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5-lipoxygenase (ALOX5) promoter polymorphism is not associated with asthma in a Caucasian isolate
Abstracts S219
J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
870
Analysis of ADORA3 as a Possible Candidate Gene for Asthma
M. N. Blumenthal, M. B. Miller, C. Reilly, W. S. Oetting, M. Brott, R. Willaert, S. Luah, R. A. King; University of Minnesota, Minneapolis, MN. RATIONALE: The ADORA3 locus is located within a chromosome region genetically linked to asthma and was considered a strong candidate gene for asthma susceptibility. METHODS: An initial genomic screen produced a NPL z-score of 4.0 at chromosome 1p21 using 3 extended families (n = 40) containing several individuals with asthma (n = 14). Further fine mapping on one family produced a NPL z-score of 8.5. An interesting candidate gene was identified (Adenosine A3 receptor, ADORA3), and analyzed using DNA sequencing. Sequence analysis included the two coding exons, intron/exon boundaries and the 5’ promoter region. Several affected and unaffected family members and several controls were sequenced. In addition to the sequencing analysis, 5 other SNPs in the ADORA3 locus, three in the 5’ promoter (rs1544223, rs2298191 and rs9728945) and two in the 3’ untranslated region (rs 923 and rs1415793) were also analyzed. RESULTS: The sequence analysis did not identify any candidate mutations in the regions sequenced. Three polymorphisms were identified in the 5’ promoter region (G/A-564, T/C-581 and T/C-1368) which created two haplotypes GTT and ACC. Analysis of both family members and unrelated controls showed no association between this haplotype and asthma. Analysis of the 5 SNPs with a second population of individuals with asthma revealed no association with asthma. CONCLUSIONS: Alterations of the ADORA3 gene, a gene within a chromosome region linked to asthma, are not associated with asthma. Funding: NIH Identification of Asthma Susceptibility Genes Using Cluster Analysis W. S. Oetting, M. N. Blumenthal, C. Reilly, M. B. Miller, R. Willaert, S. Luah, R. A. King; University of Minnesota, Minneapolis, MN. RATIONALE: Asthma is most likely a genetically heterogeneous disorder, making definition of the phenotype difficult. In defining a phenotype for asthma, we need to delineate different varieties of the disorder with the hypothesis that varying phenotypes may have different genetic sources. To test this hypothesis, we used cluster analysis to detect different types of asthmatics. METHODS: Using data from skin reactivity tests, we used k-means clustering to identify different varieties of asthmatics. Quantitative traits were defined by the distance from an individual’s collection of skin reactivity scores to each cluster center. These quantitative traits were then subjected to a genome scan using the variance components method of quantitative linkage analysis. The method is applied to a data set of 27 multigenerational families with asthma. RESULTS: We found several interesting peaks, one on chromosome 2 (D2S1776, 173 cM) with a maximum Lod score of 2.8 and two on chromosome 3 at 152 cM (D3S1764) with a maximum Lod score of 2.7 and at 102 cM (D2S2406) with a maximum Lod score of 2.0. CONCLUSIONS: The use of cluster analysis is a powerful technique for the identification of loci associated with complex genetic disorders such as asthma. Funding: NIH
871
5-Lipoxygenase (ALOX5) Promoter Polymorphism Is Not Associated With Asthma in a Caucasian Isolate M. Munoz1, S. Zhang1, A. Grant1, K. Held1, F. Chilton2, M. Surrette3, H. Allayee4, K. C. Barnes1; 1Johns Hopkins University, Baltimore, MD,
872
2Wake
Forest University School of Medicine, Winston-Salem, NC, 3Pilot Therapeutics, Inc., Charleston, SC, 4General Clinical Research Center, USC Keck School of Medicine, Los Angeles, CA. RATIONALE: Leukotrienes are biologically active lipid mediators generated from arachidonic acid at sites of inflammation. Five-lipoxygenase (5-LO) is the primary enzyme involved in the oxidative biosynthesis of leukotrienes, which in turn mediate the vascular permeability and smooth muscle contraction characteristic of asthma. Consequently, it is plausible that variants in the promoter region of the gene encoding 5-LO (ALOX5) are associated with allergic disease. Recently it has been demonstrated that dietary intake of certain fatty acids contribute to the effect of ALOX5 genotype in another inflammatory disease, atherosclerosis. METHODS: Genomic DNA was extracted from 307 Caucasian isolates from Tangier Island and screened for -147PSp1 ALOX5 promoter polymorphism. The Family-Based Association Test (FBAT) was used to test for association with asthma, atopy, and total IgE. To determine whether fasting plasma fatty acid levels confound association, we determined serum PUFA content by gas chromatography with flame ionization detection. RESULTS: Genotyping yielded 269 carriers of the -147PSp1 ALOX5 common allele (87.6%) and 38 carriers of variant alleles (12.3 %). Predominant genotype was homozygote wild-type 5/5 (62.2%) and heterozygote 4/5 (20.5%) followed by homozygote 4/4 (4.7%) and heterozygote 5/7 (3.2%). Two subjects were carriers of a very rare allele 5/8. There was no association with any of the traits and the variants. After adjusting for 6 fatty acids and the AA/EPA, AA/DGLA ratio, we did not observe any change in associations. CONCLUSIONS: Our data do not support a role for the -147PSp1 ALOX5 polymorphism and allergic disease, nor do they suggest a dietgene interaction for this trait.
