Mouse models for the functional analysis of asthma-associated human polymorphisms

描述（由申请人提供）：哮喘和过敏症的特征是Th 2应答的严重失调和Th 2细胞因子IL 5、IL 13和IL 4的表达增加。Th 2细胞因子基因座的遗传变异与过敏性炎症密切相关，但其对表型的影响还远未了解，因为远交人群中存在的遗传异质性和复杂性阻止了相关性研究揭示哪些遗传变异参与表型决定。我们的目标是表征自然变异体对人类过敏性炎症发展和易感性关键基因表达和功能影响的潜在机制。我们的模型基因是IL 13，它是哮喘和过敏的关键效应子。我们证明：（1）IL 13变异体是哮喘/变态反应的强决定因素;（2）IL 13基因座包括两个高度连锁的单核苷酸多态性（SNP）区;和（3）IL 13 + 2044 G>A在3'阻断区，导致比野生型（WT）IL 13更有活性的IL 13 R130 Q变体的表达，而5'阻断中的IL 13 - 1112 C>T增加了Th 2细胞中的IL 13转录和离体IL 13分泌。尽管取得了这些进展，但我们确信最终需要在生理基因组背景下对调节性SNP进行体内研究。在这里，我们希望探索的假设，遗传变异对人IL 13调节的影响，可以有效地建模和解剖BAC转基因（TG）小鼠携带WT或哮喘/过敏相关的人IL 13单倍型。具体而言，我们建议：具体目标1：生成并验证携带WT人IL 13基因座或最常与高加索人哮喘性状相关的IL 13单倍型的[鼠IL 13缺陷型] BAC TG小鼠系。这项工作将利用我们对IL 13变异的分析和我们在BAC重组工程中的技能，并将导致产生表现出人IL 13的忠实组织特异性和拷贝数依赖性表达，但缺乏鼠IL 13的TG系。具体目标二：通过比较和对比携带不同IL 13单倍型的[小鼠IL 13缺陷] BAC TG系中的人IL 13调节和IL 13依赖性肺应答，鉴定功能性IL 13多态性和潜在机制。这项工作将确定变异是否作用于IL 13转录和/或转录后调节或通过分泌过度活性的IL 13蛋白变体（IL 13 R130 Q）。[The鼠IL 13缺陷背景对于人IL 13依赖性表型的表征将是理想的]。通过提供受控的遗传背景，该体内模型将定义参与IL 13失调的SNP（或其阻断）及其对IL 13表达和/或功能的修饰作用，为中和IL 13介导的疾病中遗传失调的作用的成功策略铺平道路。公共卫生相关性。我们工作的总体目标是表征自然遗传变异对人类过敏性炎症的发展和易感性至关重要的基因的表达和功能的影响的机制。我们的模型是IL 13，一种在哮喘和/或过敏患者中过表达的Th 2细胞因子。我们已经相当广泛地研究了IL 13的变异，并且我们已经确定了几种在体外使该基因的表达和/或功能失调的多态性。显然，基因决定的IL 13表达和/或活性的增加可能在哮喘和变态反应的发病机制中起主要作用。尽管取得了这些进展，我们确信需要新的、更强大的生理学方法来阐明遗传变异在IL 13失调和IL 13介导的疾病中所起的作用。最终，多态性需要在生理基因组背景下进行体内研究。在该提议中，我们希望探索以下假设：（a）与哮喘相关表型相关的人IL 13多态性足以诱导IL 13表达和/或功能的明显失调，和（B）天然遗传变异对人IL 13调节的影响可以在携带确定的野生型或哮喘/变态反应相关的人IL 13单倍型的小鼠模型中有效地建模和剖析[在鼠IL 13缺陷背景下]。通过确保单倍型特异性模式的IL 13调控是衡量对一个受控的遗传背景，这种体内模型将使我们能够确定哪些多态性是必要的和足够的IL 13失调，导致分子机制负责改变IL 13的表达和/或功能。

DESCRIPTION (provided by applicant): Asthma and allergy are marked by a profound dysregulation of Th2 responses and increased expression of the Th2 cytokines IL5, IL13 and IL4. Genetic variants in the Th2 cytokine locus are strongly associated with allergic inflammation, but their impact on the phenotype is far from understood because the genetic heterogeneity and complexity existing within outbred populations prevent association studies from revealing which genetic variants are involved in phenotype determination. Our goal is to characterize the mechanism(s) underlying the impact of natural variants on the expression and function of genes critical for the development of, and the susceptibility to, human allergic inflammation. Our model gene is IL13, a key effector in asthma and allergy. We showed that: (1) IL13 variants are strong determinants of asthma/allergy; (2) the IL13 locus includes two blocks of highly linked single nucleotide polymorphisms (SNP); and (3) IL13+2044G>A in the 3' block, results in the expression of an IL13 R130Q variant more active than wild type (WT) IL13, whereas IL13-1112C>T in the 5' block increases IL13 transcription in Th2 cells and IL13 secretion ex vivo. Despite these advances, we are convinced ultimately regulatory SNPs need to be studied in vivo within a physiologic genomic context. Here we wish to explore the hypothesis that the impact of genetic variation on human IL13 regulation can be effectively modeled and dissected in BAC transgenic (TG) mice carrying WT or asthma/allergy-associated human IL13 haplotypes. More