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 (7
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. Nevertheless the data indicate possible mechanistic pathways, suggesting avenues for future study. 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
). Moreover, RXRA chr9:136355885+ is located in a region considered to contain positive regulatory elements of transcription (33
). shows the proximity of RXRA chr9:136355885+ to proposed binding sites for RXR, MAF, NF-κB, 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
); 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
), 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
), 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
). Alternatively, adipocyte proliferation is high during the first year of life but then remains low until a second proliferative phase from age 9–14 years (42
). Thus the processes that determine adiposity at age 9 years might differ from those at age 6 years. Nitric oxide synthesis by eNOS promotes preadipocyte differentiation (43
). 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
), 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
), 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.