- Email: [email protected]
Copy Number Variations in del22q11.2 Syndrome
S176 Abstracts
these, IL1B, a key cytokine in the innate immunity pathway, was significantly up-regulated in CD141 monocytes, but not alveolar macrophages, by 26.4 fold (p 5 0.009) and 19.2 fold (p 5 0.039) after LPS stimulation for 6 and 20 hours, respectively. CONCLUSIONS: Our results demonstrate the utility of genomics to identify both candidate genes within the innate immunity pathway and differential expression by cell type. Studies comparing expression profiles between atopic asthmatic and healthy subjects, and validation studies, are ongoing. Funding: NIH
689
MONDAY
GATA-3 Induces Transcriptional Activation of CRTh2 R. Quapp, T. Seibert, N. Madsen, K. Madsen, L. Cameron; University of Alberta, Edmonton, AB, CANADA. RATIONALE: CRTh2 is a receptor for PGD2, a major mediator released by mast cells following degranulation and a marker of human Th2 cells. PGD2CRTh2 signaling induces expression of IL-4, IL-13 and IL-5. CRTh2 is upregulated by IL-4 and GATA-3, however, further study is required to characterize the regulatory elements mediating CRTh2 transcription. METHODS: Luciferase promoter constructs containing a 450 base pair fragment of the CRTh2 promoter (CRTh2pro-Luc) and two conserved region (CR) from the intron (CRTh2pro-Luc-CR1 and CRTh2pro-Luc-CR2) were generated. Activity of these constructs was studied by transient transfection of Jurkat T cells following overnight stimulation with PMA and ionomycin (P/I, 20ng/ml and 1mM). RESULTS: CRTh2pro-Luc activity was induced by P/I. CRTh2pro-LucCR1 activity was higher than CRTh2pro-Luc, while CRTh2pro-Luc-CR2 was actually less active. In silico analysis revealed the presence of putative GATA, NFAT and STAT sites within the promoter. Over-expression of GATA-3 further increased P/I induction of CRTh2 promoter activity. CR1 contains 9 potential GATA motifs while CR2 contains 4 putative STAT sites, the major transcription factor downstream of IL-4. Overexpression of GATA-3 with CRTh2pro-Luc-CR1 was more active than CRTh2pro-Luc, while STAT6 did not enhance CRTh2pro-Luc-CR2 or CRTh2-pro-Luc. CONCLUSIONS: GATA-3, a Th2 differentiation factor, induces CRTh2 transcription. Although CR2 did not demonstrate transcriptional activity, even with STAT6 over-expression, this region may still be important for chromatin remodeling. Funding: Univeristy of Alberta
690
Keratins: Important Candidate genes for Asthma and Immune Responsiveness to Cockroach P. Gao1, D. Grigoryev1, L. Breslin1, C. Cheadle1, R. A. Mathias2, T. H. Beaty3, A. Togias1, K. Barnes1; 1JHAAC, Baltimore, MD, 2Center for Inherited Disease Research, NIH, Baltimore, MD, 3Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD. RATIONALE: Sensitization and exposure to cockroach allergen is one of the strongest risk factors of asthma morbidity in the US. A genome-wide linkage scan provided significant evidence of linkage to specific loci (5q31, 12q13, 17q11) for asthma only in the presence of cockroach sensitization. Here we identify target genes specific for cockroach sensitization by integrating linkage and gene expression data. METHODS: Peripheral blood mononuclear cells (PBMCs), airway brushing cells (ABCs) and bronchioalveolar lavage (BAL) were collected from asthmatic (N 5 6) and healthy (N 5 6) volunteers and processed for highthroughput (Affymetrix) gene expression analysis. Genes within the three identified chromosomal loci were analyzed for differential gene expression between asthmatics and healthy controls. RESULTS: A total of 61 genes were selected for further analyses: 10 on chromosome 5q; 38 on 12q; and 13 on chromosome 17q. Gene expression was significantly increased for 12 of these genes among the asthmatics (1.26 - 8.15 fold). Expression of a group of keratin family genes (keratin 1 (KRT1), KRT4, KRT5, KRT6A, KRT6B, KRT8, and KRT18) was significantly increased in PBMCs, ABCs and BAL. KRT1 expression increase was greatest among PBMCs (8.15 fold). Of interest, these genes are
J ALLERGY CLIN IMMUNOL JANUARY 2007
localized at the peak marker (D12S389) with the strongest evidence of linkage to asthma in the presence of cockroach sensitization (P 5 0.002). CONCLUSIONS: Our findings suggest that these keratin family genes may be important candidates for asthma and immune responsiveness to cockroach, and integration of genome-wide expression profiling with linkage analysis is a robust approach toward identifying genes underlying complex traits.
