Postnatal stability,tissue, and time specific effects of AHRR methylation change in response to maternal smoking in pregnancy

The intrauterine environment has the potential to “program” the developing fetus in a way that can be potentially deleterious to later health. While in utero environmental/stochastic factors are known to influence DNA methylation profile at birth, it has been difficult to assign specific examples of epigenetic variation to specific environmental exposures. Recently, several studies have linked exposure to smoking with DNA methylation change in the aryl hydrocarbon receptor repressor (AHRR) gene in blood. This includes hypomethylation of AHRR in neonatal blood in response to maternal smoking in pregnancy. The role of AHRR as a negative regulator of pathways involved in pleiotropic responses to environmental contaminants raises the possibility that smoking-induced hypomethylation is an adaptive response to an adverse in utero environmental exposure. However, the tissue specificity of the response to maternal smoking, and the stability of the methylation changes early in life remain to be determined. In this study we analyzed AHRR methylation in three cell types—cord blood mononuclear cells (CBMCs), buccal epithelium, and placenta tissue—from newborn twins of mothers who smoked throughout pregnancy and matched controls. Further, we explored the postnatal stability of this change at 18 months. Our results confirm the previous association between maternal smoking and AHRR methylation in neonatal blood. In addition, this study expands the region of AHRR methylation altered in response to maternal smoking during pregnancy and reveals the tissue-specific nature of epigenetic responses to environmental exposures in utero. Further, the evidence for postnatal stability of smoking-induced epigenetic change supports a role for epigenetics as a mediator of long-term effects of specific in utero exposures in humans. Longitudinal analysis of further specific exposures in larger cohorts is required to examine the extent of this phenomenon in humans.     

Postnatal stability,tissue, and time specific effects of AHRR methylation change in response to maternal smoking in pregnancy

The intrauterine environment has the potential to “program” the developing fetus in a way that can be potentially deleterious to later health. While in utero environmental/stochastic factors are known to influence DNA methylation profile at birth, it has been difficult to assign specific examples of epigenetic variation to specific environmental exposures. Recently, several studies have linked exposure to smoking with DNA methylation change in the aryl hydrocarbon receptor repressor (AHRR) gene in blood. This includes hypomethylation of AHRR in neonatal blood in response to maternal smoking in pregnancy. The role of AHRR as a negative regulator of pathways involved in pleiotropic responses to environmental contaminants raises the possibility that smoking-induced hypomethylation is an adaptive response to an adverse in utero environmental exposure. However, the tissue specificity of the response to maternal smoking, and the stability of the methylation changes early in life remain to be determined. In this study we analyzed AHRR methylation in three cell types—cord blood mononuclear cells (CBMCs), buccal epithelium, and placenta tissue—from newborn twins of mothers who smoked throughout pregnancy and matched controls. Further, we explored the postnatal stability of this change at 18 months. Our results confirm the previous association between maternal smoking and AHRR methylation in neonatal blood. In addition, this study expands the region of AHRR methylation altered in response to maternal smoking during pregnancy and reveals the tissue-specific nature of epigenetic responses to environmental exposures in utero. Further, the evidence for postnatal stability of smoking-induced epigenetic change supports a role for epigenetics as a mediator of long-term effects of specific in utero exposures in humans. Longitudinal analysis of further specific exposures in larger cohorts is required to examine the extent of this phenomenon in humans.     

