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Placental DNA Methylation Alterations Associated with Maternal Tobacco Smoking at the RUNX3 Gene are also Associated with Gestational Age
Epigenomics
Abstract
Aims:
The developmental origins of health and disease hypothesis states that later-life disease may be influenced by the quality of the in utero environment. Environmental toxicants can have detrimental effects on fetal development, potentially through effects on placental development and function. Maternal smoking during pregnancy is associated with low birth weight, preterm birth and other complications, and exposure to cigarette smoke in utero has been linked to gross pathologic and molecular changes to the placenta, including differential DNA methylation in placental tissue. The aim of this study was to investigate the relationship between maternal smoking during pregnancy, methylation changes in the placenta and gestational age.
Materials & methods:
We used Illumina(®)'s (CA, USA) Human Methylation27 BeadChip technology platform to investigate the methylation status of 21,551 autosomal, non-SNP-associated CpG loci in DNA extracted from 206 human placentas and examined loci whose variation in methylation was associated with maternal smoking during pregnancy.
Results:
We found that methylation patterns of a number of loci within the RUNX3 gene were significantly associated with smoking during pregnancy, and one of these loci was associated with decreased gestational age (p = 0.04).
Conclusion:
Our findings, demonstrating maternal smoking-induced changes in DNA methylation at specific loci, suggest a mechanism by which in utero tobacco smoke exposure could exert its detrimental effects upon the health of the fetus.
... Study selection 999 references were imported from the search. Of the 74 abstracts that met initial inclusion criteria, the fulltext review led to inclusion of 16 studies [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] that drew from 13 birth cohorts and exclusion of 58 studies ( Figure 1 & Table 1). Among excluded studies, two were excluded because they were transcriptomic [45] and metabolomic [46] studies. ...
... methylomic studies utilizing the extreme low gestational age newborns (ELGAN) cohort or other cohorts including only samples from preterm infants and thus lacking infants born at term for comparison [58][59][60][61]. 14 of the 16 included studies used a cross-sectional study design [29][30][31][32][33][34][35]38,39,44] and two were prospective [36,37]. Only three of the 16 studies evaluated epigenomic changes in maternal specimens [35,37,44], whereas the remaining utilized samples of neonatal and placental origin (both derived from the fetal compartment). ...
... Two of the included studies utilized cohorts that were uniformly White or European [32,36]. Only nine studies included both Black or non-Hispanic Black participants and White or non-Hispanic White participants [33][34][35][38][39][40][41][42][43], and of those, only seven had more than five individuals from both groups to allow for potential comparison by race and ethnicity [33][34][35]38,[40][41][42]. ...
Aim: We conducted a systematic review evaluating race/ethnicity representation in DNA methylomic studies of preterm birth. Data sources: PubMed, EMBASE, CINHAL, Scopus and relevant citations from 1 January 2000 to 30 June 2019. Study appraisal & synthesis methods: Two authors independently identified abstracts comparing DNA methylomic differences between term and preterm births that included race/ethnicity data. Results: 16 studies were included. Black and non-Hispanic Black deliveries were well represented (28%). However, large studies originating from more than 95% White populations were excluded due to unreported race/ethnicity data. Most studies were cross-sectional, allowing for reverse causation. Most studies were also racially/ethnically homogeneous, preventing direct comparison of DNA methylomic differences across race/ethnicities. Conclusion: In DNA methylomic studies, Black women and infants were well represented. However, the literature has limitations and precludes drawing definitive conclusions.
... NAP1L5, for example, has been previously identified to be differently methylated in cord blood samples characterized by intrauterine exposure to gestational diabetes mellitus [23]. L3MBTL has been reported to be differently methylated in cord blood of babies exposed to smoking during pregnancy [24] and is epigenetically associated with gestational age [25]. Increased methylation at differentially methylated regions of GNAS has been described in infants born in conditions of gestational diabetes [26]. ...
... An increase in DNA methylation at the SGCE/PEG10 DMR has been previously positively associated with paternal BMI [27], maternal stress [28], and also with maternal prenatal physical activity [29]. Methylation of PRCP gene in newborns [30] as well as RUNX3 [24] was found to be significantly associated with maternal smoking status. Hypermethylation of RUNX3 CpG sites has been also associated with decreased gestational age [24]. ...
... Methylation of PRCP gene in newborns [30] as well as RUNX3 [24] was found to be significantly associated with maternal smoking status. Hypermethylation of RUNX3 CpG sites has been also associated with decreased gestational age [24]. Taken together, this evidence hints at the presence of confounding factors in previously reported results on epigenetics of ART. ...
Background:
An increased incidence of imprint-associated disorders has been reported in babies born from assisted reproductive technology (ART). However, previous studies supporting an association between ART and an altered DNA methylation status of the conceived babies have been often conducted on a limited number of methylation sites and without correction for critical potential confounders. Moreover, all the previous studies focused on the identification of methylation changes shared among subjects while an evaluation of stochastic differences has never been conducted. This study aims to evaluate the effect of ART and other common behavioral or environmental factors associated with pregnancy on stochastic epigenetic variability using a multivariate approach.
Results:
DNA methylation levels of cord blood from 23 in vitro and 41 naturally conceived children were analyzed using the Infinium HumanMethylation450 BeadChips. After multiple testing correction, no statistically significant difference emerged in the number of cord blood stochastic epigenetic variations or in the methylation levels between in vitro- and in vivo-conceived babies. Conversely, four multiple factor analysis dimensions summarizing common phenotypic, behavioral, or environmental factors (cord blood cell composition, pre or post conception supplementation of folates, birth percentiles, gestational age, cesarean section, pre-gestational mother's weight, parents' BMI and obesity status, presence of adverse pregnancy outcomes, mother's smoking status, and season of birth) were significantly associated with stochastic epigenetic variability. The stochastic epigenetic variation analysis allowed the identification of a rare imprinting defect in the locus GNAS in one of the babies belonging to the control population, which would not have emerged using a classical case-control association analysis.
