Background

Infant birth weight is a complex quantitative trait associated with both neonatal and long-term health outcomes. Numerous studies have been published in which candidate genes (IGF1, IGF2, IGF2R, IGF binding proteins, PHLDA2 and PLAGL1) have been associated with birth weight, but these studies are difficult to reproduce in man and large cohort studies are needed due to the large inter individual variance in transcription levels. Also, very little of the trait variance is explained. We decided to identify additional candidates without regard for what is known about the genes. We hypothesize that DNA methylation differences between individuals can serve as markers of gene "expression potential" at growth related genes throughout development and that these differences may correlate with birth weight better than single time point measures of gene expression.

Methods

We performed DNA methylation and transcript profiling on cord blood and placenta from newborns. We then used novel computational approaches to identify genes correlated with birth weight.

Results

We identified 23 genes whose methylation levels explain 70-87% of the variance in birth weight. Six of these (ANGPT4, APOE, CDK2, GRB10, OSBPL5 and REG1B) are associated with growth phenotypes in human or mouse models. Gene expression profiling explained a much smaller fraction of variance in birth weight than did DNA methylation. We further show that two genes, the transcriptional repressor MSX1 and the growth factor receptor adaptor protein GRB10, are correlated with transcriptional control of at least seven genes reported to be involved in fetal or placental growth, suggesting that we have identified important networks in growth control. GRB10 methylation is also correlated with genes involved in reactive oxygen species signaling, stress signaling and oxygen sensing and more recent data implicate GRB10 in insulin signaling.

Conclusions

Single time point measurements of gene expression may reflect many factors unrelated to birth weight, while inter-individual differences in DNA methylation may represent a "molecular fossil record" of differences in birth weight-related gene expression. Finding these "unexpected" pathways may tell us something about the long-term association between low birth weight and adult disease, as well as which genes may be susceptible to environmental effects. These findings increase our understanding of the molecular mechanisms involved in human development and disease progression.

Epidemiological data indicate that children conceived in vitro have a greater relative risk of low birth-weight, major and minor birth defects, and rare disorders involving imprinted genes, suggesting that epigenetic changes may be associated with assisted reproduction. We examined DNA methylation at more than 700 genes (1536 CpG sites) in placenta and cord blood and measured gene expression levels of a subset of genes that differed in methylation levels between children conceived in vitro versus in vivo. Our results suggest that in vitro conception is associated with lower mean methylation at CpG sites in placenta and higher mean methylation at CpG sites in cord blood. We also find that in vitro conception-associated DNA methylation differences are associated with gene expression differences at both imprinted and non-imprinted genes. The range of inter-individual variation in gene expression of the in vitro and in vivo groups overlaps substantially but some individuals from the in vitro group differ from the in vivo group mean by more than two standard deviations. Several of the genes whose expression differs between the two groups have been implicated in chronic metabolic disorders, such as obesity and type II diabetes. These findings suggest that there may be epigenetic differences in the gametes or early embryos derived from couples undergoing treatment for infertility. Alternatively, assisted reproduction technology may have an effect on global patterns of DNA methylation and gene expression. In either case, these differences or changes may affect long-term patterns of gene expression.

Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART), suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs) of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth.

Author Summary

We have screened a population of children conceived in vitro for epigenetic alterations at two loci that carry parent-of-origin specific methylation marks. We made the observation that epigenetic variability was greater in extraembryonic tissues than embryonic tissues in both groups, as has also been demonstrated in the mouse. The greater level of intra-individual variation in extraembryonic tissues of the in vitro group appears to result from these embryos having fewer trophoblast stem cells. We also made the unexpected observation that variability in parental origin-dependent epigenetic marking was poorly correlated with gene expression. In fact, there is such a high level of inter-individual variation in IGF2 transcript level that the presumed half-fold reduction in IGF2 mRNA accounted for by proper transcriptional imprinting versus complete loss of imprinting would account for less than 5% of the total population variance. Given this level of variability in the expression of an imprinted gene, the presumed operation of “parental conflict” as the selective force acting to maintain imprinted gene expression at the IGF2/H19 locus in the human should be revisited.

The hypothesis that environmental factors alter somatically heritable epigenetic marks and change long-term patterns of gene expression is an exciting possibility in human disease research. Because most common diseases, and many quantitative traits, are influenced by both genetic and environmental factors, environmentally induced changes in epigenetic structures can provide a mechanistic link between genes and environment. We believe that inter-individual differences in the epigenetic modification of genes will explain a much greater fraction of inter-individual phenotypic variation than differences in genotype, alone.

Background

Sulfation plays an important role both in detoxification and in the control of steroid activity. Studies in rodents have shown that the conversion of dehydroepiandrosterone (DHEA) to DHEA-sulfate is involved in learning and the memory process.

Methods

The effects of a range of plasticizers and related compounds commonly encountered in the environment were evaluated kinetically against human DHEA sulfotransferase (SULT 2A1) and by reverse transcriptase-polymerase chain reaction (RT-PCR) against several enzymes involved in the synthesis of the sulfotransferase cofactor adenosine 3′-phosphate 5′-phosphosulfate (PAPS).

Results

We found that several of the chemicals acted as competitive inhibitors of SULT 2A1 (Ki for 4-tert-octylphenol is 2.8 μM). Additionally, after treatment of TE 671 cells with 0.005–0.5 μM 4-n-octylphenol, bis(2-ethylhexyl)phthalate, and diisodecyl phthalate, real-time RT-PCR showed dose-dependent decreases in the steady-state mRNA levels of cysteine dioxygenase type I, sulfite oxidase, and 3′-phosphate 5′-phosphosulfate synthase I.

Conclusions

These data suggest that environmental contaminants may exert effects on neuronal function both by direct inhibition of sulfotransferase enzymes and by interrupting the supply of PAPS, which has wider implications for endocrine disruption and xenobiotic metabolism.