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.