Conclusions:
We confirmed the effect of several common behavioral or environmental factors on the epigenome of newborns and described for the first time an epigenetic effect related to season of birth. Children born after ART did not appear to have an increased risk of genome-wide changes in DNA methylation either at specific loci or randomly scattered throughout the genome. The inability to identify differences between cases and controls suggests that the number of stochastic epigenetic variations potentially induced by ART was not greater than that naturally produced in response to maternal behavior or other common environmental factors.
... DNA methylation levels measured in the placenta are closely related to GA. In the human placenta, DNA methylation levels vary with pregnancy duration, gradually increasing as pregnancy progresses (Yuen et al., 2010;Novakovic et al., 2011;Maccani et al., 2013). The placentas of PTB patients exhibit more hypomethylated areas in their DNA than do individuals who experience a full-term birth (Schuster et al., 2019). ...
... In UCB, the level of CYP1A1 methylation increased in the smoking group compared with the non-smoker group (Joubert et al., 2012) In UCB, runt-related transcription factor 1 (RUNX1), myosin IG (MYO1G), neurogenin 1 (NEUROG1), and FERM domain containing 4A (FRMD4A) methylation was increased in the smoking group (Joubert et al., 2012;Küpers et al., 2015). DNA methylation of RUNX3 in the placenta of the smoking group was also higher than that in the placenta of the non-smoking group (Maccani et al., 2013). In the UCB, the level of DNA methylation of AHRR, growth factor independent 1 transcriptional repressor (GFI1), LDL receptor related protein 5 (LRP5), and contactin associated protein 2 (CNTNAP2) was lower in the smoking group than in the non-smoking group (Joubert et al., 2012;Küpers et al., 2015). ...
Preterm birth occurs worldwide and is associated with high morbidity, mortality, and economic cost. Although several risk factors associated with parturition and preterm birth have been identified, mechanisms underlying this syndrome remain unclear, thereby limiting the implementation of interventions for prevention and management. Known triggers of preterm birth include conditions related to inflammatory and immunological pathways, as well as genetics and maternal history. Importantly, epigenetics, which is the study of heritable phenotypic changes that occur without alterations in the DNA sequence, may play a role in linking social and environmental risk factors for preterm birth. Epigenetic approaches to the study of preterm birth, including analyses of the effects of microRNAs, long non-coding RNAs, DNA methylation, and histone modification, have contributed to an improved understanding of the molecular bases of both term and preterm birth. Additionally, epigenetic modifications have been linked to factors already associated with preterm birth, including obesity and smoking. The prevention and management of preterm birth remains a challenge worldwide. Although epigenetic analysis provides valuable insights into the causes and risk factors associated with this syndrome, further studies are necessary to determine whether epigenetic approaches can be used routinely for the diagnosis, prevention, and management of preterm birth.
... There are three major types of epigenetic regulations encompassing (i) posttranslational modifications of histone proteins and chromatin remodeling, (ii) noncoding RNA interference and (iii) DNA methylation, all of which create an intricate and self-reinforcing interactions converging on a common cellular process i.e. regulating gene expression (although expression-independent effects of epigenetic mechanisms are being increasingly reported) (Figure 2) (87)(88)(89). ...
... Interestingly, differentially methylated loci in relation to asthma development have been recently identified in newborns [87]. The stable CpG cg27058497 (RUNX3) has been associated with in utero tobacco smoking exposure [88], childhood asthma [89], oesophagus squamous cell carcinoma [90] and chronic fatigue syndrome [91]. Despite adjustment for maternal smoking in our gestational age EWAS model, we observed overlap between all FDR hits from our gestational age EWAS with those FDR hits presented in the maternal smoking related DNA methylation [20] with an overlap of 2302/47,324 CpGs (4.9%, P enrichment < 2.2 × 10 − 308 ). ...
Childhood cancer (CC) is the leading cause of disease-related mortality in children, with childhood leukemia (CL) being the predominant type. CC is rare, and its risk factors and molecular precursors are poorly understood and may originate in utero. Fetal life represents a sensitive period during which epigenetic regulation constitutes heritable mechanisms driving embryogenesis. We hypothesize that epigenetic (DNA methylation) deregulation in utero underlies biological pathways linking early-life factors to CL. We focus on birthweight, as a collective proxy for early-life exposure and one of the earliest phenotypes predisposing to CL, as well as its closely related intrinsic factors, gestational age and child sex. We first performed epigenome-wide analysis with optimized biostatistics methodology in large population-based cohorts and found profound associations between each of the three factors and cord blood DNA methylome. Second, we investigated, in a subset of cohorts enriched in CL cases, whether the identified birthweight biomarkers significantly associate with cancer risk and are affected by gestational age and child sex. Third, we tested the proportion by which DNA methylation mediates the effect between birthweight and CL. These steps constitute a proposed ‘three-way modelling’ aiming to identify molecular mechanisms linking early-life exposure to CC risk. This work identified epigenetics markers of early-life factors based on some of the largest studies to date, yielding insights into epigenetic deregulation in utero that could be at the origin of CL. The described framework could be useful for other exposure-outcome studies investigating underlying molecular mechanisms
... Maternal smoking during pregnancy (MSDP) is the leading preventable cause of prematurity, intrauterine growth restriction (IUGR), and perinatal death [1][2][3][4]; however, despite smoking cessation efforts, over 50% of smokers will continue to smoke during pregnancy [5,6]. MSDP affects development of multiple organ systems including placenta, lung, brain, and vasculature [7][8][9][10][11][12] and is associated with altered DNA methylation in placenta, blood, and buccal epithelium [13][14][15][16][17][18][19][20][21][22][23][24]. Perhaps the bestcharacterized effects of MSDP on long-term offspring health are respiratory outcomes. ...
