Epigenetics, Nutrition, and Growth

Berthold Koletzko

Both human growth and the response to diet, for example, the absorption and metabolism of certain nutrients, are modulated by genetic variation. Although rare monogenetic causes of obesity have been identified, the results of genome-wide association studies have demonstrated only relatively modest effects on human growth, body mass, and obesity risk. Likewise, sizeable nutrigenetic interaction effects have only
been demonstrated for some nutrients so far, for example, folic acid and polyunsaturated
fatty acids. It has been proposed that epigenetic effects may account for some of the unexplained heritability of common phenotypes and particularly for the lasting effects of exposure to environmental cues in early life on later health, performance, and disease risks (early metabolic programming of lifelong health). Epigenetic modifications of the genome are hereditary cell characteristics not determined by DNA sequence but by biochemical DNA changes, including histone modification, DNA methylation, or noncoding RNAs. Thereby, epigenetic modifications determine the accessibility of DNA to a variety of transcription and other regulatory factors and determine the activity state of underlying DNA code, that is, they can modify gene expression and hence function. Epigenetic profiles of individual cells can also act as memory of environmental exposure, particularly in early life with a high susceptibility to inducing epigenetic changes, allowing a cell to maintain its specific properties after each cell division. Most human studies to data have applied assessment of cytosine methylation given that commercial chips for high-throughput epigenome-wide analyses are available. The major challenge for such studies is not the biochemical analysis but rather the complex bioinformatic exploration of the resulting very large datasets. Some first studies have explored the potential relations of epigenetics, nutrition, and growth.

DNA methylation changes related to nutritional deprivation: a genome-wide analysis of population and in vitro data

DNA methylation has recently been identified as a mediator between in utero famine exposure and a range of metabolic and psychiatric traits. However, genome-wide analyses are scarce and cross-sectional analyses are hampered by many potential confounding factors. Moreover, causal relations are hard to identify due to the lack of controlled experimental designs. In the current study, we therefore combined a comprehensive assessment of genome-wide DNA methylation differences in people exposed to the great Chinese famine in utero with an in vitro study in which we deprived fibroblasts of nutrition.
Methods: We compared whole blood DNA methylation differences between 25 individuals in utero exposed to famine and 54 healthy control individuals using the HumanMethylation450 platform. In vitro, we analyzed DNA methylation changes in 10 fibroblast cultures that were nutritionally deprived for 72 h by withholding fetal bovine serum.
Results: We identified 3 differentially methylated regions in 4 genes (ENO2, ZNF226, CCDC51, and TMA7) that were related to famine exposure in both analyses. Pathway analysis with data from both Chinese famine samples and fibroblasts highlighted the nervous system and neurogenesis pathways as the most affected by nutritional deprivation.
Conclusions: The combination of cross-sectional and experimental data provides indications that biological adaptation to famine leads to DNA methylation changes in genes involved in the central nervous system.
This small exploratory pilot study analyzed whole blood DNA from 25 Chinese subjects
whose mothers were exposed to famine in pregnancy. A weakness of the publication
is that the approach to selecting this subgroup from the total originally included study population and the details of exposure and clinical characteristics of this subgroup are not reported. Hypomethylation of 3 differentially methylated regions in 4 gene promoters were reported with a statistical significance level of 1%. The genes involved relate to nervous system development. The sample size is very small, and the numerous statistical tests performed were not adjusted for multiple testing. Therefore, the results need to be interpreted with caution and considered as hypothesis raising, with a need for replicating the observations in larger studies.

