Author response: Identification of autosomal cis expression quantitative trait methylation (cis eQTMs) in children’s blood

The identification of expression quantitative trait methylation (eQTMs), defined as associations between DNA methylation levels and gene expression, might help the biological interpretation of epigenome-wide association studies (EWAS). We aimed to identify autosomal cis eQTMs in children's blood, using data from 832 children of the Human Early Life Exposome (HELIX) project. Blood DNA methylation and gene expression were measured with the Illumina 450K and the Affymetrix HTA v2 arrays, respectively. The relationship between methylation levels and expression of nearby genes (1 Mb window centered at the transcription start site, TSS) was assessed by fitting 13.6 M linear regressions adjusting for sex, age, cohort, and blood cell composition. We identified 39,749 blood autosomal cis eQTMs, representing 21,966 unique CpGs (eCpGs, 5.7% of total CpGs) and 8,886 unique transcript clusters (eGenes, 15.3% of total transcript clusters, equivalent to genes). In 87.9% of these cis eQTMs, the eCpG was located at <250 kb from eGene's TSS; and 58.8% of all eQTMs showed an inverse relationship between the methylation and expression levels. Only around half of the autosomal cis-eQTMs eGenes could be captured through annotation of the eCpG to the closest gene. eCpGs had less measurement error and were enriched for active blood regulatory regions and for CpGs reported to be associated with environmental exposures or phenotypic traits. In 40.4% of the eQTMs, the CpG and the eGene were both associated with at least one genetic variant. The overlap of autosomal cis eQTMs in children's blood with those described in adults was small (13.8%), and age-shared cis eQTMs tended to be proximal to the TSS and enriched for genetic variants. This catalogue of autosomal cis eQTMs in children's blood can help the biological interpretation of EWAS findings and is publicly available at https://helixomics.isglobal.org/ and at Dryad (doi:10.5061/dryad.fxpnvx0t0). The study has received funding from the European Community's Seventh Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project); the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project); from the European Union's Horizon 2020 research and innovation programme under grant agreement no 733206 (LIFECYCLE project), and from the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL and Instituto de Salud Carlos III) under the grant agreement no AC18/00006 (NutriPROGRAM project). The genotyping was supported by the projects PI17/01225 and PI17/01935, funded by the Instituto de Salud Carlos III and co-funded by European Union (ERDF, "A way to make Europe") and the Centro Nacional de Genotipado-CEGEN (PRB2-ISCIII). BiB received core infrastructure funding from the Wellcome Trust (WT101597MA) and a joint grant from the UK Medical Research Council (MRC) and Economic and Social Science Research Council (ESRC) (MR/N024397/1). INMA data collections were supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research. The Rhea project was financially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV-1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009- single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EU- FP7- HEALTH-2012 Proposal No 308333 HELIX), and the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in preschool children, in Heraklion district, Crete, Greece: 2011-2014; "Rhea Plus": Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012-15). We acknowledge support from the Spanish Ministry of Science and Innovation through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. MV-U and CR-A were supported by a FI fellowship from the Catalan Government (FI-DGR 2015 and #016FI_B 00272). MC received funding from Instituto Carlos III (Ministry of Economy and Competitiveness) (CD12/00563 and MS16/00128).

