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The role of functional polymorphims in the RANTES Gene promoter in asthma
J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 1
1!
AsthmaticPatients Exhibit Different Gene ExpressionProfiles, Depending on Their Sensitivity to Glucocorticoids
Hildur Helgag6ttir*, Unnur Steina Bjornsdottir§, Eva Halapi*, Asta Solilja Gudmundsdottir*, Thor Arnason*, Illugi Birkisson*, Asdis Elva Adalsteinsdottir*, Sigurdur lngvarsson*, Margret Andresdottir*, David Gislason~, Thorarinn Gislason¥, Jeffery Gulcher*, Karl Stefansson*, Hakon HakonarsonlE *Decode Genetics Incorporated, Reykjava'k, Iceland §University Hospital Vifilsstadir, Reykjavik, Iceland gVifilstadir, Reykjavik, Iceland ~Decode Genetics Incorporated, Reykjavik, Iceland BACKGROUND: Inhaled glucocorticoids (GCs) are widely and successfully used to control moderate to severe asthma. In spite of as much as a 10-fold variation in the effective dose for each patient, little is known about the molecular mechanisms underlying these differences in GC sensitivity. Asthma prevalence in Iceland is 6% and over 90% of patients with moderate to severe asthma use inhaled GCs on a daily basis. Given that global gene expression analysis provides a mechanism for exploring genetic variations that potentially influence drug responses, this study was set out to examine whether different gene expression profiles correlate with glucocorticoid sensitivity in moderate to severe asthma. M E T H O D S : Asthma severity was assessed by medical history, physical examination, methacholine challenge tests and PFTs. Atopy was verified by skin prick testing. Patients were classified as GC-resistant (n--25) if dependent on a daily GC inhalation dose of >_1500 mg and GC-sensitive (n=25) if on a daily GC dose of <800 mg. The two groups were comparable with respect to demographics and their asthma and atopy score. Peripheral blood mononuclear cells were collected from 50 asthmatic patients and treated in vitro with IL-I[~ and TNFtx in the absence (-GC) and presence (+GC) of pre-treatment with GC. Total RNA was extracted from the cells and gene expression was examined using Affymetrix DNA oligo array Hu95A chips which each contains over 12600 genes. RNA expression was analysed by comparing the mean difference change of signal intensity of +GC to -GC in each group. RESULTS: Comparing the mean difference change between +GC to -GC, forty genes were found to be significantly differentially expressed in the GC-resistant and GC-sensitive groups (p<0.05). These genes included various cytokines/chemokines, their receptors, transcription factors and regulators of signal transduction and apoptosis. Examples of such genes are IL-6, IL-8, CD44, NF-~z-B and jun-B, where the expression was more significandy inhibited by GC in IL-l~/TNFcz-treated cells from GC-sensitive compared to GC-resistant patients. CONCLUSION: When comparing the GC-sensitive and GC-resistant groups forty genes were identified that were significantly differently expressed between the two cohorts. The majority of these genes play an important role in the regulation and action of inflammatory responses and may contribute to the mechanisms that determine GC responsiveness, thereby rendering them candidate genes for the development of future therapies.
