BACKGROUND
dna methylation is the most extensively studied epigenetic mechanism.
an individual’s dna methylation profile is not static but subject to dynamic changes induced by genetic  <cit> , environmental  <cit>  and stochastic factors during ageing.
although some age-related epigenetic changes have been identified , how genetic and environmental factors influence methylation-change with ageing is not yet well understood.
to answer these questions, we performed an epigenome-wide longitudinal dna methylation study of  <dig> children across four european population-based birth cohorts participating in the medall  consortium epigenetic study  <cit>  using the illumina infinium humanmethylation <dig> beadchip  array.
we compared  <dig> children at ages  <dig> and 4/5 years, by investigating  <dig> paired samples of cord blood and peripheral blood dna, and another set of  <dig> children at ages  <dig> and 8 years .
we first identified a set of overlapping and consistent age-differential methylation sites  in each group and looked at their functions.
then we linked genetic variation and maternal smoking during pregnancy with the changes in these a-dmss over time to see how genetic and environmental factors regulate dynamic changes in methylation.
finally, we investigated if genes annotated to these a-dmss were enriched for disease-associated genes; we linked these a-dmss to asthma, the chronic disease with the highest prevalence in childhood.
RESULTS
identification of a-dmss
we first applied differential methylation analysis on  <dig>  cytosine-phosphate-guanine  sites that passed our quality control  in  <dig> paired samples from the inma  and eden  birth cohorts  and in  <dig> paired samples from the bamse  and piama  birth cohorts , .
the overall correlations of methylation levels between two time points in the four birth cohorts are shown in additional file 2: figure s <dig>  in the pooled analysis of all cohorts adjusting for cell-type-composition  <cit> ,  <dig>  significant a-dmss were identified in both age groups surpassing the 5% bonferroni corrected threshold  .
of these  <dig>  a-dmss,  <dig>   showed a consistent decrease in methylation with age , whereas  <dig>   showed a consistent increase in methylation with age .
only  <dig>  of the cpg sites showed opposite directions in their methylation changes between the younger and older comparison sets .
the complete list of  <dig>  consistent da-dmss and ia-dmss, and their annotation, is presented in additional file  <dig>  comparing our a-dms results with those from two independent, paediatric, age-related methylation studies  <cit> , we see more than 50% overlap in the cpg sites identified .
we observed that some of our consistent da-dmss were located in clusters, probably reflecting age associated differential methylated regions rather than one site.fig.
<dig> study design and structure of this paper.
we collected cord blood and peripheral blood samples from  <dig> children from four european birth cohorts: inma, eden, bamse and piama.
differential methylation analysis on different age groups resulted in  <dig>  consistent age-differential methylation sites .
we then linked the methylation change of these a-dmss to genetics by cis-dmeqtl, to environmental exposure from maternal smoking, and to diseases, specifically asthma
the most significant ia-dmss, cg <dig>  and cg <dig>  are located on the chromosome 4q <dig> , near the gene lrat.
mutations in this gene have been associated with early onset retinal dystrophy  <cit> .
the top associated da-dmss, cg <dig>  and cg <dig> on chromosome 11q <dig>  region were annotated in shank <dig>  mutations in this shank <dig> synaptic scaffolding gene have been associated with neurodevelopmental disorders and autism  <cit> .
characterization of da-dmss and ia-dmss
we next examined the enrichment of the  <dig>  da-dmss and  <dig>  ia-dmss in genomic regions and their predicted roles in regulating gene expression using histone modifications and chromatin state data from the roadmap epigenomics project  <cit> .
ia-dmss were situated in distinctly different functional domains compared to da-dmss, which is consistent with previous findings  <cit> .
thus, compared to all the cpg sites we tested, ia-dmss were enriched in cpg shores  and gene bodies , while da-dmss were enriched in open seas, shelves, shores, intergenic regions and transcription start site   <dig> regions.
ia-dmss were also enriched in transcriptional repression histone modification marker , while da-dmss were depleted for this marker .
both da-dmss and ia-dmss were enriched in enhancers and bivalent enhancers .
ia-dmss were also enriched in bivalent tss regions , which are thought to silence or activate expression of developmental genes  <cit> .
in contrast, da-dmss were enriched in h3k27ac, which separates active from poised enhancers and predict developmental state  <cit> .fig.