People are exposed to a multitude of environmental chemicals through air, water, food, and consumer products. A person’s total environmental exposure, acquired from conception to death, is called the “exposome.”1 The Human Early-Life Exposome (HELIX) project, a European collaboration, is an ambitious effort that will seek to characterize children’s exposomes as the children progress through early life.2 The six research stages of HELIX are described in this issue of EHP.3 HELIX, a project of 13 partner institutions, will measure environmental exposures of up to 32,000 mother–child pairs and their consequent impact on the growth, development, and health of the children. “Pregnancy and the early years of life are well recognized to be periods of high susceptibility to environmental damage with lifetime consequences. This makes early life an important starting point for development of the exposome,” says project coordinator Martine Vrijheid of the Centre for Research in Environmental Epidemiology in Barcelona, Spain. From conception onward the exposome becomes the record of every exposure a person has ever experienced. In 2005 cancer epidemiologist Christopher Wild coined the term exposome to describe the environmental counterpart of the genome. “At its most complete, the exposome encompasses life-course environmental exposures (including lifestyle factors), from the prenatal period onwards,” Wild wrote.4 He highlighted the need for complete assessments of environmental exposures in epidemiological studies of cancer. Today the exposome concept has been extended to other chronic diseases that carry large societal and economic costs.1 HELIX will use data from six ongoing, prospective European birth cohorts of mothers and children living in Spain, France, the United Kingdom, Norway, Greece, and Lithuania. Large amounts of health data already have been collected, which HELIX investigators will pool. They also plan to collect extensive biomarker data for a subset of 1,200 mother–child pairs. External exposure measures for food, water, air pollution, pesticides, noise, and ultraviolet (UV) radiation will be integrated with molecular markers from metabolomic, proteomic, transcriptomic, and other “omic” studies. Then the investigators will estimate the burden of childhood disease from multiple environmental exposures. The HELIX project will run for 4.5 years. The results are expected to help investigators identify unknown health hazards and benefits to better target future preventive measures and regulations. “Characterization of the exposome in early life can provide very effective tools for disease prevention, given that interventions at that time can reshape biological programming and shift the body’s developmental track to normal function,” says Vrijheid. Among the innovative tools created specifically for HELIX is ExpoApp, a mobile application for tracking participants’ activity levels. ExpoApp uses GPS and a smartphone’s built-in accelerometer to track a person’s location and measure physical activity every 10 seconds. Participants will wear ExpoApp-enabled smartphones for a week, along with air pollution and UV monitors. The data will be used to calculate the amount of air inhaled and an individual’s exposure to air pollutants. Because the exposome encompasses the progression of exposures over a lifetime, it’s an overwhelming research concept to understand and impractical to fully fund at one time, says Paul Lioy, a professor of environmental and occupational medicine at Robert Wood Johnson Medical School. HELIX “is a well-designed study and great first attempt to focus on one part of the exposome to demonstrate its proof-of-concept principles,” Lioy says. The methods used to study the mothers and children participating in HELIX require many different tools that cut across multiple scientific disciplines. By publishing the complex study design of HELIX in EHP, Lioy says “other environmental health and exposure scientists may be inspired to develop parallel studies of smaller populations to validate other components of the exposome concept.”

The exposome has conceptually been described to comprise three overlapping domains: (1) a general external environment including factors such as the urban environment, climate factors, social capital, stress; (2) a specific external environment including specific contaminants, diet, physical activity, tobacco, and (3) an internal environment including internal biological factors such as metabolism, gut microflora, inflammation, and oxidative stress. Here, we aim to illustrate how these three domains and their interrelations may be studied in an epidemiological study design, using the HELIX (Human Early Life Exposome) project as an example. HELIX takes pregnancy and childhood periods (“early life”) as a starting point. In six existing birth cohort studies in Europe, HELIX estimated prenatal and postnatal exposures. Exposure models for the outdoor exposome (air pollutants, noise, meteorological factors, and natural and built environment characteristics) were developed for a total of 30,000 mother–child pairs. Exposure biomarkers (for persistent organic pollutants, metals, phthalate metabolites, phenolic compounds and organophosphate pesticides) and omics markers (metabolites, proteins, mRNA, miRNA, DNA methylation) were measured in a subset of 1200 children. Nested repeat-sampling panel studies (N = 150) collected data on variability in personal exposure to air pollution and built environment measures, in biomarkers for nonpersistent chemicals (phthalates and phenolic compounds) and in all omics techniques. Outcome examinations were carried out using common protocols in the six cohorts. We will discuss some first results of the HELIX project, including a description of the correlation structure of multiple exposure data.

