Erythrocyte folate concentrations,CpG methylation at genomically imprinted domains,and birth weight in a multiethnic newborn cohort

Epigenetic mechanisms are proposed to link maternal concentrations of methyl group donor nutrients with the risk of low birth weight. However, empirical data are lacking. We have examined the association between maternal folate and birth weight and assessed the mediating role of DNA methylation at nine differentially methylated regions (DMRs) of genomically imprinted genes in these associations. Compared with newborns of women with folate levels in the lowest quartile, birth weight was higher in newborns of mothers in the second (β = 143.2, se = 63.2, P = 0.02), third (β = 117.3, se = 64.0, P = 0.07), and fourth (β = 133.9, se = 65.2, P = 0.04) quartiles, consistent with a threshold effect. This pattern of association did not vary by race/ethnicity but was more apparent in newborns of non-obese women. DNA methylation at the PLAGL1, SGCE, DLK1/MEG3 and IGF2/H19 DMRs was associated with maternal folate levels and also birth weight, suggestive of threshold effects. MEG3 DMR methylation mediated the association between maternal folate levels and birth weight (P =0.06). While the small sample size and partial scope of examined DMRs limit our conclusions, our data suggest that, with respect to birth weight, no additional benefits may be derived from increased maternal folate concentrations, especially in non-obese women. These data also support epigenetic plasticity as a key mechanistic response to folate availability during early fetal development.     

Erythrocyte folate concentrations,CpG methylation at genomically imprinted domains,and birth weight in a multiethnic newborn cohort

Epigenetic mechanisms are proposed to link maternal concentrations of methyl group donor nutrients with the risk of low birth weight. However, empirical data are lacking. We have examined the association between maternal folate and birth weight and assessed the mediating role of DNA methylation at nine differentially methylated regions (DMRs) of genomically imprinted genes in these associations. Compared with newborns of women with folate levels in the lowest quartile, birth weight was higher in newborns of mothers in the second (β = 143.2, se = 63.2, P = 0.02), third (β = 117.3, se = 64.0, P = 0.07), and fourth (β = 133.9, se = 65.2, P = 0.04) quartiles, consistent with a threshold effect. This pattern of association did not vary by race/ethnicity but was more apparent in newborns of non-obese women. DNA methylation at the PLAGL1, SGCE, DLK1/MEG3 and IGF2/H19 DMRs was associated with maternal folate levels and also birth weight, suggestive of threshold effects. MEG3 DMR methylation mediated the association between maternal folate levels and birth weight (P =0.06). While the small sample size and partial scope of examined DMRs limit our conclusions, our data suggest that, with respect to birth weight, no additional benefits may be derived from increased maternal folate concentrations, especially in non-obese women. These data also support epigenetic plasticity as a key mechanistic response to folate availability during early fetal development.     

Depression in pregnancy,infant birth weight and DNA methylation of imprint regulatory elements

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.     

DNA methylation of IGF2, GNASAS,INSIGF and LEP and being born small for gestational age

Being born small for gestational age (SGA), a proxy for intrauterine growth restriction (IUGR) and prenatal famine exposure are both associated with a greater risk of metabolic disease. Both associations have been hypothesized to involve epigenetic mechanisms. We investigated whether prenatal growth restriction early in pregnancy was associated with changes in DNA methylation at loci that were previously shown to be sensitive to early gestational famine exposure. We compared 38 individuals born preterm (<32 weeks) and with a birth weight too low for their gestational age (less than −1SDS; SGA) with 75 individuals born preterm but with a birth weight appropriate for their gestational age (greater than −1SDS) and a normal postnatal growth (greater than −1SDS at three months post term; AGA). The SGA individuals were not only lighter at birth, but also had a smaller length (p = 3.3 × 10⁻¹³) and head circumference at birth (p = 4.1 × 10⁻¹³). The DNA methylation levels of IGF2, GNASAS, INSIGF and LEP were 48.5, 47.5, 79.4 and 25.7% respectively. This was not significantly different between SGA and AGA individuals. Risk factors for being born SGA, including preeclampsia and maternal smoking, were also not associated with DNA methylation at these loci. Growth restriction early in development is not associated with DNA methylation at loci shown to be affected by prenatal famine exposure. Our and previous results by others indicate that prenatal growth restriction and famine exposure may be associated with different epigenetic changes or non-epigenetic mechanisms that may lead to similar later health outcomes.Key words: SGA, DOHAD, IUGR, DNA methylation, famine, IGF2, LEP, INS, GNASAS     

Glycogen synthase kinase-3 (Gsk-3) plays a fundamental role in maintaining DNA methylation at imprinted loci in mouse embryonic stem cells

Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α^−/−;Gsk-3β^−/− ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α^−/−;Gsk-3β^−/− ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3–dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3–mediated signal transduction in the epigenome.     

