CDiReC - Chronic Granulomatous Disease (CGD) Diagnosis and Research Center

Centre Diagnostic et Recherche sur la CGD (Granulomatose Septique Chronique)

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Rare CGD mutations identification

Federica Defendi, Cécile Martel, Michèle Mollin, Sylvain Beaumel

There are two types of CGD transmission that make up autosomal forms, with mutations in NCF1, NCF2, and CYBA genes encoding p47phox, p67phox, or p22phox proteins, respectively, and the most common X-linked CGD type (60% of CGD cases), with defects in CYBB encoding gp91phox. CGD is a very heterogeneous genetic disease, caused by a large variety of mutations such as deletions, splice site mutations, and missense or nonsense mutations located in the four genes encoding NADPH oxidase components, with no «hot-spot» location except for the NCF1 gene [Stasia et al. (2008), Stasia and Li (2008)].

All ethnic groups are equally affected. The X-linked recessive transmission type of CGD (XCGD), characterized by mutations in the CYBB gene encoding NOX2, is the most frequent form of CGD (approximately 60% of cases). Beside this X linked common form.

Most of the time, mutations in the CYBB gene lead to a lack of NOX2 expression, because of the instability of the corresponding mRNA or protein (X910CGD). In these patients, NADPH oxidase activity is always totally abolished. This phenotype, called X910 CGD, is the most frequent. It is usually caused by nonsense, missense, and splice mutations, small deletions, and insertions, sometimes associated with frameshift and early termination of protein synthesis [Stasia et al. (2005)].

X91- CGD mutants

Only 27 cases with «variant» forms of the disease, called X91- CGD have been described, in which low levels of cytochrome b558 expression are accompanied by a proportionally decreased NADPH oxidase activity. Mutations associated with this phenotype are usually located in the coding region (exons) of CYBB. These variants are of interest because they cause a structural disorganization leading either to an incomplete loss of protein or to a partial dysfunction, or both.

localisation of mutations
X91- CGD mutations Printable version

Mutations in the upstream promoter region of CYBB leading to the X91- CGD phenotype also have been described. To our knowledge only four point mutations were reported in this region (C-52T, C-53T, T-55C and A-57C [Newburger et al. (1994), Weening et al. (2000), Stasia et al. (2004)]. These mutations are located between the «CCAAT» and the «TATA» boxes in a consensus binding site for the ets family of transcription factors of the gp91phox promoter responsible for CYBB transcription. Despite the low expression of gp91phox protein and the residual NADPH oxidase activity in phagocytes, the clinical appearance of these specific X91- CGD patients varies considerably from mild forms to severe clinical phenotypes with multiple life-threatening infections during their life. This suggests that the levels of O2- production and other derived reactive oxygen species (ROS) by activated phagocytic cells are critical for effective protection against infections. A striking point is that in most of the X91- CGD cases characterized by a mutation in the CYBB promoter, the CGD diagnosis is made in adolescents (>10 years) or in adults.

We report a novel point mutation in the CYBB gene promoter (insertion of a T at position -54/-56) leading to a rare X91- CGD case in an Italian family with two members affected by CGD. The mutation caused a reduced expression of gp91phox at the mRNA and protein level associated with a decreased ability to generate O2? and H2O2 (~ 7% of control neutrophils). A decrease in the binding of nuclear factors to the mutated promoter region was found in electrophoretic mobility shift assays. However, the patient’s eosinophils were NBT-positive, displayed DHR oxidation and were gp91phox-positive. Despite a weak respiratory burst activity, the patient’s granulocytes did not kill either S. aureus or C. albicans even with long incubation times. The CGD diagnosis for both patients was made in adulthood. One of them died from a bilateral pneumonia while the other suffers from sporadic respiratory and skin infections that are kept under control by conventional antibiotic therapy. These observations raise questions about the role and the levels of ROS needed for protection against bacterial and fungal infections [Defendi et al (2009].

X91+ CGD mutants

19 mutations have been reported to cause X91+ CGD. In these variants the mutated gp91phox is normally expressed but with the absence of oxidase activity. Most of them are missense mutations, two are small deletions, and one is a deletion/insertion. They are principally located in the COOH terminus cytosolic tail of Nox2, confirming that it is an important functional part of the protein that is less involved in its structural stability. Some functional consequences of such rare mutations have been studied In some rare cases, the mutated gp91phox is normally expressed but with the absence of oxidase activity. These variants called X+CGD, have provided interesting informations about oxidase activation mechanisms [Stasia et al. (2002a), Bionda et al. (2004), Stasia (2007)].

localisation of X+ mutations
X91+ CGD mutations Printable version

However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level (cf next chapter). A cellular model (KO PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation [Dinauer (1999), Li et al. (2005), Li et al. (2007), Stasia and Li (2008)].