691
Copy Number Variations in del22q11.2 Syndrome S. A. McGhee, M. Suchard, U. Bhardwaj, Y. Zhang, E. R. B. McCabe; David Geffen School of Medicine at UCLA, Los Angeles, CA. RATIONALE: Del22q11.2 syndrome is usually due to a deletion involving the Tbx gene on chromosome 22, but not all patients have the typical deletion, and not all patients with the deletion have full expression of the disease. A whole-genome approach may be better to understand and diagnose the disorder. We evaluated the use of 500,000 SNP mapping arrays and representational oligonucleotide microarray analysis (ROMA) in the delineation of the 22q11.2 deletion and identification of additional copy number variations (CNVs) elsewhere in the del22 syndrome genomes. METHODS: We evaluated CNVs using very-high-density ROMA in five patients with del22 syndrome. Identification of CNVs and pathologic deletions was by a new normalization and segmentation procedure (which we refer to as vivaROMA) that expanded on existing methods to improve comparisons between different arrays. RESULTS: The 22q11.2 deletion is easily identified, and our results agreed with the results of clinical flouresence in situ hybridization studies in all five patients. In addition, both patients and controls had numerous other CNVs elsewhere in the genome, consistent with the findings of other groups investigating CNVs in normal genomes. Some deletions elsewhere in del22 syndrome genomes involve coding regions that could affect immune system function. CONCLUSIONS: Genetic diversity outside of the 22q11.2 region may affect the phenotype in del22q11.2 syndrome. Use of whole genome approaches to the study of this disorder could enable the identification of new genes and hypotheses about the origins of immune system dysfunction. Funding: National Institutes of Health
692
Effect of Montelukast on IL6, IL12 from Inflamatory Exudation in Gouty Arthritis Model J. A. Corado, M. Bermudez, A. Linares, L. Ponce, Z. Mora, J. Lucar, A. Acosta, R. Tovar; Universidad de Carabobo, Valencia, VENEZUELA. RATIONALE: Gouty Arthritis is a metabolic disease, characterized by the acumulation of Uric acid in joints. The increase of Uric Acid in serum leads to the MUS crystals formation. These crystals interact with Mononuclear Phagocytic System cells, causing second messengers synthesis and the activation of transcription factors wich helps genes expression, molecules releasing like proinflamatory cytokines, such as IL1, IL6, IL8, IL12 and TNF-a. METHODS: the 15 mouses sample were divided in three experimental groups A,B,C. GROUP A: received MK orally (1mg/kgP). GROUP B: received PBS (negative control of inflamation). GROUP C: received MUS into the Air Bag (positive control of inflamation). In these groups the inflamation was induced by injection of MUS crystals (2cc) into the subcutaneous cavity. RESULTS: in presence of MK, IL6 and IL12 concentrations are lower (p<0,05) than the concentration observed in presence of MUS. Comparing the inhibition percentages in the concentrations of IL6 and IL12 it is observed that IL6 had more inhibition. CONCLUSSION: MK reduced IL6 and IL12 concentrations significantly from inflamatory exudation possibly by effect on the Lipooxygenase enzime pathway.All this facts made plan ourselves that MK could be potencially a therapeutic alternative of Gouty Arthritis. Funding: Universidad de Carabobo
these, IL1B, a key cytokine in the innate immunity pathway, was significantly up-regulated in CD141 monocytes, but not alveolar macrophages, by 26.4 fold (p 5 0.009) and 19.2 fold (p 5 0.039) after LPS stimulation for 6 and 20 hours, respectively. CONCLUSIONS: Our results demonstrate the utility of genomics to identify both candidate genes within the innate immunity pathway and differential expression by cell type. Studies comparing expression profiles between atopic asthmatic and healthy subjects, and validation studies, are ongoing. Funding: NIH
