ALBERT

Description: We have restudied two kindreds that formed the basis of the original report of autosomal recessive chronic granulomatous disease (CGD) associated with leukocyte glutathione peroxidase deficiency. Case 1 from the original study and the surviving brother of the originally reported case 2 both have severe CGD, with no detectable respiratory burst activity in purified intact neutrophils. However, their leukocytes exhibit normal glutathione peroxidase enzyme activity and gene expression. Examination of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase components known to be defective in CGD reveals no detectable cytochrome b558 nor any membrane activity in a cell-free NADPH oxidase assay system. Molecular analysis of the genes encoding cytochrome b558 subunits shows, in case 1, a C--〉T substitution at nucleotide 688 of the gene encoding the gp91-phox subunit of cytochrome b558, resulting in a termination signal in place of Arginine-226. Levels of gp91-phox mRNA are markedly decreased despite normal levels of gene transcription, indicating a post- transcriptional effect of the nonsense mutation on mRNA processing or stability. The X-linked form of CGD developed in this cytogenetically normal female due to the uniform inactivation of the normal X chromosome in her granulocytes, indicated by the expression in her granulocyte mRNA of only one allele of a glucose-6-phosphate dehydrogenase polymorphisms for which she is heterozygous in genomic DNA. Case 2 (of the present study) has distinct mutations in each allele of the p22-phox gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Description: Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in activated phagocytes. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b558 (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. Gp91-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 substitutions within the first five positions of the down-stream 5′ donor splice sites. In patient 4, a similar substitution was found at position -1 of the 3′ acceptor splice site of intron 1. In carrier 5, no mutation was found in the exon 6-intron 6 boundary sequence. Instead, a single substitution was observed in exon 6 (C----A at nucleotide 633) that created a new donor splice site. Apparently, mRNA splicing occurs preferentially at this newly created splice site. We conclude that the absence of the exon sequences in the gp91-phox mRNA of these patients is due to splicing errors. Of 30 European X-linked CGD patients studied by us so far, five appear to be caused by mutations that affect correct mRNA splicing. Thus, such mutations appear to be a common cause of X-linked CGD.

Description: The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91phox, MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91phox in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91phox transcripts was also observed in primary and secondary murine colony-forming unit- spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high- level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function.

Description: Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytic cells fail to generate antimicrobial oxidants. The various forms of CGD can be classified in terms of the mode of inheritance (either X-linked or autosomal recessive), and whether the neutrophils display the absorbance spectrum of a unique b-type cytochrome important for the function of the respiratory burst oxidase. The finding that purified neutrophil cytochrome b is a heterodimer consisting of a 91kD glycosylated and a 22kD nonglycosylated polypeptide has raised the question of which subunits are absent (or defective) in the various types of CGD. To address this question we have studied the expression of the cytochrome b subunits in three genetically distinct forms of CGD: X-linked/cytochrome b-negative (X-), autosomal recessive/cytochrome b-negative (A-), and autosomal recessive/cytochrome b-positive (A+). Using polyclonal antibodies to each of the two subunits, we prepared Western blots of lysates of intact neutrophils from ten CGD patients. In the controls and three patients with A+ CGD, both cytochrome subunits were easily detected. Consistent with the previously reported finding in five X- patients, neither subunit could be identified in neutrophils from three additional X- patients. Both subunits were also undetectable in four patients with A- CGD (three females, one male). This latter group of patients most likely bears a normal 91kD gene, since the patients are genetically distinct from the 91kD-defective X- group. The mutation in A- CGD, therefore, probably involves the 22kD gene and the eventual expression of the 22kD subunit. Furthermore, the expression of the 91kD subunit in this group of patients appears to be prevented due to the 22kD mutation in a manner converse to that seen in the X- CGD patients. Based on these studies, we hypothesize that the stable of expression of either of the two cytochrome subunits is dependent upon the other.

Description: The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.

