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Biomedical subjects

P G Heyworth

Publications and source records attributed to P G Heyworth.

At least 19 recordsLinked to original sources
PubMed2003

Underlying chronic granulomatous disease in a patient with bronchocentric granulomatosis.

We present a case of bronchocentric granulomatosis in a woman with no history of asthma who was colonised with Aspergillusfumigatus. A family history of chronic granulomatous disease prompted further testing that demonstrated severely depressed neutrophil oxidant production and gp91(phox) deficiency compatible with the X linked carrier state of chronic granulomatous disease. Only one report of the association of these two rare diseases has previously appeared in the literature. We postulate that an ineffective immune response led to the prolonged colonisation of Afumigatus resulting in a hypersensitivity reaction that was manifest clinically as bronchocentric granulomatosis.

Adult↗
PubMed2001

An unusual intronic mutation in the CYBB gene giving rise to chronic granulomatous disease.

The most common, X-linked, form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene located at Xp21.1. The product of this gene is the large subunit of flavocytochrome b558, gp91phox, which forms the catalytic core of the antimicrobial superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene, or more rarely at the intron/exon borders. Alternatively, they are large (often multi-gene) deletions. In addition, four mutations have been found in the promoter region. In contrast, very few intronic mutations have been reported. Here we describe an intronic mutation that causes X-linked CGD. A single nucleotide substitution in the middle of intron V creates a novel 5' splice site and results in multiple abnormal mRNA products.

B-Lymphocytes↗
PubMed2001

Autosomal recessive chronic granulomatous disease caused by defects in NCF-1, the gene encoding the phagocyte p47-phox: mutations not arising in the NCF-1 pseudogenes.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in any one of 4 genes encoding phagocyte NADPH oxidase subunits. Unlike other CGD subtypes, in which there is great heterogeneity among mutations, 97% of affected alleles in patients previously reported with A47(0) CGD carry a single mutation, a GT deletion (DeltaGT) in exon 2 of the p47-phox gene, NCF-1. This unusually high incidence results from recombination events between NCF-1 and its highly homologous pseudogenes, in which DeltaGT originates. In 50 consecutive patients with A47(0) CGD, 4 were identified who were heterozygous for DeltaGT in NCF-1, and for the first time, 2 were identified whose DNA appeared normal at this position. To avoid co-amplification of pseudogene sequence and to enable the identification of mutations in these patients, allele-specific polymerase chain reaction was used to amplify alleles not containing DeltaGT. In each of the 4 patients who were heterozygous for DeltaGT, an additional novel mutation was identified. These were 2 missense mutations, G125 --> A in exon 2 (predicting Arg42 --> Gln) and G784 --> A in exon 8 (Gly262 --> Ser), and 2 splice junction mutations at the 5' end of intron 1, gt --> at and gtg --> gtt. The first of 2 patients who appeared normal at the GT position was a compound heterozygote with the G125 --> A transition on one allele and a deletion of G811 on the other. In the second of these patients, only a single defect was detected, G574 --> A, which predicts Gly192 --> Ser but is likely to result in defective splicing because it represents the final nucleotide of exon 6.

Adolescent↗
PubMed2001

A second case of somatic triple mosaicism in the CYBB gene causing chronic granulomatous disease.

The most common form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene that is carried on the X-chromosome and give rise to the X-linked form of the disease. The product of this gene is the large subunit of flavocytochrome b558, gp91phox, the catalytic core of the superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene or, less frequently, at the intron/exon borders. In addition, there are large, often multi-gene, deletions. Four mutations have also been found in the promoter regions. In contrast, very few intronic mutations have been reported. Here we describe an unusual intronic mutation that causes CGD. The mutation is the insertion of 12 bp in intron XI, accompanied by the deletion of exon 12. Remarkably, the grandmother of this patient is chimeric, carrying a normal allele, the patient's allele, and an allele with a 4-nucleotide insertion at a site adjacent to the patient's insertion, in combination with a 1.5-kb deletion within intron XI. The patient's mother carries a normal allele and the patient's allele. We propose that an initial mutational event during the grandmother's embryogenesis has undergone unsuccessful DNA repair and has resulted in two aberrant alleles, one of which has been inherited by the patient and his mother. Remarkably, in the only two kindreds that have been examined in detail where deletions originating within introns have led to CGD, both families have contained members with triple somatic mosaicism.

