GENETICS VARIATION IN 5LO PRODUCT PRODUCTION

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Asthma is a heterogenous syndrome with multiple potential pathways leading to the airway obstruction which is its hallmark. Among the substances which have an established role as mediators of the airway obstruction in asthma are the leukotrienes. The leukotrienes are the products of the catalytic action of a number of enzymes among which is 5-Lipoxygenase (5-LO). Given the perceived heterogeneity of the effector mechanisms in asthma and the established importance of the cysteinyl leukotrienes as one of the families of effector molecules in asthma, an understanding of the factors responsible for variations among individuals in the regulation of leukotriene synthesis has applications to both basic and clinical science. This proposal addresses one of the potential sites of regulation of 5-LO product production, i.e., regulation due to the presence of genetic mutations in the 5-LO core promoter. We have discovered a series of mutations in which there is the addition of or the deletion of Sp1/Egr-1 binding motifs (i.e.,- GGGCGG-) in the 5-LO core promoter; these mutations modify gene transcription in reporter gene constructs. Our preliminary data, reviewed herein, indicate that patients with asthma harboring the mutant forms of the core promoter have a diminished salutary therapeutic response to 5-LO inhibition. From this observation we have indirectly asserted that the lesser therapeutic response to 5- LO inhibition reflects decreased synthesis of 5-LO products by patients who are homozygous for mutant forms of the 5-LO core promoter. The goal of the proposed research is to determine if this assertion, based on analysis of the results of a large clinical trial, is true at the level of 5-LO product formation in individual patients. To test this hypothesis we will pursue two specific aims. In the first aim we will identify cohorts of patients, with atopic asthma of equivalent severity, who harbor either the wild-type or the mutant 5-LO core promoter. These subjects will be used to ascertain the quantitative production of LTE4, cysteinyl leukotrienes and lipoxins from basal and A23 187 challenged neutrophils and eosinophils isolated from the peripheral blood, or from alveolar macrophages isolated by bronchoalveolar lavage. 5-LO product production will also be studied in each of the genotypically defined cohorts of asthma patients by measurement of urinary LTE4 before and after inhaled whole lung allergen stimulation. In addition to whole lung allergen challenge, patients will undergo pulmonary segmental allergen challenge and the recovery of 5-LO products from the challenged versus the control segment compared. If the hypothesis is correct, then patients harboring the mutant forms of the core promoter should have diminished 5-LO product production when compared to patients with the wild type form of the core promoter. Furthermore, after allergen challenge (both whole lung and segmental), the yield of 5-LO products should be less in patients with the mutant forms of the core promoter than in patients with the wild type form of the core promoter.