... Placental DNAm is dysregulated with in utero exposure to heavy metals, alcohol, and air pollution [40][41][42], with some of the largest effect sizes reported in association with MSDP [15,[43][44][45][46][47][48][49]. Previous studies have reported global and gene-specific changes in placental DNAm in response to MSDP [19,42,50], and some of these changes are proposed to mediate the effect of MSDP on health outcomes, such as infant birth weight and psychiatric morbidity [13,23,51]. ...
Background
Maternal smoking during pregnancy (MSDP) affects development of multiple organ systems including the placenta, lung, brain, and vasculature. In particular, children exposed to MSDP show lifelong deficits in pulmonary function and increased risk of asthma and wheeze. Our laboratory has previously shown that vitamin C supplementation during pregnancy prevents some of the adverse effects of MSDP on offspring respiratory outcomes. Epigenetic modifications, including DNA methylation (DNAm), are a likely link between in utero exposures and adverse health outcomes, and MSDP has previously been associated with DNAm changes in blood, placenta, and buccal epithelium. Analysis of placental DNAm may reveal critical targets of MSDP and vitamin C relevant to respiratory health outcomes.
Results
DNAm was measured in placentas obtained from 72 smokers enrolled in the VCSIP RCT: NCT03203603 (37 supplemented with vitamin C, 35 with placebo) and 24 never-smokers for reference. Methylation at one CpG, cg20790161, reached Bonferroni significance and was hypomethylated in vitamin C supplemented smokers versus placebo. Analysis of spatially related CpGs identified 93 candidate differentially methylated regions (DMRs) between treatment groups, including loci known to be associated with lung function, oxidative stress, fetal development and growth, and angiogenesis. Overlap of nominally significant differentially methylated CpGs (DMCs) in never-smokers versus placebo with nominally significant DMCs in vitamin C versus placebo identified 9059 candidate “restored CpGs” for association with placental transcript expression and respiratory outcomes. Methylation at 274 restored candidate CpG sites was associated with expression of 259 genes (FDR < 0.05). We further identified candidate CpGs associated with infant lung function (34 CpGs) and composite wheeze (1 CpG) at 12 months of age (FDR < 0.05). Increased methylation in the DIP2C , APOH/PRKCA , and additional candidate gene regions was associated with improved lung function and decreased wheeze in offspring of vitamin C-treated smokers.
Conclusions
Vitamin C supplementation to pregnant smokers ameliorates changes associated with maternal smoking in placental DNA methylation and gene expression in pathways potentially linked to improved placental function and offspring respiratory health. Further work is necessary to validate candidate loci and elucidate the causal pathway between placental methylation changes and outcomes of offspring exposed to MSDP.
Clinical trial registration ClinicalTrials.gov, NCT01723696. Registered November 6, 2012. https://clinicaltrials.gov/ct2/show/record/NCT01723696 .
... In utero exposure to smoking is associated with alterations of DNA methylation patterns [82][83][84][85], and such changes may persist throughout the entire life course [85][86][87][88], leading to impaired fetal development [83,89], preterm delivery [90,91] and other chronic diseases including respiratory illness, cardiovascular disorders, and certain childhood cancers in the offspring's later life [92][93][94]. The methylation targets of maternal smoking during pregnancy could be genome-wide [85,[95][96][97][98][99][100][101] and specific such as runt-related transcription factor 3 (RUNX3), aryl hydrocarbon receptor repressor (AHRR), and cytochrome P450 1A1 (CYP1A1) in placental tissue [44,102,103]; AHRR, growth factor independent 1 (GFI1), insulin-like growth factor 2 (IGF2), PR domain containing 8 (PRDM8), discs large homolog-associated protein 2 (DLGAP2), thymic stromal lymphopoietin (TSLP), CYP1A1 in newborn umbilical cord blood samples [44,85,[104][105][106]; and Myosin 1 G (MYO1G), cortactin-associated protein-like 2 (CNTNAP2), and FRMD4A, a human epidermal stem cell marker, in children's blood [107]; MYO1G, CNTNAP2, and brain-derived neurotrophic factor (BDNF) in adolescent's peripheral blood cells [88,108]; and repetitive element satellite 2 (Sat2) in adult peripheral blood granulocytes [96], as well as AHRR, in neonatal buccal epithelium [44]. Maternal tobacco smoking has been also linked to dysregulated expression of miRNAs. ...
... Lower placental leptin promoter methylation [78] Early pregnancy Associated with placental DNA methylation of LINE1 and HSD11B2 [75] Prenatal Decreased expression of miR-21, miR-146a and miR-222; increased expression of miR-20a and miR-21 [80] Gestation Increased mtDNA methylation levels and decreased LINE-1 methylation levels [77] Prenatal Decrease in global DNA methylation for whole pregnancy [73] Prenatal Increased DNA methylation in LINE1, OGG1, APEX and PARP1 [79] Smoking Prenatal Nearly 3000 CpGs corresponding to genes differentially methylated in offspring [85] Maternal Altered DNA methylation levels at CpG sites of GFI1, AHRR and PRNP gene in male and female, differentially [110] In utero Impact key biological pathways through epigenetic modification [84] Maternal Differential methylation of MYO1G, CNTNAP2 and FRMD4A genes in children blood [107] In utero Global DNA hypomethylation; 31 CpG sites associated to 25 genes [99] Prenatal Altered methylation at 15 CpG sites [100] Prenatal Differential methylation at five CpGs in MYO1G and CNTNAP2; persist in exposed offspring for many years [88] In utero Increased CpG methylation in FRMD4A and Cllorf52; reproducible epigenetic changes persist into childhood [87] In utero Altered methylation at 185 CpGs of 110 gene regions in infants [101] In utero Hypomethylation of AHRR in the cord blood mononuclear cells, buccal epithelium and placenta tissue [44] In utero Altered methylation at TSLP promoter [106] Maternal Altered methylation patterns of a few loci within the RUNX3 gene [102] In utero Increased IGF2 DMR [105] In utero Altered LINE-1 and AluYb8 methylation levels [83] Maternal Differential DNA methylation at epigenome-wide for 26 CpGs mapped to 10 genes [104] Maternal Differential epigenome-wide placental DNA methylation [82] Gestation Decreased methylation of Sat2 [96] Maternal Increased DNA methylation in the BNDF-6 exon [108] Gestation Downregulation of miR-16, miR-21 and miR-146a in placenta [109] In utero Global DNA methylation inversely correlates with cotinine levels in cord blood [95] In utero Decreased methylation at CYPIAI promoter in the placenta [103] Prenatal Lower methylation of AluYb8; differential methylation of LINE1; increased methylation of AXL and PTPRO [98] Polycyclic aromatic hydrocarbons Prenatal Inverse relationship with LINE1 DNA methylation in cord blood [119] Prenatal Decreased global methylation in umbilical cord white blood cells [118] Prenatal Altered methylation in 5′-CpG islands of ACSL3 ...