A randomized controlled trial of folic acid intervention in pregnancy highlights a putative methylation-regulated control element at ZFP57
 Maternal blood folate concentrations during pregnancy have been previously linked with DNA methylation patterns, but this has been done predominantly through observational studies. We showed recently in an epigenetic analysis of the first randomized controlled trial of folic acid (FA) supplementation specifically in the second and third trimesters (the EpiFASSTT trial) that methylation at some imprinted genes was altered in cord blood samples in response to treatment. Here, we report on epigenome-wide screening using the Illumina EPIC array (∼ 850,000 sites) in these same samples (n = 86).
Results: The top-ranked differentially methylated promoter region (DMR) showed a gain in methylation with FA and was located upstream of the imprint regulator ZFP57. Differences in methylation in cord blood between placebo and FA treatment groups at this DMR were verified using pyrosequencing. The DMR also gains methylation in maternal blood in response to FA supplementation. We also found evidence of differential methylation at this region in an independent randomized controlled trial cohort, the AFAST trial. By altering methylation at this region in 2 model systems in vitro, we further demonstrated that it was associated with ZFP57 transcription levels.
Conclusions: These results strengthen the link between FA supplementation during later pregnancy and epigenetic changes and identify a novel mechanism for regulation of ZFP57.
This is one of the few analyses of DNA methylation effects available so far from a randomized controlled clinical intervention trial. One hundred and ninety pregnant women were randomized to receive in the second and third trimester of pregnancy until
child birth either 400 mg folic acid (FA) per day or placebo. In 86 newborn infants born
to these mothers, cord blood cells were obtained and DNA methylation analyses were
performed with a chip covering 850,000 CpG sites. Statistical analysis with a chosen
significance level of 5% showed numerous sites to have group differences in the degree of methylation, but unfortunately, an analysis of results corrected for multiple testing is not reported. Among the top 1,000 sites ranked for p levels, about 2/3 showed
hypomethylation with FA supplementation and 1/3 hypermethylation. The authors
highlight increased methylation of the imprint regulator ZFP57 in the offspring of women randomized to FA. Due to the limitations in statistical analyses, these results should be interpreted with some caution until reconfirmed in further studies.

A low glycaemic index diet in pregnancy induces DNA methylation variation in blood of newborns: results from the ROLO randomised controlled trial

 The epigenetic profile of the developing fetus is sensitive to environmental influence. Maternal diet has been shown to influence DNA methylation patterns in offspring, but research in humans is limited. We investigated the impact of a low glycaemic index dietary intervention during pregnancy on offspring DNA methylation patterns using a genome-wide methylation approach. Sixty neonates were selected from the ROLO (Randomised cOntrol trial of LOw glycaemic index diet to prevent macrosomia) study: 30 neonates from the low glycaemic index intervention arm and 30 from the control, whose mothers received no specific dietary advice. DNA methylation was investigated in 771,484 CpG sites in free DNA from cord blood serum. Principal component analysis and linear regression were carried out comparing the intervention and control groups. Gene clustering and pathway analysis were also explored. Widespread variation was identified in the newborns exposed to the dietary intervention, accounting for 11% of the total level of DNA methylation variation within the dataset. No association was found with maternal early-pregnancy body mass index, infant sex, or birthweight. Pathway analysis identified common influences of the intervention on gene clusters plausibly linked to pathways targeted by the intervention, including cardiac and immune functioning. Analysis in 60 additional samples from the ROLO study failed to replicate the original findings. Using a modest-sized discovery sample, we identified preliminary evidence of differential methylation in progeny of mothers exposed to a dietary intervention during pregnancy.
This is another one of the very few analyses of DNA methylation effects available so far
from randomized controlled clinical intervention trials. A high-risk group of pregnant women who had delivered a macrosomic infant in a preceding pregnancy were randomized to receive no intervention or a dietary education session delivered by a
trained dietician for a duration of about 2h. The focus of the training was on preferential
choice of foods with a low glycemic index and reducing the intake of foods with a high glycemic index. Written information was also provided, and the messages were reinforced in a second session with the dietician held between 28 and 34 weeks of gestation. In the overall trial, the intervention did not achieve any significant difference
between the 2 groups in infant birth weight, birth weight centile, or ponderal index.
However, women in the intervention arm had significantly less gestational weight gain
(mean difference –1.3 kg) and a lower rate of glucose intolerance (21 vs. 28%). In a sub-group of 30 infants each from the 2 intervention arms, circulating cell-free DNA was
extracted from frozen cord blood serum and analyzed for DNA methylation with a chip
covering 850,000 CpG sites. Data were evaluated with principle component and linear
regression analyses adjusted for confounders, and the Benjamin-Hochberg false discovery rate was applied to adjust for multiple testing. The results indicate preliminary
evidence for widespread but subtle changes of differentially methylated regions in the
neonatal genome in response to the dietary intervention in their mothers during pregnancy. Methylation was not associated with maternal body weight or body mass index nor with neonatal birth weight. In a further subset of another 60 infants, methylation of 3 candidate genes using the Sequenom Mass Array technology was attempted, but here the initial findings could not be confirmed. The data indicate the real potential that diet in pregnancy may modulate epigenetic signaling in the offspring, which
might impact child outcome. This should prompt further work in this promising area.