<dig> molecular enrichment of a-dmss for regulatory features.
fold enrichment of  <dig>  da-dmss  and  <dig>  ia-dmss .
a cpg islands, shore, shelf and open sea.
b gene regions including: within  <dig>  kb upstream of the transcription start site , the 5' untranslated region , the first gene exon, the gene body, the 3' utr or intergenic region.
c predicted gene expression regulatory regions based on histone modifications derived from roadmap epigenomics blood data.
d predicted chromatin state based on roadmap epigenomics blood data.
dnase: dnase i hypersensitive site, enh: enhancers, enhbiv: bivalent enhancer, tssa: active tss, tssbiv: bivalent/poised tss, tx: strong transcription, txwk: weak transcription.
the presented fold enrichments are from  <dig>  samples by χ
<dig> test and are relative to all  <dig>  cpg sites tested ; * 1 × 10−3 ≤ p <  <dig> , ** 1 × 10−6 ≤ p < 1 × 10- <dig>  *** p < 1 × 10−6
we next performed pathway analysis by genenetwork  <cit>  on genes located in close proximity to dmss probes.
in total,  <dig> genes were annotated to be located near ia-dmss probes and  <dig> genes near the da-dmss probes.
<dig> of these genes were overlapping across the two groups, suggesting complex regulation of these genes during development.
the pathway analysis of da-dmss specific genes using gene co-expression indicated enrichment of reactome pathways involved in cell signalling, including signalling by nerve growth factors, platelet derived growth factors, neurotrophin signalling and many others .
the corresponding mouse genome informatics  phenotypes pointed to cell migration, growth retardation and various abnormal tissue morphologies.
the ia-dms specific genes were enriched for developmental genes, axon guidance, regulation of insulin secretion and energy metabolism.
the enriched mgi phenotypes were related to neonatal and postnatal lethality.
this indicates that in early age methylation regulates the activity of genes, involved in basic processes of development and metabolism.
next, we inspected methylation dynamics of some well-known developmental genes and found a lot of cpgs sites which were annotated to them, such as growth differentiation factors, bone morphogeneic proteins, wnt signalling pathway  gene, paired box genes and homeotic genes  gene etc.
among them, the 5′ hoxd cluster  <cit>  that contains genes important for limb development.
the 5′ hoxd cluster consists of five genes .
we found eight ia-dmss with low methylation status within the cluster , seven of which were localized in cpg islands encompassing the five promoters and three first exons.
in contrast, meis homeobox  <dig> , a limb development regulator that forms complexes with both hoxd <dig> and hoxd <dig>  <cit> , displays a different methylation pattern.
cg <dig> lies in the gene body of meis <dig> and its methylation level decreases during ageing .
genetic variation associates with longitudinal dna methylation changes
the availability of paired samples from four different cohorts gave us the possibility to investigate the effect of genetics on methylation dynamics in a longitudinal way.
we examined whether genetic variations were associated with longitudinal dna methylation changes in childhood using cis-differential methylation quantitative trait locus  mapping.
we associated snps within ±250 kb of our  <dig>  a-dmss in gwas data available for  <dig> children from bamse and piama in the 4– <dig> years-old comparison set for discovery and from  <dig> children in the inma 0–4/5 years-old comparison set for replication .
we discovered  <dig>  snp-cpg pairs on  <dig> admss with significant cis-dmeqtls in the older comparison set , and  <dig>  snp-cpg pairs  were replicated in the younger comparison set .
<dig> potentially false-positive dmeqtls were removed due to snp-under-the-probe effects  <cit> .
the remaining  <dig>   snp-cpg pairs are listed in additional file  <dig>  consisting of  <dig> snps regulating  <dig> a-dmss  that changed with age and showed methylation change was partly under genetic control.
to understand these dmeqtls, we tested if these  <dig>  snp-cpg pairs were meqtls at age  <dig> and 8 years cross-sectionally.
most snp-cpg pairs showing dmeqtl were significant meqtls when analysed cross-sectionally at both ages .
the most significant meqtl rs7522439-cg <dig>  which are on chromosome 1p <dig> , close to gene padi <dig> .
by checking the bios qtl browser , we also found the significant cis-meqtl  in methylation data from adult blood dna samples, which is line with the recent findings that genetic effects on methylation levels are stable  <cit> .
however, we also found three cpg sites cg <dig>  cg <dig> and cg <dig> which were annotated to the ddo  gene showing significant cis-meqtl, but did not show significant cis-meqtl in the bios qtl database.
this may indicate an age specific meqtl effect since d-aspartate content in brain is decreased during adulthood  <cit> .
furthermore, we compared the meqtl-effect size at ages  <dig> and  <dig> , and found that almost all effects of genetic factors on methylation had the same direction at both ages , i.e., genetic effects on methylation remained stable with ageing.
among these  <dig>  snp-cpg pairs,  <dig> had a significantly larger effect and  <dig>  had a significantly smaller effect at age 4 years than at age  <dig> .
an example of this pattern  is shown for the rs9320331_cg <dig> snp-cpg pair in the ddo  gene, which showed a meqtl at both ages  <dig> and  <dig> , with a stronger effect at age  <dig> compared to the effect at age  <dig> .fig.
