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 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.
Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in act... more Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in activated phagocytes. Defects in at least four different genes lead t o CGD. Patients with the X-linked form of CGD have mutations in the gene for the psubunit of cytochrome b m (gp91-phox). We studied the molecular defect in four patients with X-linked CGD. In a fifth family, we studied the mother of a patient with X-linked CGD who had died before our investigations. GpSI-phox messenger RNA (mRNA) was reverse transcribed into cDNA and the coding region was amplified by polymerase chain reaction into three fragments. Sequence analysis showed the absence of the exon 7,5,3, and 2 sequences in patients 1,2,3, and 4, respectively. In carrier 5, we found both normal cDNA and cDNA that lacked 57 3'-nucleotides of exon 6. We analyzed the splice sites of the flanking introns of the missing exons. In patients 1, 2, and 3, we found single nucleotide HE SUPEROXIDE-producing NADPH:02 oxi-
Background Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects... more Background Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two X-linked CGD patients with mutations in gp91 phox that alter the regions in this membrane-bound NADPH oxidase component involved in docking of the cytosolic component p47 phox .
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 caused by defects in the nicotinamide ... more Chronic granulomatous disease (CGD) is an immunodeficiency caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex and is usually diagnosed in early childhood. CGD patients suffer from severe, recurrent infections with bacteria, fungi and yeasts. We report a 25-year-old female with protracted fever because of a Staphylococcus aureus liver abscess, which did not resolve until breakthrough into the stomach. Despite her age, CGD was considered on diagnosis on the basis of the clinical symptoms. Analysis of the NADPH-oxidase activity confirmed CGD as the underlying condition. Western blotting revealed the absence of p47 phox and subsequent sequencing of the p47 phox -encoding gene, neutrophil cytosolic factor (NCF1), identified a deletion of 837C in the maternal NCF1 allele. The paternal allele contained a stopcodon because of a conversion between NCF1 and one of its WNCF1 pseudogenes. The patient had one novel mutation, c.837delC, and one conversion in NCF1, resulting in the complete absence of the p47 phox component of the NADPH-oxidase complex. This p47 phox -deficient CGD patient had the highest age at diagnosis reported thus far.
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.
Five male patients from four different families presented with a clinical record of chronic granu... more Five male patients from four different families presented with a clinical record of chronic granulomatous disease (CGD): recurrent infections of the skin and/or respiratory tract with catalase-positive microorganisms,
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. We devised a method to recognize carriers and patients with p47 phox -deficient chroni... more Objective. We devised a method to recognize carriers and patients with p47 phox -deficient chronic granulomatous disease (A47 CGD), the most common autosomal form of the disease. CGD is characterized by the inability of phagocytic leukocytes to kill microorganisms, due to a defective NADPH oxidase system. The predominant genetic defect leading to p47 phox -deficient CGD is a GT deletion at the beginning of exon 2 in the p47 phox gene NCF1 , most likely caused by recombination events between the NCF1 and one of its pseudogenes. It is hardly possible to investigate sequences of patients, carriers, and normal individuals using standard PCR/sequencing techniques, due to greater than 99% homology between NCF1 and its pseudogenes. Methods. In our gene-scan method, a 198-bp region of genomic DNA around exon 2 of NCF1 is amplified by nonspecific PCR with one fluorochrome-labeled primer. The mixture of NCF1 and pseudogene product, which differs by two nucleotides in length, is separated according to size. The ratio between the peak heights indicates the relative number of NCF1 genes and pseudogenes within an individual's genome. Results. The method is highly reproducible (SD ϭ 4%) and sensitive (r ϭ 0.998). Of the 16 healthy individuals, 15 had a 2:4 ratio (2 genes, 4 pseudogenes), 10/12 A47 CGD carriers had a 1:5 ratio, and 34 patients had only pseudogenes. In addition, gene-scans including a 20-bp duplication in intron 2 of the pseudogenes revealed insight in the crossing-over events between NCF1 and pseudogenes. Conclusions. Our method distinguishes individuals with one NCF1 gene (carriers) from controls and from NCF1 -deficient patients.
