Mutations in the NADPH Oxidase Complex Resulting in Chronic Granulomatous Disease in Patients from the Middle East

Abstract

Chronic granulomatous disease (CGD) is an immunodeficiency disorder characterized by recurrent bacterial and fungal infections. The cause of CGD is defective neutrophil function resulting from mutations in one of the seven components of the leukocyte NADPH oxidase complex. The X-linked form is mediated by defects in CYBB encoding Nox2 (also known as gp91), while the autosomal recessive forms are mediated by mutations in CYBA encoding p22phox, NCF1 encoding p47phox, NCF2 encoding p67phox, and NCF4 encoding p40phox, which all make-up the major components of the NADPH oxidase. To function fully, these major components depend on RAC2 (encoded by RAC2), a small GTPase of the Rho family, and EROS (encoded by CYBC1) a chaperone of Nox2 that is essential for its expression. The aim of this study is to determine the genetic, biochemical, and functional defects of CGD patients in Lebanon and other Middle Eastern countries. Twenty-one Lebanese and ten non-Lebanese patients who suffer from clinical symptoms of CGD and who have defective NADPH oxidase function are included in this study. The genetic basis of their disease was determined using next generation and Sanger sequencing. The effects of the mutations on RNA synthesis and protein expression were determined by RT-PCR and flow cytometry/immunoblotting, respectively. The neutrophil function was determined using the dihydrorhodamine 123 (DHR) assay. The phenotype and function of their T and B cells were determined by activation and flow cytometry. The patients suffered from severe inflammation, bacterial, and fungal infections associated with defective NADPH oxidase function, suggestive of CGD. Whole exome sequencing (WES) of 10 patients from Lebanon revealed homozygous mutations in NCF2 and terminating hemizygous mutations in CYBB as the predominant alteration, whereas 3 non-Lebanese patients harbored homozygous deleterious mutations in CYBA, CYBB, or NCF2. The remaining 18 patients suffered from defective NADPH oxidase function but did not have a genetic diagnosis. Intracellular staining of 4 components of the NADPH oxidase complex (p22phox, p47phox, p67phox, and gp91) revealed absent p67phox in 2 genetically undiagnosed patients that were subsequently found to have a large deletion in exon 5 and a splice site mutation in exon 3 of NCF2. Interestingly, the patients with absent p67phox protein expression also showed a significant decrease in the levels of p47phox. A study of the adaptive branch of the immune system revealed normal patient T cell phenotype with diminished proliferative potential. The percentage of memory B cells was highly diminished whereas B cell function was maintained. T-regulatory cells were higher in percentage in the patients, with signs of increased activity. In this work we identified deletions in NCF2 as the most prevalent genetic defect in the patients studied using WES. Since WES is costly and time consuming, we tested the possibility of using a biochemical approach to identify the NADPH oxidase protein component that is absent in the patient, followed by targeted Sanger sequencing to determine the nature of the mutation. This led to the identification of 2 additional patients with deletions in the NCF2 gene encoding p67phox, confirming that it is the most common mutation in CGD patients from Lebanon. We also observed that absence of p67phox results in a significant decrease in the levels of p47phox but not p22phox and Nox2, suggesting that the stability of p47phox is compromised in the absence of p67phox. Furthermore, our results indicate that p67phox deficiency does not only affect neutrophil function, but it also variably affects lymphocyte homeostasis and function.