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications.     

Methylation variation at IGF2 differentially methylated regions and maternal folic acid use before and during pregnancy

Folic acid (FA) supplementation before and during pregnancy has been associated with decreased risk of neural tube defects although recent reports suggest it may also increase the risk of other chronic diseases. We evaluated exposure to maternal FA supplementation before and during pregnancy in relation to aberrant DNA methylation at two differentially methylated regions (DMRs) regulating insulin-like growth factor 2 (IGF2) expression in infants. Aberrant methylation at these regions has been associated with IGF2 deregulation and increased susceptibility to several chronic diseases. Using a self-administered questionnaire, we assessed FA intake before and during pregnancy in 438 pregnant women. Pyrosequencing was used to measure methylation at two IGF2 DMRs in umbilical cord blood leukocytes. Mixed models were used to determine relationships between maternal FA supplementation before or during pregnancy and DNA methylation levels at birth. Average methylation at the H19 DMR was 61.2%. Compared to infants born to women reporting no FA intake before or during pregnancy, methylation levels at the H19 DMR decreased with increasing FA intake (2.8%, p = 0.03 and 4.9%, p = 0.04, for intake before and during pregnancy, respectively). This methylation decrease was most pronounced in male infants (p = 0.01). Methylation alterations at the H19 DMR are likely an important mechanism by which FA risks and/or benefits are conferred in utero. Because stable methylation marks at DMRs regulating imprinted genes are acquired before gastrulation, they may serve as archives of early exposures with the potential to improve our understanding of developmental origins of adult disease.Key words: folic acid, epigenetics, IGF2, periconception, prenatal, exposure     

Depression in pregnancy,infant birth weight and DNA methylation of imprint regulatory elements

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.     

Prenatal antidepressant exposure associated with CYP2E1 DNA methylation change in neonates

Some but not all neonates are affected by prenatal exposure to serotonin reuptake inhibitor antidepressants (SRI) and maternal mood disturbances. Distinguishing the impact of these 2 exposures is challenging and raises critical questions about whether pharmacological, genetic, or epigenetic factors can explain the spectrum of reported outcomes. Using unbiased DNA methylation array measurements followed by a detailed candidate gene approach, we examined whether prenatal SRI exposure was associated with neonatal DNA methylation changes and whether such changes were associated with differences in birth outcomes. Prenatal SRI exposure was first associated with increased DNA methylation status primarily at CYP2E1(β_Non-exposed = 0.06, β_SRI-exposed = 0.30, FDR = 0); however, this finding could not be distinguished from the potential impact of prenatal maternal depressed mood. Then, using pyrosequencing of CYP2E1 regulatory regions in an expanded cohort, higher DNA methylation status—both the mean across 16 CpG sites (P < 0.01) and at each specific CpG site (P < 0.05)—was associated with exposure to lower 3rd trimester maternal depressed mood symptoms only in the SRI-exposed neonates, indicating a maternal mood x SRI exposure interaction. In addition, higher DNA methylation levels at CpG2 (P = 0.04), CpG9 (P = 0.04) and CpG10 (P = 0.02), in the interrogated CYP2E1 region, were associated with increased birth weight independently of prenatal maternal mood, SRI drug exposure, or gestational age at birth. Prenatal SRI antidepressant exposure and maternal depressed mood were associated with altered neonatal CYP2E1 DNA methylation status, which, in turn, appeared to be associated with birth weight.     

