Genetic and Epigenetic Determinants of Pediatric Obesity-Associated Asthma

肥胖相关性哮喘是一种独特的疾病，其特征是气流障碍更严重，且不理想。 对对城市儿童影响极大的传统哮喘治疗的反应。然而，它的 发病机制尚不清楚。与《经典》中发现的特应性T辅助细胞(Th)2型炎症相反 儿童哮喘，我们实验室的研究表明肥胖相关的哮喘与非特应性有关。 与下呼吸道阻塞相关的Th1炎症。此外，我们发现启动子减少了 与T细胞信号相关的基因甲基化提示表观遗传机制可能介导 肥胖儿童独特的哮喘表型。然而，非特应性的分子机制 肥胖哮喘患者的炎症情况尚不清楚。 哮喘和肥胖症是由遗传易感性决定的多因素疾病，包括与 与祖先、环境暴露引起的表观遗传修饰以及非遗传环境因素有关 影响基因表达，从而影响临床表型。因此，对基因之间的关联的研究 表达、遗传多态和表观遗传调控可能识别一个统一的分子机制 解释在城市儿童中观察到的肥胖哮喘表型。我们从中国收集了200个样品。 儿童在布朗克斯过去5年的这一奖项，以检验Th细胞基因表达的假说 肥胖哮喘患者与正常体重哮喘患者不同，是由基因多态决定的 (eQTL和祖先特有的单倍型)，并受DNA甲基化的影响。我们提出了以下建议 具体目的是为了解决这些假设：(1)量化Th细胞基因表达的差异 肥胖哮喘儿童和正常体重哮喘儿童。(2)确定表观遗传机制 调节肥胖对哮喘的影响。(3)调查是否存在遗传多态，包括 祖先特有的单倍型决定了肥胖相关哮喘的易感性。来自100的TH细胞 肥胖哮喘儿童和100名年龄和性别匹配的正常体重哮喘儿童已经被隔离。 我们同时通过定向转录组对a)基因的表达进行了全基因组分析 测序(RNA-seq)b)DNA甲基化的帮助标签和c)遗传多态和单一 核苷酸多态(SNPs)通过对这些细胞进行基于阵列的基因分型来提供祖先的信息。 基因表达与遗传多态的关联已识别出预测遗传的变异 易感性，可用于筛查高危个体。 对基因表达、DNA甲基化和遗传多态的验证研究现在将使我们能够 确认肥胖哮喘表型特异的分子机制并改善我们的 了解该病的发病机制。我们将确定与非特应性相关的关键分子 炎症可能成为治疗干预的新靶点。总之，我们的研究将提供 对肥胖相关哮喘在高危人群中发病机制的基本见解。 作为一名儿科肺科医生，我的目标是成为一名独立的翻译研究员 儿童哮喘，重点关注肥胖对肺部的影响。这位K23导师以病人为导向- 研究事业发展奖允许对遗传和表观遗传因素进行深入研究 Th细胞介导的炎症对肥胖哮喘少数民族儿童的影响这项提案的结果 我为我的R01基金提供了基础，该基金专注于对CDC42的进一步研究 途径，被确定为本K23奖中差异表达最高的途径。作为我们未来的目标，我们计划 A)在单独的儿童疾病队列中验证关键的差异表达和/或甲基化分子 控制不佳的疾病b)用分子确认关键分子在体外细胞培养系统中的作用 特定的抑制物和/或甲基化调节剂c)对 由于年龄较小，肥胖的发病、体重增加的速度、哮喘的发展和Th细胞分化 肥胖相关的哮喘困扰着城市儿童。 在本申请中提出的职业发展计划包含了)来自STABLE的指导 表观基因组学、肥胖相关疾病、T细胞生物学、哮喘、粘膜和 系统免疫，b)遗传学和表观基因组学课程，以及亲手培训新方法 调查基因表达、基因分型和DNA甲基化，以及c)在当地的参与和展示， 区域和国家会议。总而言之，这个K23奖项中提出的研究为我的 发展成为儿童肥胖相关哮喘领域的独立调查者。我现在是 重点是通过验证我们的发现在此基础上构建，以随后使用这些结果作为 进一步研究遗传易感性对儿童肥胖的贡献的初步数据- 相关性哮喘。

Obesity-associated asthma is a distinct entity characterized by worse airflow obstruction and suboptimal response to conventional asthma management that disproportionately affects urban children. However, its pathogenesis is poorly understood. In contrast to atopic T helper (Th) type 2 inflammation found in “classic” childhood asthma, studies from our lab indicate that obesity-associated asthma is associated with non-atopic Th1 inflammation that correlates with lower airway obstruction. Additionally, we found decreased promoter methylation of genes associated with T cell signaling suggesting that epigenetic mechanisms may mediate the distinct asthma phenotype in obese children. However, molecular mechanisms that underlie non-atopic inflammation in obese asthmatics are not known. Asthma and obesity are multifactorial diseases, determined by genetic susceptibility, including that related to ancestry, epigenetic modification by environmental exposures, and non-genetic environmental factors that influence gene expression and thus clinical phenotype. Hence, investigation of the association between gene expression, genetic polymorphisms, and epigenetic modulation may identify a unifying molecular mechanism to explain the obese asthma phenotype observed in urban children. We have collected 200 samples from children in the Bronx during the past 5 years of this award to test the hypothesis that Th-cell gene expression in obese asthmatics differs from that in normal-weight asthmatics, is determined by genetic polymorphisms (eQTLs and ancestry-specific haplotypes), and is influenced by DNA methylation. We proposed the following specific aims to address these hypotheses: (1) To quantify Th-cell gene expression differences between obese asthmatic and normal-weight asthmatic