Early-life exposome and lung function in children in Europe: an analysis of data from the longitudinal, population-based HELIX cohort

Dear Editor Previous research found that childhood cancer survivors of African ancestry have significantly higher morbidity and mortality than those of European ancestry [1]. However, after adjusting for socio-economic factors, the magnitudes of racial health disparities are either substantially decreased or become statistically non-significant [1], suggesting that social and economic determinants may contribute to racial health disparities. Recently, we conducted epigenome-wide association studies (EWAS) for three key social determinants of health (SDOHs), namely, personal educational attainment, personal income, and neighborhood deprivation among survivors of childhood cancer, where 130 epigenome-wide significant SDOH-CpG associations were identified among European ancestry survivors, and 25 of which were also validated in African ancestry survivors [2]. Notably, many SDOH-associated CpG sites are also associated with tobacco use. Although pulmonary impairment is an integral part of the overall disease burden, racial disparities in this specific group of conditions have not been documented, and potential underlying mechanistic causal pathways have not been studied. Moreover, other observational studies have shown that blood DNA methylation (DNAm) signature was associated with pulmonary functions [3-5]. In this cross-sectional study, we hypothesized that race and its associated SDOHs might contribute to the risk of pulmonary impairment, evaluated whether SDOH-associated CpG sites were associated with specific parameters of pulmonary function, and further applied mediation analysis to explore the potential mediating role of these DNAm sites for the association between SDOHs and risk of impaired pulmonary functions. The methods are described in Supplementary Methods and Supplementary Table S1. The occurrence rates of three adverse pulmonary outcomes, obstructive pulmonary deficit (OPD), pulmonary diffusion deficits (PDD), and restrictive pulmonary deficit (RPD), were compared between African- and European ancestry survivors in the St. Jude Lifetime Cohort (SJLIFE) study [6]. The study population is described in the Supplementary Results and Supplementary Table S2. In an unadjusted model considering common terminology criteria for adverse event (CTCAE) grade ≥2, the occurrence rates of PDD and RPD were significantly higher in African ancestry survivors than in European ancestry survivors (PDD: 25.2% vs. 18.2%, P = 0.033; RPD: 14.2% vs. 7.5%, P = 0.002), whereas OPD was comparable between the two race groups (9.8% vs. 13.1%, P = 0.206) (Supplementary Figure S1). In a multivariable model, adjusting for other covariates but without the inclusion of SDOHs (the base model), race was significantly associated with PDD (P = 0.024) and RPD (P = 0.004, Supplementary Table S3). When SDOHs were added to the model (the full model), the effect of race on pulmonary impairment became non-significant for PDD (P = 0.183), slightly attenuated for RPD (P = 0.006) and remained non-significant for OPD (P = 0.434). Notably, treatment factors, including chest RT and lung surgery, were significantly associated with all three conditions. The effect of current smokers became non-significant for OPD and PDD. Interestingly, BMI was inversely associated with PDD. Because SDOHs only accounted for the racial disparity in PDD from the above analyses, we further analyzed if SDOH-associated CpG sites could mediate the association between SDOHs and PDD. Among the 130 SDOH-CpG associations identified in our previous EWAS on European ancestry survivors [2], 61 CpGs (29 for educational attainment, 16 for personal income, and 16 for area deprivation index [ADI]) were significantly associated with the risk of PDD after adjusting for multiple comparisons (Pfalse discovery rate (FDR) < 0.050) (Supplementary Table S4), some of which have been previously reported to be associated with health conditions related to pulmonary functions. In the mediation analysis, 17 out of 29 educational attainment-associated CpGs were identified with significant average causal mediation effects (ACME) after adjusting for multiple comparisons. Using a squared pairwise Pearson correlation coefficient r2 threshold of 0.05, three independent CpGs, cg04180924 (chr3, coproporphyrinogen oxidase [CPOX], mediation = 32.9%, PFDR = 0.014), cg11205006 (chr22, HPS4, mediation = 19.0%, PFDR = 0.024), and cg27470486 (chr17, ATP citrate lyase [ACLY], mediation = 8.6%, PFDR = 0.044), were obtained by top-down pruning the 17 CpGs sorted by estimated ACME in decreasing order. For the final mediation analysis, a combined score (i.e., summation of DNAm levels of the three CpGs with the same direction of association) was used as the mediating variable, and a 48.9% mediation effect for educational attainment on PDD was achieved (Table 1). Similarly, the same single mediator, cg08064403, partially mediated the effect of personal income (mediation = 25.9%, P < 0.001) and ADI (mediation = 24.1%, P < 0.001) on PDD (Table 1). None of the SDOH-associated CpG sites was significantly associated with the risk of PDD among African ancestry survivors after adjusting for multiple comparisons because of the small number of African-ancestry survivors, hence similar mediation analysis could not be conducted among African ancestry survivors. For each CpG mediating the association between SDOHs and pulmonary conditions in European ancestry survivors, a linear regression of expression levels for Illumina-annotated genes against DNAm levels of CpGs was performed. The DNAm levels of four CpGs were negatively correlated with the gene expression levels of Illumina-annotated genes: ACLY (cg27470486), which plays a role in lipid synthesis in the lung [7]; Hermansky-Pudlak syndrome 4 (HPS4) (cg11205006), which is related to pulmonary fibrosis [8]; CPOX (cg04180924 and cg08064403) [9] and claudin domain containing 1 (CLDND1) (cg08064403) [10], two smoking-related genes (Supplementary Figures S2-S3 and Supplementary Table S5). Based on these correlations between DNAm and gene expression levels, all four CpGs were deemed as expression quantitative trait methylations. We leveraged the molecular profiling data of the well-established SJLIFE cohort and provided strong evidence supporting social-epigenetic mediators for racial disparities in pulmonary impairment among childhood cancer survivors. The present study had some limitations. First, the analysis was based on a relatively short follow-up from blood drawn for DNAm detection, so there was no clearly defined temporal association to establish the causality. Second, we attempted to take advantage of the existing whole-genome sequencing data to search for methylation quantitative trait loci (meQTL), but we did not find any strong meQTL for the genomic regions of interest that could be used in Mendelian randomization for causal inference. Third, air pollution from neighborhoods or occupational exposures and second-hand smoke, which may also contribute to pulmonary impairment, were also not considered due to the lack of data. Lastly, our analysis was based on each individual condition separately, other factors including co-morbidity (e.g., among 209 survivors with PDD, 70 had OPD, 53 had RPD, and 42 had both OPD and RPD), type and stage of primary diagnosis may also confound the results. In conclusion, the risk of pulmonary impairment among survivors of childhood cancer differs by specific condition (PDD or RPD) and race (African and Europena ancestry). SDOHs may partially explain the observed racial disparity in PDD, potentially through an epigenetic mechanism. Social-epigenetic studies like ours could inform intervention strategies, such as improving social integration and social support to counteract the elevated disease risk for social-economically disadvantaged survivors. The efficacy of this type of intervention can be objectively measured by the improvement of epigenetic markers as an intermediate outcome. Ultimately, we will close the gap of disparity in pulmonary impairment and other health outcomes due to race or social adversity among survivors of childhood cancer. ZW and ICH designed and supervised the study; MMH, KKN, KRK and LLR assisted in or provided support for data collection and recruitment of study participants; JE, HM, EP, GN, EW, and JZ supervised sample processing, and/or performed DNA/RNA extractions, carried out the Infinium MethylationEPIC array scanning and RNA sequencing; NS, QD, CC, QL, DKS, ICH, and ZW performed bioinformatic and statistical analysis; NS, QD, ICH, and ZW wrote the first draft of the manuscript. All authors contributed to data interpretation and writing and approved the final manuscript for publication. Not applicable. The authors declare that they have no competing interests. This work was supported by funding from the V Foundation (Grant # DT2020-014), the National Institutes of Health of the US (Grant # CA021765, CA195547) and the American Lebanese Syrian Associated Charities (ALSAC). The funders of the study had no role in the design and conduct of the study; were not involved in collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication. DNA methylation data are accessible at NCBI Gene Expression Omnibus database under the accession number GSE169156 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE169156). Additional clinical data about the study participants in the St. Jude Lifetime Cohort can be accessed via the survivorship portal (http://survivorship.stjude.cloud/). The SJLIFE study protocol was approved by the Institutional Review Board (IRB) at St. Jude Children's Research Hospital with a reference number (010882). All SJLIFE study participants provided written informed consent. This study was performed in accordance with the Declaration of Helsinki. Not applicable. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The role of DNA methylation for lung function, needs exploring, as it may help identify genes and pathways of importance for lung function. The aim of this study was to explore the association between blood DNA methylation and lung function in monozygotic (MZ) twins. A sample of 169 middle-aged MZ twin-pairs was included in an epigenome-wide association study. They were collected from the Danish Twin Register and examined at baseline (1998-1999) and follow-up (2008-2011). Using the twin-design, we correlated intra-pair differences in cross-sectional and longitudinal lung function with intra-pair blood DNA methylation differences at follow-up by linear regression analyses adjusted for sex, age, BMI, smoking, and blood-cell-composition. We identified several differentially methylated CpG sites associated with Forced Expiratory Volume the 1^st second (FEV₁) and Forced Vital Capacity (FVC). Three probes identified for FVC level were located in <i>GLIPR1L2</i> (p-value=7.14x10⁻⁸), a gene involved in innate immunity, and tumour-suppressor/pro-oncogenic mechanisms. Change in FEV₁ during the follow-up period was associated with methylation level in <i>TRIM27</i> (p-value=1.55x10^‑6), a negative regulator of CD4-T-cells also involved in cancer development. Several enriched pathways were identified; for FEV₁ e.g. "TGFBR" (Benjamini-Hochberg_adj p-value=0.045), the receptor for TGFβ, a growth-factor involved in normal lung tissue repair through pro-fibrotic effects. Our findings suggest that blood DNA methylation signatures are associated with lung function, identifying immunological- and cancer-related genes, as well as TGF-β-receptor related genes to be possibly involved in the level and change in lung function.

