Background Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune sys... more 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.
To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking fo... more To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking for known and possible CGD cases was mailed to paediatric, internal medicine and infectious disease departments all over Sweden. The detected patients were characterized as to genetics and the clinical presentation. Twenty–one patients (belonging to 16 different families) were found, corresponding to a prevalence of ∼ 1/450 000 individuals. The patients with X–linked disease, lacking a functional gp91phox protein (n= 12), comprised 57% and 43% of the patients had an autosomal recessive (AR) disease lacking p47phox (n= 7) or p67phox (n=1), respectively. All unrelated patients with X–linked disease displayed different gene abnormalities such as point mutations predicting nonsense (n= 3), missense (n= 1) or splice site mutations (n = 2), but also a total deletion and a unique 40 base pair duplicature insertion. The patients with p47phox–deficiency showed a GT deletion at a GTGT tandem repeat, and the p67phox–deficient patient displayed a heterozygous in–frame deletion of AAG combined with a large deletion in the other allele. Three patients died during the study period, two from Pseudomonas cepacia infections. Patients with X–linked disease had more frequent infections (mean of 1.7 per year), than the patients with AR inheritance (0.5 infections per year). The most common infections were dermal abscesses (n=111), followed by lymphadenitis (n=82) and pneumonias (n=73). Inflammatory bowel disease–like symptoms, mimicking Crohn's disease of the colon, was seen in three CGD patients.
Background Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune sys... more 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 the 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, responsible for approximately 70% of all CGD cases. The aim of the study was to evaluate the hypothesis that age-related skewing of X-chromosome inactivation, as described in several CGD families, is caused by preferential survival of bone marrow clones with an inactive NADPH oxidase.Materials and methods We studied the neutrophils from three patients and four carriers in three generations of a Turkish family with X-linked CGD. Carrier detection was carried out by the dihydrorhodamine (DHR)-1,2,3 assay, which measures on a per-cell basis the NADPH oxidase-dependent oxidation of DHR by phagocytes. The X-chromosome inactivation pattern was determined with the HUMARA assay in DNA from leucocytes as well as in DNA from a buccal smear of the four carriers.Results The three patients were identified by a negative DHR test, and the mutation in their CYBB gene was characterized by DNA sequencing. Moreover, we found an age-related degree of skewing of X-chromosome inactivation in the leucocytes of the four X-CGD carriers, both at the protein level (NADPH oxidase activity) and at the DNA level (HUMARA assay). However, similar skewing of X-chromosome inactivation was found in the buccal DNA from these women.Conclusions These novel findings indicate that the age-related degree of skewing was probably a chance finding, not related to preferential survival of NADPH oxidase-deficient precursor cells, because this enzyme is not expressed in (buccal) epithelial cells.
In the X-linked form of chronic granulomatous disease (X91° CGD), the genetic defect is linked to... more In the X-linked form of chronic granulomatous disease (X91° CGD), the genetic defect is linked to the CYBB locus on the X chromosome. We studied a family with a genetic defect in this gene, consisting of a G → A substitution at the fifth base of the 5′ donor splice site of intron 3. This mutation leads to skipping of exon 3 after transcription of the gene. The expectant mother was diagnosed as a carrier. Analysis of polymerase chain reaction (PCR)-amplified genomic DNA from a chorionic villus biopsy (CVB) showed the same mutation in the male fetus. After termination of the pregnancy, the diagnosis was confirmed by conventional methods. This is the first time that PCR has been used for prenatal diagnosis of CGD.
Chronic granulomatous Disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 ... more Chronic granulomatous Disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is essential in the process of intracellular pathogen killing by phagocytic leukocytes. Four of the five genes involved in CGD are autosomal; these are CYBA, encoding p22-phox, NCF2, encoding p67-phox, NCF1, encoding p47-phox, and NCF4, encoding p40-phox. This article lists all mutations identified in these genes in the autosomal forms of CGD. Moreover, polymorphisms in these genes are also given, which should facilitate the recognition of future disease-causing mutations.
Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 ... more Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations.
