Epigenetic predicators of asthma in neonates

描述(由申请人提供)：本申请涉及广泛的挑战领域(03)生物标记物的发现和验证，以及特定的挑战主题03-OD-101：使用血细胞中的表观遗传特征来预测疾病。哮喘目前可以控制，但不能真正治愈。因此，预防将是治疗这种疾病的理想方法。显然，在生命早期，甚至在出生时更好地发现哮喘预测指标，通过确定人口中哪些人的预防措施可能更激进，因此不太容易实施，可能是最合理和最有用的，从而极大地提高预防的有效性。由于哮喘通常在生命早期开始，而且儿童的哮喘状态与母亲的哮喘状态密切相关，因此推动这一应用的总体假设是，出生时可在表观基因组中检测到的签名，更具体地说，在甲基组中可检测到的签名，可以作为日后哮喘状态的预测指标。为了验证这一假设，我们建议评估DNA甲基化的全基因组模式，这是一个强大的和可量化的表观遗传标记。我们的分析将集中在从参加纵向婴儿免疫研究(IIS)的儿童中分离出的脐带血样本，以及已经获得的样本。IIS研究的纵向性质提供了一个独特的机会来解决和回答有关哮喘早期表观遗传学预测因素的问题。事实上，脐带血捐献者现在已经达到5-8岁，这是一个可以确定患有哮喘的年龄。最强大的候选基因的结果将通过亚硫酸氢盐测序和候选基因表达水平的测量进行定量验证。具体目的1通过询问从5岁前已患或未患哮喘的新生儿分离的脐带血细胞的甲基组，确定哮喘的候选表观遗传学预测因子。我们将对从IIS研究中登记的新生儿分离的脐带血细胞中的启动子DNA甲基化模式进行全基因组比较(每组20例)，我们将使用5岁时的哮喘状况来锚定和解释这些结果。每组将包含相同数量的新生儿样本，这些新生儿的母亲在怀孕期间患有或不患有哮喘。这些实验将产生哮喘的候选表观遗传预测因子，将在目标2中得到验证。具体目标2通过使用定量高分辨率亚硫酸氢盐测序和基因表达评估来验证目标1中确定的哮喘候选表观遗传预测因子。我们将实施严格的策略来筛选和进一步探索AIM 1中确定的哮喘的假定表观遗传学预测因子。通过测量启动子区域和单个CpG二核苷酸的DNA甲基化水平(通过亚硫酸氢盐测序)和mRNA表达水平(通过RT-PCR)，将对被全基因组启动子甲基化阵列(至少10个)发现的差异甲基化的基因进行生物学验证。这些分析将包括40个初始样本和40个额外样本。表现出与儿童哮喘后续发展相关的不同表观遗传学变化和表达的功能一致性模式的基因(例如，表达更强烈和低甲基化的基因)将被认为是真正的新生儿哮喘表观遗传学预测因子，并将被建议用于未来的前瞻性研究。 公共卫生相关性：本申请涉及广泛的挑战领域(03)生物标记物的发现和验证，以及具体的挑战主题03-OD-101：使用血细胞中的表观遗传特征来预测疾病。哮喘目前可以控制，但不能真正治愈。因此，预防将是治疗这种疾病的理想方法。显然，在生命早期，甚至在出生时更好地发现哮喘预测指标，通过确定人口中哪些人的预防措施可能更激进，因此不太容易实施，可能是最合理和最有用的，从而极大地提高预防的有效性。由于哮喘通常在生命早期开始，而且儿童的哮喘状态与母亲的哮喘状态密切相关，因此推动这一应用的总体假设是，出生时可在表观基因组中检测到的签名，更具体地说，在甲基组中可检测到的签名，可以作为日后哮喘状态的预测指标。为了验证这一假设，我们建议评估全基因组范围的启动子DNA甲基化模式，这是一个强大的和可量化的表观遗传标记。我们的分析将集中在从参加纵向婴儿免疫研究(IIS)的儿童中分离出来的脐带血样本(n=40)，并且已经可以获得。IIS研究的纵向性质提供了一个独特的机会来解决和回答有关哮喘早期表观遗传学预测因素的问题。事实上，脐带血捐献者现在已经达到5-8岁，这是一个可以确定患有哮喘的年龄。最可靠的候选基因的结果将通过亚硫酸氢盐测序和候选基因表达水平的测量进行定量验证，将分析扩展到另外40个样本。表现出与儿童哮喘后续发展相关的不同表观遗传学变化和表达的功能一致性模式的基因(例如，表达更强烈和低甲基化的基因)将被认为是真正的新生儿哮喘表观遗传学预测因子，并将被建议用于未来的前瞻性研究。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (03) Biomarker discovery and validation, and specific Challenge Topic, 03-OD-101: Use of epigenetic signatures in blood cells to predict disease. Asthma can currently be managed but not really cured. Therefore, prevention would be an ideal approach to this disease. Obviously, availability of asthma predictors detectable in early life, or even better at birth, would greatly improve the effectiveness of prevention by identifying those individuals within the population for whom more drastic, and therefore less easy to implement, preventive measures might be most justified and useful. Because asthma typically begins in early life, and the asthma status of the child is strongly related to that of the mother, the overall hypothesis driving this application is that signatures detectable in the epigenome, and more specifically, in the methylome, at birth can serve as predictors of asthma status later in life. To test this hypothesis, we propose to assess genome-wide patterns of DNA methylation, a robust and quantifiable epigenetic mark. Our analysis will focus on cord blood samples isolated from children enrolled in the longitudinal Infant Immune Study (IIS), and already available. The longitudinal nature of the IIS study offers a unique opportunity to address and answer questions about early epigenetic predictors of asthma. Indeed, the cord blood donors have now reached age 5-8 yrs, an age at which a firm diagnosis of asthma can be established. Results for the most robust candidate genes will be quantitatively validated by bisulfite sequencing and measurements of candidate gene expression levels. Specific Aim 1 To identify candidate epigenetic predictors of asthma by interrogating the methylome of cord blood cells isolated from neonates who have or have not become asthmatic by age 5 yrs. We will perform