Long polymerase chain reaction-based fluorescence in situ hybridization analysis of female carriers of X-linked chronic granulomatous disease deletions

Chronic granulomatous disease (CGD) is a rare inherited disorder in which antimicrobial activity of phagocytes is impaired due to the lack of reactive oxygen species, or oxidative burst, produced by NADPH oxidase. The X-linked form of CGD, representing approximately 70% of all cases, is caused by mutations in the cytochrome b beta subunit (CYBB) gene, which maps to chromosome Xp21.1. CYBB encodes the gp91-phox protein, a necessary component in the NADPH oxidase pathway. A wide variety of mutations have been identified in X-linked CGD patients, all of which lead to deletion of the functional protein and no oxidative burst activity. The mutations vary from single nucleotide substitutions to deletions of the entire gene. In this article, we report a mutation detection method for probands of female relatives at risk for carrier status of large deletions of the CYBB gene. Through fluorescent in situ hybridization of metaphase chromosomes, we were able to consistently distinguish carriers from noncarriers using polymerase chain reaction-derived, labeled DNA specific for exons 2 to 13 of the CYBB region at Xp21.1.     

Mutations of chronic granulomatous disease in Turkish families

BACKGROUND: Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by impairment of intracellular microbicidal activity of phagocytes. Mutations in one of four known NADPH-oxidase components preclude generation of superoxide and related antimicrobial oxidants, leading to the phenotype of CGD. Defects in gp91-phox, encoded by CYBB, lead to X-linked CGD and have been reported to be responsible for approximately 70% of all CGD cases. The aim of this study was to identify the CGD mutations in a group of Turkish CGD patients and to evaluate the predominance of CGD mutations as X-linked or autosomal recessive (AR) within the Turkish CGD families with known mutations. MATERIALS AND METHODS: Two Turkish CGD families were included in the study, and mutations were identified by sequence analysis of DNA and RNA from peripheral blood in the patients. Before mutation analysis, subgroup analysis of patients was made by flow cytometry with antibodies against NADPH oxidase components and with DHR-123 oxidase activity assay. For comparison, we included previously reported results from four other Turkish CGD families. RESULTS: Two different mutations were identified, one of them a novel mutation g.700G>T located in exon 7 of CYBB, and the other a hot-spot mutation located in exon 2 of the NCF1 gene. These mutations were detected in three patients from two Turkish families. CONCLUSIONS: Until now, we have altogether identified mutations in six Turkish CGD families. In this limited number of families our results show AR-CGD in two-thirds of the Turkish families investigated, in contrast to previous reports in the literature. This is probably due to the high rate of consanguineous marriages in Turkey. Consanguineous parents were found in 75% of the families with AR-CGD patients, which favours homozygous deficiencies.     

The human androgen receptor X-chromosome inactivation assay for clonality diagnostics of natural killer cell proliferations

Clonality is a frequently exploited characteristic of lymphoid malignancies. However, in the natural killer (NK) cell subset of large granular lymphocyte proliferations, clonality is difficult to prove because of the lack of specific genetic markers, such as immunoglobulin or T-cell receptor gene rearrangements. The human androgen receptor (HUMARA) assay, a polymerase chain reaction-based X-chromosome inactivation assay, is a potential diagnostic tool in these disorders. Although there is much experience with X-chromosome inactivation assays in myeloid proliferations, these assays have found only very limited application in clonality assessment of NK cell proliferations. We applied the HUMARA assay in laboratory diagnostics for detection of clonality in NK cell proliferations. We describe its test performance and report three cases in which clonality of NK cell populations was investigated by use of this assay. Our results demonstrate the usefulness of the HUMARA assay in the diagnostic workup of NK cell proliferations.     

Chronic granulomatous disease (CGD) and complete myeloperoxidase deficiency both yield strongly reduced dihydrorhodamine 123 test signals but can be easily discerned in routine testing for CGD

BACKGROUND: The flow cytometric dihydrorhodamine 123 (DHR) assay is used as a screening test for chronic granulomatous disease (CGD), but complete myeloperoxidase (MPO) deficiency can also lead to a strongly decreased DHR signal. Our aim was to devise simple laboratory methods to differentiate MPO deficiency (false positive for CGD) and NADPH oxidase abnormalities (true CGD). METHODS: We measured NADPH-oxidase and MPO activity in neutrophils from MPO-deficient patients, CGD patients, NADPH-oxidase-transfected K562 cells and cells with inhibited and substituted MPO. RESULTS: Eosinophils from MPO-deficient individuals retain eosinophilic peroxidase and therefore generate a normal DHR signal. The addition of recombinant human MPO enhances the DHR signal when simply added to a suspension of MPO-deficient cells but not when added to NADPH-oxidase-deficient (CGD) cells. Lucigenin-enhanced chemiluminescence (LCL) is increased in neutrophils from MPO-deficient patients, whereas neutrophils from patients with CGD show a decreased response. CONCLUSIONS: A false-positive result caused by MPO deficiency can be easily ascertained because, unlike cells from a CGD patient, cells from MPO-deficient patients (a) contain functionally normal eosinophils, (b) show a significant enhancement of the DHR signal following addition of rhMPO, and (c) generate a strong LCL signal.     

