Clinical, functional and genetic characterization of 16 patients suffering from chronic granulomatous disease variants - identification of 11 novel mutations in CYBB

Abstract

<p>Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The most common form is the X-linked CGD (X91-CGD), caused by mutations in the <em>CYBB</em> gene. Clinical, functional and genetic characterizations of 16 CGD cases of male patients and their relatives were performed. We classified them as suffering from different variants of CGD (X91⁰, X91^- or X91⁺), according to NADPH oxidase 2 (NOX2) expression and NADPH oxidase activity in neutrophils. Eleven mutations were novel (nine X91⁰-CGD and two X91^--CGD). One X91⁰-CGD was due to a new and extremely rare double missense mutation Thr208Arg-Thr503Ile. We investigated the pathological impact of each single mutation using stable transfection of each mutated cDNA in the NOX2 knock-out PLB-985 cell line. Both mutations leading to X91^--CGD were also novel; one deletion, c.-67delT, was localized in the promoter region of <em>CYBB</em>; the second c.253-1879A>G mutation activates a splicing donor site, which unveils a cryptic acceptor site leading to the inclusion of a 124-nucleotide pseudo-exon between exons 3 and 4 and responsible for the partial loss of NOX2 expression. Both X91^--CGD mutations were characterized by a low cytochrome <em>b₅₅₈</em> expression and a faint NADPH oxidase activity. The functional impact of new missense mutations is discussed in the context of a new three-dimensional model of the dehydrogenase domain of NOX2. Our study demonstrates that low NADPH oxidase activity found in both X91^--CGD patients correlates with mild clinical forms of CGD, whereas X91⁰-CGD and X91⁺-CGD cases remain the most clinically severe forms.</p>