Child, Preschool↗
PubMed2001

Primary immunodeficiency mutation databases.

Primary immunodeficiencies are intrinsic defects of immune systems. Mutations in a large number of cellular functions can lead to impaired immune responses. More than 80 primary immunodeficiencies are known to date. During the last years genes for several of these disorders have been identified. Here, mutation information for 23 genes affected in 14 immunodefects is presented. The proteins produced are employed in widely diverse functions, such as signal transduction, cell surface receptors, nucleotide metabolism, gene diversification, transcription factors, and phagocytosis. Altogether, the genetic defect of 2,140 families has been determined. Diseases with X-chromosomal origin constitute about 70% of all the cases, presumably due to full penetrance and because the single affected allele causes the phenotype. All types of mutations have been identified; missense mutations are the most common mutation type, and truncation is the most common effect on the protein level. Mutational hotspots in many disorders appear in CPG dinucleotides. The mutation data for the majority of diseases are distributed on the Internet with a special database management system, MUTbase. Despite large numbers of mutations, it has not been possible to make genotype-phenotype correlations for many of the diseases.

Alleles↗
PubMed2000

Molecular analysis of 9 new families with chronic granulomatous disease caused by mutations in CYBA, the gene encoding p22(phox).

Chronic granulomatous disease is a rare inherited disorder caused by nonexistent or severely decreased phagocyte superoxide production that results in a severe defect in host defense and consequent predisposition to microbial infection. The enzyme responsible for generating the superoxide, NADPH oxidase, involves at least 5 protein components. The absence of, or a defect in, any 1 of 4 of these proteins (p22(phox), p47(phox), p67(phox), or gp91(phox)) gives rise to the known types of chronic granulomatous disease. One of the rarest forms of the disease is due to defects in the CYBA gene encoding p22(phox), which together with gp91(phox) forms flavocytochrome b(558), the catalytic core of NADPH oxidase. To date, only 9 kindreds with p22(phox) deficiency have been described in the literature comprising 10 mutant alleles. Four polymorphisms in the CYBA gene have also been reported. Here we describe 9 new, unrelated kindreds containing 12 mutations, 9 of which are novel. In addition, we report 3 new polymorphisms. The novel mutations are (a) deletion of exons 2 and 3, (b) a missense mutation in exon 3 (T155-->C), (c) a splice site mutation at the 5' end of intron 3, (d) a missense mutation in exon 2 (G74-->T), (e) a nonsense mutation in exon 1 (G26-->A), (f) a missense mutation in exon 4 (C268-->T), (g) a frameshift in exon 3 due to the insertion of C at C162, (h) a nonsense mutation in exon 2 (G107-->A), and (i) a missense mutation in exon 2 (G70-->A).

Adolescent↗
PubMed1999

A novel mutation in the CYBB gene resulting in an unexpected pattern of exon skipping and chronic granulomatous disease.

Chronic granulomatous disease is a rare inherited disorder caused by non-existent or severely decreased phagocyte superoxide production that results in a severe defect in host defense and consequent predisposition to microbial infection. The enzyme responsible for superoxide production, NADPH oxidase, involves at least five components. An absence of, or a defect in, any one of four of these proteins (p47(phox), p67(phox), p22(phox) and gp91(phox)) gives rise to the known types of chronic granulomatous disease. The most common form of inheritance is X-linked and is due to mutations in the CYBB gene that encodes gp91(phox), the large subunit of flavocytochrome b, the terminal electron donor of the oxidase. We have recently reported a large number of mutations in this gene revealing a broad range of defects, including large and small deletions, and frameshift, nonsense, missense, splice region and regulatory region mutations. Here we report a patient who has an unusual type of mutation that results in the generation of a 'pseudo-exon' in the gp91(phox) mRNA and an unexpected pattern of splicing.

