we studied two prospective cohorts recruited antenatally in southampton , u.k . in the pah study ,
caucasian women 16 years old with singleton pregnancies < 17 weeks gestation were recruited and a validated food frequency questionnaire ( 23 ) was administered at 15 weeks gestation ; patients with diabetes and hormonally induced conceptions were excluded .
two hundred and sixteen ( 47% ) attended a clinic , and adiposity was measured using dual energy x - ray absorptiometry ( dexa ) ; 78 had dna available from an umbilical cord sample collected at birth and stored at 80c .
women aged 2034 years were recruited when not pregnant ; those who subsequently conceived were followed through pregnancy and the offspring followed up ( 22 ) . to replicate associations with adiposity found in the pah cohort
, we studied 239 children selected as having both umbilical cord dna available and childhood adiposity measurements .
both groups underwent adiposity measurement by dexa ( pah study , age 9 years : lunar dpx - l , pediatric software , version 4.7c ; ge corporation , madison , wi ; sws , age 6 years : hologic discovery , pediatric scan mode ; hologic , bedford , ma ) .
follow - up of the children and sample collection / analysis was carried out under institutional review board approval ( southampton and sw hampshire research ethics committee ) with written informed consent .
dna from 15 randomly selected pah study umbilical cords underwent methylation - specific chromatin precipitation followed by hybridization to a commercial tiled oligomer microarray ( nimblegen systems hg17_min_promoter array ) , which uses 50-mer oligonucleotides positioned on average every 100 bp from 3,750 bp to + 500 bp relative to the transcription start site of 24,134 human genes .
we focused our analysis initially on a panel of 78 candidate genes , 28 selected on the basis of animal data from our and other laboratories ( 25,26 ) and 50 selected from the array as having the highest sd between subjects relative to the sd of within - subject replicates . for this panel
we identified those with correlations between overall gene methylation status and dexa measurements of adiposity at age 9 years and from these genes chose five genes for further study based on individual oligomers showing evidence of correlation with dexa measurements at age 9 years , biological plausibility and feasibility of designing amplicons suitable for sequenom analysis ; genes selected were retinoid x receptor- ( rxra ) , endothelial nitric oxide synthase ( enos ) , superoxide dismutase-1 ( sod1 ) , interleukin-8 ( il8 ) , and phosphoinositide-3-kinase , catalytic , -polypeptide ( pi3kcd ) .
these genes all had associations of comparable strength between childhood body composition and both overall gene promoter methylation and the methylation of individual oligomers .
all chromosomal coordinates are based on university of california santa cruz , human genome march 2006 assembly ( hg18 ) .
genomic dna was purified from frozen cords by proteinase k digestion and phenol : chloroform extraction .
briefly , gene - specific amplification of bisulfite - treated dna was followed by in vitro transcription and analysis by matrix - assisted laser desorption ionization time - of - flight ( maldi - tof ) mass spectrometry ( 27,28 ) .
dna ( 1 g ) was bisulfite converted using ez dna methylation kit ( zymo research ) .
each reverse primer contained a t7-promoter tag for in vitro transcription ( 5-cagtaatacgactcactatagggagaaggct-3 ) , and the forward primer was tagged with a 10mer to balance tm ( 5-aggaagagag-3 ) .
supplementary table 1 lists amplicons , primer sequences , pcr annealing temperature , and genomic coordinates for the extended promoter regions measured .
bisulfite - treated dna was pcr amplified ( qiagen hotstar taq polymerase ) in 5 l reactions and treated with shrimp alkaline phosphatase ( sequenom ) ( 20 min at 37c ) , heat inactivated ( 85c for 5 min ) , and simultaneous in vitro transcription / uracil - cleavage reaction was carried out ( 7 l reactions ) using sequenom t - cleavage reagent mix .
transcription cleavage products were desalted with 6 mg of clean - resin and 20 nl spotted on a 384-pad spectrochip ( sequenom ) using a massarray nanodispenser ( samsung ) .
mass spectra were acquired using a massarray maldi - tof ms ( bruker - sequenom ) and peak detection , signal - to - noise calculations , and quantitative cpg site methylation performed using proprietary epityper software v1.0 ( sequenom ) .
consistent with the manufacturer s specification ( www.sequenom.com ) agreement between technical replicates was good , duplicate measures differing by < 10% methylation in almost all cases .
we excluded from analysis samples that failed to give a reliable pcr product or produced spectra with low confidence scores ( < 2.9 in epityper ) .
