A novel missense mutation in the NADPH binding domain of CYBB abolishes the NADPH oxidase activity in a male patient with increased susceptibility to infections
Introduction
Professional phagocytes generate high levels of reactive oxygen species (ROS) using a superoxide-generating NADPH oxidase, essential for intracellular killing of phagocytosed pathogens [1], [2]. NADPH oxidase is a multi-subunit enzyme, comprising both membrane and cytosolic components, encoded by five genes. The CYBB and CYBA genes encode membrane proteins (NOX2 and p22phox) respectively while NCF1, NCF2 and NCF4 genes encode cytosolic proteins (p47phox, p67phox and p40phox) respectively [3], [4]. Upon cellular activation, the cytosolic proteins in association with RAC2 combine with the membrane proteins to form an active NADPH oxidase complex responsible for the production of ROS [5].
Mutations in all of the five structural genes of the NADPH oxidase have been implicated in CGD pathology, a condition that affects 1 in 250,000 individuals. It is attributed to the malfunction of the NADPH oxidase leading to a decrease or total abolishment in the ability of monocyte-derived macrophages to generate ROS. As a result, frequent life-threatening infections of the lung, skin, lymph nodes, and liver caused by bacteria and fungi occur, accompanied by the characteristic granulomas formation in response to chronic inflammation [6], [7], [8], [9]. Importantly 65% of cases are due to mutations in the CYBB gene modulating NOX2 function, which is responsible for X-linked CGD, whereas mutations in autosomal genes, such as NCF1, CYBA, NCF2, and NCF4 cause the autosomal recessive (AR)-CGD. The most common form of AR-CGD is caused by mutations in NCF1 (25% of CGD cases) followed by rare mutations in NCF2, CYBA, and NCF4 [6]. Clinically, X-linked NOX2-deficient CGD is more severe with earlier presentation at infancy causing earlier death [9], [10]. Three variants of XCGD exist, differentiated by the varying levels of NOX2 expression. In the X910CGD form of the disease, a total lack of NOX2 expression is associated with a complete abolishment of NADPH oxidase activity, while in the X91−CGD the low level of NOX2 expression is associated with decreased NADPH activity. Conversely, X91+CGD, the third type of XCGD is usually characterized by otherwise normal expression of NOX2 accompanied by a complete lack of NADPH activity [11], [12], [13]. In light of this, we conducted this study to characterize a novel missense mutation c.1226C > A/p.A409G in the CYBB gene in a patient with X-linked CGD.
Section snippets
Patient and ethical statement
Relevant clinical data of patient with a positive family history of the disease (Fig. 3A) was reviewed. Clinically, the male patient presented severe bacterial and fungal infections such as BCG-itis due to M. bovis BCG vaccination, recurrent pneumonia caused by Streptococcus pneumonia and Candida sp respectively, TB due to M. tuberculosis and subcutaneous abscesses in the neck with unknown pathology. Typical granuloma formations were observed in lungs of the patient. The patient is 6-years old.
The patient presented CGD phenotype with no residual NADPH oxidase activity
The clinically diagnosed CGD patient was further confirmed positive for the disease via a DHR-123 assay, which demonstrated that the patient's neutrophils and monocytes produced no visible trace of H2O2 (Fig. 1A and B). To establish which component of NAPDH oxidase is defective, flow cytometry analysis of the NADPH oxidase components was performed which demonstrated the normal expression of all NADPH oxidase components including NOX2 in the patient's neutrophils compared to healthy controls (
Discussion
Here, we described a patient with X91+CGD phenotype who presented severe infections, including mycobacterial infections early in life. Genetic analysis revealed a novel missense mutation c.1226C > A/p.A409E in the CYBB gene, which impaired the oxidase activity without affecting the expression of mutated NOX2. Functional, genetic and in silico analyses suggested that the mutation c.1226C > A/p.A409E could be involved in the mechanism of pathogenesis. However, a definite confirmation at this
Conflict of interest
The authors have no financial conflicts of interest to declare.
Acknowledgements
This work was supported by grant from the Jeffrey Modell Centers Network (JMCN).
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