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

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

Morbidity and mortality from road injuries: results from the Global Burden of Disease Study 2017

BackgroundThe global burden of road injuries is known to follow complex geographical, temporal and demographic patterns. While health loss from road injuries is a major topic of global importance, there has been no recent comprehensive assessment that includes estimates for every age group, sex and country over recent years.MethodsWe used results from the Global Burden of Disease (GBD) 2017 study to report incidence, prevalence, years lived with disability, deaths, years of life lost and disability-adjusted life years for all locations in the GBD 2017 hierarchy from 1990 to 2017 for road injuries. Second, we measured mortality-to-incidence ratios by location. Third, we assessed the distribution of the natures of injury (eg, traumatic brain injury) that result from each road injury.ResultsGlobally, 1 243 068 (95% uncertainty interval 1 191 889 to 1 276 940) people died from road injuries in 2017 out of 54 192 330 (47 381 583 to 61 645 891) new cases of road injuries. Age-standardised incidence rates of road injuries increased between 1990 and 2017, while mortality rates decreased. Regionally, age-standardised mortality rates decreased in all but two regions, South Asia and Southern Latin America, where rates did not change significantly. Nine of 21 GBD regions experienced significant increases in age-standardised incidence rates, while 10 experienced significant decreases and two experienced no significant change.ConclusionsWhile road injury mortality has improved in recent decades, there are worsening rates of incidence and significant geographical heterogeneity. These findings indicate that more research is needed to better understand how road injuries can be prevented

Epigenome-wide meta-analysis of blood DNA methylation in newborns and children identifies numerous loci related to gestational age

Background Preterm birth and shorter duration of pregnancy are associated with increased morbidity in neonatal and later life. As the epigenome is known to have an important role during fetal development, we investigated associations between gestational age and blood DNA methylation in children. Methods We performed meta-analysis of Illumina's HumanMethylation450-array associations between gestational age and cord blood DNA methylation in 3648 newborns from 17 cohorts without common pregnancy complications, induced delivery or caesarean section. We also explored associations of gestational age with DNA methylation measured at 4-18 years in additional pediatric cohorts. Follow-up analyses of DNA methylation and gene expression correlations were performed in cord blood. DNA methylation profiles were also explored in tissues relevant for gestational age health effects: fetal brain and lung. Results We identified 8899 CpGs in cord blood that were associated with gestational age (range 27-42 weeks), at Bonferroni significance, P <1.06 x 10(- 7), of which 3343 were novel. These were annotated to 4966 genes. After restricting findings to at least three significant adjacent CpGs, we identified 1276 CpGs annotated to 325 genes. Results were generally consistent when analyses were restricted to term births. Cord blood findings tended not to persist into childhood and adolescence. Pathway analyses identified enrichment for biological processes critical to embryonic development. Follow-up of identified genes showed correlations between gestational age and DNA methylation levels in fetal brain and lung tissue, as well as correlation with expression levels. Conclusions We identified numerous CpGs differentially methylated in relation to gestational age at birth that appear to reflect fetal developmental processes across tissues. These findings may contribute to understanding mechanisms linking gestational age to health effects.Peer reviewe