A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox.

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patient's neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.     

The relationship between alloimmunization and posttransfusion granulocyte survival: experience in a chronic granulomatous disease cohort

BACKGROUND: The efficacy of granulocyte transfusions in patients with HLA alloimmunization is uncertain. A flow cytometric assay using dihydrorhodamine 123 (DHR), a marker for cellular NADPH oxidase activity, was used to monitor the differential survival of transfused oxidase‐positive granulocytes in alloimmunized patients with chronic granulomatous disease (CGD). STUDY DESIGN AND METHODS: Ten patients with CGD and serious infections were treated with daily granulocyte transfusions derived from steroid and granulocyte–colony‐stimulating factor–stimulated donors. The proportion of neutrophils with intact oxidase activity was quantitated by DHR fluorescence on samples drawn before and 1 hour after transfusion. The incidence of acute transfusion reactions was correlated with the results of DHR fluorescence and biweekly HLA serologic screening assays. RESULTS: Eight of 10 patients experienced acute adverse reactions in association with granulocyte transfusions. Four had only chills and/or fever, and four experienced respiratory compromise; all eight exhibited HLA alloimmunization. Mean (±SD) oxidase‐positive cell recovery was 19.7 ± 17.4% (n = 15 transfusions) versus 0.95 ± 1.59% (n = 16) in the absence and presence of HLA allosensitization, respectively (p < 0.01). Greater than 1% in vivo recovery of DHR‐enhancing donor granulocytes was strongly correlated with lack of HLA alloimmunization. CONCLUSION: The ability to detect DHR‐positive donor granulocytes by flow cytometry is strongly correlated with absence of HLA alloimmunization and lack of acute reactions to granulocyte transfusions in patients with CGD. If HLA antibodies are present and the survival of donor granulocytes is low by DHR analysis, transfusions should be discontinued, avoiding a therapy associated with high risk and unclear benefit.     

Gene therapy for chronic granulomatous disease

Chronic granulomatous disease (CGD) is a congenital immune deficiency that is a promising therapeutic target for gene replacement into haematopoietic stem cells (HSCs). CGD results from mutations in any one of four genes encoding subunits of the superoxide-generating NADPH oxidase of phagocytes. Life-threatening, recurrent bacterial and fungal infections, as well as inflammatory granulomas, are the hallmarks of the disease. NADPH oxidase activity can be reconstituted by retroviral- or lentiviral-mediated gene transfer to human CGD marrow in vitro and in xenograft transplant models. Gene transfer studies in knockout mouse models that resemble the human disease suggest that correction of oxidase activity in a minority of phagocytes will be of clinical benefit. Phase I clinical studies in unconditioned CGD patients showed transient expression of small numbers of gene-corrected neutrophils. Areas of research at present include efforts to enhance gene transfer rates into repopulating HSCs using vectors that transduce quiescent cells, and to increase the engraftment of genetically corrected HSCs using non-myeloablative conditioning and drug resistance genes for selection.     

Gene therapy for chronic granulomatous disease

Chronic granulomatous disease (CGD) is a congenital immune deficiency that is a promising therapeutic target for gene replacement into haematopoietic stem cells (HSCs). CGD results from mutations in any one of four genes encoding subunits of the superoxide-generating NADPH oxidase of phagocytes. Life-threatening, recurrent bacterial and fungal infections, as well as inflammatory granulomas, are the hallmarks of the disease. NADPH oxidase activity can be reconstituted by retroviral- or lentiviral-mediated gene transfer to human CGD marrow in vitro and in xenograft transplant models. Gene transfer studies in knockout mouse models that resemble the human disease suggest that correction of oxidase activity in a minority of phagocytes will be of clinical benefit. Phase I clinical studies in unconditioned CGD patients showed transient expression of small numbers of gene-corrected neutrophils. Areas of research at present include efforts to enhance gene transfer rates into repopulating HSCs using vectors that transduce quiescent cells, and to increase the engraftment of genetically corrected HSCs using non-myeloablative conditioning and drug resistance genes for selection.     

