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）生物标志物的发现和验证，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，预防是治疗这种疾病的理想方法。显然，在生命早期或出生时就能检测到的哮喘预测因子，将极大地提高预防的有效性，因为它能识别出人群中那些更严厉、因此更不容易实施的预防措施可能最合理、最有用的个体。因为哮喘通常在生命早期开始，孩子的哮喘状态与母亲的哮喘状态密切相关，推动这项应用的总体假设是，在表观基因组中可以检测到的特征，更具体地说，在甲基组中，出生时可以作为以后生活中哮喘状态的预测因子。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，这是一种强大且可量化的表观遗传标记。我们的分析将集中于从参加纵向婴儿免疫研究（IIS）的儿童中分离的脐带血样本，这些样本已经可以获得。IIS研究的纵向性质为解决和回答有关哮喘早期表观遗传预测因子的问题提供了独特的机会。事实上，脐带血献血者现在已经达到了5-8岁，在这个年龄可以确定哮喘的诊断。最强大的候选基因的结果将通过亚硫酸盐测序和候选基因表达水平的测量进行定量验证。通过研究从5岁前患有或未患哮喘的新生儿中分离的脐带血细胞的甲基组，确定哮喘的候选表观遗传预测因子。我们将对从IIS研究中招募的新生儿中分离的脐带血细胞的启动子DNA甲基化模式进行全基因组比较（每组n=20），我们将使用5岁时的哮喘状态来锚定和解释这些结果。每一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哮喘或没有哮喘。这些实验将产生哮喘的候选表观遗传预测因子，这将在Aim 2中得到验证。目的2：通过定量高分辨率亚硫酸盐测序和基因表达评估，验证目的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预测因子。通过全基因组启动子甲基化阵列（至少10个）发现的差异甲基化基因将通过测量启动子区域和单个CpG二核苷酸的DNA甲基化水平（通过亚硫酸盐测序）和mRNA表达水平（通过RT-PCR）进行生物学验证。这些分析将包括40个初始样本和40个附加样本。显示与儿童哮喘后续发展相关的差异表观遗传变化和表达的功能一致模式的基因（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预测因子，并将被提出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.