specifically, we propose: Specific Aim 1: To generate and validate [murine IL13-deficient] BAC TG mouse lines carrying the WT human IL13 locus or the IL13 haplotypes most commonly associated with asthma traits in Caucasians. This work will capitalize on our analysis of variation in IL13 and our skills in BAC recombineering, and will lead to the generation of TG lines exhibiting faithful tissue-specific and copy number-dependent expression of human IL13, but lacking murine IL13. Specific Aim 2: To identify functional IL13 polymorphisms, and the underlying mechanisms, by comparing and contrasting human IL13 regulation and IL13-dependent lung responses in [murine IL13-deficient] BAC TG lines carrying distinct IL13 haplotypes. This work will determine whether variation acts on IL13 transcriptional and/or post-transcriptional regulation or through the secretion of an overactive IL13 protein variant (IL13 R130Q). [The murine IL13-deficient background will be ideal for the characterization of human IL13-dependent phenotypes]. By providing a controlled genetic background, this in vivo model will define the SNPs (or blocks thereof) involved in IL13 dysregulation and their modifying effects on IL13 expression and/or function, paving the way for successful strategies to neutralize the effects of genetic dysregulation in IL13-mediated disease. PUBLIC HEALTH RELEVANCE. The overall goal of our work is to characterize the mechanisms underlying the impact of natural genetic variation on the expression and function of genes critical for the development of, and the susceptibility to, human allergic inflammation. Our model is IL13, a Th2 cytokine which is overexpressed in patients with asthma and/or allergy. We have studied variation in IL13 quite extensively, and we have identified several polymorphisms that dysregulate the expression and/or the function of this gene in vitro. Clearly, a genetically determined increase in IL13 expression and/or activity is likely to play a major role in the pathogenesis of asthma and allergy. Despite these advances, we are convinced novel, more powerful and physiologic approaches are required to elucidate the role played by genetic variation in IL13 dysregulation and IL13- mediated disease. Ultimately, polymorphisms need to be studied in vivo within the physiologic genomic context. In this proposal we wish to explore the hypothesis that (a) human IL13 polymorphisms associated with asthma-related phenotypes are sufficient to induce appreciable dysregulation of IL13 expression and/or function, and (b) the impact of natural genetic variation on human IL13 regulation can be effectively modeled and dissected in mouse models carrying defined wild type or asthma/allergy-associated human IL13 haplotypes [on a murine IL13- deficient background]. By ensuring haplotype-specific patterns of IL13 regulation are gauged against a controlled genetic background, this in vivo model will allow us to determine which polymorphisms are necessary and sufficient for IL13 dysregulation, leading to the molecular mechanisms responsible for altered IL13 expression and/or function.