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Chapter 33 : Functional Assays for the Diagnosis of Chronic Granulomatous Disease
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 (O2·−) and other related reactive oxygen species (ROS), leading to recurrent infections, granulomatous complications, and premature death. Generation of O2·− requires the assembly and activation of a multicomponent enzyme, NADPH oxidase (NOX2) or phagocyte oxidase (phox), a complex consisting of numerous cytosolic proteins, including p47phox (2), p67phox (2), and p40phox (3), and two membrane proteins, p22phox and gp91phox, that constitute cytochrome b 558 (4, 5). NOX2 catalyzes the reduction of molecular O2 to O2·− using NADPH generated by the oxidation of glucose through the pentose-phosphate pathway. O2·− is converted to H2O2 either spontaneously or enzymatically. H2O2 and O2·− 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 gp91phox (~70% of patients), p47phox (~25%), p22phox (<5%), p67phox (<5%), and p40phox (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.