<dig> genetic variation associated with longitudinal dna methylation changes by cis-dmeqtl study.
a the effect sizes of meqtl at age 4 years against those at age 8 years of  <dig> snp-cpg pairs in the age 4- <dig> group.
almost all the dots lie in the first and third quadrants, suggesting that the effect size of genetic factors on methylation from most of the snp-cpg pairs followed the same direction at both ages.
blue dots represent snp-cpg pairs with a significantly weaker effect size at age  <dig> than at age  <dig>  whereas green dots represent a stronger effect, and red dots represent a different direction of effect.
b bar plot of  <dig> snp-cpg pairs.
there are  <dig> snp-cpg pairs showing a weaker effect at age  <dig> than age  <dig> ,  <dig>  snp-cpg pairs showing stronger effect  and  <dig> snp-cpg pairs showing a different effect .
c boxplot of dmeqtl of rs9320331-cg <dig>   <dig> piama samples have been used to illustrate this dmeqtl.
d regional dmeqtl association of cg <dig> results for the age 4– <dig> group.
plots were generated using locuszoom  <cit> .
the ld estimates are color-coded as a heatmap from dark blue  to red  regional plot shows-log10
p of all snps surrounding snp rs <dig>  and the degree of linkage disequilibrium between all snps and lead snp rs <dig>  snps with lower p-values span ddo and slc22a <dig> genes within a recombination boundary.
e boxplot of meqtl of rs9320331-cg <dig> at age  <dig> and age  <dig> cross-sectionally
maternal smoking during pregnancy associates with longitudinal dna methylation changes
methylation has been found strongly associated with maternal smoking during pregnancy  <cit> .
here we further investigated whether methylation changes of the  <dig>  a-dmss between the 0–4/ <dig> and 4– <dig> years-old comparison sets were associated with maternal smoking during pregnancy.
in total, we found eight significant a-dmss in the younger set and one in the older set  associated with maternal smoking.
these nine cpg sites mapped to five genes, itga <dig>  or5b <dig>  vwf, rcbtb <dig> and creb <dig> and one pseudogene hnrnpa3p <dig>  the most significant association was for the methylation change of cg <dig> at the vwf gene , a protein-coding gene crucial for haemostasis.
both vwf and itga <dig>  are involved in regulating cell-substrate adhesion.
interestingly, all nine significant cpg sites belonged to the da-dms group, and the association of maternal smoking with methylation changes was always negative .
this means that the methylation decrease was consistently larger in the children exposed to smoke in utero compared to the non-exposed.
fig.
4a and additional file 2: figure s <dig> show that maternal smoking during pregnancy was associated with a stronger, decreasing effect on methylation change of cg <dig>  in those children.
to underpin this, we further checked the directions of effect of all nominally significant a-dmss  and found that methylation changes tended to be negatively associated with maternal smoking between  <dig> and 4/5 years  and, moreover, that 721/ <dig>  a-dmss showed a stronger decrease in methylation levels from  <dig> to 4/5 years .
in summary, we found that maternal smoking exposure tended to more strongly decrease methylation levels at the identified da-dmss.fig.
<dig> the effect of maternal smoking on methylation change.
a the effect of maternal smoking on methylation change of cg <dig>  in the age 0–4/ <dig> group.
maternal smoking shows an enhanced effect on decreasing methylation levels since the decrease from age  <dig> to age 4/5 years old of cg <dig> in the exposed group is significantly larger compared to the non-exposed group.
b density plot of effect size of all nominally significant a-dmss  with maternal smoking.
in the age 0–4/ <dig> group, methylation changes tend to be negatively associated with maternal smoking since there are more negatively associated a-dmss.
in the age 4– <dig> group, there are similar numbers of negatively- and positively associated sites
dna methylation changes related to disease development
early children development is considered to be the most important phase in life and also of great vulnerability to negative influences.
we found that many top associated a-dmss, and their annotated gene were linked to diseases, such as shank <dig>  which is related to autism, and csrp <dig>  which is linked to cardiomyopathy.
we hypothesised that these a-dmss annotated genes are crucial for the growth, and dysregulation of gene expression and eventually lead to disease development.
therefore, we further investigated if genes containing a-dmss were more likely to be associated with disease by studying the enrichment of genes on our list  in the catalogue of disease-associated genes provided by the clinical genomic database  <cit> .
indeed, we found that genes containing a-dmss were more likely to be disease-related  .
this might indicate a link between age-related methylation changes and disease.
we then investigated the associations of the a-dmss with asthma, the most common chronic pulmonary disease in childhood, and the focus of our medall study  <cit> .
since early-life exposures strongly influence asthma development, and may affect dna methylation  <cit> , we hypothesized that dynamic methylation changes occurring specifically in early childhood might be associated with asthma.
we tested the association of our  <dig>  a-dmss with asthma at age 4/ <dig> and 8 years using the medall asthma definition  <cit> .
three cpg sites were significantly associated  with asthma at age 4: cg <dig> in slc10a <dig>  member 2), cg <dig> in c10orf <dig>  and cg <dig> in znf <dig> .
we also found one significant  cpg site, cg <dig> in c11orf <dig> , to be associated with asthma at age  <dig> .
