X-linked Chronic Granulomatous Disease Patient Research Articles

Inborn Errors of Immunity With Fetal or Perinatal Clinical Manifestations.

In this article we revised the literature on Inborn Errors of Immunity (IEI) keeping our focus on those diseases presenting with intrauterine or perinatal clinical manifestations. We opted to describe our findings according to the IEI categories established by the International Union of Immunological Societies, predominantly addressing the immunological features of each condition or group of diseases. The main finding is that such precocious manifestations are largely concentrated in the group of primary immune regulatory disorders (PIRDs) and not in the group of classical immunodeficiencies. The IEI categories with higher number of immunological manifestations in utero or in perinatal period are: (i) diseases of immune dysregulation (HLH, IPEX and other Tregopathies, autosomal recessive ALPS with complete lack of FAS protein expression) and (ii) autoinflammatory diseases (NOMID/CINCA, DIRA and some interferonopathies, such as Aicardi-Goutières syndrome, AGS, and USP18 deficiency). Regarding the other IEI categories, some patients with Omenn syndrome (an atypical form of SCID), and a few X-linked CGD patients present with clinical manifestations at birth associated to immune dysregulation. The most frequent clinical features were hydrops fetalis, intrauterine growth retardation leading to fetal loss, stillbirths, and prematurity, as in HLH and IPEX. Additionally, pseudo-TORCH syndrome was observed in AGS and in USP18 deficiency. The main goal of our review was to contribute to increasing the medical awareness of IEI with intrauterine and perinatal onset, which has obvious implications for diagnosis, treatment, and genetic counseling.

High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

557. Targeted CYBB Minigene Insertion into the CYBB Locus for Correction of X-CGD iPSCs Requires Intronic Elements for Expression

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency characterized by defective phagocyte production of microbicidal reactive oxygen species (ROS), resulting in recurring, life-threatening infections and hyper-inflammation. Mutations causing X-CGD span the entire 13 exons or intronic splice sites of the >30-kb CYBB gene encoding gp91phox, resulting in a loss of gp91phox protein expression. We previously tested a TALEN-mediated targeted gene therapy approach to insert a codon-optimized CYBB minigene into the start site of endogenous CYBB. Although targeted insertion into the endogenous start site was achieved in X-CGD patient iPSCs, little or no gp91phox expression or ROS activity was observed upon granulocyte differentiation, suggesting that downstream intronic or regulatory elements may be necessary for efficient gene expression from the CYBB promoter. To test this hypothesis, we tested CRISPR-mediated targeted insertion of a codon-optimized CYBB cDNA consisting of exons 2 through 13 (CYBB2-13) together with a puromycin-resistance gene cassette into exon 2 of the CYBB locus. In iPSCs from X-CGD patients with a CYBB mutation in exon 5, exon 7, or intron 10, the efficiency of targeted insertion of the CYBB2-13 plasmid donor without random inserts in puromycin-selected clones was 50-66%. Upon granulocyte differentiation of CYBB2-13 corrected X-CGD iPSCs, gp91phox expression and ROS production were restored to levels 64-100% (gp91phox) and 68-76% (DHR) of normal healthy donor controls. As expected for expression from the endogenous CYBB promoter, expression of gp91phox was specific to CD13+ granulocytes, and was undetected in undifferentiated iPSCs. This targeted gene therapy approach should allow correction of ~90% of X-CGD patient mutations (those involving mutations in exons 2 through 13), to restore ROS activity while maintaining normal regulation of CYBB expression. Further, these findings demonstrate a key issue for the design of targeted gene insertion to capture expression from an endogenous promoter: for some endogenous promoters, the inclusion of intronic elements is necessary for efficient expression of the insert.

