CGD Patients Research Articles

Diagnóstico prenatal y nueva mutación en enfermedad granulomatosa crónica ligada al cromosoma X

BackgroundChronic Granulomatous Disease (CGD) is a rare primary immunodeficiency caused by the alteration of the enzyme complex NADPH oxidase, which affects the phagocytic function. CGD patients are susceptible to recurrent infections mainly caused by bacteria and/or fungi. MethodsWe studied a 6 year-old boy with suspicion of CGD. The diagnosis was confirmed based on the functional study of NADPH oxidase. Simultaneously, the second pregnancy of the mother was reported and genetic counselling was requested. ResultsWe identified a new disease-causing mutation by direct sequencing of the CYBB gene (X-linked CGD). The prenatal study resulted in the identification of the same mutation in the foetus. CommentsMolecular genetics characterisation of CGD is needed to obtain an accurate diagnosis of the disease and to offer prenatal diagnosis and genetic counselling in future pregnancies.

Successful Control of Preexistent Active Infection by Granulocyte Transfusions During Conditioning Induced Cytopenia In Patients with Chronic Granulomatous Disease Undergoing Hematopoietic Stem Cell Transplant

AbstractAbstract 1329Chronic granulomatous disease (CGD) is a congenital disorder resulting from decreased or absent oxidase production by neutrophils. As a result, patients with CGD are prone to bacterial and fungal infections and have a shortened life expectancy. Hematopoietic stem cell transplantation (HSCT) can be curative, and patients are increasingly referred for transplant due to an ongoing infection not amenable to cure using standard treatments. The use of granulocyte transfusions has been described in patients undergoing transplant with an underlying infection at the time of conditioning, but reports are limited to single cases. We describe here the effects of granulocyte transfusions in four patients undergoing either an HLA matched sibling or unrelated donor HSCT, using an alemtuzumab containing regimen and sirolimus as GVHD prophylaxis. Age of the patients was 6 to 25 (mean 11.5) yrs, and weight was 15.3 to 54 (mean 27.5) kg. Three of the patients had persistent Aspergillus infection involving the spine, lung, and/or brain despite long term combination antifungal therapy including a triazole, echinocandin and/or amphotericin. The fourth patient had an Actinomyces pneumonia progressive on combination antibacterial treatment. None of the patients had detectable HLA antibodies prior to the transfusions, and none received G-CSF post-transplant. The granulocytes were started on the anticipated day of neutropenia after conditioning and graft infusion and continued until evidence of graft recovery. Volunteer community donors underwent mobilization with dexamethasone 8 mg PO plus G-CSF 480 mcg SC, followed the next day by a 7-liter leukapheresis procedure (Spectra) using Tricitrasol/Hetastarch (Hespan) as the anticoagulant/sedimenting solution. Patients received a mean of 4.5 transfusions each, with three patients receiving biweekly transfusions and one receiving transfusions three times a week. The mean number of granulocytes per component transfused was 6.17 × 10e10 (range 4.0–9.12). This resulted in a mean of 2.78 × 10e9 granulocytes transfused per kg recipient weight (range 0.76–4.48). All patients tolerated the infusions well, without respiratory symptoms. The mean increase in absolute neutrophil count posttransfusion was 1.95 × 10e3/uL (range 0.5–4.61), although the timing of collection of the blood count samples was variable for each transfusion. Using a dihydrorhodamine (DHR) fluorescence based assay we were able to track the presence of oxidase positive cells to help differentiate transfused donor from residual host cells. DHR assays were performed before and after every transfusion in one patient. The number of DHR positive cells prior to infusion was 0–1.4% with a rise to 68.2% 24 hours after transfusion. 24.5% oxidase positive cells were still detectable almost 72 hours after transfusion, just prior to the next transfusion. One product required sedimentation for ABO incompatibility. The mean time to engraftment was 22 days and did not differ in patients with CGD receiving the same transplant regimen with (n=4) or without (n=6) peri-transplant granulocyte transfusions (30 days). Although all four patients had infectious processes not responding to standard antimicrobial therapy prior to transplant, there was no evidence of progressive infection during the period of neutropenia. Imaging studies of the brain, lung and/or spine suggested improvement in the infectious process in all four patients, although the timing of the scans made assessments inconclusive. Our prior experience with granulocyte transfusions in patients with CGD not undergoing transplant is that the cells do not persist in the circulation longer than 24 hours. Higher cell doses per kg in our four patients may have contributed to the sustained detection of transfused circulating cells, particularly as three of the patients weighed less than 21 kg. Peri-transplant immune suppression may also have facilitated the longer circulation time of the cells. We conclude that community-donor granulocyte transfusions are well tolerated in non-alloimmunized CGD patients undergoing HSCT, do not impact engraftment, are logistically feasible, and may provide a therapeutic bridge for patients with an underlying infection during a prescribed period of neutropenia, thus decreasing transplantation risk for such patients. Disclosures:No relevant conflicts of interest to declare.