for fragments containing a single cpg site , dna methylation was calculated by the ratio of methylated to unmethylated fragments .
limitations imposed by sequenom analysis treat cleavage products containing multiple cpg sites as single units , and the methylation values reported are weighted averages across the unit ( referred to as a cpg group ) .
dna quality and no - template controls , 0% , and 100% methylated dna were included in all assays .
supplementary table 2 shows the distributions of methylation values for cpgs and cpg groups and numbers of subjects with measurements available .
pyrosequencing analysis ( pyromark q96md ; qiagen ) was undertaken to exclude single nucleotide polymorphisms ( snps ) involving cpg dinucleotides , which would result in loss of potential for methylation .
briefly , dna was pcr amplified , and the product annealed to streptavidin - coated sepharose beads and denatured ( 0.2 m naoh ) to a single - stranded product .
nucleotides were incorporated to the open 3 dna strand in which pyrophosphate is released and used in a sulfurylase reaction emitting atp , measured in a luciferase reaction , and analyzed using pyromark md v1.0 software ( qiagen ) ( 29 ) .
using stata 11 ( statacorp ) study variables were transformed using fisher - yates ( 30 ) ( for methylation measurements ) or logarithmic transformations where necessary to satisfy statistical assumptions of normality .
a priori we restricted the analysis to cpgs with median methylation 5% and a 595% range 10% . in pah subjects we first used pearson correlation ( rp ) and linear regression to examine cpg methylation in relation to child s adiposity .
mutually adjusted regression models were built including cpg sites significantly associated with each outcome in univariate analyses ( p < 0.05 ) . because the measurements of childhood adiposity are on the log scale and the cpg measurements are z scores , the exponentiated results are interpreted as percent change in childhood adiposity measurements per sd change in methylation . to allow assessment of the influence of taking account of the child s sex , exponentiated regression coefficients are first presented excluding and including adjustment for child 's sex .
we then preadjusted all adiposity measurements for sex and took account of the mother s age , adiposity ( as continuous variables ) , and smoking during pregnancy ( as a binary variable , yes / no ) by including them as covariates in the regression model ; we next related mother s diet to cpg methylation at birth .
finally , we sought replication of the associations between cpg methylation and child s adiposity in the sws cohort .
sws measurements , taken over a wider age range than those in pah subjects , were preadjusted for age and sex .
dna from 15 randomly selected pah study umbilical cords underwent methylation - specific chromatin precipitation followed by hybridization to a commercial tiled oligomer microarray ( nimblegen systems hg17_min_promoter array ) , which uses 50-mer oligonucleotides positioned on average every 100 bp from 3,750 bp to + 500 bp relative to the transcription start site of 24,134 human genes .
we focused our analysis initially on a panel of 78 candidate genes , 28 selected on the basis of animal data from our and other laboratories ( 25,26 ) and 50 selected from the array as having the highest sd between subjects relative to the sd of within - subject replicates . for this panel
we identified those with correlations between overall gene methylation status and dexa measurements of adiposity at age 9 years and from these genes chose five genes for further study based on individual oligomers showing evidence of correlation with dexa measurements at age 9 years , biological plausibility and feasibility of designing amplicons suitable for sequenom analysis ; genes selected were retinoid x receptor- ( rxra ) , endothelial nitric oxide synthase ( enos ) , superoxide dismutase-1 ( sod1 ) , interleukin-8 ( il8 ) , and phosphoinositide-3-kinase , catalytic , -polypeptide ( pi3kcd ) .
these genes all had associations of comparable strength between childhood body composition and both overall gene promoter methylation and the methylation of individual oligomers .
all chromosomal coordinates are based on university of california santa cruz , human genome march 2006 assembly ( hg18 ) .
genomic dna was purified from frozen cords by proteinase k digestion and phenol : chloroform extraction .
briefly , gene - specific amplification of bisulfite - treated dna was followed by in vitro transcription and analysis by matrix - assisted laser desorption ionization time - of - flight ( maldi - tof ) mass spectrometry ( 27,28 ) .
dna ( 1 g ) was bisulfite converted using ez dna methylation kit ( zymo research ) .
each reverse primer contained a t7-promoter tag for in vitro transcription ( 5-cagtaatacgactcactatagggagaaggct-3 ) , and the forward primer was tagged with a 10mer to balance tm ( 5-aggaagagag-3 ) .
supplementary table 1 lists amplicons , primer sequences , pcr annealing temperature , and genomic coordinates for the extended promoter regions measured .