DNA methylation of imprinted genes at birth is associated with child weight status at birth, 1 year, and 3 years

 This study assessed the associations between 9 differentially methylated regions of imprinted genes in DNA derived from umbilical cord blood leukocytes in males and females and (1) birth weight for gestational age z score, (2) weight-for-length z score at 1 year, and (3) body mass index (BMI) z score at 3 years.
Methods: We conducted multiple linear regression in n = 567 infants at birth, n = 288 children at 1 year, and n = 294 children at 3 years from the Newborn Epigenetics Study. We stratified by sex and adjusted for race/ethnicity, maternal education, maternal pre-pregnancy BMI, prenatal smoking, maternal age, gestational age, and paternal race. We also conducted analysis restricting to infants not born small for gestational age.
Results: We found an association between higher methylation of the sequences regulating paternally expressed gene 10 and anthropometric z scores at 1 year (β = 0.84; 95% CI = 0.34–1.33; p = 0.001) and 3 years (β = 1.03; 95% CI = 0.37–1.69; p value = 0.003) in males only. Higher methylation of the differentially methylated region regulating mesoderm-specific transcript was associated with lower anthropometric z scores in females at 1 year (β = –1.03; 95% CI –1.60 to –0.45; p value = 0.001) and 3 years (β = –1.11; 95% CI –1.98 to –0.24; p value = 0.01). These associations persisted when we restricted to infants not born small for gestational age.
Conclusion: Our data support a sex-specific association between altered methylation and weight status in early life. These methylation marks can contribute to the compendium of epigenetically regulated regions detectable at birth, influencing obesity in childhood. Larger studies are required to confirm these findings.
This study associated DNA methylation in cord blood white cells with birth weight in
more than 500 newborn infants and with weight at 1 and 3 years of age in close to 300
children, respectively. Methylation of paternally expressed gene 10 (PEG10) was associated with anthropometric measures at 1 and 3 years only in boys but not in girls. These data are of interest in that they suggest a potential sex-specific effect of DNA methylation on body mass index and potentially on obesity risk, which could provide the potential for sex-specific, targeted obesity prevention strategies in the future. More inside into exposures that might affect these methylation effects would be most valuable.

Fetal growth is associated with CpG methylation in the P2 promoter of the insulin-like growth factor-1 gene