Inflammasome activation in NADPH oxidase defective mononuclear phagocytes from patients with chronic granulomatous disease

AbstractChronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent infections and deregulated inflammatory responses. CGD is caused by mutations in subunits of the NADPH oxidase, an enzyme that generates reactive oxygen species in phagocytes. To elucidate the contribution of the proinflammatory protease caspase-1 to aberrant inflammatory reactions in CGD, we analyzed cells isolated from patients with defects in the phagocyte oxidase subunits p22phox, p47phox or gp91phox. We report that mononuclear phagocytes from CGD patients activated caspase-1 and produced biologically active interleukin-1β (IL-1β) in response to danger signals. Notably, caspase-1 activation and IL-1β secretion from CGD monocytes was elevated in asymptomatic patients and strongly increased in patients with noninfectious inflammatory conditions. Treatment with IL-1 receptor antagonist reduced IL-1 production in monocytes ex vivo and during medical therapy. Our results identify phagocyte oxidase defective monocytes as a source of elevated IL-1 and provide a potential therapeutic option to ameliorate inflammatory conditions associated with CGD.

Adenosine A2A receptor activation inhibits T cell activity in a murine model of chronic granulomatous disease (87.3)

Abstract Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase system, and is characterized by an increased susceptibility to recurrent, severe bacterial and fungal infections. Early diagnosis and aggressive treatment can be effective at combating life threatening infections, but other significant complications of CGD, such as non-infectious hyperinflammatory manifestations, result in a high associated morbidity. Recent studies suggest that the hyperinflammation observed in CGD (gp91phox deficient) mice may be due in part to dysregulated T cell activity. We have broadly characterized the phenotypes of T lymphocytes collected from p91phox deficient mice and our data indicate that although the basal activation states of T cells collected from WT and CGD mice are similar, upon activation through the T cell receptor, CGD T cells produce higher levels of the pro-inflammatory cytokines IFN-γ and IL-2 and express elevated levels of the cell surface activation markers CD25, CD69 and CD40L as compared to WT T cells. This hyper-activation of CGD T cells is effectively modulated by treatment with the selective adenosine A2A receptor agonist CGS21680, which reduces the levels of IFN-γ and IL-2 by approximately 90% and down regulates CD25, CD69 and CD40L expression by approximately 50%. These data suggest that the adenosine A2A receptor is a potential pharmacological target for the treatment of the hyperinflammatory complications observed in CGD patients.

Chronic Granulomatous Disease and Mcleod Syndrome: Single Center Report of Four Cases.

