Investigating genome wide dna methylation in airway smooth muscle cells from asthmatic and non-asthmatic donors

Rationale: Genetic mechanisms fail to fully explain asthma pathogenesis and environmental factors are considered to play an important role. Environmental factors may lead to permanent changes in epigenetic patterns and contribute to asthma. Epigenetics is the study of heritable changes in gene expression that are not due to changes in DNA sequence. DNA methylation is a reversible modification of DNA structure in which a methyl group is added to cytosine residues. Parental smoking affects the methylation of buccal cell DNA from children and children with early onset wheeze have an altered blood DNA methylation profile to healthy individuals. No studies have compared DNA methylation profiles in the disease relevant cell type of airway smooth muscle (ASM) cells. Methods: DNA was isolated from ASM cells at passage 5 and bisulphite treated to convert epigenetic information into sequence-based information. Site specific, quantitative genome wide methylation was determined using the Illumina 450K Infinium Methylation BeadChip array. Hits were validated by Pyrosequencing. RNA was extracted simultaneously for mRNA expression analysis by real time PCR. Results: There were no independent CpG sites associated with asthmatic status of ASM cells following multiple test correction. Without correction over 13000 CpG sites showed a significant difference in methylation (linear modelling, p value >0.05) between asthmatic and non-asthmatic cells, and a biologically relevant difference in methylation of greater that 10% (β value >0.1 ). 10 of these sites were selected as top hits. 7 sites positively validated by pyrosequencing. They were associated with 7 different genes; LGALS3BP, ATP11A, ZNF696, KLF6, TBX1, RUNX3, and SPINT2. Expression of these genes was measured in ASM cells isolated from asthmatic and non-asthmatic donors. LGALS3BP expression was undetectable while ATP11A and ZNF696 displayed no difference in expression between cells from asthmatic and non-asthmatic donors. KLF6 and SPINT2 showed a trend towards increased expression in cells from asthmatic donors while RUNX3 and TBX1 showed a trend towards decreased expression. Conclusions: Differences in CpG methylation exist between ASM isolated from asthmatic and non-asthmatic donors. Future work will focus on identifying differentially methylated regions of DNA and further defining the association to gene and protein expression

Investigating genome wide dna methylation in airway and parenchymal fibroblasts from healthy individuals and individuals with copd

Rationale: Lung fibroblasts are implicated in respiratory disease pathology including chronic obstructive pulmonary disease (COPD). Phenotypic differences between fibroblasts isolated from the airway versus the parenchyma have been described but no studies have compared the cell types on a genome wide scale. DNA methylation is a reversible modification of the DNA structure with the ability to affect cell function via the alteration of gene expression. Here we compared genome wide DNA methylation profiles from airway and parenchymal fibroblasts and assessed modification to these profiles in cells isolated from individuals with COPD. Methods: DNA was isolated from parenchymal and airway fibroblasts at passage 4, and bisulphite treated. Site specific, quantitative genome wide methylation was determined using the Illumina 450K Infinium Methylation BeadChip array. Linear modelling and DMRcate functions identified differentially methylated sites and regions respectively between airway and parenchymal fibroblasts isolated from individuals with normal lung function versus those with COPD. Results: 3980 CpG (methylation) sites significantly differed after Bonferroni correction between airway and parenchymal fibroblasts isolated from healthy individuals. These sites had a broad distribution of effect size, with 240 CpG sites displaying a difference in methylation of >50%. 78 of these sites validated in a second cohort of 7 sets of paired airway and parenchymal fibroblasts isolated from the same individual. There was genomic proximity to these sites and DMRcate was used to refine the individual CpG sites to 5 regions of interest associated with 5 genes; HLX, TWIST1, CREB5, SKAP2 and PRDM16. Differences in methylation were less pronounced when comparing cells isolated from healthy individuals to those with COPD. In airway fibroblasts 47 DMRcate regions were identified with a maximum difference in methylation of at least 20%. In parenchymal fibroblasts 3 DMRcate regions were identified with a maximum difference in methylation of at least 20%. Conclusions: DNA methylation profiles are significantly different between airway and parenchymal fibroblasts but only small modifications are associated with COPD. Future work will focus on validating a methylation based markers of parenchymal versus airway fibroblasts and associating our differential observations with gene/protein expression

