Molecular signatures of early life exposures and complex diseases : applications using epigenetics and transcriptomics data

Environmental exposures and early life stressors may influence developmental processes and have long-term health consequences, potentially mediated by molecular mechanisms such as epigenetic modifications. The most extensively studied epigenetic mechanism is DNA methylation, which has been proposed to constitute a link between genetic and environmental factors. Epigenetic patterns established early in life (already in utero) may affect how a gene is expressed throughout life, and thereby increase susceptibility to chronic disease. Other factors like genetics and repeated airway infections also influence disease risk. Chronic obstructive pulmonary disease (COPD) is a complex disease considered a major global health problem, with tobacco smoking being one of the main risk factors. The role that deoxyribonucleic acid (DNA) methylation might play in the pathogenesis of COPD has not been comprehensively studied. Bronchoalveolar lavage (BAL) cells from the airways and alveolar space are considered key targets for COPD. Peanut allergy is another complex disease – one of the most common food allergies and the leading cause of anaphylaxis among children. Peanut oral immunotherapy (pOIT) can lead to desensitization and tolerance, and combined treatment with anti-immunoglobulin E (IgE) using omalizumab may facilitate oral immunotherapy initiation. The mechanisms of oral immunotherapy-induced tolerance, including possible changes at the transcriptional level, are not well understood. The main aim of this thesis was to identify molecular signatures of early-life exposures, chronic respiratory disease, as well as allergy treatment responses. In Study I, the association between gestational age and DNA methylation patterns (at 5´-cytosine-phosphate-guanine-3´ sites, CpGs, across the genome) was investigated in newborns and older children from the large The Pregnancy And Childhood Epigenetics (PACE) consortium meta-analysis, including 11,000 participants in 26 independent cohorts. Changes in DNA methylation associated with gestational age were explored in additional pediatric cohorts at 4–18 years. The functional follow-up and correlation analyses between DNA methylation and gene expression were performed using cord blood. In addition, we evaluated DNA methylation profiles in other relevant tissues (fetal brain and lung) related to gestational age. We found numerous epigenome-wide differentially methylated CpGs related to gestational age at birth. Notably, many of the identified CpGs had not previously been associated with gestational age. Several CpGs affected the expression of nearby genes, displayed a strong functional link with human diseases, and were enriched in biological processes essential for fetal development. The epigenetic plasticity of fetal development across tissues was captured by many methylation sites. However, the majority of methylation levels underwent changes over time and stabilized after school age. In Study II, the impact of outdoor exposure to particles of less than 2.5 micrometers in size (PM2.5) at birth and current residential address on gene expression was explored in childhood and adolescence in the MeDALL consortium encompassing three European birth cohorts. In addition, the functional molecular patterns of PM2.5 exposure were evaluated by integrating protein-protein interaction and genome-wide gene expression with matched DNA methylation. We found evidence suggestive of gene signatures in children and adolescents associated with PM2.5 exposure at birth. However, the integration of multi-omics profiles revealed several epigenetic deregulation gene module interactome hotspots where both methylation and expression levels were affected by PM2.5 exposure at birth and current address. Some of the identified genes were associated with diseases known to be caused by or worsened by air pollution exposure. In Study III, the pivotal role of DNA methylation profiles in BAL cells primarily macrophages was assessed in relation to COPD status and smoking in adults, to gain a further understanding of the disease pathogenesis. Several CpGs were associated with COPD in BAL cells, across the epigenome. Many of the identified CpGs displayed a strong functional link with gene expression and pathways enriched in cancer, various types of cell junctions, and cyclic adenosine monophosphate (cAMP) and Rap1 signaling. Notably, almost half of the CpGs co-located in the proximity of COPD-associated single nucleotide polymorphisms, which suggests that both genetic and epigenetic mechanisms are of importance at certain loci. In Study IV, the blood gene expression profiles before, during, and after pOIT and Omalizumab (O, an anti-IgE monoclonal antibody) treatment were evaluated in adolescent patients with severe peanut allergy using high-throughput ribonucleic acid (RNA) sequencing. At the first two timepoints, baseline and pOIT start, we investigated if there was an effect of omalizumab treatment on gene expression. In addition, a longitudinal analysis was performed to evaluate the combined effect of pOIT with Omalizumab (pOIT+O). We also evaluated the overlap of pOIT+O-associated genes with genes associated with acute peanut allergic reactions in a previously published clinical study by Watson et al1. First, we showed that the blood gene expression of patients with peanut allergy was not altered by omalizumab treatment alone. However, the combined effect of pOIT+O showed up- and downregulation of several genes involved in T-cell functions and immune responses. Furthermore, comparing our findings with genes previously found to be affected during acute peanut allergic reactions suggested that pOIT+O may play a role in altering the same genes (in the opposite direction). In conclusion, we demonstrated that DNA methylation profiles are related to gestational age at birth. The identified methylation sites were linked to human diseases and are likely to be involved in biological processes essential for fetal development. Most of the methylation sites also affect expression of nearby genes and reflect epigenetic plasticity of fetal development across tissues. We highlighted the added value of multi-omics analyses in relation to information on PM2.5 exposure that may enhance the understanding of molecular mechanisms and biological responses induced by air pollutants. Moreover, we revealed COPD-associated methylation changes in macrophage-dense BAL cells with a strong functional link to different pathways and gene expression. Both genetic and epigenetic mechanisms play important roles at certain loci. We also provided insights into the transcriptome profiles during pOIT and combined treatment with omalizumab