Glucose as a fetal nutrient: dynamic regulation of several glucose transporter genes by DNA methylation in the human placenta across gestation

The human placenta ensures proper fetal development through the regulation of nutrient and gas transfer from the mother to the fetus and the removal of waste products from the fetal circulation. Glucose is one of the major nutrients for the growing fetus. Its transport across the placenta to the fetus is mediated by a family of facilitative transporter proteins, known as the glucose transporters (GLUTs), encoded by the SLC2A family of genes. There are 14 members of this gene family, and the expression of several of these has been shown in human placenta; however, aside from GLUT1 and GLUT3, little is known about the role of these proteins in placental function, fetal development and disease. In this study, we analysed previously generated genome-scale DNA methylation and gene expression data to examine the role of methylation in GLUT expression throughout gestation. We found evidence that DNA methylation regulates expression of GLUT3 and GLUT10, while the constitutively expressed GLUT1 showed no promoter methylation. We further analysed the level of DNA methylation across the promoter region of GLUT3, previously shown to be involved in glucose back-flux from the fetal circulation into the placenta. Using the Sequenom EpiTYPER platform, we found increasing DNA methylation of this gene in association with decreasing expression as gestation progresses, thereby highlighting the role of epigenetic modifications in regulating the GLUT family of genes in the placenta during pregnancy. These findings warrant a reexamination of the role of additional GLUT family members in the placenta in pregnancy and disease.     

Maternal smoking during pregnancy and cord blood DNA methylation: new insight on sex differences and effect modification by maternal folate levels

Maternal smoking during pregnancy may affect newborn DNA methylation (DNAm). However, little is known about how these associations vary by a newborn’s sex and/or maternal nutrition. To fill in this research gap, we investigated epigenome-wide DNAm associations with maternal smoking during pregnancy in African American mother-newborn pairs. DNAm profiling in cord (n = 379) and maternal blood (n = 300) were performed using the Illumina HumanMethylation450 BeadChip array. We identified 12 CpG sites whose DNAm levels in cord blood were associated with maternal smoking, at a false discovery rate <5%. The identified associations in the GFI1 gene were more pronounced in male newborns than in females (P = 0.002 for maternal smoking × sex interaction at cg18146737). We further observed that maternal smoking and folate level may interactively affect cord blood DNAm level at cg05575921 in the AHRR gene (P = 5.0 × 10^?4 for interaction): compared to newborns unexposed to maternal smoking and with a high maternal folate level (>19.2 nmol/L), the DNAm level was about 0.03 lower (P = 3.6 × 10^?4) in exposed newborns with a high maternal folate level, but was 0.08 lower (P = 1.2 × 10^?8) in exposed newborns with a low maternal folate level. Our data suggest that adequate maternal folate levels may partly counteract the impact of maternal smoking on DNAm. These findings may open new avenues of inquiry regarding sex differences in response to environmental insults and novel strategies to mitigate their intergenerational health effects through optimization of maternal nutrition.     

Adaptations of placental and cord blood ABCA1 DNA methylation profile to maternal metabolic status

In utero environmental perturbations have been associated with epigenetic changes in the offspring and a lifelong susceptibility to cardiovascular diseases (CVD). DNA methylation at the ATP-binding cassette transporter A1 (ABCA1) gene was previously associated with CVD, but whether these epigenetic marks respond to changes in the maternal environment is unknown. This study was undertaken to assess the associations between the maternal metabolic profile and ABCA1 DNA methylation levels in placenta and cord blood. Placenta and cord blood samples were obtained at delivery from 100 women including 26 with impaired glucose tolerance (IGT) diagnosed following a 75 g-oral glucose tolerance test (OGTT) between week 24 and 28 of gestation. ABCA1 DNA methylation and mRNA levels were measured using bisulfite pyrosequencing and quantitative real-time PCR, respectively. We report that ABCA1 DNA methylation levels on the maternal side of the placenta are correlated with maternal high density lipoprotein cholesterol (HDL-C) levels (r < –0.21; P < 0.04) and glucose levels 2 h post-OGTT (r = 0.25; P = 0.02). On the fetal side of the placenta, ABCA1 DNA methylation levels are associated with cord blood triglyceride levels (r = –0.28; P = 0.01). ABCA1 DNA methylation variability on both sides of the placenta are also associated with ABCA1 mRNA levels (r < –0.35; P = 0.05). As opposed to placenta, cord blood DNA methylation levels are negatively correlated with maternal glucose 2 h post-OGTT (r = –0.26; P = 0.02). In conclusion, the epivariations observed in placenta and cord blood likely contribute to an optimal materno–fetal cholesterol transfer. These in utero epigenetics adaptations may also potentially trigger the long-term susceptibility of the newborn to dyslipidemia and CVD.     