 There are many reasons to think that epigenetics is a key determinant of fetal growth variability across the normal population. Since insulin-like growth factor-1 (IGF-1) and INS genes are major determinants of intrauterine growth, we examined the methylation of selected CpGs located in the regulatory region of these 2 genes.
Methods: Cord blood was sampled in 159 newborns born to mothers prospectively followed during their pregnancy. A 142-item questionnaire was filled by mothers at inclusion, during the last trimester of the pregnancy and at the delivery. The methylation of selected CpGs located in the promoters of the IGF-1 and INS genes was measured in cord blood mononuclear cells collected at birth using bisulfite-PCR-pyrosequencing.
Results: Methylation at IGF-1 CpG-137 correlated negatively with birth length (r = 0.27, p= 3.5 ×10 –4 ). The same effect size was found after adjustment for maternal age, parity, and smoking: a 10% increase in CpG-137 methylation was associated with a decrease of length by 0.23 SDS.
Conclusion: The current results suggest that the methylation of IGF-1 CpG-137 contributes to the individual variation of fetal growth by regulating IGF-1 expression in fetal tissues.
This study in a sample of 159 infants born in France explored DNA methylation in a promoter of the insulin-like growth factor-1 (IGF-1) gene and found an association of increased methylation with reduced neonatal length at birth. The effect size was not large, with an increase of CPG methylation from 40 to 70% associated with reduced length by about half a standard deviation. Nonetheless, the data suggest the exciting potential that increased methylation of this IGF1-P2 promoter may significantly impact on fetal growth, which should certainly motivate further exploration of the underlying pathways.

Meta-analysis of epigenome-wide association studies in neonates reveals widespread differential DNA methylation associated with birthweight

Birthweight is associated with health outcomes across the life course, DNA methylation may be an underlying mechanism. In this meta-analysis of epigenome-wide association studies of 8,825 neonates from 24 birth cohorts in the Pregnancy And Childhood Epigenetics Consortium, we find that DNA methylation in neonatal blood is associated with birthweight at 914 sites, with difference in birthweight ranging from –183 to 178 g per 10% increase in methylation (P Bonferroni <1.06 × 10 –7 ). In additional analyses in 7,278 participants, < 1.3% of birthweight-associated differential methylation is also observed in childhood and adolescence, but not adulthood. Birthweightrelated CpGs overlap with some Bonferroni-significant CpGs that were previously reported to be
related to maternal smoking (55/914, p = 6.12 × 10 –74 ) and body mass index in pregnancy (3/914, p = 1.13 × 10 –3 ), but not with those related to folate levels in pregnancy. Whether the associations that we observe are causal or explained by confounding or fetal growth influencing DNA methylation (i.e., reverse causality) requires further research.
This is a very impressive and well-performed, huge data analysis that combined data
of epigenome-wide association studies in almost 9,000 newborn infants from a large
number of birth cohorts, based on the established collaboration of the Pregnancy and
Childhood Epigenetic consortium. DNA methylation in neonatal blood cells showed
an association with birth weight at more than 900 genome sites, with some very large
effect sizes: a 10% change in the degree of methylation modified birth rate by as much
as up to 180 g upward and downward, respectively. These results indicate that environmental cues might markedly affect birth weight via epigenetic mechanisms. Future needs should explore which environmental cues may be of key relevance and
whether the associations might reflect causal relationships.

ExtraUterine Growth Restriction in preterm infants: growth patterns, nutrition, and epigenetic markers. A pilot study

 IntraUterine and ExtraUterine growth restriction (EUGR) may induce reprogramming mechanisms, finalized to survive before and after birth. Nutritional factors and other environmental signals could regulate gene expression through epigenetic modification, but the molecular mechanisms involved are not yet well understood. Epigenetic mechanisms could be considered as a bridge between environmental stimuli and long-lasting phenotype, acquired during the intrauterine life and the first weeks of life. Our aim was to investigate the relationship between growth patterns, nutritional determinants, and epigenetic pathways.
Methods: We enrolled 38 newborns admitted to neonatal intensive care unit at University Hospital of Pisa. Gestational age at birth was < 34 weeks and post-menstrual age was 36–42 weeks at discharge. We excluded infants with malformations or clinical syndromes. EUGR was defined as the reduction in weight z score between birth and discharge > 1 SD. We also evaluated DNA methylationof imprinting centre 1 (IC1) at birth and at discharge.
Results: We observed a decrease in SD of weight and head circumference mainly during the first weeks of life. We found a correlation between EUGR for weight and for head circumference and an increased IC1 methylation (p = 0.018 and p = 0.0028, respectively). We observed a relationship between reduced protein and lipid intake and IC1 hypermethylation (p = 0.009 and p = 0.043, respectively).
Conclusion: IC1 hypermethylation could be a reprogramming mechanism to promote a catch-up growth, by means of an increased Insulin-like growth factor 2 expression, that may have potential effects on metabolic homeostasis later in life.
This study in a small and somewhat heterogeneous group of 38 preterm infants from one neonatal intensive care in Italy determined methylation of an imprinting control region regulating the insulin growth factor-2 gene expression. There was no association
with birth weight, but increased methylation was associated with a greater proportion of children who showed a reduction in weight z-score between birth and discharge by > 1 SD. Increased methylation at this site was also inversely related to both dietary protein and lipid intake. However, statistical analysis was performed at the 5% significant level without any correction for multiple testing. Therefore, the observations need to be regarded with caution. Larger studies aiming at replication would appear highly desirable.