Long interspersed nuclear element-1 (LINE-1) or L1 elements are DNA elements present in the genom... more Long interspersed nuclear element-1 (LINE-1) or L1 elements are DNA elements present in the genome in high copy number and capable of active retrotransposition. Here we present a patient with severe chronic granulomatous disease (CGD) caused by insertion of an L1 sequence into intron 5 of the X-lined gene CYBB. Due to internal rearrangements, the insert introduced new splice sites into the intron. This resulted in a highly heterogeneous splicing pattern with introduction of two L1 fragments as new exons into the transcripts and concomitant skipping of exonic coding sequence. Because no wild-type cDNA was found, this mechanism is probably responsible for the patient's phenotype. The L1 fragment, which belongs to the Ta subset of transcriptionally active LINEs, illustrates a new mechanism by which these elements can modify the transcribed coding sequence of genes. A new exon created by LINE-1 insertion C Meischl et al y
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.
High-resolution melting analysis was applied to Xlinked chronic granulomatous disease , a rare di... more High-resolution melting analysis was applied to Xlinked chronic granulomatous disease , a rare disorder resulting from mutations in CYBB. Melting curves of the 13 PCR products bracketing CYBB exons were predicted by Poland's algorithm and compared with observed curves from 96 normal individuals. Primer plates were prepared robotically in batches and dried , greatly simplifying the 3-to 6-hour workflow that included DNA isolation , PCR , melting , and cycle sequencing of any positive products. Small point mutations or insertions/deletions were detected by mixing the hemizygous male DNA with normal male DNA to produce artificial heterozygotes , whereas detection of gross deletions was performed on unmixed samples. Eighteen validation samples and 22 clinical kindreds were analyzed for CYBB mutations. All blinded validation samples were correctly identified. The clinical probands were identified after screening for neutrophil oxidase activity. Nineteen different mutations were found , including seven near intron-exon boundaries predicting splicing defects , five substitutions within exons , three small deletions predicting premature termination , and four gross deletions of multiple exons. Ten novel mutations were found , including (c.) two missense (730T>A , 134T>G), one nonsense 90C>A) , four splice site defects (45 ؉ 1G>T , 674 ؉ 4A>G , 1461 ؉ 2delT , and 1462-2A>C), two small deletions (636delT , 1661_1662delCT), and one gross deletion of exons 6 to 8. High-resolution melting can provide timely diagnosis at low cost for effective clinical management of rare , genetic primary immunodeficiency disorders. (J Mol Diagn 2010, 12:368 -376; DOI: 10.2353/jmoldx.2010.090147)
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 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.
Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in act... more Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in activated phagocytes. Defects in at least four different genes lead t o CGD. Patients with the X-linked form of CGD have mutations in the gene for the psubunit of cytochrome b m (gp91-phox). We studied the molecular defect in four patients with X-linked CGD. In a fifth family, we studied the mother of a patient with X-linked CGD who had died before our investigations. GpSI-phox messenger RNA (mRNA) was reverse transcribed into cDNA and the coding region was amplified by polymerase chain reaction into three fragments. Sequence analysis showed the absence of the exon 7,5,3, and 2 sequences in patients 1,2,3, and 4, respectively. In carrier 5, we found both normal cDNA and cDNA that lacked 57 3'-nucleotides of exon 6. We analyzed the splice sites of the flanking introns of the missing exons. In patients 1, 2, and 3, we found single nucleotide HE SUPEROXIDE-producing NADPH:02 oxi-
Background Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects... more Background Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two X-linked CGD patients with mutations in gp91 phox that alter the regions in this membrane-bound NADPH oxidase component involved in docking of the cytosolic component p47 phox .
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 caused by defects in the nicotinamide ... more Chronic granulomatous disease (CGD) is an immunodeficiency caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex and is usually diagnosed in early childhood. CGD patients suffer from severe, recurrent infections with bacteria, fungi and yeasts. We report a 25-year-old female with protracted fever because of a Staphylococcus aureus liver abscess, which did not resolve until breakthrough into the stomach. Despite her age, CGD was considered on diagnosis on the basis of the clinical symptoms. Analysis of the NADPH-oxidase activity confirmed CGD as the underlying condition. Western blotting revealed the absence of p47 phox and subsequent sequencing of the p47 phox -encoding gene, neutrophil cytosolic factor (NCF1), identified a deletion of 837C in the maternal NCF1 allele. The paternal allele contained a stopcodon because of a conversion between NCF1 and one of its WNCF1 pseudogenes. The patient had one novel mutation, c.837delC, and one conversion in NCF1, resulting in the complete absence of the p47 phox component of the NADPH-oxidase complex. This p47 phox -deficient CGD patient had the highest age at diagnosis reported thus far.
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.