IGF2 DNA methylation is a modulator of newborn’s fetal growth and development

The insulin-like growth factor 2 (IGF2) gene, located within a cluster of imprinted genes on chromosome 11p15, encodes a fetal and placental growth factor affecting birth weight. DNA methylation variability at the IGF2 gene locus has been previously reported but its consequences on fetal growth and development are still mostly unknown in normal pediatric population. We collected one hundred placenta biopsies from 50 women with corresponding maternal and cord blood samples and measured anthropometric indices, blood pressure and metabolic phenotypes using standardized procedures. IGF2/H19 DNA methylation and IGF2 circulating levels were assessed using sodium bisulfite pyrosequencing and ELISA, respectively. Placental IGF2 (DMR0 and DMR2) DNA methylation levels were correlated with newborn’s fetal growth indices, such as weight, and with maternal IGF2 circulating concentration at the third trimester of pregnancy, whereas H19 (DMR) DNA methylation levels were correlated with IGF2 levels in cord blood. The maternal genotype of a known IGF2/H19 polymorphism (rs2107425) was associated with birth weight. Taken together, we showed that IGF2/H19 epigenotype and genotypes independently account for 31% of the newborn’s weight variance. No association was observed with maternal diabetic status, glucose concentrations or prenatal maternal body mass index. This is the first study showing that DNA methylation at the IGF2/H19 genes locus may act as a modulator of IGF2 newborn’s fetal growth and development within normal range. IGF2/H19 DNA methylation could represent a cornerstone in linking birth weight and fetal metabolic programming of late onset obesity.     

Birthweight, maternal weight trajectories and global DNA methylation of LINE-1 repetitive elements

Low birthweight, premature birth, intrauterine growth retardation, and maternal malnutrition have been related to an increased risk of cardiovascular disease, type 2 diabetes mellitus, obesity, and neuropsychiatric disorders later in life. Conversely, high birthweight has been linked to future risk of cancer. Global DNA methylation estimated by the methylation of repetitive sequences in the genome is an indicator of susceptibility to chronic diseases. We used data and biospecimens from an epigenetic birth cohort to explore the association between trajectories of fetal and maternal weight and LINE-1 methylation in 319 mother-child dyads. Newborns with low or high birthweight had significantly lower LINE-1 methylation levels in their cord blood compared to normal weight infants after adjusting for gestational age, sex of the child, maternal age at delivery, and maternal smoking during pregnancy (p = 0.007 and p = 0.036, respectively), but the magnitude of the difference was small. Infants born prematurely also had lower LINE-1 methylation levels in cord blood compared to term infants, and this difference, though small, was statistically significant (p = 0.004). We did not find important associations between maternal prepregnancy BMI or gestational weight gain and global methylation of the cord blood or fetal placental tissue. In conclusion, we found significant differences in cord blood LINE-1 methylation among newborns with low and high birthweight as well as among prematurely born infants. Future studies may elucidate whether chromosomal instabilities or other functional consequences of these changes contribute to the increased risk of chronic diseases among individuals with these characteristics.     

Maternal weight affects placental DNA methylation of genes involved in metabolic pathways in the common marmoset monkey (Callithrix jacchus)

Accumulating evidence suggests that dysregulation of placental DNA methylation (DNAm) is a mechanism linking maternal weight during pregnancy to metabolic programming outcomes. The common marmoset, Callithrix jaccus, is a platyrrhine primate species that has provided much insight into studies of the primate placenta, maternal condition, and metabolic programming, yet the relationships between maternal weight and placental DNAm are unknown. Here, we report genome-wide DNAm from term marmoset placentas using reduced representation bisulfite sequencing. We identified 74 genes whose DNAm pattern is associated with maternal weight during gestation. These genes are predominantly involved in energy metabolism and homeostasis, including the regulation of glycolytic and lipid metabolic processes pathways.     