It is thought that germ cells and preimplantation embryos during development are most susceptible to endogenous and exogenous environmental factors because the epigenome in those cells is undergoing dramatic elimination and reconstruction. Exposure to environmental factors such as nutrition, climate, stress, pathogens, toxins, and even social behavior during gametogenesis and early embryogenesis has been shown to influence disease susceptibility in the offspring. Early-life epigenetic modifications, which determine the expression of genetic information stored in the genome, are viewed as one of the general mechanisms linking prenatal exposure and phenotypic changes later in life. From atmospheric pollution, endocrine-disrupting chemicals to heavy metals, research increasingly suggests that environmental pollutions have already produced significant consequences on human health. Moreover, mounting evidence now links such pollution to relevant modification in the epigenome. The epigenetics diet, referring to a class of bioactive dietary compounds such as isothiocyanates in broccoli, genistein in soybean, resveratrol in grape, epigallocatechin-3-gallate in green tea, and ascorbic acid in fruits, has been shown to modify the epigenome leading to beneficial health outcomes. This review will primarily focus on the causes and consequences of prenatal environment pollution exposure on the epigenome, and the potential protective role of the epigenetics diet, which could play a central role in neutralizing epigenomic aberrations against environmental pollutions.
... 17−19 However, studies investigating the effects of e-cigarette exposure in mouse models of pregnancy are limited. 20−23 Although there are numerous studies that focus on offspring from maternal smoking in humans and animals, 1,7,10,24,25 the effects of maternal exposure to e-cigarette aerosols on offspring behavior and epigenetics are lacking, and epidemiological data is not yet available. In this study, we aimed to investigate the effects of maternal e-cigarette aerosol exposure on offspring in a mouse model. ...
... It has been reported that maternal tobacco smoking causes changes to DNA methylation in the offspring in cord blood 53 and placenta. 11,12,24 We report here for the first time that e-cigarette exposure in a mouse model of pregnancy results in changes in global DNA methylation in offspring brains. This finding is in line with our recent study that showed global DNA methylation in the lungs of mice offspring after maternal exposure to e-cigarette aerosols. ...
Electronic cigarette (e-cigarette) use is on the rise worldwide and is particularly attractive to young people and as a smoking substitute by pregnant woman. There is a perception in pregnant woman and women of child-bearing age that the use of e-cigarettes (vaping) is safer than smoking tobacco cigarettes during pregnancy. However, there is little evidence to support this perception. Here, we examined the offspring from mouse dams that had been exposed during and after pregnancy to ambient air (sham) (n=8), e-cigarette aerosols with nicotine (n=8) or e-cigarette aerosols without nicotine (n=8). Offspring underwent cognitive testing at 12 weeks of age and epigenetic testing of brain tissues at 1 day, 20 days and 13 weeks after birth. The findings showed deficits in short-term memory, reduced anxiety and hyperactivity in offspring using the novel object recognition and elevated plus maze tests. In addition, global DNA methylation was increased in the brains of offspring soon after birth. Using a quantitative-PCR array specific to chromatin modification enzymes on genomic DNA and histones, 13 key genes were identified to be significantly altered in the offspring brains from the e-cigarette groups compared to the non-exposed groups. The changes to genes Aurka, Aurkb, Aurkc, Kdm5c, Kdm6b, Dnmt3a, Dnmt3b and Atf2, all associated with modulating neurological activity were validated using RT-qPCR. In conclusion, in a mouse model, maternal exposure to e-cigarette aerosols resulted in both cognitive and epigenetic changes in offspring. This suggests that the use of e-cigarettes during pregnancy may have hitherto undetected neurological consequences on newborns.
... In this study, aberrant methylation of nine CpGs was found to be significantly associated with packyears of smoking in bronchial washings of the control group. Among these genes, aberrant methylation of CpGs on GATA4, EMX1, and RUNX3 genes is known to be associated with smoke exposure [33][34][35][36]. Wood smoke exposure is associated with lower percent predicted FEV1 of COPD patients in presence of aberrantly methylated GATA4 [33]. ...
... Methylation levels of EMX1 are associated with pack-years of smoking in gastric mucosa of healthy individuals [34]. Positive relationship of RUNX3 methylation to smoking has also been observed in DNA from blood leukocytes and placenta [35,36]. ...
Background
The objective of this study was to discover DNA methylation biomarkers for detecting non-small lung cancer (NSCLC) in bronchial washings and understanding the association between DNA methylation and smoking cessation.
Methods
DNA methylation was analyzed in bronchial washing samples from 70 NSCLCs and 53 hospital-based controls using Illumina HumanMethylation450K BeadChip. Methylation levels in these bronchial washings were compared to those in 897 primary lung tissues of The Cancer Genome Atlas (TCGA) data.