298. Transfection of Neutrophils from X-Linked Chronic Granulomatous Disease Patients with gp91phox mRNA Restores Oxidase Activity with High Efficiency and Viability

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to defective phagocyte NADPH oxidase required for production of antimicrobial oxidants. Fungal or bacterial infections in CGD may persist despite intensive and prolonged antibiotic therapy. In settings where conventional antibiotic therapy and/or surgical debridement do not control infection, use of allogeneic unmatched donor granulocyte transfusions has been therapeutic. However, CGD patients treated with granulocyte transfusions may develop high titers of anti-HLA antibodies, restricting use of allogeneic granulocyte transfusions and further complicating potentially curative bone marrow transplant. Using the CGD patient as an autologous donor for granulocyte transfusions would be a helpful solution.Here we demonstrate highly efficient functional correction of the functional defect in CGD by transfection of patient's own granulocytes with mRNA while retaining viability using a scalable, cGMP-compliant MaxCyte® GT™ electroporation system capable of handling clinically relevant numbers of cells (>1010cells).Freshly collected granulocytes from normal volunteers were electroporated using the MaxCyte electroporation system with eGFP-mRNA on the day of collection. Flow cytometry of demonstrated that >90% of neutrophils expressed eGFP with viability exceeding 90% at 24 hrs after electroporation. Phagocyte NADPH oxidase activity of the normal neutrophils was retained as well as bactericidal activity against Burkolderia cepacia, a CGD bacterial pathogen. Antibacterial activity of electroporated granulocytes was comparable to unmanipulated granulocytes confirming retention of cellular function following the electroporation. Finally, electroporated eGFP positive human neutrophils injected into CGD mice were shown to remain in circulation as well as migrate to the peritoneal cavity following thioglycollate IP injection and there were no adverse effects observed in the mice.Electroporation of gp91phox mRNA into human X-linked CGD (gp91phox-deficient) patient neutrophils resulted in expression of gp91phox protein in >70% of cells with >90% viability at 24 hours after treatment. Using the dihydrorhodamine assay of phagocyte NADPH oxidase activity, >70% of neutrophils remained functionally corrected 2 days after transfection. Other studies of cellular function in vitro and in vivo with electroporated corrected CGD neutrophils are in progress. In summary, we show the feasibility of restoring the function of CGD patient-autologous neutrophils via electroporation of mRNA encoding the defective gene product. This approach may have potential clinical utility in the management of severe chronic bacterial and fungal infections in CGD but also short-term correction of other hematopoietic gene diseases.

Successful Unrelated Cord Blood Stem Cell Transplantation in an X-linked Chronic Granulomatous Disease Patient with Disseminated BCG-induced Infection

A 19-month-old boy with chronic granulomatous disease (CGD) received umbilical cord blood transplantation (UCBT) from an unrelated donor after experiencing a life-threatening disseminated Bacillus Calmette-Guérin infection. After busulfan and cyclophosphamide conditioning, we performed a 5/6-matched UCBT. Engraftment and mixed chimerism was 100% in peripheral blood, and 100% of his neutrophils had normal oxidative burst activity on day 17. The patient is now 3 years old, free from infection, and growing well. To our knowledge, this is the second case of CGD treated with UCBT in Taiwan. His successful outcome illustrates that UCBT in a patient with CGD should be considered early if a human leukocyte antigen-matched donor is not available or the patient has just recovered from a severe infection.

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.

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.

671. SIV-Derived Lentiviral Vectors Pseudotyped with Modified RD114 Envelope and Containing Mutant MGMT Efficiently Transduce Human CD34+ PBSCs and Allow Selection