689
MONDAY
GATA-3 Induces Transcriptional Activation of CRTh2 R. Quapp, T. Seibert, N. Madsen, K. Madsen, L. Cameron; University of Alberta, Edmonton, AB, CANADA. RATIONALE: CRTh2 is a receptor for PGD2, a major mediator released by mast cells following degranulation and a marker of human Th2 cells. PGD2CRTh2 signaling induces expression of IL-4, IL-13 and IL-5. CRTh2 is upregulated by IL-4 and GATA-3, however, further study is required to characterize the regulatory elements mediating CRTh2 transcription. METHODS: Luciferase promoter constructs containing a 450 base pair fragment of the CRTh2 promoter (CRTh2pro-Luc) and two conserved region (CR) from the intron (CRTh2pro-Luc-CR1 and CRTh2pro-Luc-CR2) were generated. Activity of these constructs was studied by transient transfection of Jurkat T cells following overnight stimulation with PMA and ionomycin (P/I, 20ng/ml and 1mM). RESULTS: CRTh2pro-Luc activity was induced by P/I. CRTh2pro-LucCR1 activity was higher than CRTh2pro-Luc, while CRTh2pro-Luc-CR2 was actually less active. In silico analysis revealed the presence of putative GATA, NFAT and STAT sites within the promoter. Over-expression of GATA-3 further increased P/I induction of CRTh2 promoter activity. CR1 contains 9 potential GATA motifs while CR2 contains 4 putative STAT sites, the major transcription factor downstream of IL-4. Overexpression of GATA-3 with CRTh2pro-Luc-CR1 was more active than CRTh2pro-Luc, while STAT6 did not enhance CRTh2pro-Luc-CR2 or CRTh2-pro-Luc. CONCLUSIONS: GATA-3, a Th2 differentiation factor, induces CRTh2 transcription. Although CR2 did not demonstrate transcriptional activity, even with STAT6 over-expression, this region may still be important for chromatin remodeling. Funding: Univeristy of Alberta
690
Keratins: Important Candidate genes for Asthma and Immune Responsiveness to Cockroach P. Gao1, D. Grigoryev1, L. Breslin1, C. Cheadle1, R. A. Mathias2, T. H. Beaty3, A. Togias1, K. Barnes1; 1JHAAC, Baltimore, MD, 2Center for Inherited Disease Research, NIH, Baltimore, MD, 3Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD. RATIONALE: Sensitization and exposure to cockroach allergen is one of the strongest risk factors of asthma morbidity in the US. A genome-wide linkage scan provided significant evidence of linkage to specific loci (5q31, 12q13, 17q11) for asthma only in the presence of cockroach sensitization. Here we identify target genes specific for cockroach sensitization by integrating linkage and gene expression data. METHODS: Peripheral blood mononuclear cells (PBMCs), airway brushing cells (ABCs) and bronchioalveolar lavage (BAL) were collected from asthmatic (N 5 6) and healthy (N 5 6) volunteers and processed for highthroughput (Affymetrix) gene expression analysis. Genes within the three identified chromosomal loci were analyzed for differential gene expression between asthmatics and healthy controls. RESULTS: A total of 61 genes were selected for further analyses: 10 on chromosome 5q; 38 on 12q; and 13 on chromosome 17q. Gene expression was significantly increased for 12 of these genes among the asthmatics (1.26 - 8.15 fold). Expression of a group of keratin family genes (keratin 1 (KRT1), KRT4, KRT5, KRT6A, KRT6B, KRT8, and KRT18) was significantly increased in PBMCs, ABCs and BAL. KRT1 expression increase was greatest among PBMCs (8.15 fold). Of interest, these genes are
J ALLERGY CLIN IMMUNOL JANUARY 2007
localized at the peak marker (D12S389) with the strongest evidence of linkage to asthma in the presence of cockroach sensitization (P 5 0.002). CONCLUSIONS: Our findings suggest that these keratin family genes may be important candidates for asthma and immune responsiveness to cockroach, and integration of genome-wide expression profiling with linkage analysis is a robust approach toward identifying genes underlying complex traits.
