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.
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.