Five male patients from four different families presented with a clinical record of chronic granu... more Five male patients from four different families presented with a clinical record of chronic granulomatous disease (CGD): recurrent infections of the skin and/or respiratory tract with catalase-positive microorganisms,
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. We devised a method to recognize carriers and patients with p47 phox -deficient chroni... more Objective. We devised a method to recognize carriers and patients with p47 phox -deficient chronic granulomatous disease (A47 CGD), the most common autosomal form of the disease. CGD is characterized by the inability of phagocytic leukocytes to kill microorganisms, due to a defective NADPH oxidase system. The predominant genetic defect leading to p47 phox -deficient CGD is a GT deletion at the beginning of exon 2 in the p47 phox gene NCF1 , most likely caused by recombination events between the NCF1 and one of its pseudogenes. It is hardly possible to investigate sequences of patients, carriers, and normal individuals using standard PCR/sequencing techniques, due to greater than 99% homology between NCF1 and its pseudogenes. Methods. In our gene-scan method, a 198-bp region of genomic DNA around exon 2 of NCF1 is amplified by nonspecific PCR with one fluorochrome-labeled primer. The mixture of NCF1 and pseudogene product, which differs by two nucleotides in length, is separated according to size. The ratio between the peak heights indicates the relative number of NCF1 genes and pseudogenes within an individual's genome. Results. The method is highly reproducible (SD ϭ 4%) and sensitive (r ϭ 0.998). Of the 16 healthy individuals, 15 had a 2:4 ratio (2 genes, 4 pseudogenes), 10/12 A47 CGD carriers had a 1:5 ratio, and 34 patients had only pseudogenes. In addition, gene-scans including a 20-bp duplication in intron 2 of the pseudogenes revealed insight in the crossing-over events between NCF1 and pseudogenes. Conclusions. Our method distinguishes individuals with one NCF1 gene (carriers) from controls and from NCF1 -deficient patients.
Long interspersed nuclear element-1 (LINE-1) or L1 elements are DNA elements present in the genom... more Long interspersed nuclear element-1 (LINE-1) or L1 elements are DNA elements present in the genome in high copy number and capable of active retrotransposition. Here we present a patient with severe chronic granulomatous disease (CGD) caused by insertion of an L1 sequence into intron 5 of the X-lined gene CYBB. Due to internal rearrangements, the insert introduced new splice sites into the intron. This resulted in a highly heterogeneous splicing pattern with introduction of two L1 fragments as new exons into the transcripts and concomitant skipping of exonic coding sequence. Because no wild-type cDNA was found, this mechanism is probably responsible for the patient's phenotype. The L1 fragment, which belongs to the Ta subset of transcriptionally active LINEs, illustrates a new mechanism by which these elements can modify the transcribed coding sequence of genes. A new exon created by LINE-1 insertion C Meischl et al y
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.
High-resolution melting analysis was applied to Xlinked chronic granulomatous disease , a rare di... more High-resolution melting analysis was applied to Xlinked chronic granulomatous disease , a rare disorder resulting from mutations in CYBB. Melting curves of the 13 PCR products bracketing CYBB exons were predicted by Poland's algorithm and compared with observed curves from 96 normal individuals. Primer plates were prepared robotically in batches and dried , greatly simplifying the 3-to 6-hour workflow that included DNA isolation , PCR , melting , and cycle sequencing of any positive products. Small point mutations or insertions/deletions were detected by mixing the hemizygous male DNA with normal male DNA to produce artificial heterozygotes , whereas detection of gross deletions was performed on unmixed samples. Eighteen validation samples and 22 clinical kindreds were analyzed for CYBB mutations. All blinded validation samples were correctly identified. The clinical probands were identified after screening for neutrophil oxidase activity. Nineteen different mutations were found , including seven near intron-exon boundaries predicting splicing defects , five substitutions within exons , three small deletions predicting premature termination , and four gross deletions of multiple exons. Ten novel mutations were found , including (c.) two missense (730T>A , 134T>G), one nonsense 90C>A) , four splice site defects (45 ؉ 1G>T , 674 ؉ 4A>G , 1461 ؉ 2delT , and 1462-2A>C), two small deletions (636delT , 1661_1662delCT), and one gross deletion of exons 6 to 8. High-resolution melting can provide timely diagnosis at low cost for effective clinical management of rare , genetic primary immunodeficiency disorders. (J Mol Diagn 2010, 12:368 -376; DOI: 10.2353/jmoldx.2010.090147)
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