bisulfite - treated dna was pcr amplified ( qiagen hotstar taq polymerase ) in 5 l reactions and treated with shrimp alkaline phosphatase ( sequenom ) ( 20 min at 37c ) , heat inactivated ( 85c for 5 min ) , and simultaneous in vitro transcription / uracil - cleavage reaction was carried out ( 7 l reactions ) using sequenom t - cleavage reagent mix .
transcription cleavage products were desalted with 6 mg of clean - resin and 20 nl spotted on a 384-pad spectrochip ( sequenom ) using a massarray nanodispenser ( samsung ) .
mass spectra were acquired using a massarray maldi - tof ms ( bruker - sequenom ) and peak detection , signal - to - noise calculations , and quantitative cpg site methylation performed using proprietary epityper software v1.0 ( sequenom ) .
consistent with the manufacturer s specification ( www.sequenom.com ) agreement between technical replicates was good , duplicate measures differing by < 10% methylation in almost all cases .
we excluded from analysis samples that failed to give a reliable pcr product or produced spectra with low confidence scores ( < 2.9 in epityper ) .
for fragments containing a single cpg site , dna methylation was calculated by the ratio of methylated to unmethylated fragments .
limitations imposed by sequenom analysis treat cleavage products containing multiple cpg sites as single units , and the methylation values reported are weighted averages across the unit ( referred to as a cpg group ) .
dna quality and no - template controls , 0% , and 100% methylated dna were included in all assays .
supplementary table 2 shows the distributions of methylation values for cpgs and cpg groups and numbers of subjects with measurements available .
pyrosequencing analysis ( pyromark q96md ; qiagen ) was undertaken to exclude single nucleotide polymorphisms ( snps ) involving cpg dinucleotides , which would result in loss of potential for methylation .
briefly , dna was pcr amplified , and the product annealed to streptavidin - coated sepharose beads and denatured ( 0.2 m naoh ) to a single - stranded product .
nucleotides were incorporated to the open 3 dna strand in which pyrophosphate is released and used in a sulfurylase reaction emitting atp , measured in a luciferase reaction , and analyzed using pyromark md v1.0 software ( qiagen ) ( 29 ) .
using stata 11 ( statacorp ) study variables were transformed using fisher - yates ( 30 ) ( for methylation measurements ) or logarithmic transformations where necessary to satisfy statistical assumptions of normality .
a priori we restricted the analysis to cpgs with median methylation 5% and a 595% range 10% . in pah subjects we first used pearson correlation ( rp ) and linear regression to examine cpg methylation in relation to child s adiposity .
mutually adjusted regression models were built including cpg sites significantly associated with each outcome in univariate analyses ( p < 0.05 ) . because the measurements of childhood adiposity are on the log scale and the cpg measurements are z scores , the exponentiated results are interpreted as percent change in childhood adiposity measurements per sd change in methylation . to allow assessment of the influence of taking account of the child s sex , exponentiated regression coefficients are first presented excluding and including adjustment for child 's sex .
we then preadjusted all adiposity measurements for sex and took account of the mother s age , adiposity ( as continuous variables ) , and smoking during pregnancy ( as a binary variable , yes / no ) by including them as covariates in the regression model ; we next related mother s diet to cpg methylation at birth . finally , we sought replication of the associations between cpg methylation and child s adiposity in the sws cohort .
sws measurements , taken over a wider age range than those in pah subjects , were preadjusted for age and sex .
median ( interquartile range ) birth weight , age , fat mass , and % body fat at follow - up values for the pah subjects were 3,330 g ( 3,0103,790 ) , 8.66 years ( 8.528.73 ) , 5.41 kg ( 3.978.41 ) , and 18.3% ( 14.427.0 ) , respectively .
comparable values for the 239 sws children were 3,485 g ( 3,2053,773 ) , 6.59 years ( 6.416.78 ) , 4.60 kg ( 3.665.69 ) , and 23.4% ( 19.727.6 ) , respectively .
similar percentages of mothers smoked in the pah and sws cohorts ( 21 vs. 24% , respectively ) ; median maternal age and prepregnancy bmi were lower in the pah mothers ( 28 vs. 31 years and 22.3 vs. 24.3 kg / m , respectively ) .
the above characteristics are similar to the overall values for participants in the two cohorts .
supplementary table 2 shows the distribution of percent methylation of the 68 cpgs and cpg groups among the 78 pah subjects ; 31 cpgs / cpg groups met a priori criteria for further analysis .