The Impact of Pre-Transplant Flow Cytometry Crossmatches on Renal Allograft Outcome: A Pilot Study R. G. O’Brien, L. Yao, B. Arora, D. Frey, P. Boudreaux, I. Daley, R. Thiagarajan, P. Kumar; Medicine/Allergy, LSU Medical Center, New Orleans, LA. BACKGROUND: Pre-Transplant Complement Dependent Cytotoxicity (CDC) Crossmatch is an established procedure known to influence renal allograft survival rates. The Flow Cytometry Crossmatch (FCXM) is considered more sensitive, and based on literature reports, is believed to enhance renal allograft outcome. Before utilizing this procedure, some programs are requiring center specific data. PURPOSE OF THE STUDY: The purpose of this study was to investigate the impact of a positive FCXM on renal allograft outcome in a single center. METHOD: Fifty-six patients who received renal transplants at The Transplant Institute of New Orleans were investigated. The pre-transplant workup included both a CDC and FCXM. A negative CDC crossmatch against donor T Cells was required for inclusion in the study. The results of the Flow Crossmatch were not used for excluding patients for getting a transplant. The clinical outcome in terms of successful versus failed transplants was recorded over a three year period. A failed graft was defined as return to dialysis. RESULTS: Nine out of fifty-six renal allografts failed due to immunologic rejections. An additional two grafts were lost due to other reasons. Amongst those who lost the renal grafts due to rejection, thirty-three percent had a positive pre-transplant FCXM. On the other hand, the FCXM was positive in sixteen percent of those who had functioning renal grafts. CONCLUSION: Although these numbers are small, the data suggest that positive FCXMS are related to lower renal allograft survival rates at our center. Further studies will be performed on a larger patient population.
873
TUESDAY
ma severity, whereas it may not be associated with the development of asthma per se. These results suggest that IL-5 gene may be a disease-modifying gene in Korean children with atopic asthma. Funding: Ulsan University
J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
870
Analysis of ADORA3 as a Possible Candidate Gene for Asthma
M. N. Blumenthal, M. B. Miller, C. Reilly, W. S. Oetting, M. Brott, R. Willaert, S. Luah, R. A. King; University of Minnesota, Minneapolis, MN. RATIONALE: The ADORA3 locus is located within a chromosome region genetically linked to asthma and was considered a strong candidate gene for asthma susceptibility. METHODS: An initial genomic screen produced a NPL z-score of 4.0 at chromosome 1p21 using 3 extended families (n = 40) containing several individuals with asthma (n = 14). Further fine mapping on one family produced a NPL z-score of 8.5. An interesting candidate gene was identified (Adenosine A3 receptor, ADORA3), and analyzed using DNA sequencing. Sequence analysis included the two coding exons, intron/exon boundaries and the 5’ promoter region. Several affected and unaffected family members and several controls were sequenced. In addition to the sequencing analysis, 5 other SNPs in the ADORA3 locus, three in the 5’ promoter (rs1544223, rs2298191 and rs9728945) and two in the 3’ untranslated region (rs 923 and rs1415793) were also analyzed. RESULTS: The sequence analysis did not identify any candidate mutations in the regions sequenced. Three polymorphisms were identified in the 5’ promoter region (G/A-564, T/C-581 and T/C-1368) which created two haplotypes GTT and ACC. Analysis of both family members and unrelated controls showed no association between this haplotype and asthma. Analysis of the 5 SNPs with a second population of individuals with asthma revealed no association with asthma. CONCLUSIONS: Alterations of the ADORA3 gene, a gene within a chromosome region linked to asthma, are not associated with asthma. Funding: NIH Identification of Asthma Susceptibility Genes Using Cluster Analysis W. S. Oetting, M. N. Blumenthal, C. Reilly, M. B. Miller, R. Willaert, S. Luah, R. A. King; University of Minnesota, Minneapolis, MN. RATIONALE: Asthma is most likely a genetically heterogeneous disorder, making definition of the phenotype difficult. In defining a phenotype for asthma, we need to delineate different varieties of the disorder with the hypothesis that varying phenotypes may have different genetic sources. To test this hypothesis, we used cluster analysis to detect different types of asthmatics. METHODS: Using data from skin reactivity tests, we used k-means clustering to identify different varieties of asthmatics. Quantitative traits were defined by the distance from an individual’s collection of skin reactivity scores to each cluster center. These quantitative traits were then subjected to a genome scan using the variance components method of quantitative linkage analysis. The method is applied to a data set of 27 multigenerational families with asthma. RESULTS: We found several interesting peaks, one on chromosome 2 (D2S1776, 173 cM) with a maximum Lod score of 2.8 and two on chromosome 3 at 152 cM (D3S1764) with a maximum Lod score of 2.7 and at 102 cM (D2S2406) with a maximum Lod score of 2.0. CONCLUSIONS: The use of cluster analysis is a powerful technique for the identification of loci associated with complex genetic disorders such as asthma. Funding: NIH