Abstract 3595Poster Board III-532 BackgroundChronic Granulomatous Disease (CGD) is an inherited disease affecting innate immunity and leading to increased susceptibility to severe invasive fungal and bacterial infections. The X linked form of CGD is the most frequent and is caused by mutations in the gp91phox subunit of the NADPH oxidase complex encoding gene CYBB. McLeod syndrome patients present with late-onset neuromuscular troubles and a mild chronic haemolytic anaemia with acanthocytes. It is defined by the lack of expression of Kell antigens on erythrocytes and caused by mutations in the KX gene located close to the CYBB gene. The association of CGD and McLeod syndrome is a rare event. It can also be associated with Duchenne muscular dystrophy (DMD), retinitis pigmentosa or ornithine transcarbamylase deficiency. Patients : Four CGD patients patients were recently diagnosed with McLeod syndrome in Necker Hospital on a 2 year period of time. Their clinical and main biological characteristics are reviewed and a review of recent litterature Observations: In 1 patient, McLeod was evoked because of association of Duchenne myopathy and CGD revealed at 10 months with major hypotonia and recurrent infections. Initially, no acanthocytes were seen but Kell antigens testings led to confirmation of McLeod. Three other infants were diagnosed with CGD because of lung infections. One had lung biopsies revealing Nocardia sp. infection and received packed blood red cells. Four months after, an allo immunisation against red cells and a weakened Kell antigen expression led to McLeod diagnosis. For other patients, diagnosis was evoked because of anaemia and acanthocytes on blood smear analysis. All had confirmed by CYBB mutations with complete deletion. None had muscle weakness or retinal abnormality. For 2 infants, iron therapy and erythropoietin led to the correction of anemia. ConclusionManagement and evaluation of X-linked CGD patients should require the search of McLeod syndrome. Biological diagnosis is sometimes uneasy, especially because acanthocytes are sometimes hard to identify on blood smears. Kell antigen reactivity screening could be easy to perform. When McLeod syndrome is diagnosed, patients are included in the French national registry for rare erythrocyte phenotype to receive appropriate transfusion management. Erythropoietin therapy could be useful in preventing chronic anemia. Disclosures:No relevant conflicts of interest to declare.

Effective Hematopoietic Stem Cell Transplantation with Reduced Intensity Conditioning for Patients with Chronic Granulomatous Disease.

Abstract 3339Poster Board III-227Chronic granulomatous disease (CGD) is the most common inherited disorders of phagocytic function caused by abnormal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which produces reactive oxygen species. Defects in four of the NADPH components are responsible for CGD: gp91phox, p47phox, p67phox and p22phox. Due to the defects in production of superoxide, patients are highly susceptible to catalase-positive infections including fungi, as well as developing granuloma and autoimmune complications. To date, hematopoietic stem cell transplantation (HSCT) is the only curative treatment for patients with CGD. The HSCT for CGD patients using myeloablative conditioning regimen consisting of busulfan and cyclophosphamide (CY) has been proven to cure the disease. However, HSCT with this myeloablative conditioning regimen has considerable risk of transplantation-related morbidity (TRM) and mortality in patients with life-threatening infection and long-term complications including graft-versus host disease (GVHD), pulmonary late effects, and gonadal failure. To avoid the risk of TRM and long-term complications, we underwent bone marrow transplantation (BMT) with reduced-intensity conditioning (RIC) for 12 patients with CGD from 2002 to 2009 in Hiroshima University. Seven patients were transplanted from related donors and five patients from unrelated donors. Conditioning regimens consisted of fludarabine, CY and TBI (3 Gy) with or without antithymoglobulin or melphalan (L-PAM). Three of twelve patients who had severe life-threatening infections such as multiple brain abscesses, multiple liver abscesses or multiple pulmonary abscesses with high level of CRP (>15 mg/dl) underwent BMT. One of three patients who had suffered from severe fungal infection in both lungs was died from pulmonary hemorrhage due to the engraftment syndrome on day 45 after BMT. No TRM during the conditioning and early period after BMT was observed in the remaining 11 patients, irrespective of the presence of active and intractable infections/inflammation. The patients alive were eliminated from infections and/or inflammation with complete chimerism. Four patients undergoing BMT using fludarabine-based regimen without L-PAM required donor lymphocyte infusion to achieve complete donor chimerism. The addition of L-PAM to fludarabine-based RIC rapidly induced the complete engraftment of donor cells without any toxicity in 7 patients. The frequency and severity of acute or chronic GVHD were not significant and sufficiently tolerable. Karnofsky performance scale of all patients alive has been 100% after BMT. Furthermore, apparent endocrinological problems including gonadal function were not observed during the limited period after BMT. These results suggest HSCT using RIC with L-PAM is effective therapy for CGD patients for successful complete engraftment and minimal toxicity. DisclosuresNo relevant conflicts of interest to declare.