particularly marked interindividual variation in percent methylation was seen at some sites ( for example , rxra chr9:136355885 + had a median of 59% with 595th centiles 499% ; enos
correlations between methylation of different cpgs were generally low ( for example rp= 0.03 , p = 0.81 , n = 55 between the above two cpgs ) .
of the 31 cpgs with variable methylation above the a priori threshold , seven had significant associations with the child s adiposity at age 9 years ( supplementary table 3 ) .
table 1 shows the results of uni- and multivariate analyses of cpg sites with significant independent associations with the child s adiposity .
comparison of the regression coefficients for the univariate and multivariate associations of rxra chr9:136355885 + and enos chr7:150315553 + methylation with child s fat mass , % fat , and ratio of trunk to limb fat shows that mutual adjustment for each other and sex has little effect on the coefficients ; adiposity measurements were therefore preadjusted for sex in further analyses .
rxra chr9:136355885 + methylation had positive associations with childhood fat mass , % fat mass ( scatterplots shown in supplementary fig .
1 ) , and ratio of trunk to limb fat , with similar positive associations for enos chr7:150315553 + : exponentiated regression coefficients (  ) ( 95% ci ) per sd change in methylation were 17% ( 431 ) , p = 0.009 ; 10% ( 119 ) , p = 0.023 ; and 6% ( 012 ) , p = 0.039 , respectively ( n = 64 ) .
comparable values for enos chr7:150315553 + methylation were 20% ( 932 ) , p < 0.001 ; 12% ( 420 ) , p = 0.002 ; and 7% ( 213 ) , p = 0.007 , respectively ( n = 66 ) .
methylation of rxra chr9:136355885 + and enos chr7:150315553 + had similar positive associations with child s bmi (  = 3% [ 06 ] , p = 0.037 ; and 4% [ 27 ] , p = 0.001 , respectively ) .
independently of rxra chr9:136355885 + and enos chr7:150315553 + methylation , sod1 chr21:31853660/63 + methylation was also inversely related to child s trunk - to - limb fat ratio ( p = 0.037 ) .
the associations reflect clinically important shifts in adiposity such that the mean fat mass rose from 4.8 kg ( 17.3% body fat ) in the lowest quarter of rxra chr9:136355885 + methylation to 6.6 kg ( 21.3% body fat ) in the highest quarter of the distribution . taking
account of rxra chr9:136355885 + methylation and sex explained 26% of the variance in fat mass in pah children , and simultaneous analyses showed that the association between rxra chr9:136355885 + methylation and child s fat mass was not attenuated after adjustment for birth weight and mother s age , smoking , and bmi .
univariate and multivariate analyses of umbilical cord cpg methylation in relation to child s adiposity measured by dexa at age 9 years values are the percent change in outcome variable per sd change in cpg methylation and p value .
univariate analysis rows show the regression coefficients and p values relating each of enos chr7:150315553 + , rxra chr9:136355885 + , and sex to outcomes individually , whereas multivariate analysis rows show regression coefficients and p values for a combined analysis of enos chr7:150315553 + , rxra chr9:136355885 + , and sex in relation to outcomes , together with the variance explained by the multivariate model . for variances explained , n = 55 . in this population
we have previously linked lower maternal carbohydrate intake in early pregnancy with higher neonatal adiposity ( 21 ) .
relating umbilical cord cpg methylation to maternal carbohydrate intake in early pregnancy , higher methylation of rxra chr9:136355885 + , but not of enos chr7:150315553 + , was associated with a lower maternal carbohydrate intake ( fig .
the mother s early pregnancy fat and protein intakes in early pregnancy were not associated with cord rxra chr9:136355885 + methylation ( p = 0.7 and p = 0.6 , respectively ) , and no additional variance in rxra chr9:136355885 + methylation was explained by simultaneously taking account of maternal fat and protein intakes in early pregnancy or macronutrient intakes in late pregnancy .
relating umbilical cord cpg methylation to the infant s size at birth , only three cpgs showed a significant correlation with birth weight adjusted for sex and gestational age ( pik3cd chr1:9609980 + [ rp= 0.32 , p = 0.013 , n = 58 ] , pik3cd chr1:9635676/79 + [ rp= 0.36 , p = 0.028 , n = 37 ] , and sod1 chr 21:31853827 + [ rp= 0.27 , p = 0.029 , n = 65 ] ) .