Lentiviral vectors have become an attractive tool for gene transfer into hematopoietic stem cells (HSCs) because they may more efficiently transduce non-dividing multipotent HSCs. Most lentiviral gene transfer studies use human immunodeficiency virus (HIV) 1-derived vectors pseudotyped with VSV-G envelope. We investigated the ability of vectors based on simian immunodeficiency virus (SIVmac) and pseudotyped with modified RD114 envelope to efficiently transduce human HSCs using both marker constructs and a construct containing the therapeutic gene for X-linked chronic granulomatous disease (CGD) gp91phox. RD114-pseudotyped SIV vector particles transiently produced following transfection of 293T HEK cells were concentrated by high-speed centrifugation. The marker gene construct encoded a green fluorescent protein-mutant methylguanine methyltransferase fusion protein (GFP-MGMT|[ast]|) which confers resistance to O6-benzylguanine (BG) and a DNA alkylating agent such as 1,3-bis (2-chloroethyl)-1-nitrosurea (BCNU) and retains green fluorescence. Mobilized healthy human CD34+ peripheral blood stem cells (PBSCs) were efficiently transduced (>90% GFP+ on day 4 of culture) and could be selected with BG/ BCNU ex vivo on day 4 of culture (1 hour 5 |[mu]|M BG followed by 2 hours 7 |[mu]|M BCNU) to increase stable ex vivo marking to 97%. When non-selected transduced PBSCs were transplanted into NOD/ SCID mice, marking of human cells at 6 weeks post transplant was consistently greater than 50%. In vivo selection of these animals is ongoing. We next created an IRES bicistronic SIV modified RD114 pseudotyped vector encoding both gp91phox and MGMT|[ast]|, and demonstrated that we could transduce gp91phox-deficient X-linked CGD patient CD34+ PBSCs to a level of 30% ex vivo and this could be increased in culture to 55% following BG/BCNU selection at day 4. Transplant of the unselected cells into NOD/SCID mice resulted in 2.2% marking of the engrafted CGD human myeloid cells and using the ex vivo selected cells marking was 5.1%. Thus, while the therapeutic selective vector did perform, we find that it is more difficult to obtain high pre-concentration titers of this larger bicistronic construct. However, preliminary experiments using significantly higher concentrations of centrifuged vector indicate that low starting titers can be augmented by concentration to achieve much higher transduction rates similar to those seen with the marker vectors and these experiments are ongoing.

Gene expression profiling provides insight into the pathophysiology of chronic granulomatous disease.

Human polymorphonuclear leukocytes (PMNs or neutrophils) kill invading microorganisms with reactive oxygen species (ROS) and cytotoxic granule components. PMNs from individuals with X-linked chronic granulomatous disease (XCGD) do not produce ROS, thereby rendering these individuals more susceptible to infection. In addition, XCGD patients develop tissue granulomas that obstruct vital organs, the mechanism(s) for which are unknown. To gain insight into the molecular processes that contribute to the pathophysiology of XCGD, including formation of granulomas, we compared global gene expression in PMNs from XCGD patients and healthy control individuals. Genes encoding mediators of inflammation and host defense, including CD11c, CD14, CD54, FcgammaR1, FcalphaR, CD120b, TLR5, IL-4R, CCR1, p47(phox), p40(phox), IL-8, CXCL1, Nramp1, and calgranulins A and B, were up-regulated constitutively in unstimulated XCGD patient PMNs. By comparing transcript levels in normal and XCGD PMNs after phagocytosis, we discovered 206 genes whose expression changed in the presence and the absence of ROS, respectively. Notably, altered Bcl2-associated X protein synthesis accompanied defective neutrophil apoptosis in XCGD patients. We hypothesize that granuloma formation in XCGD patients reflects both increased proinflammatory activity and defective PMN apoptosis, and we conclude that ROS contribute directly or indirectly to the resolution of the inflammatory response by influencing PMN gene transcription.

Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91.

Retroviral transduction of human hematopoietic stem cells is an attractive strategy in gene therapy; however, transduction efficiency and duration of transgene expression may not be satisfactory in current protocols. Co-expression of a human multidrug resistance gene (MDR1) with a therapeutic gene affords selectable growth advantage to genetically modified cells. A bicistronic retrovirus vector, Ha-MDR-IRES-gp91, was constructed for the co-expression of MDR1 and gp91, a gene responsible for X-linked chronic granulomatous disease (X-CGD). Drug-selected co-expression of P-glycoprotein and gp91 was evaluated in transduced cells. Epstein-Barr virus-transformed B cells from X-CGD patients transduced with Ha-MDR-IRES-gp91 co-expressed human P-glycoprotein and gp91, and acquired superoxide-generating activity. Human CD34-positive cells from an X-CGD patient were transduced with Ha-MDR-IRES-gp91 and subsequently treated with 2 ng/ml vincristine. After 13 days, 20% of Ha-MDR-IRES-gp91-transduced cells were P-glycoprotein- and gp91-positive by FACS analysis. The superoxide-generating activity of the transduced population was 27% of that of normal cells. Mice transplanted with Ha-MDR-IRES-gp91-transduced bone marrow cells showed co-expression of P-glycoprotein and gp91 in peripheral blood mononuclear cells. By administering paclitaxel, the proportions of P-glycoprotein- and gp91-positive cells were increased in all the four mice examined. When mice transplanted with Ha-MDR-IRES-gp91-transduced cells were repeatedly administered paclitaxel, the ratios of P-glycoprotein- and gp91-positive cells were maintained for over 1 year. These results suggest that MDR1-bicistronic vectors may be useful to select the transduced hematopoietic cells in vivo. This may lead to the sustained expression of transgenes in the blood cells of patients treated with stem cell gene therapy.

