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

Detalhes bibliográficos
Autor(a) principal: Agudelo-Flórez,P.
Data de Publicação: 2004
Outros Autores: López,J.A., Redher,J., Carneiro-Sampaio,M.M.S., Costa-Carvalho,B.T., Grumach,A.S., Condino-Neto,A.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Brazilian Journal of Medical and Biological Research
Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000500001
Resumo: 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.
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spelling The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous diseaseSuperoxidePhagocytesPrimary immunodeficiencyRespiratory burstNeutrophilsHumanChronic 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.Associação Brasileira de Divulgação Científica2004-05-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000500001Brazilian Journal of Medical and Biological Research v.37 n.5 2004reponame:Brazilian Journal of Medical and Biological Researchinstname:Associação Brasileira de Divulgação Científica (ABDC)instacron:ABDC10.1590/S0100-879X2004000500001info:eu-repo/semantics/openAccessAgudelo-Flórez,P.López,J.A.Redher,J.Carneiro-Sampaio,M.M.S.Costa-Carvalho,B.T.Grumach,A.S.Condino-Neto,A.eng2004-04-22T00:00:00Zoai:scielo:S0100-879X2004000500001Revistahttps://www.bjournal.org/https://old.scielo.br/oai/scielo-oai.phpbjournal@terra.com.br||bjournal@terra.com.br1414-431X0100-879Xopendoar:2004-04-22T00:00Brazilian Journal of Medical and Biological Research - Associação Brasileira de Divulgação Científica (ABDC)false
dc.title.none.fl_str_mv The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease
title The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease
spellingShingle The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease
Agudelo-Flórez,P.
Superoxide
Phagocytes
Primary immunodeficiency
Respiratory burst
Neutrophils
Human
title_short The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease
title_full The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease
title_fullStr The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease
title_full_unstemmed The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease
title_sort The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease
author Agudelo-Flórez,P.
author_facet Agudelo-Flórez,P.
López,J.A.
Redher,J.
Carneiro-Sampaio,M.M.S.
Costa-Carvalho,B.T.
Grumach,A.S.
Condino-Neto,A.
author_role author
author2 López,J.A.
Redher,J.
Carneiro-Sampaio,M.M.S.
Costa-Carvalho,B.T.
Grumach,A.S.
Condino-Neto,A.
author2_role author
author
author
author
author
author
dc.contributor.author.fl_str_mv Agudelo-Flórez,P.
López,J.A.
Redher,J.
Carneiro-Sampaio,M.M.S.
Costa-Carvalho,B.T.
Grumach,A.S.
Condino-Neto,A.
dc.subject.por.fl_str_mv Superoxide
Phagocytes
Primary immunodeficiency
Respiratory burst
Neutrophils
Human
topic Superoxide
Phagocytes
Primary immunodeficiency
Respiratory burst
Neutrophils
Human
description 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.
publishDate 2004
dc.date.none.fl_str_mv 2004-05-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000500001
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000500001
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/S0100-879X2004000500001
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
dc.source.none.fl_str_mv Brazilian Journal of Medical and Biological Research v.37 n.5 2004
reponame:Brazilian Journal of Medical and Biological Research
instname:Associação Brasileira de Divulgação Científica (ABDC)
instacron:ABDC
instname_str Associação Brasileira de Divulgação Científica (ABDC)
instacron_str ABDC
institution ABDC
reponame_str Brazilian Journal of Medical and Biological Research
collection Brazilian Journal of Medical and Biological Research
repository.name.fl_str_mv Brazilian Journal of Medical and Biological Research - Associação Brasileira de Divulgação Científica (ABDC)
repository.mail.fl_str_mv bjournal@terra.com.br||bjournal@terra.com.br
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