The Use Of Reverse Transcription-pcr For The Diagnosis Of X-linked Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70% of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis--a simple, economical and rapid method--was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients.37625-3

The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70% of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis - a simple, economical and rapid method - was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients.62563

The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70% of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis - a simple, economical and rapid method - was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients

Association Of Glucose-6-phosphate Dehydrogenase Deficiency And X-linkled Chronic Granulomatous Disease In A Child With Anemia And Recurrent Infections

Patients with severe leukocyte G6PD deficiency may present with impairment of NADPH oxidase activity and a history of recurrent infections, mimicking the phenotype of chronic granulomatous disease. We report herein a child with recurrent infections who initially received the diagnosis of G6PD deficiency. His erythrocyte G6PD activity was reduced: 1.8 U/g Hb (normal: 12.1 ± 2. 1 U/g Hb). Further studies revealed that G6PD activity in neutrophils, mononuclear leukocytes, and Epstein-Barr virus-transformed B-lymphocytes from the proband was similar to healthy controls. Molecular studies showed that the G6PD deficiency was due a 202 G→A mutation, the A- variant common in African ethnic groups. The proband also exhibited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G→A substitution at the 3′ splice junction of gp91-phox exon 3. The cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable of or nonfunctional mutant gp91-phox and to the phenotype of X-linked CGD. We propose that clinicians treating a patient with G6PD deficiency during a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes' microbicidal activity and the eventual association of G6PD deficiency and chronic granulomatous disease. © 2004 Wiley-Liss, Inc.753151156Curnutte, J.T., Orkin, S.H., Dinauer, M.C., Genetic disorders of phagocyte function (1994) The Molecular Basis of Blood Diseases. 2nd Edition, pp. 493-522. , Stamatoyannopoulos G, Nienttuis A, Majerus P, and Varmus H, editors. Philadelphia: W.B. Saunders CompanyBeutler, E., G6PD deficiency (1994) Blood, 84, pp. 3613-3636Johnston Jr., R.B., Clinical aspects of chronic granulomatous disease (2001) Curr Opin Hematol, 8, pp. 17-22Roos, D., De Boer, M., Kuribayashi, F., Mutations in the X-linked and autosomal recessive forms of chronic granulomatous disease (1996) Blood, 87, pp. 1663-1681Cross, A.R., Noack, D., Rae, J., Curnutte, J.T., Heyworth, P.G., Hematologically important mutations: The autosomal recessive forms of chronic granulomatous disease (first update) (2000) Blood Cells Mol Dis, 26, pp. 561-565Heyworth, P.G., Curnutte, J.T., Rae, J., Hematologically important mutations: X-linked chronic granulomatous disease (second update) (2001) Blood Cells Mol Dis, 27, pp. 16-26Beutler, E., Vulliamy, T.J., Hematologically important mutations: Glucose-6-phosphate dehydrogenase (2002) Blood Cells Mol Dis, 28, pp. 93-103Van Bruggen, R., Bautista, J.M., Petropoulou, T., Deletion of leucine 61 in glucose-6-phosphate dehydrogenase leads to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections (2002) Blood, 100, pp. 1026-1030Roos, D., Van Zwieten, R., Wijnen, J.T., Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections (1999) Blood, 94, pp. 2955-2962Patino, P.J., Perez, J.E., Lopez, J.A., Molecular analysis of chronic granulomatous disease caused by defects in gp91-phox (1999) Hum Mutat, 13, pp. 29-37Saad, S.T., Salles, T.S., Carvalho, M.H., Costa, F.F., Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in Brazil (1997) Hum Hered, 47, pp. 17-21Loos, J.A.R.D., Weening, R.S., Houwerzijl, J., Familial deficiency of glutathione reductase in human blood cells (1976) Blood, 48, p. 53Boyun, A., Isolation of mononuclear cells and granulocytes from human blood (1968) Scand J Clin Lab Invest, 21 (SUPPL. 97), pp. 1-77Condino-Neto, A., Newburger, P.E., NADPH oxidase activity and cytochrome b558 content of human Epstein-Barr-virus-transformed B lymphocytes correlate with expression of genes encoding components of the oxidase system (1998) Arch Biochem Biophys, 360, pp. 158-164Condino-Neto, A., Newburger, P.E., Interferon-y improves splicing efficiency of CYBB gene transcripts in an interferon-responsive variant of chronic granulomatous disease due to a splice site consensus region mutation (2000) Blood, 95, pp. 3548-3554Beutler, E., Glucose-6-phosphate dehydrogenase deficiency (1983) The Metabolic Basis of Inherited Disease, pp. 1629-1653. , Stambury JBW, Fredrikson DS, Goldstein JL, and Brown MS, editors. New York: McGraw-HillConley, M.E., Notarangelo, L.D., Etzioni, A., Diagnostic criteria for primary immunodeficiencies. Representing PAGID (Pan-American Group for Immunodeficiency) and ESID (European Society for Immunodeficiencies) (1999) Clin Immunol, 93, pp. 190-197McCord, J.M., Fridovich, I., Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein) (1969) J Biol Chem, 244, pp. 6049-6055Maniatis, T., Fritsch, E.F., Sambrook, J., (1990) Molecular Cloning. A Laboratory Manual. 2nd Edition, , Plainview, NY: Cold Spring Harbor Laboratory PressBeutler, E., G6PD: Population genetics and clinical manifestations (1996) Blood Rev, 10, pp. 45-52Compri, M.B., Saad, S.T., Ramalho, A.S., Genetico-epidemiological and molecular investigation of G-6-PD deficiency in a Brazilian community (2000) Cad Saude Publ, 16, pp. 335-342Baehner, R.L., Johnston Jr., R.B., Nathan, D.G., Comparative study of the metabolic and bactericidal characteristics of severely glucose-6-phosphate dehydrogenase-deficient polymorphonuclear leukocytes and leukocytes from children with chronic granulomatous disease (1972) J Reticuloendothel Soc, 12, pp. 150-169Gray, G.R., Stamatoyannopoulos, G., Naiman, S.C., Neutrophil dysfunction, chronic granulomatous disease, and non-spherocytic haemolytic anaemia caused by complete deficiency of glucose-6-phosphate dehydrogenase (1973) Lancet, 2, pp. 530-534Corberand, J., De Larrard, B., Vergnes, H., Carriere, J.P., Chronic granulomatous disease with leukocytic glucose-6-phosphate dehydrogenase deficiency in a 28-month-old girl (1978) Am J Clin Pathol, 70, pp. 296-300Bellanti, J.A., Cantz, B.E., Schlegel, R.J., Accelerated decay of glucose 6-phosphate dehydrogenase activity in chronic granulomatous disease (1970) Pediatr Res, 4, pp. 405-411Rutenberg, W.D., Yang, M.C., Doberstyn, E.B., Bellanti, J.A., Multiple leukocyte abnormalities in chronic granulomatous disease: A familial study (1977) Pediatr Res, 11, pp. 158-163Erickson, R.P., Stites, D.P., Fudenberg, H.H., Epstein, C.J., Altered levels of glucose-6-phosphate dehydrogenase stabilizing factors in X-linked chronic granulomatous disease (1972) J Lab Clin Med, 80, pp. 644-65