Chronic Granulomatous Disease: The European Experience

CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a “respiratory burst”, essential for the clearance of phagocytosed micro-organisms. CGD patients lack this mechanism, which leads to life-threatening infections and granuloma formation. However, a clear picture of the clinical course of CGD is hampered by its low prevalence (∼1∶250,000). Therefore, extensive clinical data from 429 European patients were collected and analyzed. Of these patients 351 were males and 78 were females. X-linked (XL) CGD (gp91phox deficient) accounted for 67% of the cases, autosomal recessive (AR) inheritance for 33%. AR-CGD was diagnosed later in life, and the mean survival time was significantly better in AR patients (49.6 years) than in XL CGD (37.8 years), suggesting a milder disease course in AR patients. The disease manifested itself most frequently in the lungs (66% of patients), skin (53%), lymph nodes (50%), gastrointestinal tract (48%) and liver (32%). The most frequently cultured micro-organisms per episode were Staphylococcus aureus (30%), Aspergillus spp. (26%), and Salmonella spp. (16%). Surprisingly, Pseudomonas spp. (2%) and Burkholderia cepacia (<1%) were found only sporadically. Lesions induced by inoculation with BCG occurred in 8% of the patients. Only 71% of the patients received antibiotic maintenance therapy, and 53% antifungal prophylaxis. 33% were treated with γ-interferon. 24 patients (6%) had received a stem cell transplantation. The most prominent reason of death was pneumonia and pulmonary abscess (18/84 cases), septicemia (16/84) and brain abscess (4/84). These data provide further insight in the clinical course of CGD in Europe and hopefully can help to increase awareness and optimize the treatment of these patients.

Retroviral Gene Therapy for X-Linked Chronic Granulomatous Disease: Results from Phase I/II Trial.

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency disease caused by a defect in the gp91phox gene encoding one of the subunits of the NADPH oxidase complex. NADPH oxidase plays an important role in eradicating the pathogen engulfed by the phagocytes. Therefore, CGD patients suffer from recurring life-threatening infection by bacteria or fungi, and die before 30 in most cases. In an effort to treat this life-threatening disease, we initiated a phase I/II gene therapy trial in 2007. Two X-CGD patients were enrolled in the trial. The retroviral vector used for gene delivery was the MLV-based MT vector containing gp91 phox cDNA (Yu et al., Gene Ther 2000; 7: 797, Hong et al., J Gene Med 2004; 6: 724). Viral vectors have been produced from PG13 packaging cells in compliance with GMP. The clinical protocol was approved by the Korean FDA. G-CSF mobilized peripheral blood CD34+ cells were obtained from patients, and transduced in retronectin-coated gas-permeable bags containing SCGM media supplemented with SCF, FLT3L, TPO, and IL-3. The transduction efficiency was 10.5% for patient #1 and 28.5% for patient #2 when assessed by gp91 FACS analysis. Before receiving transduced cells, patients were treated with a conditioning regimen consisting of busulfan (3.2 mg/kg/day for 2 days) and fludarabine (40 mg/m2/day for 3 days). No adverse effects were observed from the use of busulfan and fludarabine. The percentage of superoxide-producing cells, as determined by DHR assay, was 6.4% and 14.5% at day 17, and decreased to less than 0.1% (after 1 year) and 0.4% (after 7 months). Thus far, abnormal cell expansion has not been observed.

First Report of Clinical, Functional, and Molecular Investigation of Chronic Granulomatous Disease in Nine Jordanian Families

Chronic granulomatous disease is a rare inherited immunodeficiency syndrome caused by mutations in four genes encoding essential nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex components. Clinical, functional, and molecular investigations were conducted in 15 Jordanian CGD patients from nine families. Fourteen patients were children of consanguineous parents and suffered from autosomal recessive (AR) CGD forms with mutations in the CYBA, NCF1, and NCF2 genes encoding p22phox, p47phox, and p67phox proteins, except for one patient in whom the mutation's location was not found. One patient had an extremely rare X(+)CGD subtype resulting from a novel missense mutation (G1234C) in exon 10 of CYBB. We found a genetic heterogeneity in the Jordanian families with a high frequency of rare ARCGD, probably because consanguineous marriages are common in Jordan. No clear correlation between the severity of the clinical symptoms and the CGD types could be established.