discussion
this study offers insight into the dynamic epigenetic landscape of the first 8 years of life.
we report a large number of dna methylation changes that are consistent between both our age range comparisons .
these changes with increasing age  have distinct, only partly overlapping, genetic localizations and functional annotations.
the enrichment of a-dmss in gene bodies and in enhancer regions suggest that longitudinal changes in dna methylation are not only acting as transcription repressors, but may also act as buffers to stabilize decisions by transcription factors, ensuring precise and robust transcription  <cit> .
importantly, we also demonstrate that ia-dmss occur preferentially at developmental genes  cluster and are located at bivalent chromatin domains.
these chromatin domains appear to be able to repress or activate epigenetic modifications, and are thought to be important during imprinting and development.
we focused on consistent methylation changes that are present between ages  <dig> to  <dig> as well as  <dig> to 8 years, and suggest that further analyses using even shorter time periods  may reveal further insight into the epigenetic association with specific developmental periods of a child.
for the first time, we have linked genome-wide genetic variants with longitudinal methylation changes by performing cis-dmeqtl analysis.
we find that  <dig> %  of the differentially methylated cpg sites are regulated by genetic variation, which is compatible with genetic control over epigenetic plasticity.
in most cases, meqtl were more pronounced at age  <dig> than at age  <dig>  indicating stronger genetic regulation later in childhood, and possibly linking genetic regulation of methylation with ageing.
zooming in on one dmeqtl, rs9320331-cg <dig> located on chromosome 6q <dig> close to ddo gene , we observed relationships between this snp, gene expression and methylation by combining our data with public data .
methylation of cg <dig> was reported to be negatively correlated with ddo expression in  <dig>  monocyte samples  <cit> , while snp rs <dig> has been detected as an eqtl of ddo mrna in whole blood <dig> and other tissues  <cit> .
the expression of the ddo enzyme has been reported to control the rate of brain-ageing processes by decreasing d-aspartate levels  <cit> .
therefore, a plausible mechanism for the observed meqtl is that snps regulate the expression of ddo through dna methylation to ultimately control the rate the brain ages.
in our data, we observed a significantly lower methylation of cg <dig> from age  <dig> to  <dig>  contributing to higher ddo expression and lower d-aspartate levels, and corroborating earlier findings of lower d-aspartate levels with ageing  <cit> .
moreover, we did not observe significant cis-meqtl of cg <dig> in adult data by checking bios qtl browser.
it may indicate that epigenetic mediation of genetic risk  <cit>  for ddo expression is age specific and only plays a role in childhood.
to assess the role of an important environmental factor, we examined longitudinal changes in dna methylation associated to maternal smoking during pregnancy and found association with decreasing methylation levels from birth up to age 4/ <dig>  since it has been suggested that decreasing methylation is associated with ageing  <cit> , our findings may indicate that maternal smoking during pregnancy has an immediate effect on ageing by decreasing methylation levels.
however, we did not find this “decreasing methylation” effect in our 4– <dig> year-old set.
this shift may indicate that the in utero effect of maternal smoking is most crucial during the first years of life.
we further tested the effect of smoking on horvath’s “epigenetic clock”  <cit> , which predicts age based on dna methylation data and statistical learning, but found no significant effect .
this may be because the “epigenetic clock” was trained on adult samples, making it less accurate for age predictions based on dna methylation in children .
the dohad  hypothesis proposes that early human development affects the risk of chronic, non-communicable diseases in later life  <cit> .
early life development may be expressed in epigenetic mechanisms, such as dna methylation, and this may have long-lasting effects over an individual’s life.
we reasoned that the set of a-dmss that change in the first 8 years of life could be used to test for enrichment with disease genes.
one interesting observation that came out of this analysis is that childhood a-dms-genes were more likely to be disease-linked.
we therefore tested the association of identified  <dig> a-dmss with asthma, which often starts in early life and is the most prevalent chronic disease in childhood.
we identified four a-dmss associated with asthma at ages  <dig> and  <dig>  a finding that warrants replication.
we propose that future studies could assess the association of a-dmss with other chronic, non-communicable diseases originating in early childhood.
a major strength of our study is the combination of four different european birth cohorts of general population samples.
although children from more highly educated parents may be overrepresented in our study  <cit> , our study represents a reasonable representation of european populations, forming a large dataset with good statistical power.
furthermore, the paired sample approach enables conclusions on longitudinal changes, and we specifically focused on childhood ageing to complement the focus on adult ageing populations in the literature.
however, our study had some limitations: the hm <dig> platform only covers  <dig> % of all methylation sites  <cit> , and the design of hm <dig> may be biased towards cpg-richer regions of the genome.
dna was derived from different blood cell types: cord blood at age  <dig> versus peripheral blood at older ages; this could be a potential confounder.
we therefore applied a cell-type correction  <cit>  and only used findings consistent over the two comparison sets to support our conclusions.