rxra chr9:136355885 + methylation showed a weak association with the subjects sex and age - adjusted height ( rp= 0.24 , p = 0.054 ) but was not associated with the infant s weight or ponderal index at birth .
lower maternal carbohydrate in early pregnancy is associated with higher umbilical cord rxra chr9:136355885 + methylation in the pah cohort .
we next sought to replicate the stronger associations found in the first cohort ( enos chr7:150315553 + and rxra chr9:136355885 + cpg methylation and child s adiposity ) in the sws cohort ( 20 ) . in these 239 sws children , enos
chr7:150315553 + showed no association with adiposity , but rxra chr9:136355885 + showed remarkably similar and statistically significant associations ( fat mass ,  = 6% [ 210 ] , p = 0.002 ; % fat mass ,  = 4% [ 17 ] , p = 0.002 , both
as in the pah cohort , these associations were little changed after taking account of birth weight and maternal age , smoking , and bmi .
figures 2 and 3 show the graded associations between cord rxra chr9:136355885 + methylation and child s fat mass and % fat mass in the two cohorts .
child s % fat mass and fat mass at age 9 years increase with higher umbilical cord rxra chr9:136355885 + methylation in the pah cohort .
* fat mass and percentage fat mass are preadjusted for sex . in a second independent cohort , child s % fat mass and fat mass at age 6 years increase with higher umbilical cord rxra chr9:136355885 + methylation in sws subjects .
pyrosequencing across rxra chr9:136355885 + in sws subjects showed no c allele snps that could account for differences in methylation observed between individuals .
supplementary table 2 shows the distribution of percent methylation of the 68 cpgs and cpg groups among the 78 pah subjects ; 31 cpgs / cpg groups met a priori criteria for further analysis .
particularly marked interindividual variation in percent methylation was seen at some sites ( for example , rxra chr9:136355885 + had a median of 59% with 595th centiles 499% ; enos
correlations between methylation of different cpgs were generally low ( for example rp= 0.03 , p = 0.81 , n = 55 between the above two cpgs ) .
of the 31 cpgs with variable methylation above the a priori threshold , seven had significant associations with the child s adiposity at age 9 years ( supplementary table 3 ) . table 1 shows the results of uni- and multivariate analyses of cpg sites with significant independent associations with the child s adiposity .
comparison of the regression coefficients for the univariate and multivariate associations of rxra chr9:136355885 + and enos chr7:150315553 + methylation with child s fat mass , % fat , and ratio of trunk to limb fat shows that mutual adjustment for each other and sex has little effect on the coefficients ; adiposity measurements were therefore preadjusted for sex in further analyses .
rxra chr9:136355885 + methylation had positive associations with childhood fat mass , % fat mass ( scatterplots shown in supplementary fig .
1 ) , and ratio of trunk to limb fat , with similar positive associations for enos chr7:150315553 + : exponentiated regression coefficients (  ) ( 95% ci ) per sd change in methylation were 17% ( 431 ) , p = 0.009 ; 10% ( 119 ) , p = 0.023 ; and 6% ( 012 ) , p = 0.039 , respectively ( n = 64 )
. comparable values for enos chr7:150315553 + methylation were 20% ( 932 ) , p < 0.001 ; 12% ( 420 ) , p = 0.002 ; and 7% ( 213 ) , p = 0.007 , respectively ( n = 66 ) .
methylation of rxra chr9:136355885 + and enos chr7:150315553 + had similar positive associations with child s bmi (  = 3% [ 06 ] , p = 0.037 ; and 4% [ 27 ] , p = 0.001 , respectively ) .
independently of rxra chr9:136355885 + and enos chr7:150315553 + methylation , sod1 chr21:31853660/63 + methylation was also inversely related to child s trunk - to - limb fat ratio ( p = 0.037 ) .
the associations reflect clinically important shifts in adiposity such that the mean fat mass rose from 4.8 kg ( 17.3% body fat ) in the lowest quarter of rxra chr9:136355885 + methylation to 6.6 kg ( 21.3% body fat ) in the highest quarter of the distribution . taking account of rxra chr9:136355885 + methylation and
sex explained 26% of the variance in fat mass in pah children , and simultaneous analyses showed that the association between rxra chr9:136355885 + methylation and child s fat mass was not attenuated after adjustment for birth weight and mother s age , smoking , and bmi .
univariate and multivariate analyses of umbilical cord cpg methylation in relation to child s adiposity measured by dexa at age 9 years values are the percent change in outcome variable per sd change in cpg methylation and p value .