Pregnancy exposure to atmospheric pollution and meteorological conditions and placental DNA methylation

Background:Telomere length is a molecular marker of biological aging.Objective:Here we investigated whether early-life exposure to residential air pollution was associated with leukocyte telomere length (LTL) at 8 y of age.Methods:In a multicenter European birth cohort study, HELIX (Human Early Life Exposome) (), we estimated prenatal and 1-y childhood exposure to nitrogen dioxide (), particulate matter with aerodynamic diameter (), and proximity to major roads. Average relative LTL was measured using quantitative real-time polymerase chain reaction (qPCR). Effect estimates of the association between LTL and prenatal, 1-y childhood air pollution, and proximity to major roads were calculated using multiple linear mixed models with a random cohort effect and adjusted for relevant covariates.Results:LTL was inversely associated with prenatal and 1-y childhood and exposures levels. Each standard deviation (SD) increase in prenatal was associated with a (95% CI: , ) change in LTL. Prenatal was nonsignificantly associated with LTL ( per SD increase; 95% CI: , 0.6). For each SD increment in 1-y childhood and exposure, LTL shortened by (95% CI: , ) and (95% CI: , 0.1), respectively. Each doubling in residential distance to nearest major road during childhood was associated with a 1.6% (95% CI: 0.02, 3.1) lengthening in LTL.Conclusion:Lower exposures to air pollution during pregnancy and childhood were associated with longer telomeres in European children at 8 y of age. These results suggest that reductions in traffic-related air pollution may promote molecular longevity, as exemplified by telomere length, from early life onward. https://doi.org/10.1289/EHP4148

