Clinical and molecular significance of flow cytometric analysis for reactive oxygen species production and residual p67^phox expression in p67^phox-deficient chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by molecular defects in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. p67(phox)-CGD is an autosomal recessive CGD, which is caused by a defect in the cytosolic components of NADPH oxidase, p67(phox), encoded by NCF2. We previously established a flow cytometric analysis for p67(phox) expression, which allows accurate assessment of residual protein expression in p67(phox)-CGD. We evaluated the correlation between oxidase function and p67(phox) expression, and assessed the relevancy to genotypes and clinical phenotypes in 11 patients with p67(phox)-CGD. Reactive oxygen species (ROS) production by granulocytes was evaluated using dihydrorhodamine-1,2,3 (DHR) assays. p67(phox) expression was evaluated in the monocyte population. DHR activity and p67(phox) expression were significantly correlated (r = 0.718, p < 0.0162). Additionally, DHR activity and p67(phox) expression were significantly higher in patients carrying one missense variant in combination with one nonsense or frameshift variant in the NCF2 gene than in patients with only null variants. The available clinical parameters of our patients (i.e., age at disease onset, number of infectious episodes, and each infection complication) were not linked with DHR activity or p67(phox) expression levels. In summary, our flow cytometric analysis revealed a significant correlation between residual ROS production and p67(phox) expression. More deleterious NCF2 genotypes were associated with lower levels of DHR activity and p67(phox) expression. DHR assays and protein expression analysis by using flow cytometry may be relevant strategies for predicting the genotypes of p67(phox)-CGD.