genome-wide comparisons of promoter DNA methylation patterns in cord blood cells isolated from neonates enrolled in the IIS study (n=20 per group), and we will use asthma status at age 5 yrs to anchor and interpret these results. Each group will contain equal numbers of samples from neonates whose mothers were or were not asthmatic during pregnancy. These experiments will yield candidate epigenetic predictors of asthma, which will be validated in Aim 2. Specific Aim 2 To validate the candidate epigenetic predictors of asthma identified in Aim 1 by using quantitative high resolution bisulfite sequencing and gene expression assessments. We will implement a stringent strategy to filter and further explore the putative epigenetic predictors of asthma identified in Aim 1. Genes found to be differentially methylated by genome-wide promoter methylation arrays (at least 10) will be biologically validated by measuring levels of DNA methylation at promoter regions and individual CpG dinucleotides (by bisulfite sequencing) and levels of mRNA expression (by RT-PCR). These analyses will include the 40 initial samples and 40 additional ones. Genes showing a functionally concordant pattern of differential epigenetic changes and expression (e.g., genes that are more intensely expressed and hypomethylated) correlated with subsequent development of asthma in the child will be considered as bona fide neonatal epigenetic predictors of asthma, and will be proposed for future prospective studies. PUBLIC HEALTH RELEVANCE: This application addresses broad Challenge Area (03) Biomarker discovery and validation, and specific Challenge Topic, 03-OD-101: Use of epigenetic signatures in blood cells to predict disease. Asthma can currently be managed but not really cured. Therefore, prevention would be an ideal approach to this disease. Obviously, availability of asthma predictors detectable in early life, or even better at birth, would greatly improve the effectiveness of prevention by identifying those individuals within the population for whom more drastic, and therefore less easy to implement, preventive measures might be most justified and useful. Because asthma typically begins in early life, and the asthma status of the child is strongly related to that of the mother, the overall hypothesis driving this application is that signatures detectable in the epigenome, and more specifically, in the methylome, at birth can serve as predictors of asthma status later in life. To test this hypothesis, we propose to assess genome-wide patterns of promoter DNA methylation, a robust and quantifiable epigenetic mark. Our analysis will focus on cord blood samples (n=40) isolated from children enrolled in the longitudinal Infant Immune Study (IIS), and already available. The longitudinal nature of the IIS study offers a unique opportunity to address and answer questions about early epigenetic predictors of asthma. Indeed, the cord blood donors have now reached age 5-8 yrs, an age at which a firm diagnosis of asthma can be established. Results for the most robust candidate genes will be quantitatively validated by bisulfite sequencing and measurements of candidate gene expression levels, extending the analysis to 40 additional samples. Genes showing a functionally concordant pattern of differential epigenetic changes and expression (e.g., genes that are more intensely expressed and hypomethylated) correlated with subsequent development of asthma in the child will be considered as bona fide neonatal epigenetic predictors of asthma, and will be proposed for future prospective studies.