871
5-Lipoxygenase (ALOX5) Promoter Polymorphism Is Not Associated With Asthma in a Caucasian Isolate M. Munoz1, S. Zhang1, A. Grant1, K. Held1, F. Chilton2, M. Surrette3, H. Allayee4, K. C. Barnes1; 1Johns Hopkins University, Baltimore, MD,
872
2Wake
Forest University School of Medicine, Winston-Salem, NC, 3Pilot Therapeutics, Inc., Charleston, SC, 4General Clinical Research Center, USC Keck School of Medicine, Los Angeles, CA. RATIONALE: Leukotrienes are biologically active lipid mediators generated from arachidonic acid at sites of inflammation. Five-lipoxygenase (5-LO) is the primary enzyme involved in the oxidative biosynthesis of leukotrienes, which in turn mediate the vascular permeability and smooth muscle contraction characteristic of asthma. Consequently, it is plausible that variants in the promoter region of the gene encoding 5-LO (ALOX5) are associated with allergic disease. Recently it has been demonstrated that dietary intake of certain fatty acids contribute to the effect of ALOX5 genotype in another inflammatory disease, atherosclerosis. METHODS: Genomic DNA was extracted from 307 Caucasian isolates from Tangier Island and screened for -147PSp1 ALOX5 promoter polymorphism. The Family-Based Association Test (FBAT) was used to test for association with asthma, atopy, and total IgE. To determine whether fasting plasma fatty acid levels confound association, we determined serum PUFA content by gas chromatography with flame ionization detection. RESULTS: Genotyping yielded 269 carriers of the -147PSp1 ALOX5 common allele (87.6%) and 38 carriers of variant alleles (12.3 %). Predominant genotype was homozygote wild-type 5/5 (62.2%) and heterozygote 4/5 (20.5%) followed by homozygote 4/4 (4.7%) and heterozygote 5/7 (3.2%). Two subjects were carriers of a very rare allele 5/8. There was no association with any of the traits and the variants. After adjusting for 6 fatty acids and the AA/EPA, AA/DGLA ratio, we did not observe any change in associations. CONCLUSIONS: Our data do not support a role for the -147PSp1 ALOX5 polymorphism and allergic disease, nor do they suggest a dietgene interaction for this trait.
The Impact of Pre-Transplant Flow Cytometry Crossmatches on Renal Allograft Outcome: A Pilot Study R. G. O’Brien, L. Yao, B. Arora, D. Frey, P. Boudreaux, I. Daley, R. Thiagarajan, P. Kumar; Medicine/Allergy, LSU Medical Center, New Orleans, LA. BACKGROUND: Pre-Transplant Complement Dependent Cytotoxicity (CDC) Crossmatch is an established procedure known to influence renal allograft survival rates. The Flow Cytometry Crossmatch (FCXM) is considered more sensitive, and based on literature reports, is believed to enhance renal allograft outcome. Before utilizing this procedure, some programs are requiring center specific data. PURPOSE OF THE STUDY: The purpose of this study was to investigate the impact of a positive FCXM on renal allograft outcome in a single center. METHOD: Fifty-six patients who received renal transplants at The Transplant Institute of New Orleans were investigated. The pre-transplant workup included both a CDC and FCXM. A negative CDC crossmatch against donor T Cells was required for inclusion in the study. The results of the Flow Crossmatch were not used for excluding patients for getting a transplant. The clinical outcome in terms of successful versus failed transplants was recorded over a three year period. A failed graft was defined as return to dialysis. RESULTS: Nine out of fifty-six renal allografts failed due to immunologic rejections. An additional two grafts were lost due to other reasons. Amongst those who lost the renal grafts due to rejection, thirty-three percent had a positive pre-transplant FCXM. On the other hand, the FCXM was positive in sixteen percent of those who had functioning renal grafts. CONCLUSION: Although these numbers are small, the data suggest that positive FCXMS are related to lower renal allograft survival rates at our center. Further studies will be performed on a larger patient population.
873
TUESDAY
ma severity, whereas it may not be associated with the development of asthma per se. These results suggest that IL-5 gene may be a disease-modifying gene in Korean children with atopic asthma. Funding: Ulsan University