Maternal prenatal anxiety and the fetal origins of epigenetic aging

Stress and Psychopatholog

Maternal antenatal depression and child mental health: moderation by genomic risk for attention-deficit/hyperactivity disorder

Maternal antenatal depression strongly influences child mental health but with considerable inter-individual variation that is, in part, linked to genotype. The challenge is to effectively capture the genotypic influence. We outline a novel approach to describe genomic susceptibility to maternal antenatal depression focusing on child emotional/behavioral difficulties. Two cohorts provided measures of maternal depression, child genetic variation, and child mental health symptoms. We constructed a conventional polygenic risk score (PRS) for attention-deficit/hyperactivity disorder (ADHD) (PRSADHD) that significantly moderated the association between maternal antenatal depression and internalizing problems at 60 months (p = 2.94 x 10(-4), R-2 = .18). We then constructed an interaction PRS (xPRS) based on a subset of those single nucleotide polymorphisms from the PRSADHD that most accounted for the moderation of the association between maternal antenatal depression and child outcome. The interaction between maternal antenatal depression and this xPRS accounted for a larger proportion of the variance in child emotional/behavioral problems than models based on any PRSADHD (p = 5.50 x 10(-9), R-2 = .27), with similar findings in the replication cohort. The xPRS was significantly enriched for genes involved in neuronal development and synaptic function. Our study illustrates a novel approach to the study of genotypic moderation on the impact of maternal antenatal depression on child mental health and highlights the utility of the xPRS approach. These findings advance our understanding of individual differences in the developmental origins of mental health.Stress and Psychopatholog

Systematic evaluation and validation of reference and library selection methods for deconvolution of cord blood DNA methylation data

Background: Umbilical cord blood (UCB) is commonly used in epigenome-wide association studies of prenatal exposures. Accounting for cell type composition is critical in such studies as it reduces confounding due to the cell specificity of DNA methylation (DNAm). In the absence of cell sorting information, statistical methods can be applied to deconvolve heterogeneous cell mixtures. Among these methods, reference-based approaches leverage age-appropriate cell-specific DNAm profiles to estimate cellular composition. In UCB, four reference datasets comprising DNAm signatures profiled in purified cell populations have been published using the Illumina 450 K and EPIC arrays. These datasets are biologically and technically different, and currently, there is no consensus on how to best apply them. Here, we systematically evaluate and compare these datasets and provide recommendations for reference-based UCB deconvolution. Results: We first evaluated the four reference datasets to ascertain both the purity of the samples and the potential cell cross-contamination. We filtered samples and combined datasets to obtain a joint UCB reference. We selected deconvolution libraries using two different approaches: automatic selection using the top differentially methylated probes from the function pickCompProbes in minfi and a standardized library selected using the IDOL (Identifying Optimal Libraries) iterative algorithm. We compared the performance of each reference separately and in combination, using the two approaches for reference library selection, and validated the results in an independent cohort (Generation R Study, n = 191) with matched Fluorescence-Activated Cell Sorting measured cell counts. Strict filtering and combination of the references significantly improved the accuracy and efficiency of cell type estimates. Ultimately, the IDOL library outperformed the library from the automatic selection method implemented in pickCompProbes. Conclusion: These results have important implications for epigenetic studies in UCB as implementing this method will optimally reduce confounding due to cellular heterogeneity. This work provides guidelines for future referencebased UCB deconvolution and establishes a framework for combining reference datasets in other tissues

Integrated analysis of environmental and genetic influences on cord blood DNA methylation in new-borns

Epigenetic processes, including DNA methylation (DNAm), are among the mechanisms allowing integration of genetic and environmental factors to shape cellular function. While many studies have investigated either environmental or genetic contributions to DNAm, few have assessed their integrated effects. Here we examine the relative contributions of prenatal environmental factors and genotype on DNA methylation in neonatal blood at variably methylated regions (VMRs) in 4 independent cohorts (overall n = 2365). We use Akaike’s information criterion to test which factors best explain variability of methylation in the cohort-specific VMRs: several prenatal environmental factors (E), genotypes in cis (G), or their additive (G + E) or interaction (GxE) effects. Genetic and environmental factors in combination best explain DNAm at the majority of VMRs. The CpGs best explained by either G, G + E or GxE are functionally distinct. The enrichment of genetic variants from GxE models in GWAS for complex disorders supports their importance for disease risk