CONCLUSIONS
in this research, we observed dynamic dna methylation changes in the first 8 years of life, with increasing or decreasing a-dmss having specific functional annotations and genomic localizations.
we describe that a subset of these a-dmss are under genetic control, and report how maternal smoking affects dna methylation changes in early life.
finally, we provide evidence that the set of genes annotated to a-dmss is enriched for disease development, and specifically show  <dig> cpg sites to be associated with asthma in the first 8 years of life.

 dna methylation is the most extensively studied epigenetic mechanism. an individual’s dna methylation profile is not static but subject to dynamic changes induced by genetic, environmental and stochastic factors during ageing. although some age-related epigenetic changes have been identified, how genetic and environmental factors influence methylation-change with ageing is not yet well understood. to answer these questions, we performed an epigenome-wide longitudinal dna methylation study of children across four european population-based birth cohorts participating in the medall consortium epigenetic study using the illumina infinium humanmethylation beadchip array. we compared children at ages and 4/5 years, by investigating paired samples of cord blood and peripheral blood dna, and another set of children at ages and 8 years. we first identified a set of overlapping and consistent age-differential methylation sites in each group and looked at their functions. then we linked genetic variation and maternal smoking during pregnancy with the changes in these a-dmss over time to see how genetic and environmental factors regulate dynamic changes in methylation. finally, we investigated if genes annotated to these a-dmss were enriched for disease-associated genes; we linked these a-dmss to asthma, the chronic disease with the highest prevalence in childhood.  identification of a-dmss we first applied differential methylation analysis on cytosine-phosphate-guanine sites that passed our quality control in paired samples from the inma and eden birth cohorts and in paired samples from the bamse and piama birth cohorts,. the overall correlations of methylation levels between two time points in the four birth cohorts are shown in additional file 2: figure s in the pooled analysis of all cohorts adjusting for cell-type-composition, significant a-dmss were identified in both age groups surpassing the 5% bonferroni corrected threshold. of these a-dmss, showed a consistent decrease in methylation with age, whereas showed a consistent increase in methylation with age. only of the cpg sites showed opposite directions in their methylation changes between the younger and older comparison sets. the complete list of consistent da-dmss and ia-dmss, and their annotation, is presented in additional file comparing our a-dms results with those from two independent, paediatric, age-related methylation studies, we see more than 50% overlap in the cpg sites identified. we observed that some of our consistent da-dmss were located in clusters, probably reflecting age associated differential methylated regions rather than one site.fig. study design and structure of this paper. we collected cord blood and peripheral blood samples from children from four european birth cohorts: inma, eden, bamse and piama. differential methylation analysis on different age groups resulted in consistent age-differential methylation sites. we then linked the methylation change of these a-dmss to genetics by cis-dmeqtl, to environmental exposure from maternal smoking, and to diseases, specifically asthma the most significant ia-dmss, cg and cg are located on the chromosome 4q, near the gene lrat. mutations in this gene have been associated with early onset retinal dystrophy. the top associated da-dmss, cg and cg on chromosome 11q region were annotated in shank mutations in this shank synaptic scaffolding gene have been associated with neurodevelopmental disorders and autism. characterization of da-dmss and ia-dmss we next examined the enrichment of the da-dmss and ia-dmss in genomic regions and their predicted roles in regulating gene expression using histone modifications and chromatin state data from the roadmap epigenomics project. ia-dmss were situated in distinctly different functional domains compared to da-dmss, which is consistent with previous findings. thus, compared to all the cpg sites we tested, ia-dmss were enriched in cpg shores and gene bodies, while da-dmss were enriched in open seas, shelves, shores, intergenic regions and transcription start site regions. ia-dmss were also enriched in transcriptional repression histone modification marker, while da-dmss were depleted for this marker. both da-dmss and ia-dmss were enriched in enhancers and bivalent enhancers. ia-dmss were also enriched in bivalent tss regions, which are thought to silence or activate expression of developmental genes. in contrast, da-dmss were enriched in h3k27ac, which separates active from poised enhancers and predict developmental state.fig. molecular enrichment of a-dmss for regulatory features. fold enrichment of da-dmss and ia-dmss. a cpg islands, shore, shelf and open sea. b gene regions including: within  kb upstream of the transcription start site, the 5' untranslated region, the first gene exon, the gene body, the 3' utr or intergenic region. c predicted gene expression regulatory regions based on histone modifications derived from roadmap epigenomics blood data. d predicted chromatin state based on roadmap epigenomics blood data. dnase: dnase i hypersensitive site, enh: enhancers, enhbiv: bivalent enhancer, tssa: active tss, tssbiv: bivalent/poised tss, tx: strong transcription, txwk: weak transcription. the presented fold enrichments are from samples by χ test and are relative to all cpg sites tested; * 1 × 10−3 ≤ p < , ** 1 × 10−6 ≤ p < 1 × 10- *** p < 1 × 10−6 we next performed pathway analysis by genenetwork on genes located in close proximity to dmss probes. in total, genes were annotated to be located near ia-dmss probes and genes near the da-dmss probes. of