Chronic granulomatous disease (CGD) is an innate immunodeficiency due to a genetic defect in one ... more Chronic granulomatous disease (CGD) is an innate immunodeficiency due to a genetic defect in one of the NADPH-oxidase components. In the course of 21 years, 38 Israeli CGD patients were diagnosed with 17 gene mutations, seven of which were new. Clinical, functional, and molecular studies were accomplished. Although X-linked recessive (XLR)-CGD is worldwide the most common genotype of the disease (~ 70%), in our study only 11 patients (29%) suffered from XLR-CGD. In Israel, the higher incidence of the autosomal recessive (AR) form of CGD (63%) may be related to consanguineous marriages. In three patients (8%), all four proteins of the NADPH oxidase were present. Severe clinical expression was found both in the XLR and AR forms, but in general a milder disease was evident in AR-CGD, particularly in patients with p47phox deficiency. Despite early and aggressive therapy, a mortality rate of 26% was noted. Given that bone-marrow transplantation was successful in five of seven patients, it is recommended to perform it as early as possible before tissue damage is irreversible.
Objective. The aim of this study was to analyze mutations in DNA from patients with leukocyte adh... more Objective. The aim of this study was to analyze mutations in DNA from patients with leukocyte adhesion deficiency (LAD), an immunodeficiency caused by absence of the β2 subunit (CD18) of the leukocyte integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and CR4 (CD11d/CD18).Methods. We developed genomic DNA PCR sequencing to detect mutations not only in exons but also in introns.Results. Eight LAD patients were analyzed, of which five had homozygous mutations, i.e., a 0.8-kb deletion, a branchpoint mutation in intron 5 causing mRNA missplicing, a nonsense mutation, and two missense mutations. Four of these mutations are novel. We cotransfected the two mutant CD18 proteins with normal CD11a, b, or c in COS cells. This resulted in absence of all three β2 integrins on the surface of cells transfected with CD18252Arg. However, CD18593Cys supported some LFA-1 and p150,95 formation in COS cells. The other three patients were compound heterozygotes in which only one allele had previously been characterized, because the other alleles were undetectable at the cDNA level. We identified the unknown mutations as a novel two-nucleotide deletion, a nonsense mutation, and a single nucleotide deletion.Conclusion. Our method allows identification of mutations in CD18 from genomic DNA. This opens the possibility of early prenatal diagnosis of LAD and reliable carrier detection.
Background One of the rarest forms of autosomal recessive chronic granulomatous disease (AR-CGD)... more 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 b558, (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.
Background Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune sys... more 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.
To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking fo... more To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking for known and possible CGD cases was mailed to paediatric, internal medicine and infectious disease departments all over Sweden. The detected patients were characterized as to genetics and the clinical presentation. Twenty–one patients (belonging to 16 different families) were found, corresponding to a prevalence of ∼ 1/450 000 individuals. The patients with X–linked disease, lacking a functional gp91phox protein (n= 12), comprised 57% and 43% of the patients had an autosomal recessive (AR) disease lacking p47phox (n= 7) or p67phox (n=1), respectively. All unrelated patients with X–linked disease displayed different gene abnormalities such as point mutations predicting nonsense (n= 3), missense (n= 1) or splice site mutations (n = 2), but also a total deletion and a unique 40 base pair duplicature insertion. The patients with p47phox–deficiency showed a GT deletion at a GTGT tandem repeat, and the p67phox–deficient patient displayed a heterozygous in–frame deletion of AAG combined with a large deletion in the other allele. Three patients died during the study period, two from Pseudomonas cepacia infections. Patients with X–linked disease had more frequent infections (mean of 1.7 per year), than the patients with AR inheritance (0.5 infections per year). The most common infections were dermal abscesses (n=111), followed by lymphadenitis (n=82) and pneumonias (n=73). Inflammatory bowel disease–like symptoms, mimicking Crohn's disease of the colon, was seen in three CGD patients.
Background Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune sys... more 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 the 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, responsible for approximately 70% of all CGD cases. The aim of the study was to evaluate the hypothesis that age-related skewing of X-chromosome inactivation, as described in several CGD families, is caused by preferential survival of bone marrow clones with an inactive NADPH oxidase.Materials and methods We studied the neutrophils from three patients and four carriers in three generations of a Turkish family with X-linked CGD. Carrier detection was carried out by the dihydrorhodamine (DHR)-1,2,3 assay, which measures on a per-cell basis the NADPH oxidase-dependent oxidation of DHR by phagocytes. The X-chromosome inactivation pattern was determined with the HUMARA assay in DNA from leucocytes as well as in DNA from a buccal smear of the four carriers.Results The three patients were identified by a negative DHR test, and the mutation in their CYBB gene was characterized by DNA sequencing. Moreover, we found an age-related degree of skewing of X-chromosome inactivation in the leucocytes of the four X-CGD carriers, both at the protein level (NADPH oxidase activity) and at the DNA level (HUMARA assay). However, similar skewing of X-chromosome inactivation was found in the buccal DNA from these women.Conclusions These novel findings indicate that the age-related degree of skewing was probably a chance finding, not related to preferential survival of NADPH oxidase-deficient precursor cells, because this enzyme is not expressed in (buccal) epithelial cells.