ROS-deficient monocytes have aberrant gene expression that correlates with inflammatory disorders of chronic granulomatous disease

Chronic granulomatous disease is an immunodeficiency caused by an inability to produce reactive oxygen species. While the mechanism of hyper-sensitivity to infection is well understood in CGD, the basis for debilitating inflammatory disorders that arise in the absence of evident infection has not been fully explained. Herein it is demonstrated that resting and TLR-activated monocytes from individuals with CGD expressed significantly higher levels of inflammatory mediators than control cells; the expression in CGD cells resembled normal cells stimulated with lipopolysaccharide. The lack of acute illness, infection or circulating endotoxin in the blood of the CGD patients at the time of sampling was consistent with infection-free inflammation. The enhanced expression of inflammatory mediators correlated with elevated expression of NF-κB and was dependent on ERK1/2 signalling. The results are consistent with the hypothesis that ROS are anti-inflammatory mediators that control gene expression and potentially limit the development of sterile inflammatory disorders.

Genome Sequence Analysis of the Emerging Human Pathogenic Acetic Acid Bacterium Granulibacter bethesdensis

Chronic granulomatous disease (CGD) is an inherited immune deficiency characterized by increased susceptibility to infection with Staphylococcus, certain gram-negative bacteria, and fungi. Granulibacter bethesdensis, a newly described genus and species within the family Acetobacteraceae, was recently isolated from four CGD patients residing in geographically distinct locales who presented with fever and lymphadenitis. We sequenced the genome of the reference strain of Granulibacter bethesdensis, which was isolated from lymph nodes of the original patient. The genome contains 2,708,355 base pairs in a single circular chromosome, in which 2,437 putative open reading frames (ORFs) were identified, 1,470 of which share sequence similarity with ORFs in the nonpathogenic but related Gluconobacter oxydans genome. Included in the 967 ORFs that are unique to G. bethesdensis are ORFs potentially important for virulence, adherence, DNA uptake, and methanol utilization. GC% values and best BLAST analysis suggested that some of these unique ORFs were recently acquired. Comparison of G. bethesdensis to other known CGD pathogens demonstrated conservation of some putative virulence factors, suggesting possible common mechanisms involved in pathogenesis in CGD. Genotyping of the four patient isolates by use of a custom microarray demonstrated genome-wide variations in regions encoding DNA uptake systems and transcriptional regulators and in hypothetical ORFs. G. bethesdensis is a genetically diverse emerging human pathogen that may have recently acquired virulence factors new to this family of organisms.

One of three successfully treated CGD patients in a Swiss‐German gene therapy trial died due to his underlying disease

One of three successfully treated CGD patients in a Swiss‐German gene therapy trial died due to his underlying disease

Autoimmune Disease in Chronic Granulomatous Disease (CGD): A Diverse Spectrum.