Association of glucose-6-phosphate dehydrogenase deficiency and X-linked chronic granulomatous disease in a child with anemia and recurrent infections

Patients with severe leukocyte G6PD deficiency may present with impairment of NADPH oxidase activity and a history of recurrent infections, mimicking the phenotype of chronic granulomatous disease. We report herein a child with recurrent infections who initially received the diagnosis of G6PD deficiency. His erythrocyte G6PD activity was reduced: 1.8 U/g Hb (normal: 12.1 +/- 2.1 U/g Hb). Further studies revealed that G6PD activity in neutrophils, mononuclear leukocytes, and Epstein-Barr virus-transformed B-lymphocytes from the proband was similar to healthy controls. Molecular studies showed that the G6PD deficiency was due a 202 G-->A mutation, the A(-) variant common in African ethnic groups. the proband also exhibited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G-->A substitution at the 3' splice junction of gp91-phox exon 3. the cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable or nonfunctional mutant gp91-phox and to the phenotype of X-linked CGD. We propose that clinicians treating a patient with G6PD deficiency during a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes' microbicidal activity and the eventual association of G6PD deficiency and chronic granulomatous disease. Am. J. Hematol. 75:151-156, 2004. (C) 2004 Wiley-Liss, Inc.Univ Estadual Campinas, Sch Med, Ctr Invest Pediat, BR-13081970 Campinas, SP, BrazilUniv Estadual Campinas, Sch Med, Dept Pediat, BR-13081970 Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Pediat, Div Allergy Immunol & Rheumatol, São Paulo, BrazilUniv Massachusetts, Sch Med, Dept Pediat, Worcester, MA USAState Univ Campinas, Sch Med, Dept Med, Div Hematol, Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Pediat, Div Allergy Immunol & Rheumatol, São Paulo, BrazilWeb of Scienc

Association of glucose-6-phosphate dehydrogenase deficiency and X-linked chronic granulomatous disease in a child with anemia and recurrent infections

Patients with severe leukocyte G6PD deficiency may present with impairment of NADPH oxidase activity and a history of recurrent infections, mimicking the phenotype of chronic granulomatous disease. We report herein a child with recurrent infections who initially received the diagnosis of G6PD deficiency. His erythrocyte G6PD activity was reduced: 1.8 U/g Hb (normal: 12.1 +/- 2.1 U/g Hb). Further studies revealed that G6PD activity in neutrophils, mononuclear leukocytes, and Epstein-Barr virus-transformed B-lymphocytes from the proband was similar to healthy controls. Molecular studies showed that the G6PD deficiency was due a 202 G-->A mutation, the A(-) variant common in African ethnic groups. The proband also exhibited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G-->A substitution at the 3' splice junction of gp91-phox exon 3. The cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable or nonfunctional mutant gp91-phox and to the phenotype of X-linked CGD. We propose that clinicians treating a patient with G6PD deficiency during a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes' microbicidal activity and the eventual association of G6PD deficiency and chronic granulomatous disease. Am. J. Hematol. 75:151-156, 2004. (C) 2004 Wiley-Liss, Inc.75315115