We present preliminary evidence for a unifying new hypothesis in which epigenetic modification induced in early development may contribute to an increased susceptibility to age-related diseases by prematurely advancing the normal aging process. Using a genome-wide, high-resolution cytosine methylation assay (testing ~1.32 million loci throughout the genome), we avoid the necessity for any a priori assumptions about candidate loci and reveal potential new candidates in mediating the fetal origin of adult disease. We identified dysregulation of DNA methylation in purified populations of cord blood-derived CD34+ stem cells from IUGR neonates compared with matched controls, and find that these changes occur throughout the genome, although they are of a more modest degree and relatively limited extent compared with the epigenomic dysregulation observed in conditions such as cancer. As our study was based on a limited number of subjects, such subtle differences in cytosine methylation challenge the ability of genome-wide approaches to identify genuinely significant loci even at standard significance levels (0.05–0.001) which do not reflect multiple testing. In large-scale studies, when thousands to millions of loci are measured, the threshold to declare any locus significant is typically based on a more stringent level of significance (e.g. Bonferroni correction or similar) in order to minimize the possibility of false positives, therefore we have displayed the power at two such thresholds. Based on these simulations, a more expansive approach, with sample size of at least 25 subjects per group is recommended for studies of methylation changes in conditions where such subtle effects are expected.
Nonetheless, we found that the IUGR subjects were distinctive for having a number of consistent differences in methylation near genes involved in processes critical for stem cell function, including cell cycle and cellular maintenance. Quantitative bisulphite validation studies confirmed our ability to discriminate differences in methylation in these samples, and the biological coherence of results in terms of functional pathway relatedness is suggestive of underlying changes in epigenetic regulation as a response to IUGR.
A locus that emerged consistently in this pathway analysis was the HNF4A
gene, already implicated in T2DM 
, but not previously demonstrated to undergo epigenetic dysregulation as a response to IUGR. Best known for its implications as a monogenic, autosomal dominant form of maturity onset diabetes of the young (MODY) 
, HNF4A is involved in development and function of both the liver and the pancreas 
and actively coordinates gene expression of many important metabolic pathways in both tissues 
. We find differences in DNA methylation targeted to only one of the HNF4A
promoters, supporting a model of isoform variation of the gene being related to susceptibility to T2DM, a major age-related disease.
Other loci identified in this study were found to be related functionally to HNF4A
and also include ATG5
, which may have roles in mediating susceptibility to later disease. ATG5
is an essential component of autophagy that, when depleted, renders cells more susceptible to starvation and starvation-induced cell death 
. We found that ATG5
is relatively methylated in IUGR at a CpG island-containing site just downstream of the transcription start site, potentially reducing expression in IUGR compared with controls, in parallel with increased sensitivity to starvation-induced cell death. Transcriptional adaptor 3 (TADA3L
) is associated with and is required for full p53 activity, causing growth arrest, senescence, and p53-mediated apoptosis 
isoforms are highly expressed in CD34+ stem cells 
, but we find that TADA3L
is relatively methylated in IUGR at a CpG island-containing bidirectional promoter, potentially downregulating its expression and altering CD34+ stem cell population dynamics.
However, the modest level of differences in methylation that we and others have observed 
raises an important question: what is the biological significance of changes in methylation on the order of ~6%? We note that such a change must represent a difference in a proportion of cells and/or alleles undergoing methylation within the broader population of CD34+ cells. Because CD34+ stem cells are multipotent progenitors, the presence of an epigenetically dysregulated subpopulation may go on to mediate susceptibility to chronic disease, with potentially greater effects over time should this subpopulation expand. Alternatively, this stem cell population may serve to define loci susceptible to constitutive “epimutations” 
that are likely to exist in descendent cell types or unrelated lineages (e.g.
liver or pancreatic progenitors) where they may have the chance to induce functional changes in critical cell types or tissues. Adding further information about epigenetic and transcriptional regulators other than cytosine methylation plus transcriptional profiling studies will be very valuable in gaining a greater understanding of the epigenetic dysregulation and its functional consequences in IUGR.
We hypothesize that the changes we observe by focused studies of hematopoietic (CD34+) stem cells are representative of the influence of the intrauterine environment on epigenetic regulation and independent cellular programs throughout the developing fetus. While all cells are believed to accumulate epimutations over time 
, periods of rapid cell division (e.g.
during fetal development) represent the most vulnerable windows for cellular injury and potential dysregulation of the epigenome, with associated decreases in cellular fitness, function, and, of particular note for multipotent stem cells, replicative capacity. We therefore propose that adverse intrauterine conditions are more likely to contribute to replicative senescence and early exhaustion of regenerative pools of stem cell precursors throughout the body, increasing susceptibility to and speeding onset of age-related diseases like T2DM.
Although the exact mechanism remains unclear, the results of this study are indicative of epigenetic dysregulation associated with intrauterine growth restriction. Moreover, these findings suggest that epigenetic changes serve as a steward of cellular memory of aberrant intrauterine environments, and that site-specific changes in DNA methylation may mediate the increased susceptibility to age-related diseases observed later in life.
GEO database, accession number GSE17727.