CGD is an immunodeficiency caused by defects in phagocyte oxidase with increased infections. A major characteristic is extensive granuloma formation associated with infection. However, unusual autoinflammatory processes have been reported in CGD patients that may be autoimmune disease. We outline the spectrum of autoimmune disorders in CGD, making a case for these patients actually having an autoimmune disorder, rather than this being extremes of CGD granuloma formation. The most common autoimmune disease candidate in CGD is inflammatory bowel disease affecting almost 20% of CGD patients, satisfying diagnostic criteria established for Crohn's disease. X-linked CGD carriers and patients can develop discoid lupus. There are many reports about Crohn's disease and discoid lupus in CGD. We previously reported in detail 2 CGD patients with sarcoidosis (Pediatrics 2006 Mar;117(3):e590–5) making note of this here, but do not re-describe the cases. Here we present 3 additional cases of CGD patients with other unusual autoinflammatory processes responsive to immunesuppression, that extend the spectrum of autoimmune diseases observed in CGD. Patient #1: X-linked CGD patient presenting at age 14yrs with acute swelling, pain and redness of the left thigh secondary to deep venous thrombosis. Several months later while on warfarin 3·5mg/d daily with INR of 3, he developed extensive intracerebral sinus thrombosis, and subsequent further extension of clots in his left calf. A trial of oral prednisone at 0.5mg/kg/day was commenced. Within 24 hours, symptoms improved dramatically. Prednisone was tapered, with intermittent increases for symptom flares, to current maintenance of 5mg/d. Patient #2: X-linked CGD at age 9mo presented with respiratory distress and a pericardial effusion without fevers, trauma or infection. The pericardial effusion resolved with combined therapy of antibiotics and oral prednisone. 6 months later, the pericardial effusion recurred, a repeat infectious work-up was negative, and he promptly responded to oral prednisone alone. This pattern recurred every few months without precipitating events until he was 4 years of age and then had a recurrence at age 10yrs. Patient #3: Female with p47phox-deficient CGD who at age 15yrs developed arthralgias that progressed to involve elbows, knees, metacarpal-phalangeal, and proximal phalangeal joints bilaterally. Erythematous maculopapules were present on arms and forehead, with bilateral swelling in metacarpal-phalangeal and proximal interphalangeal joints, as well as development of rheumatoid nodules. A positive rheumatoid factor serology was found. Treatment with non-steroidal anti-inflammatory medications and minocycline resulted in short-lived improvement, but remission was achieved with etanercept. The 3 patients above who have not been previously reported, extend the unusual spectrum of autoimmune diseases associated with CGD. We raise the possibility that CGD may be a co-factor in the overt development of autoimmune diseases in patients with an underlying genetic predisposition, rather than this being merely extremes of the spectrum of granuloma formation specific to CGD. This represents a paradigm shift, and has significant implications for diagnosis and management. Maintaining a high index of suspicion for autoimmune disorders in patients with CGD will minimize unnecessary invasive procedures, and allows early institution of appropriate therapy reducing risks of long-term complications.

Gene Correction Reduces Cutaneous Inflammation and Granuloma Formation in Murine X-Linked Chronic Granulomatous Disease

Our laboratory previously demonstrated that X-linked chronic granulomatous disease (X-CGD) mice develop exaggerated inflammatory responses and form granulomas following intradermal challenge with sterile Aspergillus fumigatus (AF) hyphae. In this study, we examined the efficacy of retroviral-mediated gene transfer (RMGT) into X-CGD bone marrow stem cells in preventing this abnormal inflammatory response. Sterile AF or saline was injected subcutaneously into the ears of wild-type, female X-CGD carrier, X-CGD, or X-CGD mice chimeric for varying numbers of either wild-type or RMGT-corrected neutrophils. Intradermal AF induced marked inflammation at both 3 and 30 d in the X-CGD mice, but not in the carriers or the wild-type mice. Similar to wild-type mice, chimeric X-CGD mice with >20% oxidase-positive neutrophils displayed a minimal and self-limited inflammatory response. Inflammation in chimeric (both wild-type and RMGT-corrected) mice with <15% oxidase-positive neutrophils was also improved compared to X-CGD mice, although still abnormal. This is the first evidence that partial correction of NADPH oxidase activity by gene therapy is likely to be beneficial in reducing or preventing the chronic inflammatory complications of CGD patients if sufficient numbers of RMGT-corrected neutrophils are obtained.

Mobilization and transduction of CD34(+) peripheral blood stem cells in patients with X-linked chronic granulomatous disease.