these genes were overlapping across the two groups, suggesting complex regulation of these genes during development. the pathway analysis of da-dmss specific genes using gene co-expression indicated enrichment of reactome pathways involved in cell signalling, including signalling by nerve growth factors, platelet derived growth factors, neurotrophin signalling and many others. the corresponding mouse genome informatics phenotypes pointed to cell migration, growth retardation and various abnormal tissue morphologies. the ia-dms specific genes were enriched for developmental genes, axon guidance, regulation of insulin secretion and energy metabolism. the enriched mgi phenotypes were related to neonatal and postnatal lethality. this indicates that in early age methylation regulates the activity of genes, involved in basic processes of development and metabolism. next, we inspected methylation dynamics of some well-known developmental genes and found a lot of cpgs sites which were annotated to them, such as growth differentiation factors, bone morphogeneic proteins, wnt signalling pathway gene, paired box genes and homeotic genes gene etc. among them, the 5′ hoxd cluster that contains genes important for limb development. the 5′ hoxd cluster consists of five genes. we found eight ia-dmss with low methylation status within the cluster, seven of which were localized in cpg islands encompassing the five promoters and three first exons. in contrast, meis homeobox, a limb development regulator that forms complexes with both hoxd and hoxd, displays a different methylation pattern. cg lies in the gene body of meis and its methylation level decreases during ageing. genetic variation associates with longitudinal dna methylation changes the availability of paired samples from four different cohorts gave us the possibility to investigate the effect of genetics on methylation dynamics in a longitudinal way. we examined whether genetic variations were associated with longitudinal dna methylation changes in childhood using cis-differential methylation quantitative trait locus mapping. we associated snps within ±250 kb of our a-dmss in gwas data available for children from bamse and piama in the 4– years-old comparison set for discovery and from children in the inma 0–4/5 years-old comparison set for replication. we discovered snp-cpg pairs on admss with significant cis-dmeqtls in the older comparison set, and snp-cpg pairs were replicated in the younger comparison set. potentially false-positive dmeqtls were removed due to snp-under-the-probe effects. the remaining snp-cpg pairs are listed in additional file consisting of snps regulating a-dmss that changed with age and showed methylation change was partly under genetic control. to understand these dmeqtls, we tested if these snp-cpg pairs were meqtls at age and 8 years cross-sectionally. most snp-cpg pairs showing dmeqtl were significant meqtls when analysed cross-sectionally at both ages. the most significant meqtl rs7522439-cg which are on chromosome 1p, close to gene padi. by checking the bios qtl browser, we also found the significant cis-meqtl in methylation data from adult blood dna samples, which is line with the recent findings that genetic effects on methylation levels are stable. however, we also found three cpg sites cg cg and cg which were annotated to the ddo gene showing significant cis-meqtl, but did not show significant cis-meqtl in the bios qtl database. this may indicate an age specific meqtl effect since d-aspartate content in brain is decreased during adulthood. furthermore, we compared the meqtl-effect size at ages and, and found that almost all effects of genetic factors on methylation had the same direction at both ages, i.e., genetic effects on methylation remained stable with ageing. among these snp-cpg pairs, had a significantly larger effect and had a significantly smaller effect at age 4 years than at age. an example of this pattern is shown for the rs9320331_cg snp-cpg pair in the ddo gene, which showed a meqtl at both ages and, with a stronger effect at age compared to the effect at age.fig. genetic variation associated with longitudinal dna methylation changes by cis-dmeqtl study. a the effect sizes of meqtl at age 4 years against those at age 8 years of snp-cpg pairs in the age 4- group. almost all the dots lie in the first and third quadrants, suggesting that the effect size of genetic factors on methylation from most of the snp-cpg pairs followed the same direction at both ages. blue dots represent snp-cpg pairs with a significantly weaker effect size at age than at age whereas green dots represent a stronger effect, and red dots represent a different direction of effect. b bar plot of snp-cpg pairs. there are snp-cpg pairs showing a weaker effect at age than age, snp-cpg pairs showing stronger effect and snp-cpg pairs showing a different effect. c boxplot of dmeqtl of rs9320331-cg piama samples have been used to illustrate this dmeqtl. d regional dmeqtl association of cg results for the age 4– group. plots were generated using locuszoom. the ld estimates are color-coded as a heatmap from dark blue to red regional plot shows-log10 p of all snps surrounding snp rs and the degree of linkage disequilibrium between all snps and lead snp rs snps with lower p-values span ddo and slc22a genes within a recombination boundary. e boxplot of meqtl of rs9320331-cg at age and age cross-sectionally maternal smoking during pregnancy associates with longitudinal dna methylation changes methylation has been found strongly associated with maternal smoking during pregnancy. here we further investigated whether methylation changes of the a-dmss between the 0–4/ and 4– years-old comparison sets were associated with maternal smoking during pregnancy. in total, we found eight significant a-dmss in the younger set and one in the older set associated with maternal smoking. these nine cpg sites mapped to five genes, itga or5b vwf, rcbtb and creb and one pseudogene hnrnpa3p the most significant association was for the methylation change of cg at the vwf gene, a protein-coding gene crucial for haemostasis. both vwf and