Six different CYBA mutations including three novel mutations in ten families from Turkey, resulting in autosomal recessive chronic granulomatous disease

Background  One of the rarest forms of autosomal recessive chronic granulomatous disease (AR-CGD) is attributable to mutations in the CYBA gene, which encodes the alpha polypeptide of cytochrome b ₅₅₈, (also known as p22- phox ), a key transmembrane protein in the phagocyte NADPH oxidase system. This gene is localized on chromosome 16q24, encompasses 8·5 kb and contains six exons.
Materials and methods  We report here the clinical and molecular characterization of 12 AR-CGD patients from 10 consanguineous, unrelated Turkish families with clinical CGD and positive family history. The ages of the six male and six female patients were between 1and 18 years. Before mutation analysis, subgroup analysis of patients was made by flow cytometry with antibodies against NADPH-oxidase components and with the DHR assay (flow cytometric assay of NADPH oxidase activity in leucocytes).
Results  Mutation analysis of CYBA showed six different mutations: a frameshift insertion in exon 3 (C after C166); a missense mutation in exon 2 (p.Gly24Arg), a splice-site deletion in intron 1 (4-bp deletion +4_+7 AGTG), a novel nonsense mutation in exon 6 (p.Cys113X), a novel large deletion of exons 3–6 and a novel 1-bp deletion in exon 6 (c.408delC). All mutations were present in homozygous form and all parents investigated were found to be heterozygotes for these mutations.
Conclusions  In our series of 40 CGD families, approximately 25% of the families have p22- phox defects, with six different mutations, including three novel mutations. The high rate of consanguineous marriages seems to be the underlying aetiology.     

A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease.

A membrane-bound cytochrome b, a heterodimer formed by a 91-kD glycoprotein and a 22-kD polypeptide, is a critical component of the phagocyte NADPH-oxidase responsible for the generation of superoxide anion. Mutations in the gene for the 91-kD chain of this cytochrome result in the X-linked form of chronic granulomatous disease (CGD), in which phagocytes are unable to produce superoxide. Typically, there is a marked deficiency of the 91-kD subunit and the cytochrome spectrum is absent (X- CGD). In a variant form of CGD with X-linked inheritance, affected males have a normal visible absorbance spectrum of cytochrome b, yet fail to generate superoxide (X+ CGD). The size and abundance of the mRNA for the 91-kD subunit and its encoded protein were examined and appeared normal. To search for a putative mutation in the coding sequence of the 91-kD subunit gene, the corresponding RNA from an affected X+ male was amplified by the polymerase chain reaction and sequenced. A single nucleotide change, a C----A transversion, was identified that predicts a nonconservative Pro----His substitution at residue 415 of the encoded protein. Hybridization of amplified genomic DNA with allele-specific oligonucleotide probes demonstrated the mutation to be specific to affected X+ males and the carrier state. These results strengthen the concept that all X-linked CGD relates to mutations affecting the expression or structure of the 91-kD cytochrome b subunit. The mechanism by which the Pro 415----His mutation renders the oxidase nonfunctional is unknown, but may involve an impaired interaction with other components of the oxidase.     

Use of an X-linked human neutrophil marker to estimate timing of lyonization and size of the dividing stem cell pool.

In families with X-linked chronic granulomatous disease (CGD), heterozygous females have two stable populations of polymorphonuclear leukocytes (PMN) in their blood; one normal, the other, deficient in oxygen metabolism. The two types of PMN can be distinguished by the ability or lack of ability to reduce nitroblue tetrazolium dye. The variation in the percent normal PMN among 11 CGD heterozygotes was shown to follow a binomial distribution based on eight independent trials and a chance of success of 50%. This is consistent with the occurrence of X-chromosome inactivation (lyonization) when eight embryonic founder cells for the hematopoietic system are present. Serial determinations of the percent normal PMN in individual heterozygotes showed very limited variability (standard deviations ranged from 2.0% to 5.2%) most of which could be ascribed to experimental error. An estimate of the remaining variation (residual variance) was introduced into a well-known formula to calculate the appropriate number of pluripotent stem cells necessary to support hematopoiesis and a figure exceeding 400 was obtained. Thus, the data indicate that in humans there is a highly polyclonal system of hematopoiesis.     

NADPH oxidase deficiency in X-linked chronic granulomatous disease.

We measured the cyanide-insensitive pyridine nucleotide oxidase activity of fractionated resting and phagocytic neutrophils from 11 normal donors, 1 patient with hereditary deficiency of myeloperoxidase, and 7 patients with X-linked chronic granulomatous disease (CGD). When measured under optimal conditions (at pH 5.5 and in the presence of 0.5 mM Mn++), NADPH oxidase activity increased fourfold with phagocytosis and was six-fold higher than with NADH. Phagocytic neutrophils from patients with CGD were markedly deficient in NADPH oxidase activity.     