Base Sequence↗
PubMed1999

Activation of human neutrophil NADPH oxidase by phosphatidic acid or diacylglycerol in a cell-free system. Activity of diacylglycerol is dependent on its conversion to phosphatidic acid.

The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg(2+) and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation.

Cell-Free System↗
PubMed1999

Autosomal recessive chronic granulomatous disease caused by novel mutations in NCF-2, the gene encoding the p67-phox component of phagocyte NADPH oxidase.

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency disease that leads to severe recurrent infections. CGD is caused by defects in the phagocyte NADPH oxidase, a multiprotein enzyme that reduces oxygen to superoxide, a precursor of microbicidal oxidants. Less than 6% of CGD patients have an autosomal recessive form of the disease caused by mutations in NCF-2. This gene encodes p67-phox, a cytosolic oxidase subunit that associates with membrane-bound flavocytochrome b558 and regulates electron transfer. We studied six patients from five families with p67-phox deficiency and identified seven different mutant alleles. Patients from three of the kindreds were homozygous for their respective mutation, although the parents of only one family were known to be related. Five of the mutations have not previously been identified: (1) a missense mutation (383C-->T) in exon 5, (2) a nonsense mutation (196C-->T) in exon 3, (3) a missense mutation (230G-->A) in exon 3, (4) a nonsense mutation (298C-->T) in exon 4, and (5) a dinucleotide deletion (835-836 AC) from exon 9. Phagocytes from each of the patients analyzed failed to generate a measurable respiratory burst and had no detectable p67-phox protein. Our results further demonstrate that there is great heterogeneity among the mutations in p67-phox-deficient CGD patients, with no evidence for mutational hot-spots or a founder effect. Our data also support the hypothesis that the stability of p67-phox is particularly sensitive to missense mutations that cause amino acid substitutions within its N-terminal domain. In contrast, mutations predicting single amino acid changes elsewhere in the protein generally represent benign polymorphisms.

Adolescent↗
PubMed1997

Cofilin undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase complex. Evidence for a novel cycle of phosphorylation and dephosphorylation.

Neutrophils contain a 21-kDa phosphoprotein that undergoes rapid dephosphorylation upon stimulation of these cells with the chemoattractant N-fMet-Leu-Phe (fMLP), activators of protein kinase C [e.g., 4 beta-phorbol 12-myristate 13-acetate (PMA)] or the calcium ionophore A23187. This phosphoprotein was identified as the non-muscle form of cofilin by peptide sequencing and immunoblotting with specific antibodies. Evidence is presented that in neutrophils cofilin is regulated by a continual cycle of phosphorylation and dephosphorylation, and that the phosphatase undergoes activation during cell stimulation. Experiments with a wide variety of antagonists further suggested that the protein kinase that participates in these reactions may be a novel enzyme. The kinetics of cofilin dephosphorylation in neutrophils stimulated with fMLP or PMA were very similar to those observed for superoxide (O2-) release. Immunofluorescent studies revealed that cofilin was present throughout the cytosol of resting neutrophils and underwent rapid translocation to the F-actin-rich, ruffled membranes of stimulated cells. Cytochemical analysis further revealed that the ruffled membranes also contained large amounts of hydrogen peroxide (H2O2), a product of the O2-/H2O2-generating activity of stimulated neutrophils (NADPH oxidase). Cofilin is therefore well placed to participate in the continual polymerization and depolymerization of F-actin that is thought to give rise to the oscillatory pattern of H2O2 production observed under certain conditions.

Actin Depolymerizing Factors↗
PubMed1996

Protein phosphorylation in neutrophils from patients with p67-phox-deficient chronic granulomatous disease.