In the X-linked form of chronic granulomatous disease (X91° CGD), the genetic defect is linked to... more In the X-linked form of chronic granulomatous disease (X91° CGD), the genetic defect is linked to the CYBB locus on the X chromosome. We studied a family with a genetic defect in this gene, consisting of a G → A substitution at the fifth base of the 5′ donor splice site of intron 3. This mutation leads to skipping of exon 3 after transcription of the gene. The expectant mother was diagnosed as a carrier. Analysis of polymerase chain reaction (PCR)-amplified genomic DNA from a chorionic villus biopsy (CVB) showed the same mutation in the male fetus. After termination of the pregnancy, the diagnosis was confirmed by conventional methods. This is the first time that PCR has been used for prenatal diagnosis of CGD.
Chronic granulomatous Disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 ... more Chronic granulomatous Disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is essential in the process of intracellular pathogen killing by phagocytic leukocytes. Four of the five genes involved in CGD are autosomal; these are CYBA, encoding p22-phox, NCF2, encoding p67-phox, NCF1, encoding p47-phox, and NCF4, encoding p40-phox. This article lists all mutations identified in these genes in the autosomal forms of CGD. Moreover, polymorphisms in these genes are also given, which should facilitate the recognition of future disease-causing mutations.
Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 ... more Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations.
Chronic granulomatous disease (CGD) is an innate immunodeficiency due to a genetic defect in one ... more Chronic granulomatous disease (CGD) is an innate immunodeficiency due to a genetic defect in one of the NADPH-oxidase components. In the course of 21 years, 38 Israeli CGD patients were diagnosed with 17 gene mutations, seven of which were new. Clinical, functional, and molecular studies were accomplished. Although X-linked recessive (XLR)-CGD is worldwide the most common genotype of the disease (~ 70%), in our study only 11 patients (29%) suffered from XLR-CGD. In Israel, the higher incidence of the autosomal recessive (AR) form of CGD (63%) may be related to consanguineous marriages. In three patients (8%), all four proteins of the NADPH oxidase were present. Severe clinical expression was found both in the XLR and AR forms, but in general a milder disease was evident in AR-CGD, particularly in patients with p47phox deficiency. Despite early and aggressive therapy, a mortality rate of 26% was noted. Given that bone-marrow transplantation was successful in five of seven patients, it is recommended to perform it as early as possible before tissue damage is irreversible.
Objective. The aim of this study was to analyze mutations in DNA from patients with leukocyte adh... more Objective. The aim of this study was to analyze mutations in DNA from patients with leukocyte adhesion deficiency (LAD), an immunodeficiency caused by absence of the β2 subunit (CD18) of the leukocyte integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and CR4 (CD11d/CD18).Methods. We developed genomic DNA PCR sequencing to detect mutations not only in exons but also in introns.Results. Eight LAD patients were analyzed, of which five had homozygous mutations, i.e., a 0.8-kb deletion, a branchpoint mutation in intron 5 causing mRNA missplicing, a nonsense mutation, and two missense mutations. Four of these mutations are novel. We cotransfected the two mutant CD18 proteins with normal CD11a, b, or c in COS cells. This resulted in absence of all three β2 integrins on the surface of cells transfected with CD18252Arg. However, CD18593Cys supported some LFA-1 and p150,95 formation in COS cells. The other three patients were compound heterozygotes in which only one allele had previously been characterized, because the other alleles were undetectable at the cDNA level. We identified the unknown mutations as a novel two-nucleotide deletion, a nonsense mutation, and a single nucleotide deletion.Conclusion. Our method allows identification of mutations in CD18 from genomic DNA. This opens the possibility of early prenatal diagnosis of LAD and reliable carrier detection.
Background One of the rarest forms of autosomal recessive chronic granulomatous disease (AR-CGD)... more 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 b558, (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.
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