As a single-gene defect in phagocytes, the X-linked form of chronic granulomatous disease (X-CGD) is a disorder potentially amenable to gene therapy by transfer of a functional copy of the gp91(phox) gene into hematopoietic stem cells (HSC). Although antimicrobial agents and interferon-gamma (IFN-gamma) have significantly improved its prognosis, CGD is still associated with high morbidity and mortality. The disease can be cured by bone marrow transplantation (BMT); however, BMT in CGD has been associated with unacceptably high rates of morbidity, mortality, and graft failure, except in very selected cases in which an HLA-identical donor is available. Prerequisites for a clinical gene therapy of CGD are an efficient mobilization of peripheral blood stem cells (PBSC) as well as the preservation of their viability and hematopoietic potential following transduction and ex vivo culture. We show that (i) mobilization and collection of CD34(+) cells after a 4-week IFN-gamma-free period by G-CSF results in sufficient numbers of cells for transplantation; (ii) the quality of collected stem cells is not altered in comparison to cells obtained from healthy volunteers as assessed by long-term culture initiating cells (LTC-IC) and progenitor cell expansion; (iii) retroviral transfer of the gp91(phox) gene under defined, serum-free conditions leads to high and stable reconstitution of the respiratory burst activity in X-CGD neutrophils derived from transduced CD34(+) progenitor and LTC-IC. Withdrawal of IFN-gamma in CGD patients may improve mobilization of CD34(+) stem cells by G-CSF. The gene transfer conditions established here are applicable to a clinical approach for gene therapy of X-CGD.

Molecular and functional characterization of a new X-linked chronic granulomatous disease variant (X91 +) case with a double missense mutation in the cytosolic gp91 phox C-terminal tail

We report here two atypical cases of X-linked CGD patients (first cousins) in which cytochrome b 558 is present at a normal level but is not functional (X91 +). The mutations were localized by single-strand conformational polymorphism of reverse transcriptase–polymerase chain reaction amplified fragments and then identified by sequence analysis. They consisted in two base substitutions (C919 to A and C923 to G), changing His303 to Asn and Pro304 to Arg in the cytosolic gp91 phox C-terminal tail. Mismatched polymerase chain reaction and genomic DNA sequencing showed that mothers had both wild-type and mutated alleles, confirming that this case was transmitted in an X-linked fashion. A normal amount of FAD was found in neutrophil membranes, both in the X91 + patients and their parents. Epstein–Barr virus-transformed B lymphocytes from the X91 + patients acidified normally upon stimulation with arachidonic acid, indicating that the mutated gp91 phox still functioned as a proton channel. A cell-free translocation assay demonstrated that the association of the cytosolic factors p47 phox and p67 phox with the membrane fraction was strongly disrupted. We concluded that residues 303 and 304 are crucial for the stable assembly of the NADPH oxidase complex and for electron transfer, but not for its proton channel activity.

Essential Requirement of Cytosolic Phospholipase A2for Stimulation of NADPH Oxidase-associated Diaphorase Activity in Granulocyte-like Cells

We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation. In this study we used this model to investigate the physiological role of cPLA(2) in regulation of NADPH oxidase-associated diaphorase activity. A novel diaphorase activity assay, using 4-iodonitrotetrazolium violet as an electron acceptor, was used in permeabilized neutrophils and PLB-985 cells differentiated toward the granulocytic or monocytic phenotypes. Phorbol 12-myristate 13-acetate, guanosine 5'-3-O- (thio)triphosphate (GTP gamma S), or FMLP stimulated a similar diphenylene iodonium-sensitive diaphorase activity pattern in neutrophils and in differentiated parent PLB-985 cells. This diaphorase activity was not detected in undifferentiated cells, but developed during differentiation. Furthermore, diaphorase activity could not be stimulated in permeabilized neutrophils from X-linked CGD patients and in differentiated gp91(phox)-targeted PLB-985 cells that lacked normal expression of gp91(phox), but was restored to these cells following transduction with retrovirus encoding gp91(phox). The differentiated PLB-D cells showed no diaphorase activity when stimulated by either GTP gamma S or FMLP, and only partial activation when stimulated with phorbol 12-myristate 13-acetate. Diaphorase activity in response to either agonists was fully restored by the addition of 10 microm free AA. The permeabilized cell 4-iodonitrotetrazolium violet reduction assay offers a unique tool for the evaluation of NADPH oxidase-associated diaphorase activity in stimulated whole cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in activation of electron transfer through the FAD reduction center of NADPH oxidase.