itga are involved in regulating cell-substrate adhesion. interestingly, all nine significant cpg sites belonged to the da-dms group, and the association of maternal smoking with methylation changes was always negative. this means that the methylation decrease was consistently larger in the children exposed to smoke in utero compared to the non-exposed. fig. 4a and additional file 2: figure s show that maternal smoking during pregnancy was associated with a stronger, decreasing effect on methylation change of cg in those children. to underpin this, we further checked the directions of effect of all nominally significant a-dmss and found that methylation changes tended to be negatively associated with maternal smoking between and 4/5 years and, moreover, that 721/ a-dmss showed a stronger decrease in methylation levels from to 4/5 years. in summary, we found that maternal smoking exposure tended to more strongly decrease methylation levels at the identified da-dmss.fig. the effect of maternal smoking on methylation change. a the effect of maternal smoking on methylation change of cg in the age 0–4/ group. maternal smoking shows an enhanced effect on decreasing methylation levels since the decrease from age to age 4/5 years old of cg in the exposed group is significantly larger compared to the non-exposed group. b density plot of effect size of all nominally significant a-dmss with maternal smoking. in the age 0–4/ group, methylation changes tend to be negatively associated with maternal smoking since there are more negatively associated a-dmss. in the age 4– group, there are similar numbers of negatively- and positively associated sites dna methylation changes related to disease development early children development is considered to be the most important phase in life and also of great vulnerability to negative influences. we found that many top associated a-dmss, and their annotated gene were linked to diseases, such as shank which is related to autism, and csrp which is linked to cardiomyopathy. we hypothesised that these a-dmss annotated genes are crucial for the growth, and dysregulation of gene expression and eventually lead to disease development. therefore, we further investigated if genes containing a-dmss were more likely to be associated with disease by studying the enrichment of genes on our list in the catalogue of disease-associated genes provided by the clinical genomic database. indeed, we found that genes containing a-dmss were more likely to be disease-related. this might indicate a link between age-related methylation changes and disease. we then investigated the associations of the a-dmss with asthma, the most common chronic pulmonary disease in childhood, and the focus of our medall study. since early-life exposures strongly influence asthma development, and may affect dna methylation, we hypothesized that dynamic methylation changes occurring specifically in early childhood might be associated with asthma. we tested the association of our a-dmss with asthma at age 4/ and 8 years using the medall asthma definition. three cpg sites were significantly associated with asthma at age 4: cg in slc10a member 2), cg in c10orf and cg in znf. we also found one significant cpg site, cg in c11orf, to be associated with asthma at age. discussion this study offers insight into the dynamic epigenetic landscape of the first 8 years of life. we report a large number of dna methylation changes that are consistent between both our age range comparisons. these changes with increasing age have distinct, only partly overlapping, genetic localizations and functional annotations. the enrichment of a-dmss in gene bodies and in enhancer regions suggest that longitudinal changes in dna methylation are not only acting as transcription repressors, but may also act as buffers to stabilize decisions by transcription factors, ensuring precise and robust transcription. importantly, we also demonstrate that ia-dmss occur preferentially at developmental genes cluster and are located at bivalent chromatin domains. these chromatin domains appear to be able to repress or activate epigenetic modifications, and are thought to be important during imprinting and development. we focused on consistent methylation changes that are present between ages to as well as to 8 years, and suggest that further analyses using even shorter time periods may reveal further insight into the epigenetic association with specific developmental periods of a child. for the first time, we have linked genome-wide genetic variants with longitudinal methylation changes by performing cis-dmeqtl analysis. we find that % of the differentially methylated cpg sites are regulated by genetic variation, which is compatible with genetic control over epigenetic plasticity. in most cases, meqtl were more pronounced at age than at age indicating stronger genetic regulation later in childhood, and possibly linking genetic regulation of methylation with ageing. zooming in on one dmeqtl, rs9320331-cg located on chromosome 6q close to ddo gene, we observed relationships between this snp, gene expression and methylation by combining our data with public data. methylation of cg was reported to be negatively correlated with ddo expression in monocyte samples, while snp rs has been detected as an eqtl of ddo mrna in whole blood and other tissues. the expression of the ddo enzyme has been reported to control the rate of brain-ageing processes by decreasing d-aspartate levels. therefore, a plausible mechanism for the observed meqtl is that snps regulate the expression of ddo through dna methylation to ultimately control the rate the brain ages. in our data, we observed a significantly lower methylation of cg from age to contributing to higher ddo expression and lower d-aspartate levels, and corroborating earlier findings of lower d-aspartate levels with ageing. moreover, we did not observe significant cis-meqtl of cg in adult data by checking bios qtl browser. it may indicate that epigenetic mediation of genetic risk for ddo expression is age specific and only plays a role in childhood. to assess the role of an important