Introduction
The ‘developmental origins of adult disease susceptibility’ hypothesis posits that environmental exposures early in the development permanently alter the phenotype, influencing susceptibility to lifetime risk of obesity and several cancers [
1]. These alterations are thought to be mechanistically driven by epigenetic alterations. The most stable and well-studied epigenetic mechanism is DNA methylation, which involves the covalent attachment of a methyl group to the 5-carbon position of the pyrimidine ring of cytosine residues when in 5′-CpG-3′ context. This epigenetic modification is faithfully transmitted to nascent DNA molecules during DNA replication and is maintained during somatic cell proliferation. DNA methylation contributes to the induction and maintenance of transcriptional gene silencing, propagating shifts in gene expression that orchestrate long-term susceptibility to several adenocarcinoma including colon [
2,
3], ovary [
4], prostate [
5,
6], stomach [
7], and esophagus [
8]. Epigenetic mechanisms have also been hypothesized to link birth weight, a proxy for the well-being of the offspring in utero, with some of these cancers in adulthood [
9‐
15]. However, genomic regions in which DNA methylation increases risk of these chronic diseases are still unknown. Ultimately, cancer prevention will require identification of aberrant epigenetic profiles driving risk, demonstration of their functional significance as well as environmental exposures associated with their perturbation and ‘windows’ of susceptibility during life’s course.
Identification of aberrant DNA methylation marks has been hampered by a lack of stable targets with known baseline levels, allowing for deviations from baseline to be detected. The need for epidemiologic studies to use specimens obtained from non-invasive means, such as peripheral blood leukocytes or buccal cells, which are often the only tissues available in population-based studies, has presented additional hurdles; epigenetic regulation and dysregulation can vary by tissue and cell type. Genomically imprinted genes are among several major classes of genes that are regulated by DNA methylation [
16]. Unlike most autosomal genes that follow Mendelian laws of inheritance where both parentally derived alleles are expressed, imprinted genes are monoallelically expressed, with their functional haploidy controlled by parental allele-specific DNA methylation at differentially methylated regions (DMRs). Because these methylation marks are stable covalent modifications that are established before germ layer specification [
17], these methylation profiles should be maintained in all germ layers, resulting in quantifiable systemic homogeneity in locus-specific epigenetic regulation [
17,
18]. As such, imprinted DMRs stably exhibit ~50% methylation in somatic tissues.
Insulin-
like-
growth factor-
2 (
IGF2) is perhaps the most intensively studied imprinted gene and is paternally expressed, with at least two regulatory DMRs located upstream of exon 3 and upstream of neighboring maternally expressed
H19 [
19]. Hypermethylation (methylation fractions higher than the expected ~50%) at the
H19 DMR [
2,
4] and CpG hypomethylation (methylation fractions lower than the expected ~50%) at the
IGF2 DMR [
2] have been associated with higher
IGF2 mRNA levels suggesting increased transcriptional activity, presumably via relaxation of imprint controls. Such increased transcriptional activity is a common feature in adenocarcinomas of the ovary, esophagus and prostate [
4,
8,
20‐
25].
IGF2 DMR loss of methylation was observed in DNA that was obtained from peripheral blood leukocytes of adults exposed to severe caloric restriction periconeptionally, suggesting methylation shifts in response to adverse events occur early and are stable over many decades [
26]. Based on these observations, methylation status at regulatory sequences of imprinted genes has been proposed as ‘archives’ or ‘biosensors’ of early exposure [
27,
28]. However, the functional significance of these DMR methylation marks has not previously been demonstrated in epidemiologic studies. The objective of these analyses was to examine variation in DMR methylation at sequences regulating
IGF2/H19, in relation to UCB plasma IGF2 protein concentrations. We also determined whether IGF2 protein concentrations are associated with variation in birth weight, an indicator of maternal and fetal well-being.
Discussion
We examined DNA methylation within two regulatory regions of imprinted
IGF2 in relation to circulating IGF2 concentrations in UCB of newborns. We observed a significant increase in IGF2 protein concentrations with decrease in methylation levels at the
IGF2 DMR. This association was strongest among infants born to women with a pre-pregnancy BMI > 30, similar in African Americans and Caucasians. Effect sizes of a similar magnitude have been previously reported at the
IGF2 DMR and other regions [
26,
34,
35]. We also evaluated IGF2 protein concentrations in relation to birth weight, and observed elevated IGF2 protein concentrations were associated with a higher birth weight. We found no evidence for associations between CpG methylation at the
H19 DMR and IGF2 protein concentration after adjusting for maternal obesity and race/ethnicity. We provide the first evidence for an association between DNA methylation at the
IGF2 DMR and IGF2 protein concentrations. These findings are consistent with the interpretation that DNA methylation at the
IGF2 DMR is functionally relevant to the production of the potent mitogenic growth factor IGF2 and that soluble IGF2 independently contributes to variation in birth weight.
