The Functional Consequences of the 17q12 Asthma Susceptibility Locus

 描述（由申请人提供）：本提案的目的是确定负责染色体17 q12上与哮喘遗传相关的哮喘基因。染色体17 q12上的一个常见单倍型是全基因组关联研究中出现的最强、最一致可重复的哮喘易感性位点。17 q12哮喘风险单倍型赋予三个相邻基因的区域染色质重塑：ORMDL 3，GSDMB和ZPBP 2。然而，仅从现有的遗传数据来看，不可能肯定地得出结论，这三个中的哪一个负责遗传关联。根据我们的初步数据，我们观察到ORDML 3和GSDMB都在支气管上皮中组成型表达，并且它们的过表达导致不连续的支气管炎。 分子和细胞在哮喘中的重要作用。相比之下，我们发现ZPBP 2在支气管上皮或其他哮喘相关细胞类型中均不表达。因此，我们假设ORMDL 3或GSDMB，而不是ZPBP 2，赋予哮喘风险。为了验证这一假设，我们提出了三个具体目标。在具体目标1中，我们将研究我们实验室产生的诱导型转基因小鼠，这些小鼠在支气管上皮中有条件地过表达ORMDL 3或GSDMB。我们将评估每个基因的过度表达在一个已建立的过敏性哮喘小鼠模型的影响，检查生理，组织学和代谢的后果。在具体目标2中，我们将通过实验评估（通过体外敲低和过表达研究）ORMDL 3和GSDMB在人支气管上皮细胞中表达的独立细胞后果，这些细胞来源于风险和保护性17 q12单倍型纯合子个体。在具体目标3中，我们将以几种方式研究17 q12单倍型与鞘脂代谢的关系（i）我们将测量来自我们在目标1中的小鼠研究的气道和肺匀浆中的鞘脂水平，以确定ORMDL 3表达的体内效应;（二）我们将通过遗传关联来研究功能性17 q12调节变体是否有助于200例患者血浆鞘脂水平的变化。哮喘;以及（iii）我们将这些血浆鞘脂水平 对沙丁胺醇有支气管扩张反应，对乙酰甲胆碱有气道高反应性。通过这些努力获得的知识将促进我们对这一最重要的哮喘易感性位点的理解，并将为开发新的哮喘治疗方法提供必要的基础。

 DESCRIPTION (provided by applicant): The objective of this proposal is to identify the asthma gene that is responsible for the genetic associations on chromosome 17q12 with asthma. A common haplotype on chromosome 17q12 is the strongest, most consistently reproducible asthma-susceptibility locus to emerge from genome-wide association studies. The 17q12 asthma-risk haplotype confers regional chromatin remodeling of three neighboring genes: ORMDL3, GSDMB, and ZPBP2. However, from the available genetic data alone, it is impossible to conclude with any certainty which of these three is responsible for the genetic association. From our preliminary data, we observe that both ORDML3 and GSDMB are constitutively expressed in bronchial epithelium, and that their overexpression results in discrete molecular and cellular consequences of importance in asthma. In contrast, we find that ZPBP2 is not expressed in either bronchial epithelium or other asthma-relevant cell types. We thus hypothesize that ORMDL3 or GSDMB, but not ZPBP2, confers asthma risk. To test this hypothesis, we propose three Specific Aims. In Specific Aim 1, we will study inducible transgenic mice generated in our lab that conditionally over express ORMDL3 or GSDMB in bronchial epithelium. We will assess the effects of each gene's over expression in an established murine model of allergic asthma, examining the physiological, histological, and metabolic consequences. In Specific Aim 2, we will experimentally assess (via in vitro knockdown and over expression studies) the independent cellular consequences of ORMDL3 and GSDMB expression in human bronchial epithelial cells derived from individuals homozygous for the risk and protective 17q12 haplotypes. In Specific Aim 3, we will study the relationship of 17q12 haplotype with sphingolipid metabolism in several ways (i) we will measure sphingolipid levels in the airways & lung homogenates from our murine studies in Aim 1 to determine the in vivo effects of ORMDL3 expression; (ii) we will examine by genetic association whether the functional 17q12 regulatory variant contributes to variations in plasma sphingolipid levels in 200 asthmatics; and (iii) we will correlate these plasma sphingolipid levels with bronchodilator response to albuterol and airways hyperreactivity to methacholine. The knowledge gained through these proposed efforts will advance our understanding of this most important asthma-susceptibility locus, and will provide the requisite foundations for the development of novel asthma therapies.