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Chapter 33 : Functional Assays for the Diagnosis of Chronic Granulomatous Disease

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Functional Assays for the Diagnosis of Chronic Granulomatous Disease, Page 1 of 2

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Abstract:

Chronic granulomatous disease (CGD) is a rare genetic disease (~1 in 200,000 in the U.S.) first described in the 1950s (1). It is characterized by a failure of phagocytes (polymorphonuclear neutrophils [PMN], monocytes, macrophages, and eosinophils) to generate superoxide (O·) and other related reactive oxygen species (ROS), leading to recurrent infections, granulomatous complications, and premature death. Generation of O· requires the assembly and activation of a multicomponent enzyme, NADPH oxidase (NOX2) or phagocyte oxidase (phox), a complex consisting of numerous cytosolic proteins, including p47 (2), p67 (2), and p40 (3), and two membrane proteins, p22 and gp91, that constitute cytochrome (4, 5). NOX2 catalyzes the reduction of molecular O to O· using NADPH generated by the oxidation of glucose through the pentose-phosphate pathway. O· is converted to HO either spontaneously or enzymatically. HO and O· can react to form the highly reactive hydroxyl radical, OH•. The molecular defect in CGD results from mutations in any one of 5 protein subunits of NOX2, that include gp91 (~70% of patients), p47 (~25%), p22 (<5%), p67 (<5%), and p40 (one case identified). Because the molecular defect in CGD is the inability to generate ROS, most of the assays used in the diagnosis of CGD that are described below are based on assessments of ROS production using different probes and different detection platforms. The last two assays—flow cytometric analysis of NOX2 expression and immunoblot analysis of phox subunits—focus on identifying the specific protein defect and defining the target for genetic sequencing.

Citation: Priel D, Kuhns D. 2016. Functional Assays for the Diagnosis of Chronic Granulomatous Disease, p 310-320. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch33
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Figures

Image of FIGURE 1
FIGURE 1

Analysis of HO production by dihydrorhodamine-123 staining. PMN populations of buffer-treated (Basal) and PMA-treated (PMA) samples were gated using FS × SS shown in the contour plots on the left panel for each condition. The middle panel for each condition represents the DHR histogram which is presented on a log scale. The right panel for each condition represents a dot plot of the back-gated region of the DHR histogram to identify the nature of the population analyzed. Stimulation with PMA induces a shift in the FS × SS pattern of the PMN population in all subjects tested: a normal subject (A), a patient with gp91 CGD (B), an X-linked carrier of CGD (C), and a patient with p47 CGD (D). DHR cells are indicated in red in back-gated FS × SS dot plots.

Citation: Priel D, Kuhns D. 2016. Functional Assays for the Diagnosis of Chronic Granulomatous Disease, p 310-320. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch33
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Image of FIGURE 2
FIGURE 2

Histochemical staining of PMN with NBT. (A) PMN from a normal subject under basal condition exhibit only the safranin counterstain of the cytosol and nucleus. (B) PMN from a normal subject stimulated with PMA exhibit blue-black formazan deposits in the cytosol and the safranin counterstain in the nucleus. (C) PMN from a patient with gp91 CGD stimulated with PMA fail to generate ROS and exhibit only the safranin counterstain of the cytosol and nucleus. (D) PMN from an X-linked carrier of CGD stimulated with PMA exhibit both an NBT population and an NBT population of PMN. In this case, the X-linked carrier was 52% NBT.

Citation: Priel D, Kuhns D. 2016. Functional Assays for the Diagnosis of Chronic Granulomatous Disease, p 310-320. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch33
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Image of FIGURE 3
FIGURE 3

Analysis of PMN ROS generation by luminol-enhanced chemiluminescence. PMN (1 × 10 cells/200 μl HBSS/HEPES with 100 μM luminol) were incubated for 120 min in the presence of PMA (100 ng/ml). Luminescence of each well was determined every 2 min. The data from each subject were performed in triplicate. Blue circles represent the mean response of normal subjects ( = 20); yellow circles, an X-linked carrier of gp91 CGD; green circles, a patient with p47 CGD; red circles, a patient with gp91 CGD.

Citation: Priel D, Kuhns D. 2016. Functional Assays for the Diagnosis of Chronic Granulomatous Disease, p 310-320. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch33
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Image of FIGURE 4
FIGURE 4

Immunoblot analysis of phox subunits of NOX2. DFP-treated PMN (5 × 10 cells) were sonicated in 100 μl of sample buffer. For each subject, an aliquot (20 μl or 1 × 10 cell equivalents) of PMN lysate was loaded into a well of the precast BisTris SDS-PAGE gel. In this composite figure, the bands corresponding to each phox subunit from each subject have been aligned. Lane 1 represents PMN from a normal subject and defines the position and the expression of each phox subunit. Lanes 2 and 3 represent CGD patients with missense mutations in gp91 and illustrate the proportional expression observed with gp91 and p22. Lane 4 represents a CGD patient with a nonsense mutation in gp91 with undetectable expression of gp91 but faint expression of p22. Lane 5 represents a CGD patient with p22 deficiency with undetectable expression of both p22 and gp91. Lane 6 represents a CGD patient with undetectable p47. Lane 7 represents a CGD patient with undetectable expression of p67.

Citation: Priel D, Kuhns D. 2016. Functional Assays for the Diagnosis of Chronic Granulomatous Disease, p 310-320. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch33
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References

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11. Kuhns DB, Alvord WG, Heller T, Feld JJ, Pike KM, Marciano BE, Uzel G, DeRavin SS, Long Priel DA, Soule BP, Zarember KA, Malech HL, Holland SM, Gallin JI. 2010. Residual NADPH oxidase and survival in chronic granulomatous disease. N Engl J Med 363:26002610.[CrossRef].[PubMed]
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13. Yamauchi A, Yu L, Pötgens AJ, Kuribayashi F, Nunoi H, Kanegasaki S, Roos D, Malech HL, Dinauer MC, Nakamura M. 2001. Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox. Microbiol Immunol 45:249257.[PubMed].[CrossRef]

Tables

Generic image for table
TABLE 1

Experimental design for quantitative analysis of O· generation using superoxide dismutase-inhibitable ferricytochrome c reduction

Citation: Priel D, Kuhns D. 2016. Functional Assays for the Diagnosis of Chronic Granulomatous Disease, p 310-320. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch33

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