In vitro molecular reconstitution of the respiratory burst in B lymphoblasts from p47-phox-deficient chronic granulomatous disease.

Abstract

Epstein-Barr virus-transformed lymphocytes generate superoxide in response to various agonists in an enzymatic reaction similar to that which occurs in stimulated phagocytes. We generated transformed B lymphoblast cell lines from controls, from four patients with p47-phox-deficient chronic granulomatous disease, and from three parents. The cells from controls and from the parents generated 7.0-35 nmol of O°/107 cells per 30 min in response to phorbol myristate acetate. None of the patient cell lines generated any detectable superoxide. Both p47-phox and p67-phox were detected by immunoblot in the cytosol of control and parent cell lines and, as in neutrophils, these proteins had affinity for GTP-agarose. The patients' cell lines contained no detectable p47-phox by immunoblot. mRNA for both cytosolic proteins was detected in all cell lines. We generated cDNA and obtained multiple clones from two patients by polymerase chain reaction. One patient was a compound heterozygote with each allele resulting in an early stop codon. Clones derived from the other patient demonstrated only a GT deletion at base 75. The cDNA for p47-phox was inserted into an EBV-expression vector and stably transfected cell lines were obtained using hygromycin B selection. Transfected cell lines from a p47-phox-deficient patient generated normal levels of superoxide and had readily detectable cytosolic p47-phox. Thus, B lymphoblasts provide an excellent model system for studies of the NADPH oxidase, for expression of functional recombinant forms ofoxidase components, and for initial experimental approaches to genetic reconstitution in CGD. (J. Clin. Invest. 1993.91:201-207.)

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

“…1 In contrast to X91, A22, and A67 CGD, in which there is a high degree of heterogeneity among mutations, many of them family-specific, 5-9 a single common mutation has been reported in 60 patients worldwide with A47 0 CGD and was identified in 97% of the affected alleles. 5,[10][11][12][13] Fifty-six (93%) of these persons were homozygous for a dinucleotide deletion (⌬GT) at a GTGT repeat at the beginning of exon 2, which predicts a frameshift and premature stop codon at amino acid 51. 10 In one patient who was heterozygous for ⌬GT, a single nucleotide deletion (G502) was also observed and remains the only non-⌬GT mutation reported in A47 0 CGD before this study.…”

Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease

“…Mutations in neutrophil cytosolic factor 1 (NCF1) are responsible for autosomal-recessive p47 phox -deficient CGD (p47-CGD), comprising ;25% of CGD cases. Notably, while mutations span across the genes for all other CGD patient phox genes, .80% of p47-CGD patients are homozygous for a 2-nt deletion (DGT) from the GTGT start of exon 2, [1][2][3][4][5][6] resulting in a codon frame shift and premature termination, abrogating p47 phox expression. The same DGT is constitutive to the start of presumptive exon 2 in the NCF1 pseudogenes NCF1B and NCF1C.…”

Identification of a novel NCF-1 (p47-phox) pseudogene not containing the signature GT deletion: significance for A47° chronic granulomatous disease carrier detection

“…Ninety-five of 104 unrelated patients reported to date were homozygous and a further 7 were heterozygous for a dinucleotide deletion (⌬GT) in a GTGT sequence at the beginning of exon 2 of NCF-1. [3][4][5][6][7][8][9] The deletion predicts a frameshift and a premature stop codon at residue 51 and leads to complete absence of p47-phox protein from the patients' phagocytes (A47°CGD). 3 Only 8 other mutations have been identified in NCF-1.…”

“…3 Only 8 other mutations have been identified in NCF-1. 4,8,10,11 In contrast, the mutations that cause the other forms of CGD are highly heterogeneous, with many of them being specific to each affected family (mutations and primary references are tabulated by Cross et al 11 and Heyworth et al 12 ). A47°CGD is also unusual in that it is at least 4 times more common than the other autosomal recessive forms of the disease.…”