A mutation located at the 5' splice junction sequence of intron 3 in the p67phox gene causes the lack of p67phox mRNA in a patient with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is due to a functional defect of the O2(-)-generating NADPH oxidase of neutrophils. Mutations resulting in CGD have been shown to occur in only four genes, thus identifying the main components of the oxidase complex, namely the two subunits of a membrane-bound cytochrome b and two cytosolic factors of activation of 67 kD (p67phox) and 47 kD (p47phox). The present study deals with the biochemical and genetic analysis of the defect in a patient suffering from a p67phox-deficient form of CGD. The p67phox deficiency was ascertained by immunochemistry and the ability of recombinant p67phox to restore NADPH oxidase activity using a cell-free system of oxidase activation. The cellular extracts from the proband contained no p67phox protein and no p67phox mRNA when assayed by Western and Northern blot analysis. However, reverse transcription of mRNA and subsequent cDNA amplification by polymerase chain reaction using specific p67phox primers showed that trace amounts of a p67phox mRNA deleted for exon 3 were synthesized in the patient immortalized B lymphocytes. Sequence analysis of the genomic DNA showed a T-to-C transition at position +2 of intron 3. This point mutation in the consensus 5' splice site of the intron 3 was probably responsible for lack of accumulation of mRNA and also for the skipping of exon 3 detected in the few mRNA molecules that escaped cellular degradation.

[1]  B. Babior,et al.  The respiratory burst oxidase , 1994, Basic life sciences.

[2]  M. de Boer,et al.  Autosomal recessive chronic granulomatous disease with absence of the 67-kD cytosolic NADPH oxidase component: identification of mutation and detection of carriers. , 1994, Blood.

[3]  A. Harris,et al.  The stop mutation R553X in the CFTR gene results in exon skipping. , 1994, Genomics.

[4]  H. Malech,et al.  Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease. , 1993, Blood.

[5]  J. Seigneurin,et al.  The O2- generating oxidase of B lymphocytes: Epstein-Barr virus-immortalized B lymphocytes as a tool for the identification of defective components of the oxidase in chronic granulomatous disease. , 1993, Biochimica et biophysica acta.

[6]  David Valle,et al.  The skipping of constitutive exons in vivo induced by nonsense mutations , 1993, Science.

[7]  A. de Klein,et al.  Cytochrome b558-negative, autosomal recessive chronic granulomatous disease: two new mutations in the cytochrome b558 light chain of the NADPH oxidase (p22-phox). , 1992, American journal of human genetics.

[8]  S. Baserga,et al.  Beta-globin nonsense mutation: deficient accumulation of mRNA occurs despite normal cytoplasmic stability. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[9]  G. Bokoch,et al.  Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. , 1991, Science.

[10]  J. Seigneurin,et al.  Activation of O2(-)-generating oxidase in an heterologous cell-free system derived from Epstein-Barr-virus-transformed human B lymphocytes and bovine neutrophils. Application to the study of defects in cytosolic factors in chronic granulomatous disease. , 1991, European journal of biochemistry.

[11]  F. Morel,et al.  The superoxide-generating oxidase of phagocytic cells , 1991 .

[12]  A. Abo,et al.  Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1 , 1991, Nature.

[13]  P. Vignais,et al.  Cytosolic factors in bovine neutrophil oxidase activation. Partial purification and demonstration of translocation to a membrane fraction. , 1990, Biochemistry.

[14]  W. Nauseef,et al.  Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. , 1990, The Journal of clinical investigation.

[15]  T. Hashimoto,et al.  Molecular analysis of human acatalasemia. Identification of a splicing mutation. , 1990, Journal of molecular biology.

[16]  J. Donelson,et al.  Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[17]  H. Malech,et al.  Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease. , 1989, Science.

[18]  P. Vignais,et al.  Parameters of activation of the membrane-bound O2- generating oxidase from bovine neutrophils in a cell-free system. , 1989, Biochemical and biophysical research communications.

[19]  J. Gauchat,et al.  Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines. , 1989, Journal of immunology.

[20]  S. Avrameas,et al.  Polyspecific natural antibodies and autoantibodies secreted by human lymphocytes immortalized with Epstein-Barr virus. , 1988, Blood.

[21]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[22]  A. Jesaitis,et al.  Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000. , 1987, The Journal of clinical investigation.

[23]  F D Ledley,et al.  GT to AT transition at a splice donor site causes skipping of the preceding exon in phenylketonuria. , 1987, Nucleic acids research.

[24]  A. Fauci,et al.  B cell lines as models for inherited phagocytic diseases: abnormal superoxide generation in chronic granulomatous disease and giant granules in Chediak-Higashi syndrome. , 1984, Journal of immunology.

[25]  F. Morel,et al.  Examination of the oxidase function of the b-type cytochrome in human polymorphonuclear leucocytes. , 1984, Biochimica et biophysica acta.

[26]  Stephen M. Mount,et al.  A catalogue of splice junction sequences. , 1982, Nucleic acids research.

[27]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Curnutte Jt.  Molecular basis of the autosomal recessive forms of chronic granulomatous disease. , 1992 .

[29]  S. Orkin,et al.  Molecular genetics of chronic granulomatous disease. , 1988, Immunodeficiency reviews.