univariate analysis rows show the regression coefficients and p values relating each of enos chr7:150315553 + , rxra chr9:136355885 + , and sex to outcomes individually , whereas multivariate analysis rows show regression coefficients and p values for a combined analysis of enos chr7:150315553 + , rxra chr9:136355885 + , and sex in relation to outcomes , together with the variance explained by the multivariate model . for variances explained , n = 55 . in this population
we have previously linked lower maternal carbohydrate intake in early pregnancy with higher neonatal adiposity ( 21 ) .
relating umbilical cord cpg methylation to maternal carbohydrate intake in early pregnancy , higher methylation of rxra chr9:136355885 + , but not of enos chr7:150315553 + , was associated with a lower maternal carbohydrate intake ( fig .
the mother s early pregnancy fat and protein intakes in early pregnancy were not associated with cord rxra chr9:136355885 + methylation ( p = 0.7 and p = 0.6 , respectively ) , and no additional variance in rxra chr9:136355885 + methylation was explained by simultaneously taking account of maternal fat and protein intakes in early pregnancy or macronutrient intakes in late pregnancy .
relating umbilical cord cpg methylation to the infant s size at birth , only three cpgs showed a significant correlation with birth weight adjusted for sex and gestational age ( pik3cd chr1:9609980 + [ rp= 0.32 , p = 0.013 , n = 58 ] , pik3cd chr1:9635676/79 + [ rp= 0.36 , p = 0.028 , n = 37 ] , and sod1 chr 21:31853827 + [ rp= 0.27 , p = 0.029 , n = 65 ] ) .
rxra chr9:136355885 + methylation showed a weak association with the subjects sex and age - adjusted height ( rp= 0.24 , p = 0.054 ) but was not associated with the infant s weight or ponderal index at birth .
lower maternal carbohydrate in early pregnancy is associated with higher umbilical cord rxra chr9:136355885 + methylation in the pah cohort .
we next sought to replicate the stronger associations found in the first cohort ( enos chr7:150315553 + and rxra chr9:136355885 + cpg methylation and child s adiposity ) in the sws cohort ( 20 ) . in these 239 sws children , enos
chr7:150315553 + showed no association with adiposity , but rxra chr9:136355885 + showed remarkably similar and statistically significant associations ( fat mass ,  = 6% [ 210 ] , p = 0.002 ; % fat mass ,  = 4% [ 17 ] , p = 0.002 , both
as in the pah cohort , these associations were little changed after taking account of birth weight and maternal age , smoking , and bmi .
figures 2 and 3 show the graded associations between cord rxra chr9:136355885 + methylation and child s fat mass and % fat mass in the two cohorts .
child s % fat mass and fat mass at age 9 years increase with higher umbilical cord rxra chr9:136355885 + methylation in the pah cohort .
* fat mass and percentage fat mass are preadjusted for sex . in a second independent cohort ,
child s % fat mass and fat mass at age 6 years increase with higher umbilical cord rxra chr9:136355885 + methylation in sws subjects .
pyrosequencing across rxra chr9:136355885 + in sws subjects showed no c allele snps that could account for differences in methylation observed between individuals .
this study provides novel evidence for the importance of the developmental contribution to later adiposity .
we found that greater methylation of rxra chr9:136355885 + measured at birth was strongly correlated with greater adiposity in later childhood in two independent cohorts .
although we studied a subset of children in both cohorts these were selected on the basis of subject and specimen availability , so it is unlikely that selection bias could explain the relationships observed unless the association between rxra methylation and adiposity was different in the remainder of the cohort .
the data build on animal experiments suggesting that the developmental environment acts through epigenetic processes to exert a strong influence on postnatal body composition and metabolic function ( 711 ) .
our study shows that specific components of the epigenetic state at birth predict later childhood adiposity .
the associations with adiposity were linked to specific cpgs 5 to the start site of the selected candidate genes . although some of the cpgs studied were either within the proximal promoter or close to it , others were more distal and may be exerting effects through the regulation of other genes .
our observation that adjacent or nearby cpgs within the same promoter showed differences in the strength of association with child s adiposity suggests highly specific changes in the transcriptional regulation of these genes induced by the developmental environment , rather than generalized changes in promoter methylation . both cpg hyper- ( enos chr7:150315553 + and rxra chr9:136355885 + ) and hypomethylation ( sod1 chr21:31853660/63 + ) at different sites were associated with body fat distribution , again indicating complexity in transcriptional control .