512Sp1 Displays Differential Binding and Transcriptional Activity With |nterleukin-10 -571 Promoter Polymorphism
John W Steinke, Angela Owens, Larry Borish University of Virginia, Charlottesville, VA Alterations in transcription of the interleukin (IL)-10 gene may contribute to the development and severity of autoimmune, infectious, neoplastic and allergic diseases. Previously, we have reported a C-to-A base substitution at bp -571 in the IL-10 promoter that results in increased promoter strength. The transcription factor Spl was found to bind immediately upstream of the polymorphism through competition With consensus Spl oligonucleotides and Spl-specific antisera. In electrophoretic mobility shift assays, we did not observe differences in binding affinity of recombinant Spl to the C or A form of the IL- 10 promoter. However, when nuclear extract from an IL-10 producing cell line was used, several Sp 1-containing
Abstracts
S173
multimeric complexes were observed which appeared in a dose-dependant manner. At the highest protein concentration used, binding of the Spl complex was identical for the C and A forms of the IL-10 promoter. At lower protein concentrations, two lower molecular weight (MW) Spl complexes were present. The lowest M W complex formed more rapidly on the C form of the IL-10 promoter while the second lowest MW ~complex formed a higher affinity complex with the A form of the promoter. Using specific antisera, we found that one of the proteins in complex with Spl was the transcription factor Sp3. Methylation protection footprinting demonstrated that binding of Spl to the IL-10 promoter created a hypersensitive site on the C form, but not the A form of the promoter. IL-10 reporter constructs containing the C and A form of the promoter were transfected into the Drosophilia Schneiders cell line which lacks endogenous Sp 1. Titration of a Sp 1 expression vector resulted in decreased transcription from the C form of the promoter, while no inhibition of transcription was observed at low doses of Spl for the A form of the promoter. High doses resulted in stimulation of transcription for the A form of the 1L- 10 promoter. In summary, the transcription factor Sp 1 can bind to the IL- 10 promoter upstream of a C-toA polymorphism. Sp 1, with or without Sp3, can form different multidimeric complexes on the two forms of the promoter that results in different transcriptional activities. Individuals carrying this allelic form of the IL- 10 promoter may display increased synthesis of IL-10 that could alter their immune response and lead to an increased severity of atopic disease.
5t
Je~The Role of FunctionalPnlymorphismsin the RANTESGene Pro| ~]1 rooter in Asthma
Nobuyuki Hizawa, Etsuro Yamaguchi, Satoshi Konno, Eisei Jinushi, Masaharu Nishimura School of Medicine, Hokkaido University, Sapporo, Japan Eosinophil infiltration of the airways in response to allergen exposure is a characteristic of asthma. RANTES is involved in the recruitment of eosinophils into the asthmatic airways, and .asthmatic individuals have increased expression ofRANTES mRNA and protein in their airways. Both the -403A and the -28G alleles of the RANTES promoter region strongly enhance promoter activity in reporter constructs in vitro. Thus, they may influence susceptibility to asthma. We investigated the genetic influence o f these functional alleles on the development of asthma using case-control analysis in a Japanese population (341 asthmatics and 312 controls). Asthma was diagnosed based on episodic symptoms of airflow obstruction that was at least partially reversible, and increased airway responsiveness to methacholine (Mch). We defined atopy as positive RAST (30.35 UA/ml) or MAST scores (31.0 lumicount) to at least one of 10 common allergens. All subjects in the study gave informed consent for enrollment in the study and blood drawing. Given the evidence for genetic heterogeneity of asthma according to age at onset of the disease, we divided asthmatics into 3 subgroups according to age at onset: 134 late-onset asthmatics (onset at >40 years old); 104 middle-onset asthmatics (onset at 20 to 40 years old); and 103 early-onset asthmatics (onset at <20 years old). The fractions of these subgroups that were atopic were as follows: late-onset, 48% (65/134); middle-onset, 77% (80/104); early-onset, 89% (92/103). No significant deviation from Hardy-Weinberg equilibrium was observed in control subjects for either of the RANTES promoter polymorphisms. The -28G allele was significantly associated with late-onset asthma (OR=1.92, [95%CI: 1.32, 2.80], pc<0.005). However, this allele was not associated with the other 2 asthma subgroups. The -403A allele was not associated with any of the 3 asthma subgroups. Evidence for association between the -28G allele and late-onset asthma remained significant when multivariate analysis was performed using a logistic regression model including 2 polymorphisms at the RANTES promoter region, sex, age, atopic status and smoking status (OR=2.73, [1.38, 5.44], p<0.005). Our findings suggested that, among Japanese, the -28G allele of the RANTES promoter region confers susceptibility to late-onset asthma.