Results
Twenty-four CpGs (p < 1.03E−07) were significantly methylated in bronchial washings from 70 NSCLC patients compared to those from 53 controls. The CpGs also had significant methylation in the TCGA cohort. The 123 participants were divided into a training set (N = 82) and a test set (N = 41) to build a classification model. Logistic regression model showed the best performance for classification of lung cancer in bronchial washing samples: the sensitivity and specificity of a marker panel consisting of seven CpGs in TFAP2A, TBX15, PHF11, TOX2, PRR15, PDGFRA, and HOXA11 genes were 87.0 and 83.3% in the test set, respectively. The area under the curve (AUC) was equal to 0.87 (95% confidence interval = 0.73–0.96, p < 0.001). Methylation levels of two CpGs in RUNX3 and MIR196A1 genes were inversely associated with duration of smoking cessation in the controls, but not in NSCLCs, after adjusting for pack-years of smoking.
Conclusions
The present study suggests that NSCLC may be detected by analyzing methylation changes of seven CpGs in bronchial washings. Furthermore, smoking cessation may lead to decreased DNA methylation in nonmalignant bronchial epithelial cells in a gene-specific manner.
Electronic supplementary material
The online version of this article (10.1186/s13148-018-0498-8) contains supplementary material, which is available to authorized users.
... Our results suggest that maternal smoking during pregnancy affects primarily the fetal side of placenta, so analyses of another part of the placenta or from a full thickness biopsy will likely result in different observations. Differential methylation between exposed and unexposed placentas was previously reported for RUNX3, 36 an observation that could not be reproduced in our study even though our primers were designed to analyze the same CpG locus. Compared to mothers who did not smoke, differential DNA methylation was only observed in the placentas exposed to maternal smoking throughout pregnancy for the affected CpG loci in the CYP1A1 and AHRR genes, while the CpG locus in the GFI1 promoter was differentially methylated in the placentas of mothers who quit smoking while pregnant. ...
... The first set of primers were designed to cover CpG loci that were reported previously by methylation arrays to exhibit differential methylation between those exposed to maternal smoking during pregnancy vs. controls in either cord blood, maternal blood or placenta. [13][14][15][16]36 These CpG loci were located in the gene body for the genes AHRR, GFI1, GLANT2, RUNX3, and TSLP1. A second set of primers included CpG islands near the transcriptional start site in the same set of genes, plus CYP1A1. ...
We investigated the impact of maternal smoking during pregnancy on placental DNA methylation and how this may mediate the association between maternal smoking and pro-inflammatory proteins in cord blood. The study population consisted of 27 individuals exposed to maternal smoking throughout pregnancy, 32 individuals exposed during a proportion of the pregnancy, and 61 unexposed individuals. Methylation of 11 regions within 6 genes in placenta tissue was assessed by pyrosequencing. Levels of 7 pro-inflammatory proteins in cord blood were assessed by electrochemiluminescence. Differential methylation was observed in the CYP1A1 promoter and AHRR gene body regions between women who smoked throughout pregnancy and non-smokers on the fetal-side of the placenta and in the GFI1 promoter between women who quit smoking while pregnant and non-smokers on the maternal-side of the placenta. Maternal smoking resulted in elevated levels of IL-8 protein in cord blood, which was not mediated by DNA methylation of our candidate regions at either the maternal or the fetal side of the placenta. Placental DNA methylation was associated with levels of inflammatory proteins in cord blood. Our observations suggest that maternal smoking during pregnancy affects both placental DNA methylation and the neonate's immune response.
... Modification of DNAm during embryonic and fetal development may lead to structural and functional alterations in the developing lung and/or predispose risk for respiratory infections later in life through changes in the immune response. Exposure to MSDP is associated with altered DNA methylation in placenta, blood, and buccal epithelium [18][19][20][21][22][23][24][25][26][27][28][29], and may provide a mechanistic link between in utero exposures and future health outcomes [30][31][32][33]. Previous studies have reported DNAm as a mediator of the effects of MSDP on health outcomes such as reduced birth weight and psychiatric morbidity [18,28,34]. ...
Background
We previously reported in the “Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function” randomized clinical trial (RCT) that vitamin C (500 mg/day) supplementation to pregnant smokers is associated with improved respiratory outcomes that persist through 5 years of age. The objective of this study was to assess whether buccal cell DNA methylation (DNAm), as a surrogate for airway epithelium, is associated with vitamin C supplementation, improved lung function, and decreased occurrence of wheeze.
Methods
We conducted epigenome-wide association studies (EWAS) using Infinium MethylationEPIC arrays and buccal DNAm from 158 subjects (80 placebo; 78 vitamin C) with pulmonary function testing (PFT) performed at the 5-year visit. EWAS were performed on (1) vitamin C treatment, (2) forced expiratory flow between 25 and 75% of expired volume (FEF25–75), and (3) offspring wheeze. Models were adjusted for sex, race, study site, gestational age at randomization (≤ OR > 18 weeks), proportion of epithelial cells, and latent covariates in addition to child length at PFT in EWAS for FEF25–75. We considered FDR p < 0.05 as genome-wide significant and nominal p < 0.001 as candidates for downstream analyses. Buccal DNAm measured in a subset of subjects at birth and near 1 year of age was used to determine whether DNAm signatures originated in utero, or emerged with age.
Results
Vitamin C treatment was associated with 457 FDR significant (q < 0.05) differentially methylated CpGs (DMCs; 236 hypermethylated; 221 hypomethylated) and 53 differentially methylated regions (DMRs; 26 hyper; 27 hypo) at 5 years of age. FEF25–75 was associated with one FDR significant DMC (cg05814800), 1,468 candidate DMCs (p < 0.001), and 44 DMRs. Current wheeze was associated with 0 FDR-DMCs, 782 candidate DMCs, and 19 DMRs (p < 0.001). In 365/457 vitamin C FDR significant DMCs at 5 years of age, there was no significant interaction between time and treatment.
Conclusions
Vitamin C supplementation to pregnant smokers is associated with buccal DNA methylation in offspring at 5 years of age, and most methylation signatures appear to be persistent from the prenatal period. Buccal methylation at 5 years was also associated with current lung function and occurrence of wheeze, and these functionally associated loci are enriched for vitamin C associated loci.