Umbilical cord blood and placental mercury,selenium and selenoprotein expression in relation to maternal fish consumption

Seafood is an important source of nutrients for fetal neurodevelopment. Most individuals are exposed to the toxic element mercury through seafood. Due to the neurotoxic effects of mercury, United States government agencies recommend no more than 340 g (12 oz) per week of seafood consumption during pregnancy. However, recent studies have shown that selenium, also abundant in seafood, can have protective effects against mercury toxicity. In this study, we analyzed mercury and selenium levels and selenoprotein mRNA, protein, and activity in placenta of a cohort of women in Hawaii in relation to maternal seafood consumption assessed with dietary surveys. Fish consumption resulted in differences in mercury levels in placenta and cord blood. When taken as a group, those who consumed no fish exhibited the lowest mercury levels in placenta and cord blood. However, there were numerous individuals who either had higher mercury with no fish consumption or lower mercury with high fish consumption, indicating a lack of correlation. Placental expression of selenoprotein mRNAs, proteins and enzyme activity was not statistically different in any region among the different dietary groups. While the absence of seafood consumption correlates with lower average placental and cord blood mercury levels, no strong correlations were seen between seafood consumption or its absence and the levels of either selenoproteins or selenoenzyme activity.     

Altered gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA) placentae

Imprinted genes are known to be crucial for placental development and fetal growth in mammals, but no primary epigenetic abnormality in placenta has been documented to compromise human fetal growth. Imprinted genes demonstrate parent-of-origin-specific allelic expression that is epigenetically regulated i.e. extrinsic to the primary DNA sequence. To undertake an epigenetic analysis of poor fetal growth in placentae and cord blood tissues, we first established the tissue-specific patterns of methylation and imprinted gene expression for two imprinting clusters (KvDMR and H19 DMR) on chromosome 11p15 in placentae and neonatal blood for 20 control cases and 24 Small for Gestational Age (SGA) cases. We confirmed that, in normal human placenta, the H19 promoter is unmethylated. In contrast, most other human tissues show paternal methylation. In addition, we showed that the IGF2 DMR2, also paternally methylated in most human tissues, exhibits hypomethylation in placentae. However, in neonatal blood DNA, these two regions maintain the differential methylation status seen in most other tissues. Significantly, we have been able to demonstrate that placenta does maintain differential methylation at the imprinting control regions H19 DMR and KvDMR. Of note, in one SGA placenta, we found a methylation alteration at the H19 DMR and concomitant biallelic expression of the H19 gene, suggesting that loss of imprinting at H19 is one cause of poor fetal growth in humans. Of particular interest, we demonstrated also a decrease in IGF2 mRNA levels in all SGA placentae and showed that the decrease is, in most cases, independent of H19 regulation.     

LINE-1 DNA methylation is inversely correlated with cord plasma homocysteine in man: A preliminary study

Folic acid supplementation during pregnancy has known beneficial effects. It reduces risk of neural tube defects and low birth weight. Folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. However, most data on the effects of folate on the epigenome is derived from animal or in vitro models. We examined the relationship between cord blood methylation and maternal folic acid intake, cord blood folate and homocysteine using data from 24 pregnant women. Genome-wide methylation was determined by the level of methylation of LINE-1 repeats using Pyrosequencing. We show that cord plasma homocysteine (p = 0.001, r = -0.688), but not serum folate or maternal folic acid intake, is inverse correlated with LINE-1 methylation. This remained significant after correction for potential confounders (p = 0.004). These data indicate that levels of folate-associated intermediates in cord blood during late pregnancy have significant consequences for the fetal epigenome.     