Neutrophils are known to contain a major 67-kD protein that undergoes enhanced phosphorylation and translocation to the membrane during cell stimulation. Recent studies have assumed that this 67-kD phosphoprotein is the 67-kD subunit of the phagocyte oxidase (p67-phox). We compare here the protein phosphorylation patterns in lysates of normal neutrophils and neutrophils from patients with chronic granulomatous disease (CGD) that are completely deficient in p67-phox. The phosphoproteins were labeled by incubation of the cells with radioactive inorganic phosphate (32Pi) or by the addition of [gamma-32P]ATP to electropermeabilized neutrophils. With either method, stimulation of the normal or CGD cells always resulted in an enhanced incorporation of 32p into two proteins in the 67-kD area. The extent of phosphorylation of these two proteins was very similar in the normal and CGD cells when permeabilized neutrophils loaded with [gamma -32P]ATP were compared. Moreover, no overall differences in the protein phosphorylation patterns were observed between the normal and CGD cells. Our data indicate that the major 67-kD phosphoproteins observed in stimulated neutrophils are clearly different from p67-phox.

Amino Acid Sequence↗
PubMed1995

Naphthalenesulphonamides block neutrophil superoxide production by intact cells and in a cell-free system: is myosin light chain kinase responsible for these effects?

Selective antagonists of myosin light chain kinase (MLCK) [e.g. ML-7; 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] were found to inhibit superoxide (O2-) release from stimulated neutrophils. The concentrations of ML-7 that were inhibitory were substantially lower than those reported for a selective antagonist of protein kinase C [i.e. H-7; 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride]. ML-7 also reduced the phosphorylation of the 47 kDa subunit of the NADPH-oxidase system (p47-phox) and blocked translocation of this protein to the Triton X-100-insoluble fraction in stimulated cells. Interestingly, ML-7 also inhibited O2- production in a cell-free system derived from neutrophils at concentrations similar to those that were effective in vivo. This cell-free system does not require ATP and is insensitive to all other inhibitors of protein kinases tested, including some highly effective against MLCK (i.e. staurosporine). Thus, the data suggest that ML-7 does not block O2- release by inhibiting a protein kinase but instead may interact directly with a subunit of the oxidase. The binding site for ML-7 may provide a valuable target for inhibiting the inflammatory properties of phagocytic leucocytes by naphthalenesulphonamides designed to lack activity against protein kinases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

A variant X-linked chronic granulomatous disease patient (X91+) with partially functional cytochrome b.

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

Amino Acid Sequence↗
PubMed1995

Investigation of neutrophil signal transduction using a specific inhibitor of phosphatidylinositol 3-kinase.

Neutrophils contain a multicomponent NADPH oxidase system that is involved in the production of microbicidal oxidants. Stimulation of human neutrophils with the peptide FMLP activates this respiratory burst enzyme to produce superoxide and also has been shown to result in activation of phosphatidylinositol (Ptdlns) 3-kinase. Treatment of human neutrophils with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a potent and specific inhibitor of Ptdlns 3-kinase, resulted in complete inhibition of Ptdlns 3-kinase activity as well as in inhibition of superoxide production in FMLP-treated neutrophils in suspension; FMLP-stimulated oxidant production in adherent cells was also abolished. Treatment of human neutrophils with PMA resulted in production of superoxide without activation of Ptdlns 3-kinase; LY294002 did not block superoxide production in neutrophils exposed to PMA. In addition, LY294002 did not inhibit cellfree NADPH oxidase activation, CD11b-dependent adhesion, actin polymerization in response to FMLP, or FMLP-induced calcium flux. These results suggest that the signal transduction pathway of the FMLP-receptor involves activation of Ptdlns 3-kinase, which is required for subsequent superoxide production induced by the chemotactic peptide. Furthermore, Ptdlns 3-kinase may be located directly upstream of protein kinase C or other protein kinases, which in turn activate the NADPH oxidase system.

Actins↗