Associations of maternal dietary inflammatory potential and quality with offspring birth outcomes: An individual participant data pooled analysis of 7 European cohorts in the ALPHABET consortium

BackgroundAdverse birth outcomes are major causes of morbidity and mortality during childhood and associate with a higher risk of noncommunicable diseases in adult life. Maternal periconception and antenatal nutrition, mostly focusing on single nutrients or foods, has been shown to influence infant birth outcomes. However, evidence on whole diet that considers complex nutrient and food interaction is rare and conflicting. We aim to elucidate the influence of whole-diet maternal dietary inflammatory potential and quality during periconceptional and antenatal periods on birth outcomes.Methods and findingsWe harmonized and pooled individual participant data (IPD) from up to 24,861 mother–child pairs in 7 European mother–offspring cohorts [cohort name, country (recruitment dates): ALSPAC, UK (1 April 1991 to 31 December 1992); EDEN, France (27 January 2003 to 6 March 2006); Generation R, the Netherlands (1 April 2002 to 31 January 2006); Lifeways, Ireland (2 October 2001 to 4 April 2003); REPRO_PL, Poland (18 September 2007 to 16 December 2011); ROLO, Ireland (1 January 2007 to 1 January 2011); SWS, United Kingdom (6 April 1998 to 17 December 2002)]. Maternal diets were assessed preconceptionally (n = 2 cohorts) and antenatally (n = 7 cohorts). Maternal dietary inflammatory potential and quality were ranked using the energy-adjusted Dietary Inflammatory Index (E-DII) and Dietary Approaches to Stop Hypertension (DASH) index, respectively. Primary outcomes were birth weight and gestational age at birth. Adverse birth outcomes, i.e., low birth weight (LBW), macrosomia, small-for-gestational-age (SGA), large-for-gestational-age (LGA), preterm and postterm births were defined according to standard clinical cutoffs. Associations of maternal E-DII and DASH scores with infant birth outcomes were assessed using cohort-specific multivariable regression analyses (adjusted for confounders including maternal education, ethnicity, prepregnancy body mass index (BMI), maternal height, parity, cigarettes smoking, and alcohol consumption), with subsequent random-effects meta-analyses.Overall, the study mothers had a mean ± SD age of 29.5 ± 4.9 y at delivery and a mean BMI of 23.3 ± 4.2 kg/m². Higher pregnancy DASH score (higher dietary quality) was associated with higher birth weight [β(95% CI) = 18.5(5.7, 31.3) g per 1-SD higher DASH score; P value = 0.005] and head circumference [0.03(0.01, 0.06) cm; P value = 0.004], longer birth length [0.05(0.01, 0.10) cm; P value = 0.010], and lower risk of delivering LBW [odds ratio (OR) (95% CI) = 0.89(0.82, 0.95); P value = 0.001] and SGA [0.87(0.82, 0.94); P value < 0.001] infants. Higher maternal prepregnancy E-DII score (more pro-inflammatory diet) was associated with lower birth weight [β(95% CI) = −18.7(−34.8, −2.6) g per 1-SD higher E-DII score; P value = 0.023] and shorter birth length [−0.07(−0.14, −0.01) cm; P value = 0.031], whereas higher pregnancy E-DII score was associated with a shorter birth length [−0.06(−0.10, −0.01) cm; P value = 0.026] and higher risk of SGA [OR(95% CI) = 1.18(1.11, 1.26); P value < 0.001]. In male, but not female, infants higher maternal prepregnancy E-DII was associated with lower birth weight and head circumference, shorter birth length, and higher risk of SGA (P-for-sex-interaction = 0.029, 0.059, 0.104, and 0.075, respectively). No consistent associations were observed for maternal E-DII and DASH scores with gestational age, preterm and postterm birth, or macrosomia and LGA. Limitations of this study were that self-reported dietary data might have increased nondifferential measurement error and that causality cannot be claimed definitely with observational design.ConclusionsIn this cohort study, we observed that maternal diet that is of low quality and high inflammatory potential is associated with lower offspring birth size and higher risk of offspring being born SGA in this multicenter meta-analysis using harmonized IPD. Improving overall maternal dietary pattern based on predefined criteria may optimize fetal growth and avert substantial healthcare burden associated with adverse birth outcomes.

In this cohort analysis, Ling-Wei Chen and colleagues explore associations of maternal dietary patterns with offspring birth outcomes.     