1!
AsthmaticPatients Exhibit Different Gene ExpressionProfiles, Depending on Their Sensitivity to Glucocorticoids
Hildur Helgag6ttir*, Unnur Steina Bjornsdottir§, Eva Halapi*, Asta Solilja Gudmundsdottir*, Thor Arnason*, Illugi Birkisson*, Asdis Elva Adalsteinsdottir*, Sigurdur lngvarsson*, Margret Andresdottir*, David Gislason~, Thorarinn Gislason¥, Jeffery Gulcher*, Karl Stefansson*, Hakon HakonarsonlE *Decode Genetics Incorporated, Reykjava'k, Iceland §University Hospital Vifilsstadir, Reykjavik, Iceland gVifilstadir, Reykjavik, Iceland ~Decode Genetics Incorporated, Reykjavik, Iceland BACKGROUND: Inhaled glucocorticoids (GCs) are widely and successfully used to control moderate to severe asthma. In spite of as much as a 10-fold variation in the effective dose for each patient, little is known about the molecular mechanisms underlying these differences in GC sensitivity. Asthma prevalence in Iceland is 6% and over 90% of patients with moderate to severe asthma use inhaled GCs on a daily basis. Given that global gene expression analysis provides a mechanism for exploring genetic variations that potentially influence drug responses, this study was set out to examine whether different gene expression profiles correlate with glucocorticoid sensitivity in moderate to severe asthma. M E T H O D S : Asthma severity was assessed by medical history, physical examination, methacholine challenge tests and PFTs. Atopy was verified by skin prick testing. Patients were classified as GC-resistant (n--25) if dependent on a daily GC inhalation dose of >_1500 mg and GC-sensitive (n=25) if on a daily GC dose of <800 mg. The two groups were comparable with respect to demographics and their asthma and atopy score. Peripheral blood mononuclear cells were collected from 50 asthmatic patients and treated in vitro with IL-I[~ and TNFtx in the absence (-GC) and presence (+GC) of pre-treatment with GC. Total RNA was extracted from the cells and gene expression was examined using Affymetrix DNA oligo array Hu95A chips which each contains over 12600 genes. RNA expression was analysed by comparing the mean difference change of signal intensity of +GC to -GC in each group. RESULTS: Comparing the mean difference change between +GC to -GC, forty genes were found to be significantly differentially expressed in the GC-resistant and GC-sensitive groups (p<0.05). These genes included various cytokines/chemokines, their receptors, transcription factors and regulators of signal transduction and apoptosis. Examples of such genes are IL-6, IL-8, CD44, NF-~z-B and jun-B, where the expression was more significandy inhibited by GC in IL-l~/TNFcz-treated cells from GC-sensitive compared to GC-resistant patients. CONCLUSION: When comparing the GC-sensitive and GC-resistant groups forty genes were identified that were significantly differently expressed between the two cohorts. The majority of these genes play an important role in the regulation and action of inflammatory responses and may contribute to the mechanisms that determine GC responsiveness, thereby rendering them candidate genes for the development of future therapies.