children. (2) To determine if epigenetic mechanisms mediate the effect of obesity on asthma. (3) To investigate if genetic polymorphisms, including ancestry-specific haplotypes, determine susceptibility to obesity-associated asthma. Th cells from 100 obese asthmatic and 100 normal-weight asthmatic children matched for age and gender have been isolated. We have simultaneously conducted genome-wide assays of a) gene expression by Directional Transcriptome Sequencing (RNA-seq) b) DNA methylation by HELP-tagging and c) genetic polymorphisms and single nucleotide polymorphisms (SNPs) informative of ancestry by array-based genotyping on these cells. Association of gene expression with genetic polymorphisms has identified variants predicting genetic susceptibility, which may be used to screen at-risk individuals. Verification studies of gene expression, DNA methylation, and genetic polymorphisms will now allow us to confirm the mechanistic molecular pathways specific to the obese asthma phenotype and improve our understanding of the disease pathogenesis. We will identify key molecules associated with non-atopic inflammation that could be novel targets for therapeutic intervention. Together, our study will provide fundamental insights into the pathogenesis of obesity-associated asthma among high-risk ethnicities. As a pediatric pulmonologist, my goal is to become an independent translational investigator in the field of pediatric asthma with a focus on the pulmonary effects of obesity. This K23 Mentored Patient Oriented- Research Career Development Award has allowed an in-depth study of genetic and epigenetic factors influencing Th cell mediated inflammation in obese asthmatic minority children. Findings from this proposal have provided the foundation for my R01 funding, which is focused on further investigation of the CDC42 pathway, identified as the top differentially expressed pathway in this K23 award. As our future goals, we plan to a) validate the key differentially expressed and/or methylated molecules in a separate cohort of children with poorly controlled disease b) confirm the role of key molecules in in-vitro cell culture systems with molecule- specific inhibitors and/or methylation modulators c) conduct a prospective investigation into links between obesity onset, rapidity of weight gain, development of asthma and Th cell differentiation, given the young age at which obesity-associated asthma afflicts urban children. The career development plan proposed in this application incorporated a) mentorship from established researchers in the field of epigenomics, obesity-related diseases, T cell biology, asthma, and mucosal and systemic immunity, b) coursework in genetics and epigenomics, and hand on training in novel methods to investigate gene expression, genotyping and DNA methylation, and c) participation and presentation at local, regional and national meetings. Together, the research proposed in this K23 award laid the foundation for my development into an independent investigator in the field of pediatric obesity-associated asthma. I am now focused on building on this foundation by verifying our findings to subsequently use these results as preliminary data for further investigation of the contribution of genetic susceptibility to childhood obesity- associated asthma.