Placental FKBP5 Genetic and Epigenetic Variation Is Associated with Infant Neurobehavioral Outcomes in the RICHS Cohort

Adverse maternal environments can lead to increased fetal exposure to maternal cortisol, which can cause infant neurobehavioral deficits. The placenta regulates fetal cortisol exposure and response, and placental DNA methylation can influence this function. FK506 binding protein (FKBP5) is a negative regulator of cortisol response, FKBP5 methylation has been linked to brain morphology and mental disorder risk, and genetic variation of FKBP5 was associated with post-traumatic stress disorder in adults. We hypothesized that placental FKBP5 methylation and genetic variation contribute to gene expression control, and are associated with infant neurodevelopmental outcomes assessed using the Neonatal Intensive Care Unit (NICU) Network Neurobehavioral Scales (NNNS). In 509 infants enrolled in the Rhode Island Child Health Study, placental FKBP5 methylation was measured at intron 7 using quantitative bisulfite pyrosequencing. Placental FKBP5 mRNA was measured in a subset of 61 infants by quantitative PCR, and the SNP rs1360780 was genotyped using a quantitative allelic discrimination assay. Relationships between methylation, expression and NNNS scores were examined using linear models adjusted for confounding variables, then logistic models were created to determine the influence of methylation on membership in high risk groups of infants. FKBP5 methylation was negatively associated with expression (P = 0.08, r = −0.22); infants with the TT genotype had higher expression than individuals with CC and CT genotypes (P = 0.06), and those with CC genotype displayed a negative relationship between methylation and expression (P = 0.06, r = −0.43). Infants in the highest quartile of FKBP5 methylation had increased risk of NNNS high arousal compared to infants in the lowest quartile (OR 2.22, CI 1.07–4.61). TT genotype infants had increased odds of high NNNS stress abstinence (OR 1.98, CI 0.92–4.26). Placental FKBP5 methylation reduces expression in a genotype specific fashion, and genetic variation supersedes this effect. These genetic and epigenetic differences in expression may alter the placenta’s ability to modulate cortisol response and exposure, leading to altered neurobehavioral outcomes.     

Differentially methylated regions of imprinted genes in prenatal, perinatal and postnatal human tissues

Epigenetic plasticity in relation to in utero exposures may mechanistically explain observed differences in the likelihood of developing common complex diseases including hypertension, diabetes and cardiovascular disease through the cumulative effects of subtle alterations in gene expression. Imprinted genes are essential mediators of growth and development and are characterized by differentially methylated regulatory regions (DMRs) that carry parental allele-specific methylation profiles. This theoretical 50% level of methylation provides a baseline from which endogenously- or exogenously-induced deviations in methylation can be detected. We quantified DNA methylation at imprinted gene DMRs in a large panel of human conceptal tissues, in matched buccal cell specimens collected at birth and at one year of age, and in the major cell fractions of umbilical cord blood to assess the stability of methylation at these regions. DNA methylation was measured using validated pyrosequencing assays at seven DMRs regulating the IGF2/H19, DLK1/MEG3, MEST, NNAT and SGCE/PEG10 imprinted domains. DMR methylation did not significantly differ for the H19, MEST and SGCE/PEG10 DMRs across all conceptal tissues analyzed (ANOVA p>0.10). Methylation differences at several DMRs were observed in tissues from brain (IGF2 and MEG3-IG DMRs), liver (IGF2 and MEG3 DMRs) and placenta (both DLK1/MEG3 DMRs and NNAT DMR). In most infants, methylation profiles in buccal cells at birth and at one year of age were comparable, as was methylation in the major cell fractions of umbilical cord blood. Several infants showed temporal deviations in methylation at multiple DMRs. Similarity of inter-individual and intra-individual methylation at some, but not all of the DMRs analyzed supports the possibility that methylation of these regions can serve as useful biosensors of exposure.     

An epigenome-wide association meta-analysis of prenatal maternal stress in neonates: A model approach for replication

Prenatal maternal stress exposure has been associated with neonatal differential DNA methylation. However, the available evidence in humans is largely based on candidate gene methylation studies, where only a few CpG sites were evaluated. The aim of this study was to examine the association between prenatal exposure to maternal stress and offspring genome-wide cord blood methylation using different methods. First, we conducted a meta-analysis and follow-up pathway analyses. Second, we used novel region discovery methods [i.e., differentially methylated regions (DMRs) analyses]. To this end, we used data from two independent population-based studies, the Generation R Study (n = 912) and the Avon Longitudinal Study of Parents and Children (ALSPAC, n = 828), to (i) measure genome-wide DNA methylation in cord blood and (ii) extract a prenatal maternal stress composite. The meta-analysis (n_total = 1,740) revealed no epigenome-wide (meta P <1.00e-07) associations of prenatal maternal stress exposure with neonatal differential DNA methylation. Follow-up analyses of the top hits derived from our epigenome-wide meta-analysis (meta P <1.00e-04) indicated an over-representation of the methyltransferase activity pathway. We identified no Bonferroni-corrected (P <1.00e-06) DMRs associated with prenatal maternal stress exposure. Combining data from two independent population-based samples in an epigenome-wide meta-analysis, the current study indicates that there are no large effects of prenatal maternal stress exposure on neonatal DNA methylation. Such replication efforts are essential in the search for robust associations, whether derived from candidate gene methylation or epigenome-wide studies.     