512Sp1 Displays Differential Binding and Transcriptional Activity With |nterleukin-10 -571 Promoter Polymorphism
John W Steinke, Angela Owens, Larry Borish University of Virginia, Charlottesville, VA Alterations in transcription of the interleukin (IL)-10 gene may contribute to the development and severity of autoimmune, infectious, neoplastic and allergic diseases. Previously, we have reported a C-to-A base substitution at bp -571 in the IL-10 promoter that results in increased promoter strength. The transcription factor Spl was found to bind immediately upstream of the polymorphism through competition With consensus Spl oligonucleotides and Spl-specific antisera. In electrophoretic mobility shift assays, we did not observe differences in binding affinity of recombinant Spl to the C or A form of the IL- 10 promoter. However, when nuclear extract from an IL-10 producing cell line was used, several Sp 1-containing
Abstracts
S173
multimeric complexes were observed which appeared in a dose-dependant manner. At the highest protein concentration used, binding of the Spl complex was identical for the C and A forms of the IL-10 promoter. At lower protein concentrations, two lower molecular weight (MW) Spl complexes were present. The lowest M W complex formed more rapidly on the C form of the IL-10 promoter while the second lowest MW ~complex formed a higher affinity complex with the A form of the promoter. Using specific antisera, we found that one of the proteins in complex with Spl was the transcription factor Sp3. Methylation protection footprinting demonstrated that binding of Spl to the IL-10 promoter created a hypersensitive site on the C form, but not the A form of the promoter. IL-10 reporter constructs containing the C and A form of the promoter were transfected into the Drosophilia Schneiders cell line which lacks endogenous Sp 1. Titration of a Sp 1 expression vector resulted in decreased transcription from the C form of the promoter, while no inhibition of transcription was observed at low doses of Spl for the A form of the promoter. High doses resulted in stimulation of transcription for the A form of the 1L- 10 promoter. In summary, the transcription factor Sp 1 can bind to the IL- 10 promoter upstream of a C-toA polymorphism. Sp 1, with or without Sp3, can form different multidimeric complexes on the two forms of the promoter that results in different transcriptional activities. Individuals carrying this allelic form of the IL- 10 promoter may display increased synthesis of IL-10 that could alter their immune response and lead to an increased severity of atopic disease.
5t
Je~The Role of FunctionalPnlymorphismsin the RANTESGene Pro| ~]1 rooter in Asthma
Nobuyuki Hizawa, Etsuro Yamaguchi, Satoshi Konno, Eisei Jinushi, Masaharu Nishimura School of Medicine, Hokkaido University, Sapporo, Japan Eosinophil infiltration of the airways in response to allergen exposure is a characteristic of asthma. RANTES is involved in the recruitment of eosinophils into the asthmatic airways, and .asthmatic individuals have increased expression ofRANTES mRNA and protein in their airways. Both the -403A and the -28G alleles of the RANTES promoter region strongly enhance promoter activity in reporter constructs in vitro. Thus, they may influence susceptibility to asthma. We investigated the genetic influence o f these functional alleles on the development of asthma using case-control analysis in a Japanese population (341 asthmatics and 312 controls). Asthma was diagnosed based on episodic symptoms of airflow obstruction that was at least partially reversible, and increased airway responsiveness to methacholine (Mch). We defined atopy as positive RAST (30.35 UA/ml) or MAST scores (31.0 lumicount) to at least one of 10 common allergens. All subjects in the study gave informed consent for enrollment in the study and blood drawing. Given the evidence for genetic heterogeneity of asthma according to age at onset of the disease, we divided asthmatics into 3 subgroups according to age at onset: 134 late-onset asthmatics (onset at >40 years old); 104 middle-onset asthmatics (onset at 20 to 40 years old); and 103 early-onset asthmatics (onset at <20 years old). The fractions of these subgroups that were atopic were as follows: late-onset, 48% (65/134); middle-onset, 77% (80/104); early-onset, 89% (92/103). No significant deviation from Hardy-Weinberg equilibrium was observed in control subjects for either of the RANTES promoter polymorphisms. The -28G allele was significantly associated with late-onset asthma (OR=1.92, [95%CI: 1.32, 2.80], pc<0.005). However, this allele was not associated with the other 2 asthma subgroups. The -403A allele was not associated with any of the 3 asthma subgroups. Evidence for association between the -28G allele and late-onset asthma remained significant when multivariate analysis was performed using a logistic regression model including 2 polymorphisms at the RANTES promoter region, sex, age, atopic status and smoking status (OR=2.73, [1.38, 5.44], p<0.005). Our findings suggested that, among Japanese, the -28G allele of the RANTES promoter region confers susceptibility to late-onset asthma.