Clinical and molecular significance of flow cytometric analysis for reactive oxygen species production and residual p67phox expression in p67phox-deficient chronic granulomatous disease

Hanae Miyazawa; Masahiro Muraoka; Yusuke Matsuda; Tomoko Toma; Tomohiro Morio; Tomonari Shigemura; Kohei Haraguchi; Tadashi Matsubayashi; Toshinao Kawai; Yuya Shirai; Taizo Wada

doi:10.1111/sji.13372

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

Scand J Immunol. 2024 Apr 23:e13372. doi: 10.1111/sji.13372. Online ahead of print.

Authors

Affiliations

¹ Department of Pediatrics, School of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan.
² Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University Graduate School of Medical and Dental Sciences, Tokyo, Japan.
³ Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan.
⁴ Department of Pediatrics, Nagasaki University Hospital, Nagasaki, Japan.
⁵ Department of Pediatrics, Seirei Hamamatsu General Hospital, Hamamatsu, Japan.
⁶ Division of Immunology, National Center for Child Health and Development, Tokyo, Japan.
⁷ Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Japan.

PMID: 38654426
DOI: 10.1111/sji.13372

Abstract

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.

Keywords: NCF2; chronic granulomatous disease; p67phox.