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

Neutrophils And Mononuclear Cells From Patients With Chronic Granulomatous Disease Release Nitric Oxide

Chronic granulomatous disease (CGD) is a group of genetic disorders characterised by recurrent severe suppurative infections due to impaired microbial killing. The principal biochemical defect is an impairment in the production of reactive oxygen intermediates by phagocytes. Nitric oxide (NO) is synthesised from the guanidino nitrogen atom(s) of L-arginine and has recently been proposed to be involved in defence mechanisms. The aim of this study was to investigate the involvement of the oxidative burst in the biosynthesis of NO by neutrophils and mononuclear cells from patients with CGD. NO synthesis was assayed by the ability of neutrophils and mononuclear cells to inhibit thrombin-induced washed platelet aggregation while superoxide anion (O2-) production was measured spectrophotometrically by the superoxide dismutase inhibitable reduction of cytochrome c. Neutrophils and mononuclear cells from patients with CGD released NO. This release was inhibited by nitro-L-arginine methyl ester but could be reversed by L-arginine. Zymosan- and PMA-induced O2- production was less than 10% as compared with health controls. These results indicate that O2- production is not essential for NO synthesis in human leucocytes.35548549

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

Antibody response to pneumococcal capsular polysaccharide vaccine in Down syndrome patients

The majority of children with Down syndrome (DS) tend to have frequent bacterial infections including recurrent respiratory infections. Our objective was to evaluate the production of antibodies to pneumococcal polysaccharide antigens after active immunization in DS subjects. IgG antibodies to pneumococcal serotypes (1, 3, 6B, 9V, and 14) were measured before and 6 weeks after immunization with a 23-valent pneumococcal vaccine (Pneumo23®, Pasteur-Merrieux) in 6- to 13-year-old DS children (N = 17) and in aged-matched normal controls (N = 30). An adequate response was defined as a 4-fold increase over baseline or a post-immunization level of specific pneumococcal serotype antibody > or = 1.3 µg/mL. After immunization, all DS children had an increase in post-immunization levels against all serotypes analyzed. A 4-fold or more increase was observed in all DS children concerning serotypes 1 and 14, in 90% of subjects for serotypes 3 and 9V, and in 65% for serotype 6B. Regarding this increase, 8 of the 17 DS children had an adequate response to all serotypes analyzed, 8/17 patients to 4 serotypes and 1/17 to 3 serotypes. However, when we compared post-immunization levels between DS children and controls, we observed lower levels in the former group (P < 0.05) for all serotypes except serotype 3. We conclude that pneumococcal polysaccharide immunization could be beneficial for these DS children