The exact mechanism by which loss of methyl groups at the I
GF2 DMR increases IGF2 protein concentrations, exacerbated by maternal obesity, is still unknown, although changes in DNA methylation on the maternally derived allele has been shown to relax imprint controls, reactivating the otherwise silent maternally derived allele, which has been shown in previous studies to increase
IGF2 mRNA and presumably protein products. Because imprinted genes are usually found in clusters [
36] and their regulation may be networked [
37], methylation alterations of this DMR have the potential for regulating (and dysregulating) multiple genes on different chromosomes. Therefore, methylation at this single DMR can provide information on epigenetic dysregulation elsewhere in the genome [
18]. Using DNA obtained from leukocytes, methylation differences of a similar magnitude were reported among Gambians [
17] and Dutch [
26] adults who were exposed to nutritional challenges periconceptionally. Together, these findings support the idea that methylation marks acquired in the periconceptional period is similar across tissue types and is stable over many years. The stability of DNA methylation marks at the
IGF2 DMR during a 3-year period was also recently reported among adult population controls [
38]. The small magnitude of differences is also consistent with the contention that methylation marks acquired as a result of adjustments to perceived environment may not be as large as those found in cancer [
39,
40]). Strong inverse associations between DNA methylation levels at the
IGF2 DMR using DNA obtained from leukocytes, biallelic expression of this otherwise monoallelically expressed gene, and elevated colon cancer risk [
2,
38,
41] have been reported. In tissue specimens, hypermethylation at the
H19 DMR is also frequent in cancers of the ovary [
42], breast [
43], esophagus [
44], and prostate [
45]. The
IGF2 and
H19 DMRs have been previously proposed as biomarkers, biosensors, or archives of early exposures [
2,
27,
28]. Therefore, our findings of a strong relationship between lower methylation at the
IGF2 DMR and elevated IGF2 protein levels support the use of DNA methylation levels at this and perhaps other DMRs as biosensors or archives of early exposure. Differences observed by maternal obesity may be partly explained by genetic factors that also play a role in modulating IGF2 levels in the offspring.
Findings that protein levels of the mitogenic IGF2 are strongly associated with decreased methylation at the
IGF2 DMR and with birth weight support the interpretation that variation in birth weight is modulated, at least in part, by
IGF2 plasticity. This may also be occurring at other DMRs, if hypothesized co-regulation with other imprinted regions [
37] can be demonstrated. Previous studies have reported an association between circulating IGF2 protein concentrations and high birth weight [
46] as well as risk of obesity [
47] and several cancers [
48,
49] in adulthood, suggesting early exposures may contribute to the variation in birth weight via epigenetic alterations.
Our inability to find a consistent association between
H19 DMR methylation and IGF2 protein levels except among the 188 infants born to women with BMI < 30 was unexpected. Current thinking for transcriptional control of the imprinted
IGF2/H19 domain correlates increased DNA methylation at the
H19 DMR with failure to bind methylation sensitive CTCF, which can transcriptionally activate
IGF2 on the normally silent maternal chromosome [
50‐
52]. We cannot exclude the possibility that these were chance findings, since the statistical power to conduct multivariable analyses may have been inadequate. However, others have also failed to find a relationship between methylation at the
H19 DMR and
IGF2 transcription among children conceived in vitro and in vivo [
52] suggesting that at the
H19 DMR, other processes including genetic factors or other unmeasured environmental stimuli may contribute to increased IGF2 protein concentrations and body size, independent of DNA methylation alterations.
The main limitation of this study is the small sample size which, although more than adequate for examining crude relationships among DMR methylation fractions, protein IGF2 concentrations and birth weight, may have been inadequate for multivariable analyses, especially when testing interaction by race and maternal BMI. Despite the need for larger studies to clarify these relationships, the analyses raised the possibility that maternal obesity, which may be driven by other factors including genetic variation, also plays a role in modulating IGF2 concentrations. This suggests that a more detailed understanding of these epigenetic targets will require genetic analyses within the sequences under evaluation. Another limitation is that DNA obtained from unfractionated blood containing multiple cell types may have distinct epigenetic profiles depending on the cell population analyzed. However, we have previously described the analysis of fractionated cord blood for methylation at the same
H19 and
IGF2 DMRs, and the major cell types showed no significant differences in methylation levels [
30]. Additionally, overall white blood cell counts vary and could confound the methylation fraction measures. However, inclusion of chorioamnionitis during parturition into statistical models did not alter our findings, suggesting variation in total cell counts may not contribute to differences in methylation at these DMRs. Furthermore, although we demonstrated an association between DMR methylation and protein levels downstream, we were limited by a lack of transcription data. However, a positive correlation between the levels of
IGF2 mRNA and DNA methylation at these DMRs has been previously reported. These limitations notwithstanding, our findings suggest that
IGF2 DMR plasticity is an important mechanism by which IGF2 protein levels are modulated.
In summary, among 300 newborns, we found a strong inverse association between DNA methylation at the IGF2 regulatory sequence and elevated plasma protein concentrations of the encoded mitogenic growth factor. This relationship appeared to be stronger in infants born to women who were obese before pregnancy, but not weight gain. Higher IGF2 protein concentrations were also associated with higher birth weight. Herein, we provide evidence in support of the functional significance of aberrant methylation profiles at regulatory elements controlling the expression of imprinted IGF2, supporting the use of IGF2 DMR methylation as an archive or biosensor of early exposure. If co-regulation of imprinted domains can be demonstrated in humans, and transcripts are also associated with DNA methylation and protein levels for other imprinted genes in the same population, our findings would support the use of methylation marks in an imprint regulatory network to improve exposure assessment in studies of cancer and other chronic diseases.
Acknowledgments
We thank the parents and children of the Newborn Epigenetics Study, research nurse Tammy Bishop, and research personnel: Stacy Murray, Carole Grenier, Darby Kroyer, Natasha Duggan, Suba Narasimhan, and James Nathan Yarnall for enrolling and tracing participants, and processing the specimens; and the obstetrics faculty and staff at Duke University and Durham regional Hospitals, Durham, NC. This work was supported in part by R21ES014947, R01ES016772, R01DK085173, K01CA104517 the American Cancer Society (grant number ACS-IRG-83-006) and the Fred and Alice Stanback Foundation. Dr. Susan Murphy was also supported by the Duke Comprehensive Cancer Center and Duke Environmental Health Sciences Research Center.