Childhood internalizing symptoms and epigenetic aging

Item does not contain fulltext1 p

Internalizing symptoms associate with the pace of epigenetic aging in childhood

Childhood psychiatric symptoms may be associated with advanced biological aging. This study examined whether epigenetic age acceleration (EAA) associates with internalizing and externalizing symptoms that were prospectively collected across childhood in a longitudinal cohort study. At age 6 buccal epithelial cells from 148 children (69 girls) were collected to survey genome-wide DNA methylation. EAA was estimated using the Horvath clock. Internalizing symptoms at ages 2.5 and 4 years significantly predicted higher EAA at age 6, which in turn was significantly associated with internalizing symptoms at ages 6-10 years. Similar trends for externalizing symptoms did not reach statistical significance. These findings indicate advanced biological aging in relation to child mental health and may help better identify those at risk for lasting impairments associated with internalizing disorders

Cumulative risk exposure and child cellular aging in a Dutch low-risk community sample

One of the proposed mechanisms linking childhood stressor exposure to negative mental and physical health outcomes in later life is cellular aging. In this prospective, longitudinal, and pre-registered study, we examined the association between a cumulative pattern of childhood risk exposure from age 6 to age 10 (i.e., poor maternal mental health, parental relationship problems, family/friend death, bullying victimization, poor quality friendships) and change in two biomarkers of cellular aging (i.e., telomere length, epigenetic age) from age 6 to age 10 in a Dutch low-risk community sample (n = 193). We further examined the moderating effect of cortisol reactivity at age 6. Ordinary Least Squares regression analyses revealed no significant main effects of childhood risk exposure on change in cellular aging, nor a moderation effect of child cortisol reactivity. Secondary findings showed a positive correlation between telomere length and cortisol reactivity at age 6, warranting further investigation. More research in similar communities is needed before drawing strong conclusions based on the null results

The PedBE clock accurately estimates DNA methylation age in pediatric buccal cells

Contains fulltext : 219496.pdf (Publisher’s version ) (Closed access)DNA methylation is the most studied modification in human population epigenetics. Its information content can be explored in 2 principal ways - epigenome-wide association studies and epigenetic age. The latter likely reflects cellular/biological age and works with impressive accuracy across most tissues. In adults, it associates with various environments and health. However, current epigenetic clocks are not very accurate in the pediatric age range perhaps because DNA methylation changes much faster in children. Addressing this crucial gap, we created a precise tool to estimate DNA methylation age specific to pediatric buccal epithelial cells. This tool has the potential to become the standard reference for epigenetic studies broadly relevant to child development across the spectrum from health to disease.The development of biological markers of aging has primarily focused on adult samples. Epigenetic clocks are a promising tool for measuring biological age that show impressive accuracy across most tissues and age ranges. In adults, deviations from the DNA methylation (DNAm) age prediction are correlated with several age-related phenotypes, such as mortality and frailty. In children, however, fewer such associations have been made, possibly because DNAm changes are more dynamic in pediatric populations as compared to adults. To address this gap, we aimed to develop a highly accurate, noninvasive, biological measure of age specific to pediatric samples using buccal epithelial cell DNAm. We gathered 1,721 genome-wide DNAm profiles from 11 different cohorts of typically developing individuals aged 0 to 20 y old. Elastic net penalized regression was used to select 94 CpG sites from a training dataset (n = 1,032), with performance assessed in a separate test dataset (n = 689). DNAm at these 94 CpG sites was highly predictive of age in the test cohort (median absolute error = 0.35 y). The Pediatric-Buccal-Epigenetic (PedBE) clock was characterized in additional cohorts, showcasing the accuracy in longitudinal data, the performance in nonbuccal tissues and adult age ranges, and the association with obstetric outcomes. The PedBE tool for measuring biological age in children might help in understanding the environmental and contextual factors that shape the DNA methylome during child development, and how it, in turn, might relate to child health and disease.7 p