Exposure to prenatal social stressors during pregnancy is associated with adverse birth outcomes and has been linked to epigenetic changes in DNA methylation (DNAm); however, less understood is the effect of neighborhood-level stressors like crime during pregnancy on offspring DNAm. Using data from the Newborn Epigenetic Study, we conducted epigenome-wide and regional analyses of the association between exposure to neighborhood crime and DNAm in offspring cord blood using Illumina’s HumanMethylation450k BeadChip among 185 mother-offspring pairs. Prenatal exposure to neighborhood crime at the census block group level was mapped to participants' residential addresses during the gestational window from the date of last menstrual period to delivery. Models for the epigenome-wide and regional analyses were adjusted for maternal age, race/ethnicity, education, smoking, cell-type composition, and offspring sex. Genetic influence and gene expression enrichment were assessed using methylation quantitative trait loci (mQTLs) and expression quantitative trait methylation (eQTMs) analyses. Functional enrichment was determined using Gene Ontology and KEGG databases. We did not find evidence of epigenome-wide associations between prenatal neighborhood crime exposure and DNAm; however, we identified nine differentially methylated regions (DMRs) comprising 51 CpG sites associated with neighborhood crime. CpG sites within significant differentially methylated regions were associated with mQTLs at birth and eQTMs upon further examination. KEGG analysis identified a significant Th1 and Th2 cell differentiation pathway. Our results suggest potential links between prenatal neighborhood crime exposure and offspring DNAm; however, additional research is needed in larger cohorts across wider geographic areas to confirm our results.

BACKGROUND AND AIM: DNA methylation is a potential biological mechanism through which residential greenness affects health, but little is known about its association with greenness and whether the association could be modified by genetic background. We aimed to evaluate the association between surrounding greenness and genome-wide DNA methylation and potential gene-greenness interaction effects on DNA methylation. METHODS: We measured blood-derived DNA methylation using the HumanMethylation450 BeadChip array (Illumina) for 479 Australian women, including 66 monozygotic, 66 dizygotic twin pairs, and 215 sisters of these twins. Surrounding greenness was represented by Normalized Difference Vegetation Index (NDVI) and Enhanced Vegetation Index (EVI) within 300, 500, 1000 or 2000 metres surrounding participants’ home addresses. For each cytosine-guanine dinucleotide (CpG), the association between its methylation level and NDVI or EVI were evaluated by generalized estimating equations, adjusting for age, education, marital status, area-level socioeconomic status, smoking behavior, cell-type proportions and familial clustering. We used comb-p and DMRcate to identify significant differentially methylated regions (DMRs). For each significant CpG, we evaluated the interaction effects of greenness and single-nucleotide polymorphisms (SNPs) within ±1Mb window on its methylation level. RESULTS:We found associations between surrounding greenness and blood DNA methylation for one CpG (cg04720477, mapped to the promoter region of CNP gene) with false discovery rate [FDR]0.05, and for another 9 CpGs with 0.05≤FDR0.10. For two of these CpGs, we found 33 SNPs significantly (FDR0.05) modified the greenness-methylation association. There were 35 significant DMRs related to surrounding greenness that were identified by both comb-p (Sidak p-value0.01) and DMRcate (FDR0.01). Those CpGs and DMRs were mapped to genes related to many human diseases, such as mental health disorders and neoplasms as well as nutritional and metabolic diseases. CONCLUSIONS:Surrounding greenness was associated with blood DNA methylation of many loci across human genome, and this association could be modified by genetic variations. KEYWORDS: Green space, Epigenomics, Molecular epidemiology, Environmental epidemiology