Missense mutation in exon 7 of the common gamma chain gene causes a moderate form of X-linked combined immunodeficiency.

Clinical and immunologic features of a recently recognized X-linked combined immunodeficiency disease (XCID) suggested that XCID and X-linked severe combined immunodeficiency (XSCID) might arise from different genetic defects. The recent discovery of mutations in the common gamma chain (gamma c) gene, a constituent of several cytokine receptors, in XSCID provided an opportunity to test directly whether a previously unrecognized mutation in this same gene was responsible for XCID. The status of X chromosome inactivation in blood leukocytes from obligate carriers of XCID was determined from the polymorphic, short tandem repeats (CAG), in the androgen receptor gene, which also contains a methylation-sensitive HpaII site. As in XSCID, X-chromosome inactivation in obligate carriers of XCID was nonrandom in T and B lymphocytes. In addition, X chromosome inactivation in PMNs was variable. Findings from this analysis prompted sequencing of the gamma c gene in this pedigree. A missense mutation in the region coding for the cytoplasmic portion of the gamma c gene was found in three affected males but not in a normal brother. Therefore, this point mutation in the gamma c gene leads to a less severe degree of deficiency in cellular and humoral immunity than that seen in XSCID.     

Restitution of superoxide generation in autosomal cytochrome-negative chronic granulomatous disease (A22(0) CGD)-derived B lymphocyte cell lines by transfection with p22phax cDNA

The respiratory burst oxidase of phagocytes and B lymphocytes is a multicomponent enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2-production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or physiologic stimuli, such as phagocytosis in phagocytes or cross- linking of surface immunoglobulin in B lymphocytes. The activity of this enzyme is greatly diminished or absent in patients with chronic granulomatous disease (CGD), an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every CGD patient studied so far, an abnormality has been found in a gene encoding one of the four components of the respiratory burst oxidase: the membrane proteins p22phox or gp91phox which together form the cytochrome b558 protein, or the cytosolic proteins p47phox or p67phox. Autosomal recessive cytochrome-negative CGD (A22(0) CGD) is associated with mutations in the gene coding for p22phox. We report here that the capacity for O2- production and cytochrome b558 protein expression were restored to Epstein-Barr virus-transformed B lymphocytes from two A22(0) CGD patients by transfection with an expression plasmid containing a p22phox cDNA. No detectable O2- was generated by untransfected p22phox-deficient lymphocytes. The genetic reconstitution of the respiratory burst in A22(0) CGD B lymphocytes by transfer of the wild-type p22phox cDNA represents a further step towards somatic gene therapy for this subgroup of A22(0) CGD. This system will also be useful for expression of genetically engineered mutant p22phox proteins in intact cells, facilitating the structure-function analysis of cytochrome b558.     

Increased phagocytic activity of polymorphonuclear leukocytes of chronic granulomatous disease as determined with flow cytometric assay.

Using flow cytometry, we compared the phagocytic activity of polymorphonuclear leukocytes (PMNs) from healthy donors with that of PMNs from 10 patients with chronic granulomatous disease (CGD), eight carriers of X-linked CGD, and one patient with myeloperoxidase deficiency. Ingestion of fluorescent bacteria by CGD and myeloperoxidase-deficient PMNs was significantly increased, that is, about 1.5 times that of normal controls. In CGD carriers, two PMN populations were found: one population consisted of PMNs with enhanced phagocytosis, and the other consisted of PMNs with normal phagocytic activity. With a two-dimensional analysis, we also demonstrated in CGD carriers that phagocytosis of the PMNs that failed to generate hydrogen peroxide was significantly elevated, as was phagocytosis of PMNs in patients with CGD, while on the other hand, the PMNs that normally produced hydrogen peroxide exhibited a normal phagocytic activity. When sodium azide, an inhibitor of myeloperoxidase and catalase, was added to control and carrier PMNs, phagocytic activity was significantly increased, p less than 0.01 in both, but not to the level seen with CGD PMNs. Phagocytosis of CGD and myeloperoxidase-deficient PMNs, however, remained unchanged by the azide treatment. On the basis of above findings, we speculate that phagocytosis of CGD PMNs is increased because the H2O2-myeloperoxidase-halide system, which may modulate phagocytic activity of PMNs, fails to operate.     

