Diagnostic Modalities Based on Flow Cytometry forChronic Granulomatous Disease: A MulticenterStudy in a Well-Defined Cohort

TARAMAWOSWOS:000588386800037TARAMAPUBMEDTARAMASCOPUSBackground: Chronic granulomatous disease (CGD) is characterized by defective microbial killing due to mutations affecting subunits of thenicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Definitive genetic identificationof disease subtypes may be delayed or notreadily available. Objective: We sought to investigate the role of intracellular staining of NADPH oxidase enzyme subunits in predicting the respective genetic defectsin CGD patients and carriers. Methods: Thirty-fourgenetically inherited CGD patients, including twelve patients with X-linked CGD (gp91phoxdeficiency due to CYBB mutations) and 22 patients with autosomal recessive CGD (p22phox, p47phox and p67phoxdeficiency due toCYBA, NCF1 and NCF2mutations, respectively)were recruited from different immunology centers and followed up prospectively.Dihydrorhodamine (DHR) testingand NADPH oxidase subunit expression in white blood cells were determined by flow cytometry. Results:gp91phoxand p22phoxdefects, which result in simultaneous loss of both proteins due to their complex formation, were only differentiated by comparative analysis ofpatients’and mothers’ intracellular staining. p47phox and p67phox protein expression was almost undetectable in patients compared to carrier mothers and healthy controls. The expression values of the respective subunits were found to be significantly higherin all controlsas compared to carrier mothers, which in turn were higher than those of patients. Conclusion: Analysis of NADPH oxidase enzyme subunits byflow cytometry in patients and carriers is useful in the rapid predictionof the genetic defect of patients with CGD,thus guiding targeted sequencing and aiding in their early diagnosis