The standard procedure for diagnosing allergic contact dermatitis is to perform a patch test. Because this has several disadvantages, the development of a new in vitro test system would be of immense value. Gene trans...The standard procedure for diagnosing allergic contact dermatitis is to perform a patch test. Because this has several disadvantages, the development of a new in vitro test system would be of immense value. Gene transcripts that distinguish allergies from non-allergies may have the potential to serve as the molecular basis for such a diagnostic tool. In this study, we use the microarray technology in the identification of differentially expressed genes in allergen-stimulated peripheral blood mononuclear cells (PBMCs) from 3 chromium-allergic patients versus 3 healthy controls. Using an Affymetrix GeneChip., the gene expression was analysed in PBMC cultures grown with 100 μg/ml CrCl3 or in media alone for 24 hr. A total of 26 genes were differentially expressed by more than twofold (P < 0.01) in allergen-activated PBMCs from patients compared with controls. 18 of these were upregulated, whereas 8 were downregulated. The expression of 1 downregulated gene, CASP8, was also found specifically and significantly reduced in an expanded population including 4 additional chromium allergic patients and 1 additional control subject by real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The expression of 2 upregulated genes, ETS2 and CISH, correlated with a high-proliferative response following CrCl3 exposure. Additionally, real-time RT-PCR analysis indicated that the same gene expression changes are valid for nickel allergies, potentially making the expression profile more widely available. The 26 differentially expressed genes identified in this study may potentially function as diagnostic markers for contact sensitivity.展开更多
Background:Werner syndrome (WS) is a rare autosomal recessive progeroid disorder caused by mutations of the WRN gene encoding a protein of the RecQ-type family of DNA helicases. Objectives:To develop a rapid and simpl...Background:Werner syndrome (WS) is a rare autosomal recessive progeroid disorder caused by mutations of the WRN gene encoding a protein of the RecQ-type family of DNA helicases. Objectives:To develop a rapid and simple reverse transcription-polymerase chain reaction (RT-PCR) strategy for mutation analysis of the WRN gene, to identify pathogenic mutations in a German patient with WS and to determine the effects of the pathogenic mutations on WRN mRNA stability. Methods:Allele-specific RT-PCR, semiquantitative RT-PCR, DNA sequencing. Results:We describe a novel and rapid RT-PCR-based method for mutation analysis in WS and report a German patient with WS carrying a previously reported (1396delA) as well as a novel nonsense mutation (2334delAC)of the WRN gene. By semiquantitative RT-PCR analysis we demonstrate that this compound heterozygous genotype leads to WRN transcript decay. Conclusions:In previous studies WS was primarily attributed to a loss of function of stable truncated WRN gene products. Our findings indicate that mutations can also lead to markedly decreased WRN transcript stability.展开更多
文摘The standard procedure for diagnosing allergic contact dermatitis is to perform a patch test. Because this has several disadvantages, the development of a new in vitro test system would be of immense value. Gene transcripts that distinguish allergies from non-allergies may have the potential to serve as the molecular basis for such a diagnostic tool. In this study, we use the microarray technology in the identification of differentially expressed genes in allergen-stimulated peripheral blood mononuclear cells (PBMCs) from 3 chromium-allergic patients versus 3 healthy controls. Using an Affymetrix GeneChip., the gene expression was analysed in PBMC cultures grown with 100 μg/ml CrCl3 or in media alone for 24 hr. A total of 26 genes were differentially expressed by more than twofold (P < 0.01) in allergen-activated PBMCs from patients compared with controls. 18 of these were upregulated, whereas 8 were downregulated. The expression of 1 downregulated gene, CASP8, was also found specifically and significantly reduced in an expanded population including 4 additional chromium allergic patients and 1 additional control subject by real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The expression of 2 upregulated genes, ETS2 and CISH, correlated with a high-proliferative response following CrCl3 exposure. Additionally, real-time RT-PCR analysis indicated that the same gene expression changes are valid for nickel allergies, potentially making the expression profile more widely available. The 26 differentially expressed genes identified in this study may potentially function as diagnostic markers for contact sensitivity.
文摘Background:Werner syndrome (WS) is a rare autosomal recessive progeroid disorder caused by mutations of the WRN gene encoding a protein of the RecQ-type family of DNA helicases. Objectives:To develop a rapid and simple reverse transcription-polymerase chain reaction (RT-PCR) strategy for mutation analysis of the WRN gene, to identify pathogenic mutations in a German patient with WS and to determine the effects of the pathogenic mutations on WRN mRNA stability. Methods:Allele-specific RT-PCR, semiquantitative RT-PCR, DNA sequencing. Results:We describe a novel and rapid RT-PCR-based method for mutation analysis in WS and report a German patient with WS carrying a previously reported (1396delA) as well as a novel nonsense mutation (2334delAC)of the WRN gene. By semiquantitative RT-PCR analysis we demonstrate that this compound heterozygous genotype leads to WRN transcript decay. Conclusions:In previous studies WS was primarily attributed to a loss of function of stable truncated WRN gene products. Our findings indicate that mutations can also lead to markedly decreased WRN transcript stability.