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.

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.

A Variant X-linked Chronic Granulomatous Disease Patient (X91+) with Partially Functional Cytochrome b

Genetic analysis of a patient with the variant cytochrome b-245-positive form of chronic granulomatous disease revealed a missense mutation resulting in a Arg54-->Ser substitution in the gp91phox subunit of cytochrome b-245. As a consequence, although no O2- is made, NADPH oxidase-associated FAD accepts electrons from NADPH in the cell-free activation system and becomes reduced. The reduced flavin exhibits normal levels of iodonitrotetrazolium violet diaphorase activity, and the patient's neutrophils exhibit high levels of intracellular oxidant production and show a low level of NBT staining in the NBT slide test. Thus, this mutation appears to render the heme center of NADPH oxidase present but nonfunctional, while leaving the flavin center fully functional.

Point mutations in the beta-subunit of cytochrome b558 leading to X- linked chronic granulomatous disease

The NADPH:O2 oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b558 is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb- CGD) the phagocytes are defective in the beta-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b558 with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N-terminal half of the protein, suggesting that this part of cytochrome b558 is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron.

Point Mutations in the β-Subunit of Cytochrome b558 Leading to X-Linked Chronic Granulomatous Disease

Abstract The NADPH:O2 oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b558 is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb- CGD) the phagocytes are defective in the beta-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b558 with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N-terminal half of the protein, suggesting that this part of cytochrome b558 is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron.

Phorbol myristate acetate induced oxidation of 2',7'-dichlorofluorescin by neutrophils from patients with chronic granulomatous disease.

The oxidative metabolic burst of stimulated human polymorphonuclear leukocytes (PMNs) has been evaluated by the measurement of oxygen consumption, chemiluminescence, and oxygen radicals (O2-, H2O2, OH-) derived from activation of the hexose monophosphate shunt (HMPS). PMNs from patients with chronic granulomatous disease (CGD) are shown to lack functional NADPH oxidase and undetectable oxygen radical generation. However, using single cell analysis by flow cytometry and 2',7'-dichlorofluorescin (DCFH) oxidation by H2O2, significant DCFH oxidation by the PMA stimulated CGD PMNs was observed. Furthermore, 1mM potassium cyanide enhanced DCFH oxidation by control and CGD PMNs. DCFH oxidation by cells from an obligate heterozygous mother of an X-linked CGD patient was intermediate. These observations suggest that a PMA induced oxidase enzyme is present in CGD cells.

Complementation in monocyte hybrids revealing genetic heterogeneity in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare syndrome, found predominantly in male children and characterized by life-threatening, recurrent infections. The superoxide (O2-)/hydrogen peroxide (H2O2) generating system in the granulocytes and monocytes of CGD patients is completely defective. Furthermore, a novel type of cytochrome b, detected by the optical spectrum of phagocytes from healthy subjects, is lacking in those of most male CGD patients. In female CGD patients, the cytochrome b is present, but cannot, as in normal cells, be reduced on metabolic stimulation of the phagocytes in anaerobic conditions. Here, to demonstrate the importance of cytochrome b in this system and to investigate the genetic background of the various forms of CGD, we have hybridized monocytes from a cytochrome b negative, X-linked male CGD patient with monocytes from a cytochrome b positive, male CGD patient with unknown genetic background. Monocytes were used because they are the only blood phagocytes that show an active protein synthesis, whereas fibroblasts or lymphocytes do not express the O2-/H2O2 generating system. The heterologous hybrids were positive in the nitroblue tetrazolium (NBT) slide test, indicating the complementation of the O2-/H2O2 generating system, whereas the homologous hybrids remained negative, as did the non-fused cells of these patients. We thus conclude that cytochrome b is part of the O2-/H2O2 generating system and that somatic cell hybridization experiments with monocytes provide a means of studying the genetic background of CGD patients. We believe this to be the first report of genetic complementation by somatic cell hybridization experiments using monocytes instead of fibroblasts.