environmental factor, we examined longitudinal changes in dna methylation associated to maternal smoking during pregnancy and found association with decreasing methylation levels from birth up to age 4/ since it has been suggested that decreasing methylation is associated with ageing, our findings may indicate that maternal smoking during pregnancy has an immediate effect on ageing by decreasing methylation levels. however, we did not find this “decreasing methylation” effect in our 4– year-old set. this shift may indicate that the in utero effect of maternal smoking is most crucial during the first years of life. we further tested the effect of smoking on horvath’s “epigenetic clock”, which predicts age based on dna methylation data and statistical learning, but found no significant effect. this may be because the “epigenetic clock” was trained on adult samples, making it less accurate for age predictions based on dna methylation in children. the dohad hypothesis proposes that early human development affects the risk of chronic, non-communicable diseases in later life. early life development may be expressed in epigenetic mechanisms, such as dna methylation, and this may have long-lasting effects over an individual’s life. we reasoned that the set of a-dmss that change in the first 8 years of life could be used to test for enrichment with disease genes. one interesting observation that came out of this analysis is that childhood a-dms-genes were more likely to be disease-linked. we therefore tested the association of identified a-dmss with asthma, which often starts in early life and is the most prevalent chronic disease in childhood. we identified four a-dmss associated with asthma at ages and a finding that warrants replication. we propose that future studies could assess the association of a-dmss with other chronic, non-communicable diseases originating in early childhood. a major strength of our study is the combination of four different european birth cohorts of general population samples. although children from more highly educated parents may be overrepresented in our study, our study represents a reasonable representation of european populations, forming a large dataset with good statistical power. furthermore, the paired sample approach enables conclusions on longitudinal changes, and we specifically focused on childhood ageing to complement the focus on adult ageing populations in the literature. however, our study had some limitations: the hm platform only covers % of all methylation sites, and the design of hm may be biased towards cpg-richer regions of the genome. dna was derived from different blood cell types: cord blood at age versus peripheral blood at older ages; this could be a potential confounder. we therefore applied a cell-type correction and only used findings consistent over the two comparison sets to support our conclusions.  in this research, we observed dynamic dna methylation changes in the first 8 years of life, with increasing or decreasing a-dmss having specific functional annotations and genomic localizations. we describe that a subset of these a-dmss are under genetic control, and report how maternal smoking affects dna methylation changes in early life. finally, we provide evidence that the set of genes annotated to a-dmss is enriched for disease development, and specifically show cpg sites to be associated with asthma in the first 8 years of life. 
maternal smoking tended to decrease methylation levels at the identified da-dmss. by analysing paired dna blood samples from children at birth and at 4– years old and paired samples from children at and 8 years old from four european birth cohorts using the illumina infinium human methylation 450 k chip, we have identified consistent age-differential methylation sites at epigenome-wide significance of p <   × 10− genes with an increase in age-differential methylation were enriched in pathways related to ‘development’, and were more often located in bivalent transcription start site regions, which can silence or activate expression of developmental genes. dna methylation has been found to associate with disease, aging and environmental exposure, but it is unknown how genome, environment and disease influence dna methylation dynamics in childhood. dna methylationagingmethylation quantitative trait locimaternal smokingissue-copyright-statement© the author 2017 we also found a-dmss that were regulated by genetic variants using cis-differential methylation quantitative trait locus mapping. our study provides new insights into the dynamic epigenetic landscape of the first 8 years of life.
 dna methylation has been found to associate with disease, aging and environmental exposure, but it is unknown how genome, environment and disease influence dna methylation dynamics in childhood.  by analysing paired dna blood samples from children at birth and at 4– years old and paired samples from children at and 8 years old from four european birth cohorts using the illumina infinium human methylation 450 k chip, we have identified consistent age-differential methylation sites at epigenome-wide significance of p <   × 10− genes with an increase in age-differential methylation were enriched in pathways related to ‘development’, and were more often located in bivalent transcription start site regions, which can silence or activate expression of developmental genes. genes with a decrease in age-differential methylation were involved in cell signalling, and enriched on h3k27ac, which can predict developmental state. maternal smoking tended to decrease methylation levels at the identified da-dmss. we also found a-dmss that were regulated by genetic variants using cis-differential methylation quantitative trait locus mapping. moreover, a-dms-associated genes during early development were significantly more likely to be linked with disease.  our study provides new insights into the dynamic epigenetic landscape of the first 8 years of life. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords dna methylationagingmethylation quantitative trait locimaternal smokingissue-copyright-statement© the author 2017 