the specificity of the associations between methylation of an individual cpg and both maternal diet and child s phenotype endorses the concept of a fine control of development by environmental factors via epigenetic processes .
our observation indicates one potential mechanistic pathway involved , because induction of transcription by rxra is dependent on its binding to ligands including the peroxisome proliferator  activated receptors , involved in insulin sensitivity , adipogenesis , and fat metabolism ( 31,32 ) .
moreover , rxra chr9:136355885 + is located in a region considered to contain positive regulatory elements of transcription ( 33 ) .
figure 4 shows the proximity of rxra chr9:136355885 + to proposed binding sites for rxr , maf , nf-b , and ap1 .
retinoid receptor biology is complex , and increased rxra methylation might be acting through a variety of pathways ( 34 ) ; however , an association between increased rxra methylation and adiposity is consistent with the observation of strongly diminished rxra expression in visceral white adipose tissue from obese mice ( 35 ) .
moreover , a role for retinoid receptor methylation in developmental influences on later metabolic risk is supported by recent experimental data showing an influence of maternal diet during pregnancy on methylation of lxra , a heterodimeric partner of rxra ( 36 )
. schematic diagram of the rxra promoter region , showing the position of the cpg group at rxra chr9:136355885 + ( underlined ) and of neighboring transcription factor binding sites .
genome - wide association studies suggest that fixed genetic variation makes a relatively small contribution to risk of obesity , heart disease , and diabetes ( 1,2 ) ; our findings raise the possibility that the developmental environment component may be equally or more important .
we excluded the presence of a snp at rxra chr9:136355885 + by sequencing , but without genome - wide analysis it is not possible to exclude a genetic effect of distant snps , which could influence both dna methylation of a particular sequence and child s phenotype .
however , even if this were the case , our data clearly indicate that epigenetic measures at birth may have prognostic value .
although epigenetic changes can be dynamic , experimental studies have shown that environmental factors acting on the genotype during development relate to epigenetic profile in adulthood ( 7,15 ) , and there are longitudinal human studies showing that dna methylation is often stable over time ( 37 ) .
such changes can be tissue specific , and in this respect the umbilical cord may be advantageous because it contains a high proportion of fetal vascular tissue and mesenchymal cells , which may be relevant to later adiposity .
furthermore , unlike the placenta it is a tissue in which consistency of sampling between individuals is more likely .
although experimental work in the rat suggests that methylation changes induced by maternal diet can be similar in the umbilical cord and liver ( 38 ) , further work is needed to determine the relevance of epigenetic changes in human umbilical cord tissue .
recent data show that for some genomic regions methylation appears largely independent of tissue of origin , whereas for others there is a clear tissue - specific dependence ( 39 ) .
many epidemiological studies have shown associations between fetal development , through the proxy measure of birth size , and later adiposity and metabolic function ( 3,4 ) , but the developmental contribution to such phenotypic characteristics has remained uncertain and controversial .
this study provides the first estimate of the developmental contribution to phenotype associated with human disease risk based on measures of the underpinning biology : our data for rxra chr9:136355885 + suggest that a substantial proportion of the variation in adiposity in prepubertal children can be explained by epigenetic measurements made at birth .
although our data are correlative and thus can only imply an association between dna methylation at birth and later phenotype , the importance of the observation stands irrespective of whether the rxra methylation is causally related to the development of adiposity . even if it is simply a noncausal association , the changed epigenetic status provides an objective marker of altered developmental trajectory by the time of birth .
despite the limitations of dietary intake assessment tools , the instrument we used is both validated and provides information that can be used to rank the nutrient intakes of individuals ( 23 ) .
methylation of enos chr7:150315553 + was associated with later adiposity in the initial cohort only .
this may reflect a chance finding , or different maternal characteristics in the two cohorts , such as the greater maternal adiposity and maternal folate supplementation in the sws cohort ( 22,40,41 ) .
alternatively , adipocyte proliferation is high during the first year of life but then remains low until a second proliferative phase from age 914 years ( 42 ) .
thus the processes that determine adiposity at age 9 years might differ from those at age 6 years .
therefore , a further possible explanation is that enos methylation in umbilical cord marks capacity for adipogenesis which has a greater net contribution to adiposity at age 9 years than age 6 years .
our findings show strong associations between epigenetic markers and childhood total and central body fat . beyond these simple associations ,
multivariate analysis indicates that the associations explain substantial proportions of the variances in outcomes , emphasizing an important developmental contribution to phenotypes associated with metabolic dysfunction and disease risk .