Clinical trial registration ClinicalTrials.gov, NCT01723696 and NCT03203603.
... H3ac, H3K27me (Onishchenko et al., 2008) 5mC (Bose et al., 2012;Onishchenko et al., 2008) N/A Depression like behaviour (Onishchenko et al., 2008) Methamphetamine N/A 5mC (Itzhak et al., 2015) N/A Increased cocaine reward and hyperlocomotion as well as diminished conditional fear (Itzhak et al., 2015) Nicotine H3ac (Levine et al., 2011) 5mC (Breton et al., 2009;Maccani et al., 2013;Suter et al., 2011Suter et al., , 2010 miR16, miR21, miR146a (Maccani et al., 2010) • Increased aggression, locomotion in adult male (Yochum et al., 2014) • Birth weight reduction (Suter et al., 2011) Opioid N/A 5mC (Chorbov et al., (Watson et al., 2015) DNA methylation at promoter Adult male rats ↓ DNA methylation of synaptic Dlgap2 in nucleus accumbens of adult brains (Watson et al., 2015) WIN-55,212-2 cannabinoid agonist ...
Abstract: Changes in gene expression are involved in many brain functions. Epigenetic processes modulate gene expression by histone modification and DNA methylation or RNA-mediated processes, which is important for brain function. Consequently, epigenetic changes are also a part of brain diseases such as mental illness and addiction. Understanding the role of different factors on the brain epigenome may help us understand the function of the brain. This review discussed the effects of caffeine, lipids, addictive substances, physical activity, and pollutants on the epigenetic changes in the brain and their modulatory effects on brain function.
... As explained lifestyle-derived epigenetic regulators: environmental tobacco smoke and ethanol consumption, cigarette smoking interferes with epigenetic regulation. Maternal tobacco smoking leads to placental DNA methylation that is directly linked with the gestational age (Maccani et al., 2013). Enormous studies revealed that changes in DNA methylation appear from in utero exposure to cigarette smoking. ...
... The difference in smoking status may indicate that the polymorphism is highly associated with smoking; tobacco may contribute to RUNX3 aberrant expression and thus may elevate the risk of CRC. 21 However, the exact mechanism is still unclear. RUNX3 expression was downregulated in high-grade tumor stage groups and the oncogene-repressor role was attenuated; thus, the tumor progression was enhanced. ...
Background
Accumulating evidence has indicated that runt-related transcription factor 3 ( RUNX3) gene polymorphism (rs7528484) is associated with an alimentary system cancer risk. However, the role of rs7528484 in colorectal cancer is still unclear. The present study aimed to explore the association between rs7528484 and colorectal cancer susceptibility in a Chinese Han population.
Material and methods
We firstly investigated the effect of the polymorphism rs7528484 in distal promoter of RUNX3 polymorphism on colorectal cancer risk in a Chinese Han population comprising 427 colorectal cancer patients and 503 controls. We then carried out a phenotype–genotype association analysis to validate its influence on the adjacent gene RUNX3.
Results
Logistic regression analysis demonstrated that the T allele of rs7528484 was significantly associated with an increased risk for colorectal cancer occurrence in our case-control study (odds ratio = 1.33; 95% confidence interval = 1.09–1.65; P = 0.005). In stratified analysis, the susceptibility of colorectal cancer in the T allele carriers increased among the smokers, III and IV tumor stage, and at the rectum. Furthermore, the T allele was significantly correlated with lower expression of RUNX3 in vitro.
Conclusion
In summary, the current case-control and genotype–phenotype study provides convincing evidence that functional RUNX3 polymorphism (rs7528484) is related to colorectal cancer risk and is a plausible marker for the prediction of colorectal cancer.
... Current evidence suggests that epigenetic mechanisms, such as DNA methylation (DNAm), may mediate the effect of MSDP on the children's health outcomes. MSDP-induced alterations in DNAm have been observed in the placenta, umbilical cord blood and buccal cells of the offspring [20,21]. In addition, a dose-response effect of MSDP has been reported on the methylation levels of 15 CpG sites located in seven gene regions [22]. ...
Background: To investigate the intergenerational effects of grandmaternal smoking during pregnancy (GMSDP) on the DNA methylation of grandchildren. Methods: Data from the Isle of Wight birth cohort with information regarding GMSDP and DNA methylation profiling at the birth of grandchildren (n = 161) were used. Differentially methylated CpG sites related to GMSDP were identified using testing–training screening, analysis of variance and multivariate analysis of covariance. The association between identified CpG sites and expression levels of neighboring genes was tested by linear regression. Results: Twenty-three CpG sites were differentially methylated in grandchildren because of GMSDP, and eight of these were associated with expression levels of 13 neighboring genes. Conclusion: GMSDP has an intergenerational effect on the DNA methylation profile of grandchildren independent of maternal smoking during pregnancy.
... Another group that used this same BeadChip reported CpG sites mapping to GTF2H2C and GTF2H2D in the placental methylome strongly associated with maternal smoking (Chhabra et al., 2014). Usage of the Illumina HumanMethylation BeadChip technology in another cohort population showed that methylation patterns within the RUNX3 gene were linked to maternal smoking during pregnancy with one of the loci correlating with decreased gestational age (Maccani et al., 2013). ...
