Pregnancy and infancy are critical periods for nutritional programming of metabolic health. Epigenetic changes such as DNA methylation seem to have a crucial role linking early-life nutrition to metabolic health across the life course. The aim of NutriPROGRAM was to identify early-life nutrition-related factors and related epigenetic pathways leading to metabolic adaptations and disease across the life course. 

During the project, we have evaluated the newly developed Dutch toddler diet quality index. We have identified associations of nutrition-related factors and in early life with DNA methylation at a large number of sites across the genome. These include maternal dietary glycemic index and load, Mediterranean diet adherence, vitamin B12 and fatty acid levels, as well as breastfeeding, infant protein intake and child eating behaviour. Also, we found associations, although in some cases a limited number, of DNA methylation with child health outcomes, such as body and fat mass index and lipid levels. Some of these were shown to differ depending on other factors, such as maternal obesity or smoking.

We have described DNA methylation trajectories from birth into adolescence and have created a publicly available website through which these can be visualized. In addition, a publicly available catalogue of blood autosomal cis-eQTMs in children was created. Both of these resources will help in designing and interpreting future DNA methylation studies.  

Ongoing work focuses on the use of DNA methylation to predict child health outcomes, as well as on discovery of tissue-specific mQTLs in buccal and placental tissue, which will contribute to the understanding of biological pathways and to inform studies into causality. 

As such, NutriPROGRAM has achieved its expected impacts, which were: 

1) Create a set of harmonised and integrated nutrition-related factors that can be used by scientists studying the relationships between nutrition and health; 

2) Lead to a better understanding of the role of DNA methylation in the pathways underlying the relation of early-life nutrition and metabolic health; 

3) Lead to a better understanding of causality and modification of DNA methylation in these pathways; 

4) Contribute to the development of methods to help identify the most promising findings from DNA methylation studies for further research; 

5) Form a sustainable, international network of researchers on early-life nutrition, epigenetics and long-term metabolic outcomes that will lay the foundation for further research in this area for many years to come.

Trajectories of DNA methylation from birth to adolescence 

DNA methylation is known to play an important role in child health and development, but not much is known about how DNA methylation changes with age in this important period of life. Using information from over 2300 children, we have characterized trajectories of DNA methylation from birth into adolescence. We found that DNA methylation changes with age at over half of the examined CpG sites and that there are differences between individuals in terms of patterns of change. These findings support a developmental role for DNA methylation and will be important for future studies in this area. We have created a publicly available website where the DNA methylation trajectories can be visualized: http://epidelta.mrcieu.ac.uk.

DNA methylation in relation to gene expression 

For the biological interpretation of studies on DNA methylation, it is important to understand the associations of DNA methylation and gene expression. In over 800 children, we characterized these associations. We identified 39,749 blood autosomal cis eQTMs,expression quantitative trait methylation, defined as correlations between gene expression and DNA methylation levels, representing 21,966 unique CpGs and 8,886 unique genes (transcript clusters). The overlap of child blood cis-eQTMs with those previously described in adults was small. Only half of the cis-eQTMs could be captured through annotation to the closest gene. This catalogue of blood autosomal cis-eQTMs in children can help the biological interpretation of EWAS findings. It is publicly available at: https://helixomics.isglobal.org/. 

DNA methylation and body mass index from birth to adolescence 

DNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. We examined whether DNA methylation in cord blood and whole blood in childhood and adolescence was associated with BMI in the age range from 2 to 18 years using data from up to 4133 children from 23 studies. Overall, there were only minimal associations of DNA methylation with childhood and adolescent BMI. With the advancing age of the participants across childhood and adolescence, we observed increasing overlap with altered DNA methylation loci reported in association with adults BMI. These findings may be compatible with the hypothesis that DNA methylation differences are mostly a consequence rather than a cause of obesity, but this needs further study.

Paternal body mass index and child DNA methylation 

Previous small, mostly candidate gene studies have shown associations between paternal pre-pregnancy body mass index (BMI) and offspring-blood DNA methylation. In NutriPROGRAM and the Pregnancy and Childhood Epigenetics (PACE) consortium, we co-ordinated epigenome-wide association studies of paternal BMI in relation to DNA methylation and then meta-analysed the results across 13 data sets at birth (4894 babies) and six data sets in childhood (1982 babies). We found little evidence to support previous findings, even at imprinted regions. However, this does not rule out the possibility of a paternal epigenetic effect in different tissues, at regions not covered by the 450k array, via different mechanisms or in populations with greater extremes of paternal BMI. More research is warranted to help in understanding the size and nature of contributions of paternal adiposity to offspring epigenetics and health outcomes.

Maternal dietary glycemic index and load during pregnancy and child DNA methylation

Suboptimal nutrition in pregnancy is associated with worse offspring cardiometabolic health. DNA methylation may be an underlying mechanism. We meta-analyzed epigenome-wide association studies (EWAS) of maternal dietary glycemic index and load with cord blood DNA methylation in a total of 2003 mother-child pairs from three studies. We found that maternal glycemic index and load were associated with cord blood DNA methylation at 41 sites, mostly in mothers with overweight/obesity. We did not observe overlap with CpGs associated with maternal glycemic traits, BMI, or child birth weight or BMI. DNA methylation at a limited number of sites was associated with gene expression in blood or adipose tissue. This indicates potential functional effects, which need to be further examined. 

Maternal adherence to the Mediterranean diet in pregnancy and child DNA methylation

Higher adherence to the Mediterranean diet during pregnancy is related to a lower risk of preterm birth and to better offspring cardiometabolic health. DNA methylation may be an underlying biological mechanism. We evaluated whether maternal adherence to the Mediterranean diet was associated with offspring cord blood DNA methylation, by analysing epigenome-wide association studies of maternal adherence to the Mediterranean diet during pregnancy and offspring cord blood DNA methylation in 2802 mother-child pairs from five cohorts. We found that adherence to a Mediterranean diet was associated with cord blood DNA methylation at one site. The functional relevance and potential link with childhood health outcomes should be taken forward to future studies.

Maternal vitamin B12 concentrations in pregnancy and child DNA methylation

Circulating vitamin B12 concentrations during pregnancy are associated with offspring health. Fetal DNA methylation changes could underlie these associations. We analysed if circulating vitamin B12 concentrations in mothers during pregnancy (2420 mother-child pairs) or in cord blood (1029 infants) are associated with DNA methylation in cord blood. Maternal and newborn vitamin B12 concentrations were associated with DNA methylation at 109 and 7 CpGs, respectively. Some of these have been previously associated with health outcomes, such as birth weight, gestational age, childhood cognitive skills or nonverbal IQ. Around one-fifth were associated with nearby gene expression. This study provides important information about DNA methylation loci potentially underlying associations of vitamin B12 concentrations with child health outcomes.

Animal protein intake during infancy and childhood DNA methylation

In a joint analysis of 2 longitudinal studies in the NutriPROGRAM consortium coordinated in Germany and the Netherlands, animal protein intake in infancy was examined in relation to DNA methylation in early and in late childhood. Results showed that infant animal protein intake was not associated with DNA methylation in early childhood, but was associated with DNA methylation in late childhood.