BackgroundDNA methylation is a potential biological mechanism through which residential greenness affects health, but little is known about its association with greenness and whether the association could be modified by genetic background. We aimed to evaluate the association between surrounding greenness and genome-wide DNA methylation and potential gene-greenness interaction effects on DNA methylation. MethodsWe measured blood-derived DNA methylation using the HumanMethylation450 BeadChip array (Illumina) for 479 Australian women, including 66 monozygotic, 66 dizygotic twin pairs, and 215 sisters of these twins. Surrounding greenness was represented by Normalized Difference Vegetation Index (NDVI) and Enhanced Vegetation Index (EVI) within 300, 500, 1000 or 2000 m surrounding participants’ home addresses. For each cytosine-guanine dinucleotide (CpG), the associations between its methylation level and NDVI or EVI were evaluated by generalized estimating equations, after adjusting for age, education, marital status, area-level socioeconomic status, smoking behavior, cell-type proportions, and familial clustering. We used comb-p and DMRcate to identify significant differentially methylated regions (DMRs). For each significant CpG, we evaluated the interaction effects of greenness and single-nucleotide polymorphisms (SNPs) within ±1 Mb window on its methylation level. ResultsWe found associations between surrounding greenness and blood DNA methylation for one CpG (cg04720477, mapped to the promoter region of CNP gene) with false discovery rate [FDR] < 0.05, and for another 9 CpGs with 0.05 ≤ FDR < 0.10. For two of these CpGs, we found 33 SNPs significantly (FDR < 0.05) modified the greenness-methylation association. There were 35 significant DMRs related to surrounding greenness that were identified by both comb-p (Sidak p-value < 0.01) and DMRcate (FDR < 0.01). Those CpGs and DMRs were mapped to genes related to many human diseases, such as mental health disorders and neoplasms as well as nutritional and metabolic diseases. ConclusionsSurrounding greenness was associated with blood DNA methylation of many loci across human genome, and this association could be modified by genetic variations.

ABSTRACTDNA methylation in blood may adapt to conditions affecting our health, such as inflammation, and multiple studies have identified differential DNA methylation related to smoking, obesity and various diseases. The purpose of this study was to evaluate previously reported, and explore possible new, associations between levels of inflammatory markers and DNA methylation in blood. We used a well-characterized study population consisting of 127 individuals, all of whom were participants in the population-based Västerbotten Intervention Programme cohort and had provided two blood samples, ten years apart. Levels of CRP and 160 other proteins were measured in plasma, and DNA methylation levels (assessed using the 850K Illumina Infinium MethylationEPIC BeadChip) were measured in white blood cell DNA. Associations between CpG methylation and protein levels were estimated using linear mixed models. In the study we were able to confirm the direction for 85 of 102 previously reported protein-methylation associations. Depicting associations in a network allowed us to identify CpG sites with associations to multiple proteins, and ten CpG sites were each associated with three or more inflammatory markers. Furthermore, two genetic regions included nine additional unreported CpG sites that may represent trans-acting methylation sites. Our study supports a complex interaction between DNA methylation and circulating proteins involved in the inflammatory response. The notion of trans-acting methylation sites affecting, or being affected by, the expression of genes on completely different chromosomes should be taken into account when interpreting results from epigenome-wide association studies.

Dna Methylation Mediates the Effect of Maternal Smoking During Pregnancy on Birthweight

Early life tobacco exposure and children’s telomere length: The HELIX project

DNA methylation has become increasingly recognized in the etiology of complex diseases, including thrombotic disorders. Blood is often collected in epidemiological studies for genotyping and has recently also been used to examine DNA methylation in epigenome-wide association studies. DNA methylation patterns are often tissue-specific, thus, peripheral blood may not accurately reflect the methylation pattern in the tissue of relevance. Here, we collected paired liver and blood samples concurrently from 27 individuals undergoing liver surgery. We performed targeted bisulfite sequencing for a set of 35 hemostatic genes primarily expressed in liver to analyze DNA methylation levels of >10,000 cytosine-phosphate-guanine (CpG) dinucleotides. We evaluated whether DNA methylation in blood could serve as a proxy for DNA methylation in liver at individual CpGs. Approximately 30% of CpGs were nonvariable and were predominantly hypo- (<25%) or hypermethylated (>70%) in both tissues. While blood can serve as a proxy for liver at these CpGs, the low variability renders these unlikely to explain phenotypic differences. We therefore focused on CpG sites with variable methylation levels in liver. The level of blood–liver tissue correlation varied widely across these variable CpGs; moderate correlations (0.5 ≤ r < 0.75) were detected for 6% and strong correlations (r ≥ 0.75) for a further 4%. Our findings indicate that it is essential to study the concordance of DNA methylation between blood and liver at individual CpGs. This paired blood–liver dataset is intended as a resource to aid interpretation of blood-based DNA methylation results.