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 Taj Ali Khan a, * , Kalsoom Kalsoom b , Asif Iqbal c , Huma Asif c , Hazir Rahman a , Syed Omar Farooq d , Hassan Naveed a , Umar Nasir d , Muhammad Usman Amin d , Mubashir Hussain a , Hamid Nawaz Tipu e , Andrei Florea f a Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan b Department of Food Science & Technology, University of Agriculture, Peshawar, Pakistan c Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, Brazil d Khyber Medical University, Institute of Medical Sciences, Kohat, Pakistan e Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan f The Department of Chemistry, University of Oxford, Oxford, United Kingdom article info Article history: Received 5 July 2016 Received in revised form 11 September 2016 Accepted 21 September 2016 Available online 23 September 2016 Keywords: Chronic granulomatous disease Immunodeficiency Mycobacterium tuberculosis Monocyte-derived macrophages abstract Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the five structural genes (CYBB, CYBA, NCF1, NCF2, and NCF4) that typically results in a decrease in function or inability to generate a respiratory burst, leading to defective killing of pathogens, including fungi and intracellular bacteria. Mutations in CYBB, encoding the gp91phox (also known as NOX2) result in X- linked CGD account for approximately 65% of CGD cases. Here, we aimed the characterization of a novel missense mutation c.1226C > A/p.A409E in the CYBB gene in a patient with X-linked CGD. Relevant clinical data of a male patient whose family was positive for XCGD was reviewed. Oxidative burst and NADPH protein expression was evaluated by flow cytometry, while Genetic analysis was performed by Sanger sequencing. Monocyte-derived macrophages (MDMs) were evaluated for their capacity for phagocytosis and growth suppression of the intracellular Mycobacterium tuberculosis (M. tuberculosis). We thus report the absence of an oxidative burst in the phagocytes of the patient. Flow cytometry evaluation revealed a normal expression of NADPH oxidase components in neutrophils and genetic analysis proved the existence of a novel missense c.1226C > A mutation in the CYBB gene resulting in p.A409E. Further, we have showed that the patient's MDMs were unhindered in their ability to take up mycobacteria normally. Instead, the MDMs failed to control the intracellular proliferation of M. tuberculosis, a phenotype that improved in the presence of recombinant human interferon-gamma (rhIFN-g). This work expands the genetic spectrum of X-linked CGD and demonstrates improvement in macrophage function in X91 þ CGD patient by rhIFN-g. © 2016 Elsevier Ltd. All rights reserved. 1. 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 oxi- dase have been implicated in CGD pathology, a condition that af- fects 1 in 250,000 individuals. It is attributed to the malfunction of * Corresponding author. Department of Microbiology, Kohat University of Science and Technology, Kohat, KPK, 26000, Pakistan. E-mail address: microbiologist63@yahoo.com (T.A. Khan). Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath http://dx.doi.org/10.1016/j.micpath.2016.09.020 0882-4010/© 2016 Elsevier Ltd. All rights reserved. Microbial Pathogenesis 100 (2016) 163e169