DNA methylation in imprinted genes IGF2 and GNASXL is associated with prenatal maternal stress

Epigenetic regulation of imprinted genes during embryonic development is influenced by the prenatal environment. Our aim was to examine the effect of maternal emotional stress and cortisol levels during pregnancy on methylation of imprinted genes, insulin‐like growth factor 2 (IGF2) and guanine nucleotide‐binding protein, alpha stimulating extra‐large (GNASXL), using umbilical cord blood DNA. Maternal depressed mood (Edinburgh Depression Scale; EDS), pregnancy‐related anxiety questionnaire (PRAQ) and cortisol day profiles were assessed throughout pregnancy. At birth, a cord blood sample (n = 80) was taken to study DNA methylation of IGF2 DMR0 (differentially methylated region), IGF2 anti‐sense (IGF2AS) and GNASXL using Sequenom Epi TYPER. Linear mixed models were used to examine the relationship between DNA methylation and maternal stress, while correcting for confounders. We also studied the association of DNA methylation with the child ponderal index at birth. We found a cytosine–guanine dinucleotide (CpG)‐specific association of PRAQ subscales with IGF2 DMR0 (CpG5, P < 0.0001) and GNASXL (CpG11, P = 0.0003), while IGF2AS was associated with maternal EDS scores (CpG33, P = 0.0003) and cortisol levels (CpG33, P = 0.0006; CpG37‐38, P = 0.0005). However, there was no association of methylation with ponderal index at birth. In conclusion, maternal stress during pregnancy, as defined by cortisol measurements, EDS and PRAQ scores, is associated with DNA methylation of imprinted genes IGF2 and GNASXL. Our results provide further evidence that prenatal adversity can influence imprinted gene methylation, although future studies are needed to unravel the exact mechanisms.     

Association of maternal and nutrient supply line factors with DNA methylation at the imprinted IGF2/H19 locus in multiple tissues of newborn twins

Epigenetic events are crucial for early development, but can be influenced by environmental factors, potentially programming the genome for later adverse health outcomes. The insulin-like growth factor 2 (IGF2)/H19 locus is crucial for prenatal growth and the epigenetic state at this locus is environmentally labile. Recent studies have implicated maternal factors, including folate intake and smoking, in the regulation of DNA methylation at this locus, although data are often conflicting in the direction and magnitude of effect. Most studies have focused on single tissues and on one or two differentially-methylated regions (DMRs) regulating IGF2/H19 expression. In this study, we investigated the relationship between multiple shared and non-shared gestational/maternal factors and DNA methylation at four IGF2/H19 DMRs in five newborn cell types from 67 pairs of monozygotic and 49 pairs of dizygotic twins. Data on maternal and non-shared supply line factors were collected during the second and third trimesters of pregnancy and DNA methylation was measured via mass spectrometry using Sequenom MassArray EpiTyper analysis. Our exploratory approach showed that the site of umbilical cord insertion into the placenta in monochorionic twins has the strongest positive association with methylation in all IGF2/H19 DMRs (p < 0.05). Further, evidence for tissue- and locus-specific effects were observed, emphasizing that responsiveness to environmental exposures in utero cannot be generalized across genes and tissues, potentially accounting for the lack of consistency in previous findings. Such complexity in responsiveness to environmental exposures in utero has implications for all epigenetic studies investigating the developmental origins of health and disease.     

Heritability and role for the environment in DNA methylation in AXL receptor tyrosine kinase

DNA methylation in AXL, a receptor tyrosine kinase relevant in cancer and immune function, is reportedly highly heritable. We present evidence to suggest that heritability of DNA methylation in AXL is variable, dependent on population characteristics and cell type studied. Moreover, environmental exposures in utero, particularly exposure to maternal smoking, contributes to variation in DNA methylation of select CpG loci that can affect calculations of heritability. Children exposed to maternal smoking in utero had a 2.3% increase (95 % CI 0.3, 4.2) in DNA methylation in AXL, which was magnified in girls as compared to boys. These results present compelling evidence that environmental exposure to tobacco smoke during pregnancy may alter DNA methylation levels in subtle but potentially important ways, and that these changes are persistent years after birth.