Description: X-linked chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a recombinant retrovirus vector was constructed and evaluated for its expression of human gp91phox in a human X-CGD myeloid cell line in which the endogenous gp91phox gene had been disrupted by gene targeting. The retrovirus construct, Zip/PGKgp91, was first introduced into the GP+envAm12 amphotropic packaging line and yielded virus producer clones with estimated titers of up to 1 x 10(5) cfu/mL. Coculture infection of X- CGD myeloid cells with Zip/PGKgp91 resulted in restoration of respiratory burst activity to 15% of the cells. Isolated clonal infectants expressed relatively low levels of recombinant gp91phox (〈 or = 12% of wild-type), but exhibited considerable superoxide- generating activity (up to nearly 60% of wild-type). These results show the feasibility of phenotypic correction of CGD using gene replacement therapy and suggest that even modest levels of gp91phox expression may lead to considerable functional correction of X-CGD neutrophils.

Description: X-linked chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a recombinant retrovirus vector was constructed and evaluated for its expression of human gp91phox in a human X-CGD myeloid cell line in which the endogenous gp91phox gene had been disrupted by gene targeting. The retrovirus construct, Zip/PGKgp91, was first introduced into the GP+envAm12 amphotropic packaging line and yielded virus producer clones with estimated titers of up to 1 x 10(5) cfu/mL. Coculture infection of X- CGD myeloid cells with Zip/PGKgp91 resulted in restoration of respiratory burst activity to 15% of the cells. Isolated clonal infectants expressed relatively low levels of recombinant gp91phox (〈 or = 12% of wild-type), but exhibited considerable superoxide- generating activity (up to nearly 60% of wild-type). These results show the feasibility of phenotypic correction of CGD using gene replacement therapy and suggest that even modest levels of gp91phox expression may lead to considerable functional correction of X-CGD neutrophils.

Description: The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.

Description: Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in activated phagocytes. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b558 (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. Gp91-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 substitutions within the first five positions of the down-stream 5′ donor splice sites. In patient 4, a similar substitution was found at position -1 of the 3′ acceptor splice site of intron 1. In carrier 5, no mutation was found in the exon 6-intron 6 boundary sequence. Instead, a single substitution was observed in exon 6 (C----A at nucleotide 633) that created a new donor splice site. Apparently, mRNA splicing occurs preferentially at this newly created splice site. We conclude that the absence of the exon sequences in the gp91-phox mRNA of these patients is due to splicing errors. Of 30 European X-linked CGD patients studied by us so far, five appear to be caused by mutations that affect correct mRNA splicing. Thus, such mutations appear to be a common cause of X-linked CGD.

Description: Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytic cells fail to generate antimicrobial oxidants. The various forms of CGD can be classified in terms of the mode of inheritance (either X-linked or autosomal recessive), and whether the neutrophils display the absorbance spectrum of a unique b-type cytochrome important for the function of the respiratory burst oxidase. The finding that purified neutrophil cytochrome b is a heterodimer consisting of a 91kD glycosylated and a 22kD nonglycosylated polypeptide has raised the question of which subunits are absent (or defective) in the various types of CGD. To address this question we have studied the expression of the cytochrome b subunits in three genetically distinct forms of CGD: X-linked/cytochrome b-negative (X-), autosomal recessive/cytochrome b-negative (A-), and autosomal recessive/cytochrome b-positive (A+). Using polyclonal antibodies to each of the two subunits, we prepared Western blots of lysates of intact neutrophils from ten CGD patients. In the controls and three patients with A+ CGD, both cytochrome subunits were easily detected. Consistent with the previously reported finding in five X- patients, neither subunit could be identified in neutrophils from three additional X- patients. Both subunits were also undetectable in four patients with A- CGD (three females, one male). This latter group of patients most likely bears a normal 91kD gene, since the patients are genetically distinct from the 91kD-defective X- group. The mutation in A- CGD, therefore, probably involves the 22kD gene and the eventual expression of the 22kD subunit. Furthermore, the expression of the 91kD subunit in this group of patients appears to be prevented due to the 22kD mutation in a manner converse to that seen in the X- CGD patients. Based on these studies, we hypothesize that the stable of expression of either of the two cytochrome subunits is dependent upon the other.