GENETICS VARIATION IN 5LO PRODUCT PRODUCTION

5LO 产品生产中的遗传变异

• 批准号: 2897325
• 负责人: Jeffrey Mark Drazen
• 金额: $ 21.68万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2897325
• 关键词: asthma; blood chemistry; clinical research; diagnostic respiratory lavage; enzyme inhibitors; fatty acid biosynthesis; gene deletion mutation; gene expression; gene mutation; genetic mapping; genetic promoter element; genetic susceptibility; genetic transcription; human subject; leukotrienes; lipoxygenase; nucleic acid sequence; respiratory airflow disorder

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.

哮喘是一种异质性综合征，具有多种导致气道阻塞的潜在途径，这是其标志。已确定作为哮喘气道阻塞介质的物质是白三烯。白三烯是多种酶催化作用的产物，其中包括5-脂氧合酶(5-LO)。 鉴于哮喘效应机制的异质性以及半胱氨酰白三烯作为哮喘效应分子家族之一的重要性，了解导致个体间白三烯合成调节差异的因素可应用于以下两个方面：和临床科学。 该提案解决了 5-LO 产品生产调控的潜在位点之一，即由于 5-LO 核心启动子中存在基因突变而进行的调控。我们发现了一系列突变，其中在 5-LO 核心启动子中添加或删除了 Sp1/Egr-1 结合基序（即-GGGCGG-）；这些突变改变了报告基因构建体中的基因转录。我们在此回顾的初步数据表明，携带核心启动子突变形式的哮喘患者对 5-LO 抑制的有益治疗反应减弱。根据这一观察，我们间接断言，对 5-LO 抑制的较小治疗反应反映了 5-LO 核心启动子突变形式纯合的患者的 5-LO 产物合成减少。拟议研究的目的是根据大型临床试验结果的分析，确定这一断言在个体患者 5-LO 产物形成水平上是否成立。为了检验这个假设，我们将追求两个具体目标。在第一个目标中，我们将确定具有同等严重程度的特应性哮喘的患者群体，他们携带野生型或突变型 5-LO 核心启动子。这些受试者将用于确定从外周血分离的基础和 A23 187 攻击的中性粒细胞和嗜酸性粒细胞或通过支气管肺泡灌洗分离的肺泡巨噬细胞中 LTE4、半胱氨酰白三烯和脂氧素的定量产生。还将通过测量吸入全肺过敏原刺激前后的尿液 LTE4，在每个基因型定义的哮喘患者群体中研究 5-LO 产品的生产。 除了全肺过敏原激发之外，患者还将接受肺节段过敏原激发，并将激发部位与对照部位的 5-LO 产品回收率进行比较。如果假设正确，那么与具有野生型核心启动子的患者相比，携带突变型核心启动子的患者应该减少 5-LO 产物的产生。此外，在过敏原攻击（全肺和节段）之后，具有核心启动子突变形式的患者中5-LO产物的产量应低于具有核心启动子野生型形式的患者。