Virulence of catalase-deficient aspergillus nidulans in p47(phox)-/- mice. Implications for fungal pathogenicity and host defense in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare genetic disorder in which phagocytes fail to produce superoxide because of defects in one of several components of the NADPH oxidase complex. As a result, patients develop recurrent life-threatening bacterial and fungal infections. The organisms to which CGD patients are most susceptible produce catalase, regarded as an important factor for microbial pathogenicity in CGD. To test the role of pathogen-derived catalase in CGD directly, we have generated isogenic strains of Aspergillus nidulans in which one or both of the catalase genes (catA and catB), have been deleted. We hypothesized that catalase negative mutants would be less virulent than the wild-type strain in experimental animal models. CGD mice were produced by disruption of the p47(phox) gene which encodes the 47-kD subunit of the NADPH oxidase. Wild-type A. nidulans inoculated intranasally caused fatal infection in CGD mice, but did not cause disease in wild-type littermates. Surprisingly, wild-type A. nidulans and the catA, catB, and catA/catB mutants were equally virulent in CGD mice. Histopathological studies of fatally infected CGD mice showed widely distributed lesions in the lungs regardless of the presence or absence of the catA and catB genes. Similar to the CGD model, catalase-deficient A. nidulans was highly virulent in cortisone-treated BALB/c mice. Taken together, these results indicate that catalases do not play a significant role in pathogenicity of A. nidulans in p47(phox)-/- mice, and therefore raise doubt about the central role of catalases as a fungal virulence factor in CGD.     

Four different NCF2 mutations in six families from Turkey and an overview of NCF2 gene mutations

Background  One of the rarest forms of autosomal recessive chronic granulomatous disease (AR-CGD) is attributable to mutations in the NCF2 gene, which encodes the polypeptide p67^phox, a key cytoplasmic protein in the phagocyte NADPH oxidase system. NCF2 is localized on chromosome 1q25, encompasses 40 kb and contains 16 exons.
Materials and methods  We report here the clinical and molecular characterization of six patients with CGD from six consanguineous Turkish families. The ages of the five female patients were between 3 and 22 years and a male patient was 2 years old; all patients showed clear clinical symptoms of CGD.
Results  The mothers of the patients did not show a bimodal histogram pattern specific for X-CGD in the dihydrorhodamine-1,2,3 (DHR) assay. Moreover, p67^phox protein expression was not detectable using flow cytometric analysis of the patients' neutrophils except in those from patient 6, which had a diminished expression. Mutation analysis of NCF2 revealed four different homozygous mutations: a novel nonsense mutation in exon 3 c.229C>T, p.Arg77X; a novel missense mutation in exon 4 c.279C>G, p.Asp93Glu; a nonsense mutation in exon 4 c.304C>T, p.Arg102X; and a novel missense mutation in exon 6 c.605C>T, p.Ala202Val. The parents were found to be heterozygotes for these mutations.
Conclusions  The prevalence of NCF2 mutant families is approximately 15% in our series of 40 CGD families. This high incidence of A67 CGD in Turkey is undoubtedly caused by the high incidence of consanguineous marriages. We found three new mutations in NCF2 and one previously described. These are presented together with an overview of all NCF2 mutations now known.     

Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency: difficulties in the diagnosis

ObjectivesTo search for biochemical and molecular markers for the diagnosis of patients and carriers with 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency.Design and methodsOrganic acids in urine, MHBD activity in fibroblasts, immunoblotting and molecular studies were performed in seven patients. Seven carriers were also studied.ResultsUnder low protein diet or poor feeding all the patients showed only a slightly altered organic acid profile. Measurement of 2-methyl-3-hydroxybutyric acid and tiglylglycine after an isoleucine loading test, failed to demonstrate the carrier status of one patient. However, measurement of 2-ethylhydracrylic acid (EHA) was positive in all the carriers tested. MHBD activity was clearly deficient in males and in one female patient. We identified four missense mutations, two of them were novel.ConclusionsQuantification of EHA may be of help for the diagnosis of the heterozygous condition. The carrier females showed the classical biochemical variability of X-linked diseases due to random X-chromosome inactivation.     

A skewed view of X chromosome inactivation

X chromosome inactivation involves a random choice to silence either X chromosome early in mammalian female development. Once silenced the inactive X is stably inherited through subsequent somatic cell divisions, and thus, females are generally mosaics, having a mixture of cells with one or the other parental X active. While in most females the number of cells with either X being active is roughly equal, skewing of X chromosome inactivation is observed in a percentage of women. In this issue of the JCI, Bolduc and colleagues address whether skewing of X chromosome inactivation in humans is influenced by an X-linked locus that can alter this initial random inactivation (see the related article beginning on page 333). Their data indicate that most of the skewing observed in humans results from secondary events rather than being due to an inherited tendency to inactivate a particular X chromosome.