691
Copy Number Variations in del22q11.2 Syndrome S. A. McGhee, M. Suchard, U. Bhardwaj, Y. Zhang, E. R. B. McCabe; David Geffen School of Medicine at UCLA, Los Angeles, CA. RATIONALE: Del22q11.2 syndrome is usually due to a deletion involving the Tbx gene on chromosome 22, but not all patients have the typical deletion, and not all patients with the deletion have full expression of the disease. A whole-genome approach may be better to understand and diagnose the disorder. We evaluated the use of 500,000 SNP mapping arrays and representational oligonucleotide microarray analysis (ROMA) in the delineation of the 22q11.2 deletion and identification of additional copy number variations (CNVs) elsewhere in the del22 syndrome genomes. METHODS: We evaluated CNVs using very-high-density ROMA in five patients with del22 syndrome. Identification of CNVs and pathologic deletions was by a new normalization and segmentation procedure (which we refer to as vivaROMA) that expanded on existing methods to improve comparisons between different arrays. RESULTS: The 22q11.2 deletion is easily identified, and our results agreed with the results of clinical flouresence in situ hybridization studies in all five patients. In addition, both patients and controls had numerous other CNVs elsewhere in the genome, consistent with the findings of other groups investigating CNVs in normal genomes. Some deletions elsewhere in del22 syndrome genomes involve coding regions that could affect immune system function. CONCLUSIONS: Genetic diversity outside of the 22q11.2 region may affect the phenotype in del22q11.2 syndrome. Use of whole genome approaches to the study of this disorder could enable the identification of new genes and hypotheses about the origins of immune system dysfunction. Funding: National Institutes of Health
692
Effect of Montelukast on IL6, IL12 from Inflamatory Exudation in Gouty Arthritis Model J. A. Corado, M. Bermudez, A. Linares, L. Ponce, Z. Mora, J. Lucar, A. Acosta, R. Tovar; Universidad de Carabobo, Valencia, VENEZUELA. RATIONALE: Gouty Arthritis is a metabolic disease, characterized by the acumulation of Uric acid in joints. The increase of Uric Acid in serum leads to the MUS crystals formation. These crystals interact with Mononuclear Phagocytic System cells, causing second messengers synthesis and the activation of transcription factors wich helps genes expression, molecules releasing like proinflamatory cytokines, such as IL1, IL6, IL8, IL12 and TNF-a. METHODS: the 15 mouses sample were divided in three experimental groups A,B,C. GROUP A: received MK orally (1mg/kgP). GROUP B: received PBS (negative control of inflamation). GROUP C: received MUS into the Air Bag (positive control of inflamation). In these groups the inflamation was induced by injection of MUS crystals (2cc) into the subcutaneous cavity. RESULTS: in presence of MK, IL6 and IL12 concentrations are lower (p<0,05) than the concentration observed in presence of MUS. Comparing the inhibition percentages in the concentrations of IL6 and IL12 it is observed that IL6 had more inhibition. CONCLUSSION: MK reduced IL6 and IL12 concentrations significantly from inflamatory exudation possibly by effect on the Lipooxygenase enzime pathway.All this facts made plan ourselves that MK could be potencially a therapeutic alternative of Gouty Arthritis. Funding: Universidad de Carabobo