Neuropeptide S (NPS) variants modify the signaling and risk effects of NPS Receptor 1 (NPSR1) variants in asthma

Single nucleotide polymorphisms (SNPs) close to the gain-of-function substitution, Asn(107) Ile (rs324981, A>T), in Neuropeptide S Receptor 1 (NPSR1) have been associated with asthma. Furthermore, a functional SNP (rs4751440, G>C) in Neuropeptide S (NPS) encodes a Val(6)Leu substitution on the mature peptide that results in reduced bioactivity. We sought to examine the effects of different combinations of these NPS and NPSR1 variants on downstream signaling and genetic risk of asthma. In transfected cells, the magnitude of NPSR1-induced activation of cAMP/PKA signal transduction pathways and downstream gene expression was dependent on the combination of the NPS and NPSR1 variants with NPS-Val(6)/NPSR1-Ile(107) resulting in strongest and NPS-Leu(6)/NPSR1-Asn(107) in weakest effects, respectively. One or two copies of the NPS-Leu(6) (rs4751440) were associated with physician-diagnosed childhood asthma (OR: 0.67, 95% CI 0.49-0.92, p = 0.01) and together with two other linked NPS variants (rs1931704 and rs10830123) formed a protective haplotype (p = 0.008) in the Swedish birth cohort BAMSE (2033 children). NPS rs10830123 showed epistasis with NPSR1 rs324981 encoding Asn(107)Ile (p = 0.009) in BAMSE and with the linked NPSR1 rs17199659 (p = 0.005) in the German MAGIC/ISAAC II cohort (1454 children). In conclusion, NPS variants modify asthma risk and should be considered in genetic association studies of NPSR1 with asthma and other complex diseases.Peer reviewe

DNA Methylation Levels in Mononuclear Leukocytes from the Mother and Her Child Are Associated with IgE Sensitization to Allergens in Early Life

DNA methylation changes may predispose becoming IgE-sensitized to allergens. We analyzed whether DNA methylation in peripheral blood mononuclear cells (PBMC) is associated with IgE sensitization at 5 years of age (5Y). DNA methylation was measured in 288 PBMC samples from 74 mother/child pairs from the birth cohort ALADDIN (Assessment of Lifestyle and Allergic Disease During INfancy) using the HumanMethylation450BeadChip (Illumina). PBMCs were obtained from the mothers during pregnancy and from their children in cord blood, at 2 years and 5Y. DNA methylation levels at each time point were compared between children with and without IgE sensitization to allergens at 5Y. For replication, CpG sites associated with IgE sensitization in ALADDIN were evaluated in whole blood DNA of 256 children, 4 years old, from the BAMSE (Swedish abbreviation for Children, Allergy, Milieu, Stockholm, Epidemiology) cohort. We found 34 differentially methylated regions (DMRs) associated with IgE sensitization to airborne allergens and 38 DMRs associated with sensitization to food allergens in children at 5Y (Sidak p ≤ 0.05). Genes associated with airborne sensitization were enriched in the pathway of endocytosis, while genes associated with food sensitization were enriched in focal adhesion, the bacterial invasion of epithelial cells, and leukocyte migration. Furthermore, 25 DMRs in maternal PBMCs were associated with IgE sensitization to airborne allergens in their children at 5Y, which were functionally annotated to the mTOR (mammalian Target of Rapamycin) signaling pathway. This study supports that DNA methylation is associated with IgE sensitization early in life and revealed new candidate genes for atopy. Moreover, our study provides evidence that maternal DNA methylation levels are associated with IgE sensitization in the child supporting early in utero effects on atopy predisposition.</p

Changes of DNA methylation are associated with changes in lung function during adolescence

Background Adolescence is a significant period for the gender-dependent development of lung function. Prior studies have shown that DNA methylation (DNA-M) is associated with lung function and DNA-M at some cytosine-phosphate-guanine dinucleotide sites (CpGs) changes over time. This study examined whether changes of DNA-M at lung-function-related CpGs are associated with changes in lung function during adolescence for each gender, and if so, the biological significance of the detected CpGs. Methods Genome-scale DNA-M was measured in peripheral blood samples at ages 10 (n = 330) and 18 years (n = 476) from the Isle of Wight (IOW) birth cohort in United Kingdom, using Illumina Infinium arrays (450 K and EPIC). Spirometry was conducted at both ages. A training and testing method was used to screen 402,714 CpGs for their potential associations with lung function. Linear regressions were applied to assess the association of changes in lung function with changes of DNA-M at those CpGs potentially related to lung function. Adolescence-related and personal and family-related confounders were included in the model. The analyses were stratified by gender. Multiple testing was adjusted by controlling false discovery rate of 0.05. Findings were further examined in two independent birth cohorts, the Avon Longitudinal Study of Children and Parents (ALSPAC) and the Children, Allergy, Milieu, Stockholm, Epidemiology (BAMSE) cohort. Pathway analyses were performed on genes to which the identified CpGs were mapped. Results For females, 42 CpGs showed statistically significant associations with change in FEV1/FVC, but none for change in FEV1 or FVC. No CpGs were identified for males. In replication analyses, 16 and 21 of the 42 CpGs showed the same direction of associations among the females in the ALSPAC and BAMSE cohorts, respectively, with 11 CpGs overlapping across all the three cohorts. Through pathway analyses, significant biological processes were identified that have previously been related to lung function development. Conclusions The detected 11 CpGs in all three cohorts have the potential to serve as the candidate epigenetic markers for changes in lung function during adolescence in female