DNA Methylation of IGF2DMR and H19 Is Associated with Fetal and Infant Growth: The Generation R Study

Changes in epigenetic programming of embryonic growth genes during pregnancy seem to affect fetal growth. Therefore, in a population-based prospective birth cohort in the Netherlands, we examined associations between fetal and infant growth and DNA methylation of IGF2DMR, H19 and MTHFR. For this study, we selected 69 case children born small-for-gestational age (SGA, birth weight <-2SDS) and 471 control children. Fetal growth was assessed with serial ultrasound measurements. Information on birth outcomes was retrieved from medical records. Infant weight was assessed at three and six months. Methylation was assessed in DNA extracted from umbilical cord white blood cells. Analyses were performed using linear mixed models with DNA methylation as dependent variable. The DNA methylation levels of IGF2DMR and H19 in the control group were, median (90% range), 53.6% (44.5–61.6) and 30.0% (25.6–34.2) and in the SGA group 52.0% (43.9–60.9) and 30.5% (23.9–32.9), respectively. The MTHFR region was found to be hypomethylated with limited variability in the control and SGA group, 2.5% (1.4–4.0) and 2.4% (1.5–3.8), respectively. SGA was associated with lower IGF2DMR DNA methylation (β = −1.07, 95% CI −1.93; −0.21, P-value = 0.015), but not with H19 methylation. A weight gain in the first three months after birth was associated with lower IGF2DMR DNA methylation (β = −0.53, 95% CI −0.91; −0.16, P-value = 0.005). Genetic variants in the IGF2/H19 locus were associated with IGF2DMR DNA methylation (P-value<0.05), but not with H19 methylation. Furthermore, our results suggest a possibility of mediation of DNA methylation in the association between the genetic variants and SGA. To conclude, IGF2DMR and H19 DNA methylation is associated with fetal and infant growth.     

In utero arsenic exposure and epigenome-wide associations in placenta,umbilical artery,and human umbilical vein endothelial cells

Exposure to arsenic early in life has been associated with increased risk of several chronic diseases and is believed to alter epigenetic programming in utero. In the present study, we evaluate the epigenome-wide association of arsenic exposure in utero and DNA methylation in placenta (n = 37), umbilical artery (n = 45) and human umbilical vein endothelial cells (HUVEC) (n = 52) in a birth cohort using the Infinium HumanMethylation450 BeadChip array. Unadjusted and cell mixture adjusted associations for each tissue were examined along with enrichment analyses relative to CpG island location and omnibus permutation tests of association among biological pathways. One CpG in artery (cg26587014) and 4 CpGs in placenta (cg12825509; cg20554753; cg23439277; cg21055948) reached a Bonferroni adjusted level of significance. Several CpGs were differentially methylated in artery and placenta when controlling the false discovery rate (q-value<0.05), but none in HUVEC. Enrichment of hypomethylated CpG islands was observed for artery while hypermethylation of open sea regions were present in placenta relative to prenatal arsenic exposure. The melanogenesis pathway was differentially methylated in artery (Max F P < 0.001), placenta (Max F P < 0.001), and HUVEC (Max F P = 0.02). Similarly, the insulin-signaling pathway was differentially methylated in artery (Max F P = 0.02), placenta (Max F P = 0.02), and HUVEC (Max F P = 0.02). Our results show that prenatal arsenic exposure can alter DNA methylation in artery and placenta but not in HUVEC. Further studies are needed to determine if these alterations in DNA methylation mediate the effect of prenatal arsenic exposure and health outcomes later in life.     

