phagocytic cells plays a central role in cellular host defense (1). Phagocytes release large amounts of superoxide in the respiratory extraburst after stimulation with microorganisms. The production and subsequent conversion of superoxide to microbicidal reactive oxygen metabolites [(ROMs); e.g., H2O2] are critical for the elimination of pathogens such as Staphylococcus aureus, Serratia marcescens, and Aspergillus (2).
After activation of phagocytes, p47-phox and p67-phox, together with other proteins such as Rac, translocate from cytosol to the membrane, where they associate with two membrane-bound subunits of cytochrome b558, p22-phox and gp91-phox (3). This initiates an electron flux from NADPH via the activated phox to molecular oxygen leading to the generation of superoxide (4).
The essential role of this oxidase in cellular host defense is clearly demonstrated in patients suffering from a rare inherited disorder known as chronic granulomatous disease (CGD) (5). Mutations affecting any of the four subunits gp91-phox, p22-phox, p47-phox, or p67-phox either render phagocytes from CGD patients incapable of superoxide generation after stimulation or strongly decrease its production (6). Typically, these patients suffer from recurrent and life-threatening bacterial and fungal infections (3).
Here we present the case of a 43-year-old female patient with recurrent serious conditions that are typical of CGD, including Aspergillus fumigatus infection and formation of intestinal granulomas. The history of the patient is remarkable because for the first 17 years of her life, no typical CGD infections and no typical symptoms of CGD occurred. In the following years, the patient experienced cutaneous abscesses in the anogenital and back region and recurrent bacterial pneumonia (ages 29, 36, 41, 42, and 43 years). At age 29 years, the patient suffered from a severe therapy-resistant salmonella sepsis with ensuing severe damage of the liver and hepatic coma.
The symptoms and history led to the differential diagnosis of CGD. Stimuli-induced ROM formation of her neutrophils was tested with dihydrorhodamine 123[Phago-Burst; Orpegen; see Ref. (7)]. Most of the patient’s neutrophils (98%) were unable to produce ROMs aftermaximal stimulation with phorbol-myristate-acetate. Only 2% at most of her neutrophils produced H2O2 at concentrations within the reference interval (Fig. 1
). To identify the affected subunit of the patient’s NADPH oxidase, the expression of cytochrome b558 was measured by staining the neutrophils with the monoclonal antibody 7D5 followed by flow cytometric analysis (8). Cytochrome b558 was not expressed on most neutrophils, indicating a defect of gp91-phox or p22-phox. The gp91-phox gene is located on the X chromosome, and carriers show a mosaic of nonpathogenic and defective cells, as our patient did, attributable to lyonization. The p22-phox gene is located on chromosome 16q24, and therefore a mosaic of nonpathologic and defective cells is not likely because of a mutation in this gene. Defects of p47-phox or p67-phox are not to be expected because they do not affect the expression of cytochrome b558.
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To verify the patient’s carrier status, a molecular analysis of the gp91-phox gene was performed. The genomic DNA was prepared by standard methods with a commercially available product (QIAamp; Qiagen) (9). All exons of the gp91-phox gene (CYBB) and their intronic border regions were amplified by PCR and sequenced (cycle sequencing; BIG-DYE PE and ABI 377; Perkin-Elmer) (9). A microdeletion of a base pair was detected on one of the two alleles of the X chromosomes, 
-A 409 in exon 5 of the gp91-phox gene (CYBB), which led to a shift of the reading frame and to a premature stop codon at position amino acid 139, Val139
stop. This carrier status, together with an extreme lyonization, appears to explain the clinical phenotype of CGD with severe recurrent infections.
The late onset of this patient’s CGD could perhaps be explained by age-related skewing of lyonization (10). Alternatively, the patient may have had functional compensation of her phagocyte defect during her childhood, or infections may have triggered some discrete organ damage or alteration of further parts of her immune system, leading to enhanced susceptibility to further infections. This case supports the view that appropriate testing for CGD should be performed at any age whenever suspicious symptoms occur.
References
The following articles in journals at HighWire Press have cited this article:
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  M. C. B. Godoy, P. M. Vos, P. L. Cooperberg, C. P. Lydell, P. Phillips, and N. L. Muller Chest Radiographic and CT Manifestations of Chronic Granulomatous Disease in Adults Am. J. Roentgenol., November 1, 2008; 191(5): 1570 - 1575. [Abstract] [Full Text] [PDF]
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 L. Mauch, A. Lun, M. R.G. O'Gorman, J. S. Harris, I. Schulze, A. Zychlinsky, T. Fuchs, U. Oelschlagel, S. Brenner, D. Kutter, et al. Chronic Granulomatous Disease (CGD) and Complete Myeloperoxidase Deficiency Both Yield Strongly Reduced Dihydrorhodamine 123 Test Signals but Can Be Easily Discerned in Routine Testing for CGD Clin. Chem., May 1, 2007; 53(5): 890 - 896. [Abstract] [Full Text] [PDF]
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 B. Wolach, Y. Scharf, R. Gavrieli, M. de Boer, and D. Roos Unusual late presentation of X-linked chronic granulomatous disease in an adult female with a somatic mosaic for a novel mutation in CYBB Blood, January 1, 2005; 105(1): 61 - 66. [Abstract] [Full Text] [PDF]
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