it is noteworthy that the genes for which we report effects are not imprinted . in vitro fertilization increases risk of imprinting disorders ( 44 ) , and methylation effects on imprinted genes
have been reported in offspring of mothers exposed to famine during various periods of pregnancy ( 45,46 ) , but with no associations with phenotype reported .
the current study implicates the human prenatal environment with epigenetic changes in nonimprinted genes and is the first to link epigenetic status at birth with clinically relevant later phenotypic variation .
variation in the degree of methylation of nonimprinted genes and in later cardiovascular and metabolic physiology can be induced experimentally by manipulation of the developmental environment , for example by altering maternal nutrition or administering glucocorticoids during pregnancy ( 8,9 ) , often without necessarily affecting the birth size of the offspring .
although we found associations between some epigenetic markers and birth weight , these were weaker associations than those with later phenotype and were for different markers ; moreover , previous reports on the same cohort showed only modest associations between birth weight and later body composition ( 6 ) . because birth weight in humans is influenced by multiple factors including the mother s own birth weight and height and by gestational length ( 40 ) , epigenetic changes
may provide a more sensitive index than birth weight of environmentally induced effects on fetal development .
there are potentially important implications of the strong and replicated association between rxra chr9:136355885 + methylation and later adiposity .
first , the effect is considerably greater than that of factors such as birth weight or maternal body composition , suggesting that epigenetic measurements made in the neonate may be useful predictors of later obesity and other phenotypic outcomes .
second , the association between cpg methylation and child s adiposity operates within the normal ranges of maternal nutritional state and birth size ; this supports the argument that developmental programming is the consequence of an evolved and potentially adaptive process involving the mechanisms of developmental plasticity ( 10 ) .
indeed the data provide strong evidence supporting a role for developmental plasticity in determining individual risk of metabolic disease .
third , the data suggest that developmental factors may be more significant in contributing to phenotypic variation and disease risk than generally considered .
fourth , the association between rxra chr9:136355885 + methylation and mother s carbohydrate intake raises the possibility that conditions in early pregnancy could affect child s adiposity through this pathway .
this provides additional support for the argument that all women of reproductive age should have appropriate nutritional , education , and lifestyle support to improve the health of the next generation .
finally , our data suggest that epigenetic measures at birth may have prognostic value and potential utility for monitoring programs to optimize maternal health and nutrition for long - term benefits to the offspring ; however , evaluation of this possibility will require further research correlating methylation measurements in early life with those in later life .
 objectivefixed genomic variation explains only a small proportion of the risk of adiposity . in animal models , 
 maternal diet alters offspring body composition , accompanied by epigenetic changes in metabolic control genes . 
 little is known about whether such processes operate in humans.research design and methodsusing sequenom massarray we measured the methylation status of 68 cpgs 5 from five candidate genes in umbilical cord tissue dna from healthy neonates . 
 methylation varied greatly at particular cpgs : for 31 cpgs with median methylation 5% and a 595% range 10% , we related methylation status to maternal pregnancy diet and to child s adiposity at age 9 years . 
 replication was sought in a second independent cohort.resultsin cohort 1 , retinoid x receptor- ( rxra ) chr9:136355885 + and endothelial nitric oxide synthase ( enos ) chr7:150315553 + methylation had independent associations with sex - adjusted childhood fat mass ( exponentiated regression coefficient [  ] 17% per sd change in methylation [ 95% ci 431 ] , p = 0.009 , n = 64 , and  = 20% [ 932 ] , p < 0.001 , n = 66 , respectively ) and % fat mass (  = 10% [ 119 ] , p = 0.023 , n = 64 and  = 12% [ 420 ] , p = 0.002 , n = 66 , respectively ) . 
 regression analyses including sex and neonatal epigenetic marks explained > 25% of the variance in childhood adiposity . 
 higher methylation of rxra chr9:136355885 + , but not of enos chr7:150315553 + , was associated with lower maternal carbohydrate intake in early pregnancy , previously linked with higher neonatal adiposity in this population . in cohort 2 , 
 cord enos chr7:150315553 + methylation showed no association with adiposity , but rxra chr9:136355885 + methylation showed similar associations with fat mass and % fat mass (  = 6% [ 210 ] and  = 4% [ 17 ] , respectively , both p = 0.002 , n = 239).conclusionsour findings suggest a substantial component of metabolic disease risk has a prenatal developmental basis . 
 perinatal epigenetic analysis may have utility in identifying individual vulnerability to later obesity and metabolic disease . 