Non-germ Line Restoration of Genomic Imprinting for a Small Subset of Imprinted Genes in Ubiquitin-like PHD and RING Finger Domain-Containing 1 (Uhrf1) Null Mouse Embryonic Stem Cells

The underlying mechanism for the establishment and maintenance of differential DNA methylation in imprinted genes is largely unknown. Previous studies using Dnmt1 knock-out embryonic stem (ES) cells demonstrated that, although re-expression of DNMT1 restored DNA methylation in the non-imprinted regions, the methylation patterns of imprinted genes could be restored only through germ line passage. Knock-out of Uhrf1, an accessory factor essential for DNMT1-mediated DNA methylation, in mouse ES cells also led to impaired global DNA methylation and loss of genomic imprinting. Here, we demonstrate that, although re-expression of UHRF1 in Uhrf1^−/− ES cells restored DNA methylation for the bulk genome but not for most of the imprinted genes, it did rescue DNA methylation for the imprinted H19, Nnat, and Dlk1 genes. Analysis of histone modifications at the differential methylated regions of the imprinted genes by ChIP assays revealed that for the imprinted genes whose DNA methylation could be restored upon re-expression of UHRF1, the active histone markers (especially H3K4me3) were maintained at considerably low levels, and low levels were maintained even in Uhrf1^−/− ES cells. In contrast, for the imprinted genes whose DNA methylation could not be restored upon UHRF1 re-expression, the active histone markers (especially H3K4me3) were relatively high and became even higher in Uhrf1^−/− ES cells. Our study thus supports a role for histone modifications in determining the establishment of imprinting-related DNA methylation and demonstrates that mouse ES cells can be a valuable model for mechanistic study of the establishment and maintenance of differential DNA methylation in imprinted genes.     

DNA Methyltransferase Candidate Polymorphisms,Imprinting Methylation,and Birth Outcome

Background

Birth weight and prematurity are important obstetric outcomes linked to lifelong health. We studied a large birth cohort to look for evidence of epigenetic involvement in birth outcomes.

Methods

We investigated the association between birth weight, length, placental weight and duration of gestation and four candidate variants in 1,236 mothers and 1,073 newborns; DNMT1 (rs2162560), DNMT3A (rs734693), DNMT3B (rs2424913) and DNMT3L (rs7354779). We measured methylation of LINE1 and the imprinted genes, PEG3, SNRPN, and IGF2, in cord blood.

Results

The minor DNMT3L allele in the baby was associated with higher birth weight (+54 95% CI 10,99 g; p = 0.016), birth length (+0.23 95% CI 0.04,0.42 cm; p = 0.017), placental weight, (+18 95% CI 3,33 g; p = 0.017), and reduced risk of being in the lowest birth weight decile (p = 0.018) or requiring neonatal care (p = 0.039). The DNMT3B minor allele in the mother was associated with an increased risk of prematurity (p = 0.001). Placental size was related to PEG3 (p<0.001) and IGF2 (p<0.001) methylation. Birth weight was related to LINE1 and IGF2 methylation but only at p = 0.052. The risk of requiring neonatal treatment was related to LINE1 (p = 0.010) and SNRPN (p = 0.001) methylation. PEG3 methylation was influenced by baby DNMT3A genotype (p = 0.012) and LINE1 by baby 3B genotype (p = 0.044). Maternal DNMT3L genotype was related to IGF2 methylation in the cord blood but this effect was only seen in carriers of the minor frequency allele (p = 0.050).

Conclusions

The results here suggest that epigenetic processes are linked birth outcome and health in early life. Our emerging understanding of the role of epigenetics in health and biological function across the lifecourse suggests that these early epigenetic events could have longer term implications.     

Disruption of imprinted gene expression and DNA methylation status in porcine parthenogenetic fetuses and placentas

Parthenogenetically activated oocytes cannot develop to term in mammals due to the lack of paternal gene expression and failed X chromosome inactivation (XCI). To further characterize porcine parthenogenesis, the expression of 18 imprinted genes was compared between parthenogenetic (PA) and normally fertilized embryos (Con) using quantitative real-time PCR (qRT-PCR). The results revealed that maternally expressed genes were over-expressed, whereas paternally expressed genes were significantly reduced in PA fetuses and placentas. The results of bisulfite sequencing PCR (BSP) demonstrated that PRE-1 and Satellite were hypermethylated in both Con and PA fetuses and placentas, while XIST DMRs were hypomethylated only in PA samples. Taken together, these results suggest that the aberrant methylation profile of XIST DMRs and abnormal imprinted gene expression may be responsible for developmental failure and impaired growth in porcine parthenogenesis.