DNA Methylation Trajectories During Pregnancy

There is emerging evidence on DNA methylation (DNAm) variability over time; however, little is known about dynamics of DNAm patterns during pregnancy. We performed an epigenome-wide longitudinal DNAm study of a well-characterized sample of young women from the Swedish Born into Life study, with repeated blood sampling before, during and after pregnancy (n = 21), using the Illumina Infinium MethylationEPIC array. We conducted a replication in the Isle of Wight third-generation birth cohort (n = 27), using the Infinium HumanMethylation450k BeadChip. We identified 196 CpG sites displaying intra-individual longitudinal change in DNAm with a false discovery rate (FDR) P 3 differentially methylated CpGs: HOXB3, AVP, LOC100996291, and MicroRNA 10a. Of 36 CpGs available in the replication cohort, 17 were replicated, all but 2 with the same direction of association (replication P <.05). Biological pathway analysis demonstrated that FDR-significant CpGs belong to genes overrepresented in metabolism-related pathways, such as adipose tissue development, regulation of insulin receptor signaling, and mammary gland fat development. These results contribute to a better understanding of the biological mechanisms underlying important physiological alterations and adaptations for pregnancy and lactation.Peer reviewe

17q21 variant increases the risk of exacerbations in asthmatic children despite inhaled corticosteroids use

_To the Editor,_ Approximately 25% of the asthmatic children suffer from uncontrolled asthma despite regular use of inhaled corticosteroids (ICS). Variation within the 17q21 locus is the strongest genetic determinant for childhood‐onset asthma. Recently, the influence of this locus on treatment outcomes has been shown in several studies. The Pharmacogenomics in Childhood Asthma (PiCA) consortium is a multiethnic consortium that brings together data from ≥14 000 asthmatic children/young adults from 12 different countries to study the pharmacogenomics of uncontrolled asthma despite treatment. In 14 PiCA populations (with over 4000 asthmatic patients), we studied the association between variation in the 17q21 locus, and asthma exacerbations despite ICS use. We specifically focused on rs7216389, a single nucleotide polymorphism (SNP) in the 17q21 locus strongly associated with childhood asthma and initially identified by Moffatt et al. [...

The Ser82 RAGE variant affects lung function and serum RAGE in smokers and sRAGE production in vitro

Introduction: Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. Methods: Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. Results: Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. Conclusions: This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism

DNA methylation in childhood asthma : an epigenome-wide meta-analysis

Background DNA methylation profiles associated with childhood asthma might provide novel insights into disease pathogenesis. We did an epigenome-wide association study to assess methylation profiles associated with childhood asthma. Methods We did a large-scale epigenome-wide association study (EWAS) within the Mechanisms of the Development of ALLergy (MeDALL) project. We examined epigenome-wide methylation using Illumina Infinium Human Methylation450 BeadChips (450K) in whole blood in 207 children with asthma and 610 controls at age 4-5 years, and 185 children with asthma and 546 controls at age 8 years using a cross-sectional case-control design. After identification of differentially methylated CpG sites in the discovery analysis, we did a validation study in children (4-16 years; 247 cases and 2949 controls) from six additional European cohorts and meta-analysed the results. We next investigated whether replicated CpG sites in cord blood predict later asthma in 1316 children. We subsequently investigated cell-type-specific methylation of the identified CpG sites in eosinophils and respiratory epithelial cells and their related gene-expression signatures. We studied cell-type specificity of the asthma association of the replicated CpG sites in 455 respiratory epithelial cell samples, collected by nasal brushing of 16-year-old children as well as in DNA isolated from blood eosinophils (16 with asthma, eight controls [age 2-56 years]) and compared this with whole-blood DNA samples of 74 individuals with asthma and 93 controls (age 1-79 years). Whole-blood transcriptional profiles associated with replicated CpG sites were annotated using RNA-seq data of subsets of peripheral blood mononuclear cells sorted by fluorescence-activated cell sorting. Findings 27 methylated CpG sites were identified in the discovery analysis. 14 of these CpG sites were replicated and passed genome-wide significance (p Interpretation Reduced whole-blood DNA methylation at 14 CpG sites acquired after birth was strongly associated with childhood asthma. These CpG sites and their associated transcriptional profiles indicate activation of eosinophils and cytotoxic T cells in childhood asthma. Our findings merit further investigations of the role of epigenetics in a clinical context.Peer reviewe