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

Abstract

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 CYBB 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 CYBB; 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 b₅₅₈ 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.

Keywords: NADPH oxidase; NOX; X-linked CGD variants; clinical severity.

Fig. 1

Phenotypical analysis of the neutrophils from X‐linked chronic granulomatous disease (CGD) patients having new CYBB mutations. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) expression of patients and their mothers’ neutrophils was measured by flow cytometry, as described in Materials and methods. Solid grey curve corresponds to dihydrorhodamine (DHR)‐loaded resting neutrophils, empty black curve corresponds to DHR‐loaded neutrophils after phorbol myristate acetate (PMA) stimulation (a). For patient P12, the absence of gp91^phox (or NOX2) and p22^phox expression was shown by Western blotting using the primary specific antibodies 48, 449, respectively (b). Note that all the subunits of the NADPH oxidase complex are expressed in patient P15’s neutrophils (X91⁺CGD) by Western blotting using specific antibodies, as described in Material and methods (c).

Fig. 2

Functional analysis of patient P6’s T208R and T503I substitutions in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) by means of transgenic PLB‐985 cells. Measurement of the NOX2 oxidase activity of neutrophils of patient P6 and of his mother by resorufine oxidation (Amplex Red^R) after opsonized zymosan, phorbol myristate acetate (PMA) and platelet‐activating factor/N‐formyl‐Met‐Leu‐Phe (PAF/fMLF) activation, as described in Materials and methods (a). Expression of NADPH oxidase subunits NOX2, p67^phox and p47^phox p40^phox and p22^phox in patient P6 and his mother’s neutrophils by Western blot analysis (b). Cytochrome b ₅₅₈ differential spectra of 1% Triton X100 soluble extract from patient P6 and his mother’s neutrophils (c). NOX2 expression in differentiated T208R–NOX2 and T503I–NOX2 transgenic PLB‐985 cells by flow cytometry using the 7D5 monoclonal antibody against NOX2 and phycoerythrin (PE)‐coupled polyclonal antibody anti‐IgG1 (empty black curve). Solid grey curve corresponds to resting neutrophils labeled with monoclonal mouse IgG1 isotype as an irrelevant antibody (d). NADPH oxidase activity of differentiated T208R–NOX2 and T503I–NOX2 transgenic PLB‐985 cells measured by chemiluminescence in presence of luminol, horseradish peroxidase (HRP) after 80 ng/ml PMA stimulation (e).

Fig. 3

(a) Schematic representation of X‐linked chronic granulomatous disease (CGD) point mutations within the NOX2 protein – new mutations are shown in red and known mutations in blue. New mutations in the promoter (patient P1) or in intron sequences (patients P4a and P12) of CYBB are not represented. (b) NOX2 dehydrogenase model and modeling of CGD mutations. The DH model is represented as ribbons, dark green for the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) binding domain – corresponding to the 3A1F Protein Data Bank (db) structure – and purple‐blue for the modelled FAD binding domain ([37]). NOX2 NIS, in light green, is an approximate structural model but is partly disordered, flexible as suggested by the absence of density in the 3A1F structure for this sequence. Some of the residues corresponding to X91⁰ and X91⁺ mutations are highlighted as shown in stick (V327, G412, W516 and T503). Three zooms highlight the V327, G412 and W516 residues with emphasis on the neighboring residues around the considered mutations. In each case, the mutated position is presented with both the wild‐type side chain and the corresponding mutation, in orange, superimposed. Molecular graphic images were produced using PyMOL software

Fig. 4

Phenotypical and genotypical characterization of the X91⁻ ‐chronic granulomatous disease (CGD) of patient P1 due to a new point mutation in the CYBB promoter. Flow cytometry dot‐plots of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) oxidase activity [dihydrorhodamine (DHR)] and CD294 expression (eosinophil marker) in neutrophils and eosinophils of granulocyte preparations from a healthy subject (bottom panels) and patient P1 (top panels) at rest (left panels) or after stimulation with phorbol myristate acetate (PMA) (right panels). Left panels correspond to dot‐plots of cells labeled with isotype control antibodies and loaded with DHR (a). Flow cytometry dot‐plots of NOX2 and CD294 expression (eosinophil marker) in neutrophils and eosinophils of granulocyte preparations from a healthy subject (bottom panels) and patient P1 (top panels) using 7D5 monoclonal antibody and anti‐CD294 antibody (right panels) compared to dot‐plots of isotype controls (left panels) (b). NOX2 and p22^phox expression in patient P1 and his mother’s granulocytes by Western blot analysis. A faint NOX2 and p22^phox expression are visible probably due to the presence of eosinophils in the granulocyte preparation (c). Cytochrome b ₅₅₈ differential spectra of 1% Triton X100 soluble extract from patient P1 and his mother’s neutrophils (d). Thymidine deletion at position −67 detected in the CYBB promoter region of patient P1 and his mother as a carrier, compared to a healthy donor sequence (e). Schematic representation of part of the CYBB promoter region, with localization of the ‘CCAAT’ and ‘TATA’ boxes, the ATG starting codon and the point mutation described in panel (e) (f).

Fig. 5

Phenotypical analysis of the neutrophils of patient P4a suffering from an X91⁻‐chronic granulomatous disease (CGD) and having a new CYBB mutation. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) oxidase activity of neutrophils from patient P4a, his sister and his mother was measured by flow cytometry after stimulation during 15 min with 200 ng/ml phorbol myristate acetate (PMA) in the presence of the dihydrorodamine (DHR) probe, as described in Materials and methods. Solid grey curve corresponds to DHR‐loaded resting neutrophils, empty black curve corresponds to DHR‐loaded neutrophils after PMA stimulation (a). The NADPH oxidase activity of neutrophils from patient P4a, his sister and his mother was also measured using the nitro blue‐tetrazolium (NBT) reduction test (b). NOX2 and p22^phox expression was shown by Western blot using the primary specific antibodies 48 and 449, respectively (c). Nox2 (d) and p22^phox expression (e) was shown by flow cytometry using the primary specific antibodies 7D5 and 449, respectively. Solid grey curve corresponds to resting neutrophils labeled with irrelevant antibodies; empty black curve corresponds to neutrophils labeled with the 7D5 (anti‐NOX2) or 449 antibodies (p22^phox) and secondary antibodies coupled with phycoerythrin (PE). Genetic analysis of the mutation of patient P4a in CYBB gene, leading to an X91⁻‐CGD. NOX2 cDNA was amplified in three overlapping fragments, as described in Materials and methods. A 124 base pair (bp) of intron 3 was inserted between exons 3 and 4 leading to the creation of a pseudoexon. Intronic regions surrounding the inserted region of intron 3 in the NOX2 cDNA was amplified and sequenced. A c.253‐1879A>G hemizygous mutation was found creating a splicing donor site, which unveils a cryptic acceptor site leading the inclusion of 124 nucleotides as a pseudo‐exon responsible for the partial loss of the NOX2 expression. The consequence in the NOX2 protein is a missense mutation at Cys85→Leu and a frameshift leading to the generation of a stop codon located 32 amino acids further on (p.Cys85LeufsX32) (f).