Phthalate exposures, DNA methylation and adiposity in Mexican children through adolescence

 Phthalates are a class of endocrine disrupting chemicals with near ubiquitous exposure to populations around the world. Phthalates have been associated with children’s adiposity in previous studies, though discrepancies exist across studies that may be due to timing of exposure or outcome assessment and population differences (i.e., genetics, other confounders). DNA methylation, an epigenetic modification involved in gene regulation, may mediate the effects of early life phthalate exposures on health outcomes. This study aims to evaluate the mediating effect of DNA methylation at growth-related genes on the association between phthalate exposure and repeat measures of adiposity (body mass index [BMI]-for-age z-score, waist circumference, and skinfolds thickness) in Mexican children. Urinary phthalate metabolite concentrations were quantified in mothers at each of the 3 trimesters of pregnancy and in children at the first peri-adolescent study visit. Blood leukocyte DNA methylation at H19 and HSD11B2 was quantified during the first periadolescent visit, and adiposity was measured at the first visit and again ∼ 3 years later among participants (n = 109 boys, 114 girls) from the Early Life Exposure in Mexico to Environmental Toxicants project. Associations between phthalates or DNA methylation and repeat outcome measures were assessed separately in boys and girls using generalized estimating equation models including covariates (urinary specific gravity, maternal education, and child’s age). Sobel tests were used to
assess DNA methylation as a mediator in models adjusting for the same covariates. Associations between phthalates and adiposity varied by phthalate and timing of exposure. Early gestation MBP, MIBP, and MBzP were associated with adiposity among girls. For example, among girls first trimester maternal urine concentrations of MIBP were associated with increases in skinfold thickness, BMI-for-age, and waist circumference (p < 0.01). Second trimester and adolescent MBzP were associated with adiposity among boys in opposite directions. In girls, H19 methylation was positively
associated with skinfold thickness. No significant mediation of phthalate exposure on adiposity by DNA methylation of H19 or HSD11B2 was observed (Sobel p > 0.05). However, the mediation analysis was underpowered to detect small to medium effect sizes, and the role of DNA methylation as a mediator between phthalates and outcomes merits further study.
The authors explored the hypothesis that phthalates, widespread industrial plasticizers
and additives with endocrine disruption properties DNA methylation, might induce
epigenetic modifications that exert effects on childhood obesity. In a subsample of 109 boys and 114 girls from a Mexican longitudinal other-child cohort study up to the age of 14 years, and many of them also with follow-up to age 17 years, phthalates were measured in maternal and child urine samples by liquid chromatography triple mass spectrometry. DNA methylation was determined in DNA extracted from children’s whole blood samples collected at an age between 8 and 14 years. While some phthalate measures in mothers and children were associated with measures of child obesity and adiposity, no significant mediation effects for DNA methylation were detected for the association between phthalates and outcomes in the total study sample, but there non-significant for DNA methylation as a mediator between phthalate exposure and adiposity in the Sobel test for non-zero mediation. These observations indicate the potential of exploring epigenetics as a mediator influencing susceptibility to effects from toxicant exposures, which should be tested in larger studies with greater statistical power.