The second most common form of CGD is autosomal recessive (ARCGD), accounting for approximately 30% of the cases. Most of the time, it is caused by the deletion of a GT from a GTGT tandem repeat at the first splice junction in the NCF1 gene encoding p47phox (A47 CGD). In addition to these usual CGD types, mutations in the CYBA and NCF2 genes encoding p22phox and p67phox, respectively, account for rare A220 CGD [Stasia et al. (2002b)] and A670 CGD, each accounting for less than 5% of cases. However we recently found a genetic heterogeneity in the Jordanian families with a high frequency of rare ARCGD, probably because consanguineous marriages are common in Jordan [Bakri et al. (2008)].

X+CGD and Nox2 superoxidase mutants as powerful tools to study NADPH oxidase activation

Antoine Picciocchi, Franck Debeurme, Sylvain Beaumel, Laure Carrichon, Federica Defendi Research program from March 2007 to March 2009 Supported by grants from USIDNET (US Immunodeficiency network) a NIH consortium, Towson, Maryland,U.S.A

The main goal of this project is to identify new sequences of Nox2 essential for the functioning and the assembly of the NADPH oxidase phagocyte complex. This will be useful to modify the sequence of Nox2 in order to improve its oxidase activity and to facilitate its activatibility in future CGD protein therapy approaches.

This research program is under investigation.

1. Published X+-CGD mutants will be modeled in KO PLB985 cells (X0CGD PLB-985) to identify Nox2 domains essential for oxidase activity and activation/assembly (X+CGD). Phenotypes of each mutant will be studied (in vivo and in vitro oxidase activity, Nox2 expression, oxidase component assembly, electron transfer, cofactors and substrate binding).

2. The molecular mechanism by which the Nox2 super-mutants recently constructed in our laboratory produce high oxidase activity will be studied. The bactericidal power of such mutants will be evaluated against common CGD germs and fungi (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans).

superoxidase activity
D-loopNOX4-NOX2 Super-oxidase mutant expressed in PLB-985 cells [Li et al. (2005)]

3. Constitutively active Nox2 mutants that do not require cytosolic factor assembly for activation, such as some ferredoxin reductase analogs, will be constructed and studied based on the deletion of the alpha-helix sequence of Nox2.

alpha helix of Nox2
Representation of the alpha-helix (in gold) in the cytosolic tail of Nox2 [Li et al. (2007)]

Recombinant cytochrome b558

Marie-Claire Dagher, Antoine Picciocchi

Production of large amounts of cytochrome b558 and mutants for biochemical and structural characterization requires an efficient overexpression system. The baculovirus/insect cell was chosen. Co-expression of both subunits (Nox2 and p22phox) in the same cell is obtained after cloning into the pFastBac-dual vector and generation of recombinant baculovirus (BacToBac expression system, Invitrogen). The presence and position of the tag for purification was systematically studied using the Ligation Independant Cloning (LIC) method and a web-based software called «LIC generator» was designed for this purpose and used successfully. With improved knowledge of factors affecting the assembly and targeting of the two subunits, production of an active protein is expected in the close future.

cytochrome b558 typical red/ox spectrum
Typical cytochrome b558 spectrum in insect cells membranes expressing gp91phox and p22phox

Native gene therapy

Federica Defendi, Stephanie Phelps, Nicholas Heintz

Chronic granulomatous disease (CGD) is a rare genetic condition characterized by functional inadequacy of the phagocyte NADPH oxidase that severely compromises the anti-microbial capacity of the innate immune system. Approximately 60% of CGD patients lack functional Nox2 (i.e., gp91phox), the catalytic subunit of the NADPH oxidase complex. Nox2 is encoded by the CYBB gene on the X chromosome. Viral gene therapy for CGD has had limited success, but with sometimes serious side effects. The intent of this proposal is to refine gene transfer technology for introducing the entire CYBB gene into CYBB knock-out (KO) PLB-985 cells as an experimental model for restoring normal Nox2 expression in hematopoetic stem cells from CGD patients.

This research project will investigate novel technology for introducing entire human genes into immune cells that lack the functional Nox2 subunit of the NADPH oxidase complex that produces reactive oxygen species required for killing bacteria and other microbes. The long term aim is to use replacement of entire human genes to restore function of the phox complex, and thereby development a safe and effective gene therapy for CGD.

This project) is a collaborative effort between our team and Nicholas Heintz at the University of Vermont, Burlington, USA. Dr. Heintz’s group has refined technology for introducing bacterial artificial chromosomes (BACs) that encompass entire gene loci into cultured cells in order to restore gene function or alter gene copy number. Refining BAC transfection technology has been aided by the work of Stephanie Phelps, who has optimized protocols for retrofitting BACs with markers (Illenye and Heintz 2004).

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Directeur de publication:  Marie-José Stasia   Webmaster:   Marie-Claire Dagher