Chronic granulomatous disease: more than the lack of superoxide?

Chronic granulomatous disease (CGD) is an inherited disease characterized by severe and recurrent bacterial and fungal infections manifested in most cases in early childhood. Phagocytic cells of CGD patients are unable to produce superoxide anions, and their efficiency in bacterial killing is significantly impaired. Recent work has shown alterations in the electrophysiological properties of CGD granulocytes, which might contribute to the pathogenesis of the disease. The new aspects that we discuss in this review concern the proton channel function of gp91phox (the electron-transporting subunit of the NADPH oxidase) and the electrogenic activity of the active enzyme complex, which can affect the transmembrane trafficking of several ions. Based on the reviewed data, we also propose a hypothesis that the absence of a functional NADPH oxidase in CGD neutrophils could result in altered ion compositions within intracellular and intraphagosomal spaces during the process of phagocytosis.

Recombination events between the p47-phoxgene and its highly homologous pseudogenes are the main cause of autosomal recessive chronic granulomatous disease

AbstractChronic granulomatous disease (CGD) is an inherited disease caused by defects in the superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytes. Genetic lesions in any of 4 components of this antimicrobial enzyme have been detected. Family-specific mutations are found in 3 of 4 forms of CGD due to deficiencies of the gp91-phox, p22-phox, andp67-phox genes. In p47-phox–deficient CGD (autosomal recessive form A47°) patients, a GT deletion (▵GT) at the beginning of exon 2 of the p47-phox gene has been reported in 19 of 20 alleles. This GT deletion is also characteristic for the recently identified p47-phox pseudogenes. To explore a possible link between these findings, a sequence analysis of 28 unrelated, racially diverse A47° CGD patients and 37 healthy individuals was performed. The GT deletion in exon 2 was present on all alleles in 25 patients. Only 3 patients but all healthy individuals contained the GTGT and ▵GT sequences. A total of 22 patients carried additional pseudogene-specific intronic sequences on all alleles, either only in intron 1 or in intron 1 and intron 2, which lead to different types of chimeric DNA strands. It is concluded that recombination events between the p47-phox gene and its highly homologous pseudogenes result in the incorporation of ▵GT into the p47-phox gene, thereby leading to the high frequency of GT deletion in A47° CGD patients.

Uncommon missense and splice mutations and resulting biochemical phenotypes in German patients with X-linked chronic granulomatous disease

Chronic granulomatous disease is an inherited disease characterized by the inability of phagocytes to generate normal amounts of superoxide, leaving patients susceptible to opportunistic, life-threatening infections. In the majority of cases, cytochrome b 558 is absent in the X-chromosomal form of CGD. However, the neutrophils from six of nine X-linked CGD patients, reported here, expressed normal or decreased amounts of this cytochrome and are referred to as “variant” forms. In three of these six variant patients, a roughly proportional decrease in cytochrome b 558 expression and production of H 2O 2 were found. In two cases this phenotype could be well explained by special splice mutations, whereas in the third case it was caused by a missense mutation, predicting Ser 193 → Phe. In the other three variant patients, cytochrome b 558 expression and H 2O 2 production were clearly disproportionate as the generation of H 2O 2 was much more decreased than cytochrome expression. Missense mutations also were found in these cases. One of these mutations, predicting Leu 546 → Pro and affecting the putative nicotinamide adenine dinucleotide phosphate binding site, led to normal levels of cytochrome b 558 expression and reduced H 2O 2 production. In the other two mutations, predicting Pro 339 → His and His 338 → Tyr, the putative flavin adenine dinucleotide binding site was affected. This could explain the corresponding uncommon phenotypes, characterized by zero or trace amounts of H 2O 2 production and the expression of relatively high amounts of nonfunctional or low functional cytochrome b 558, respectively. The only missense mutation found that prevented the expression of any cytochrome b 558 was caused by a predicted His 222 → Arg exchange in one of the three classic cases. The two other classic phenotypes were caused by splice mutations.