The conceptus is most vulnerable to developmental perturbation during its early stages when the events that create functional organ systems are being launched. As the placenta is in direct contact with maternal tissues, it readily encounters any xenobiotics in her bloodstream. Besides serving as a conduit for solutes and waste, the placenta possesses a tightly regulated endocrine system that is, of itself, vulnerable to pharmaceutical agents, endocrine disrupting chemicals (EDCs), and other environmental toxicants. To determine whether extrinsic factors affect placental function, transcriptomics and other omics approaches have become more widely used. In casting a wide net with such approaches, they have provided mechanistic insights into placental physiological and pathological responses and how placental responses may impact the fetus, especially the developing brain through the placenta-brain axis. This review will discuss how such omics technologies have been utilized to understand effects of EDCs, including the widely prevalent plasticizers bisphenol A (BPA), bisphenol S (BPS), and phthalates, other environmental toxicants, pharmaceutical agents, maternal smoking, and air pollution on placental gene expression, DNA methylation, and metabolomic profiles. It is also increasingly becoming clear that miRNA (miR) are important epigenetic regulators of placental function. Thus, the evidence to date that xenobiotics affect placental miR expression patterns will also be explored. Such omics approaches with mouse and human placenta will assuredly provide key biomarkers that may be used as barometers of exposure and can be targeted by early mitigation approaches to prevent later diseases, in particular neurobehavioral disorders, originating due to placental dysfunction.
... However, the placental epigenome has not been as thoroughly studied, although the placenta is likely a critical target organ of MSDP-associated toxicity. A handful of prior studies have examined the relationships between MSDP and (DNAm) in human placenta [11][12][13][14] , identifying MSDP-associated CpGs some of which have been suggested to partially mediate the effects of MSDP on lower birth weight (BW) 15 . ...
Maternal smoking during pregnancy (MSDP) contributes to poor birth outcomes, in part through disrupted placental functions, which may be reflected in the placental epigenome. Here we present a meta-analysis of the associations between MSDP and placental DNA methylation (DNAm) and between DNAm and birth outcomes within the Pregnancy And Childhood Epigenetics (PACE) consortium (N = 1700, 344 with MSDP). We identify 443 CpGs that are associated with MSDP, of which 142 associated with birth outcomes, 40 associated with gene expression, and 13 CpGs are associated with all three. Only two CpGs have consistent associations from a prior meta-analysis of cord blood DNAm, demonstrating substantial tissue-specific responses to MSDP. The placental MSDP-associated CpGs are enriched for environmental response genes, growth-factor signaling, and inflammation, which play important roles in placental function. We demonstrate links between placental DNAm, MSDP and poor birth outcomes, which may better inform the mechanisms through which MSDP impacts placental function and fetal growth. Maternal smoking during pregnancy contributes to poor birth outcomes. Here the authors perform a meta-analysis of the associations between maternal smoking during pregnancy and placental DNA methylation and identify links between these and poor birth outcomes, which may better inform the mechanisms through which smoking impacts placental function and fetal growth.
... Thus, prenatal smoke exposure at critical points of fetal development may exert important and persistent effects on DNA methylation which in turn influence gene expression and phenotypes later in life. As such, DNA methylation profiles may have utility as biomarkers of MS, as estimators of risk for behavioral deficits and disease, and as therapeutic targets [288][289][290]. In conjunction with its critical roles in brain and behavioral phenotypes during development and across the lifespan, the DNA methylome also mediates intergenerational phenotypic transmission via the germline. ...
Maternal smoking during pregnancy is associated with an ensemble of neurodevelopmental consequences in children and therefore constitutes a pressing public health concern. Adding to this burden, contemporary epidemiological and especially animal model research suggests that grandmaternal smoking is similarly associated with neurodevelopmental abnormalities in grandchildren, indicative of intergenerational transmission of the neurodevelopmental impacts of maternal smoking. Probing the mechanistic bases of neurodevelopmental anomalies in the children of maternal smokers and the intergenerational transmission thereof, emerging research intimates that epigenetic changes, namely DNA methylome perturbations, are key factors. Altogether, these findings warrant future research to fully elucidate the etiology of neurodevelopmental impairments in the children and grandchildren of maternal smokers and underscore the clear potential thereof to benefit public health by informing the development and implementation of preventative measures, prophylactics, and treatments. To this end, the present review aims to encapsulate the burgeoning evidence linking maternal smoking to intergenerational epigenetic inheritance of neurodevelopmental abnormalities, to identify the strengths and weaknesses thereof, and to highlight areas of emphasis for future human and animal model research therein.
... The exposed GDM and NGT women had a significantly higher level of methylation at CpG1 and CpG3 site compared with unexposed ones. The association between tobacco smoking and altered DNA methylation patterns has been shown for a number of single genes, mostly related to inflammation, oxidative stress, and hypoxia, as well as in genome-wide methylation studies [69][70][71] showing some of the CpG sites associated with low birthweight in the offspring or decreased gestational age [72,73]. So far, these studies have suggested that the maternal smoking deregulates the placental methylation in CpGs which correlates with alterations in gene expression along signature pathways [69,74,75]. ...
Aims
Gestational diabetes mellitus (GDM) can lead to short- and long-term complications for the child. Epigenetic alterations could contribute to explaining the metabolic disturbances associated with foetal programming. Although the role of the FTO gene remains unclear, it affects metabolic phenotypes probably mediated by epigenetic mechanisms. The aim of this study was to assess whether placental DNA epigenetic modifications at FTO promoter-associated cysteine–phosphate–guanine (CpG) sites are correlated with GDM. A secondary aim was to evaluate the association between the placental FTO DNA methylation and the maternal metabolic traits in women with and without GDM.
Methods
Socio-demographic characteristics, clinical parameters at the third trimester of pregnancy, Mediterranean diet adherence, and physical activity were assessed in 33 GDM women and 27 controls. Clinical information about the newborns was registered at birth. The FTO rs9939609 (T > A) was genotyped.
Results
No association between FTO DNA methylation and GDM was found. DNA methylation on the maternal side at the CpG1 was associated with maternal smoking in GDM ( p = 0.034), and DNA methylation at the CpG3 was correlated with smoking or former smoking in controls ( p = 0.023). A higher level of TGs was correlated with higher foetal placental DNA methylation at the CpG2 ( p = 0.036) in GDM. An inverse association between HDL-C and maternal placental DNA methylation at the CpG3 in controls ( p = 0.045) was found. An association between FTO rs9939609 and neonatal birthweight ( p = 0.033) was detected.
Conclusions
In the awareness that the obesity pathophysiology is complex, the study adds a piece to this intricate mosaic.
... Furthermore, in response to prenatal cigarette exposure, two other genes, NR3C1, a glucocorticoid receptor [50], and HSD11B2, a corticosteroid dehydrogenase isozyme [51], are found to be epigenetically regulated with hypermethylation in the placenta, associated with poor neurodevelopment in the offspring [52]. There are several differential methylation patterns identified in genes such as RUNX3 [46], PURA, GTF2H2, HKR1 [49], GCA, and GPR13 [53] that are associated with in-utero exposure to nicotine. The functions of these genes are not yet well understood in the developing placenta, but they may play important roles in one or the other metabolic pathways essential for healthy fetal development. ...
Maternal diabetes alters the global epigenetic mechanisms and expression of genes involved in neural tube development in mouse embryos. Since DNA methylation is a critical epigenetic mechanism that regulates gene functions, gene-specific DNA methylation alterations were estimated in human neural progenitor cells (hNPCs) exposed to high glucose (HG) in the present study. The DNA methylation pattern of genes involved in several signalling pathways including axon guidance (SLIT1-ROBO2 pathway), and Hippo pathway (YAP and TAZ) was altered in hNPCs exposed to HG. The expression levels of SLIT1-ROBO2 pathways genes (including its effectors, SRGAP1 and CDC42) which mediates diverse cellular processes such as proliferation, neurogenesis and axon guidance, and Hippo pathway genes (YAP and TAZ) which regulates proliferation, stemness, differentiation and organ size were downregulated in hNPCs exposed to HG. A recent report suggests a possible cross-talk between SLIT1-ROBO2 and TAZ via CDC42, a mediator of actin dynamics. Consistent with this, SLIT1 knockdown downregulated the expression of its effectors and TAZ in hNPCs, suggesting that HG perturbs the cross-talk between SLIT1-ROBO2 and TAZ in hNPCs. Overall, this study demonstrates that HG epigenetically alters the SLIT1-ROBO2 and Hippo signalling pathways in hNPCs, forming the basis for neurodevelopmental disorders in offspring of diabetic pregnancy.
... However, following the prenatal programming paradigm regarding prenatal epigenetic changes such as alterations in DNA methylation patterns and their role in onset of diseases, it is suggested that changes in DNA methylation could be one of the contributing mechanisms in dependency establishment. Thus far, a number of birth cohort studies indeed reported aberrantly methylated CpG sites in peripheral blood [26], placenta [27] and buccal cells [28] from children of smoking mothers. Furthermore, in the mouse studies from our group, PSE was shown to induce differential gene regulation and DNA methylation profiles of a variety of genes [29,30], but this effect was not shown by other studies [31]. ...
Prenatal smoke exposure (PSE) is a risk factor for nicotine dependence. One susceptibility gene for nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher activity of the CYP2A6 enzyme is associated with nicotine dependence, but no research has addressed the PSE effects on the CYP2A6 gene or its mouse homologue Cyp2a5. We hypothesized that PSE affects Cyp2a5 promoter methylation, Cyp2a5 mRNA levels, and nicotine metabolism in offspring. We used a smoke-exposed pregnant mouse model. RNA, DNA, and microsomal protein were isolated from liver tissue of foetal, neonatal, and adult offspring. Enzyme activity, Cyp2a5 mRNA levels, and Cyp2a5 methylation status of six CpG sites within the promoter region were analysed via HPLC, RT-PCR, and bisulphite pyrosequencing. Our data show that PSE induced higher cotinine levels in livers of male neonatal and adult offspring compared to controls. PSE-induced cotinine levels in neonates correlated with Cyp2a5 mRNA expression and promoter methylation at CpG-7 and CpG+45. PSE increased methylation in almost all CpG sites in foetal offspring, and this effect persisted at CpG-74 in male neonatal and adult offspring. Our results indicate that male offspring of mothers which were exposed to cigarette smoke during pregnancy have a higher hepatic nicotine metabolism, which could be regulated by DNA methylation. Given the detected persistence into adulthood, extrapolation to the human situation suggests that sons born from smoking mothers could be more susceptible to nicotine dependence later in life.
... Interestingly, differentially methylated loci in relation to asthma development have been recently identified in newborns [87]. The stable CpG cg27058497 (RUNX3) has been associated with in utero tobacco smoking exposure [88], childhood asthma [89], oesophagus squamous cell carcinoma [90] and chronic fatigue syndrome [91]. Despite adjustment for maternal smoking in our gestational age EWAS model, we observed overlap between all FDR hits from our gestational age EWAS with those FDR hits presented in the maternal smoking related DNA methylation [20] with an overlap of 2302/47,324 CpGs (4.9%, P enrichment < 2.2 × 10 − 308 ). ...
Background:
Preterm birth and shorter duration of pregnancy are associated with increased morbidity in neonatal and later life. As the epigenome is known to have an important role during fetal development, we investigated associations between gestational age and blood DNA methylation in children.
Methods:
We performed meta-analysis of Illumina's HumanMethylation450-array associations between gestational age and cord blood DNA methylation in 3648 newborns from 17 cohorts without common pregnancy complications, induced delivery or caesarean section. We also explored associations of gestational age with DNA methylation measured at 4-18 years in additional pediatric cohorts. Follow-up analyses of DNA methylation and gene expression correlations were performed in cord blood. DNA methylation profiles were also explored in tissues relevant for gestational age health effects: fetal brain and lung.
Results:
We identified 8899 CpGs in cord blood that were associated with gestational age (range 27-42 weeks), at Bonferroni significance, P < 1.06 × 10- 7, of which 3343 were novel. These were annotated to 4966 genes. After restricting findings to at least three significant adjacent CpGs, we identified 1276 CpGs annotated to 325 genes. Results were generally consistent when analyses were restricted to term births. Cord blood findings tended not to persist into childhood and adolescence. Pathway analyses identified enrichment for biological processes critical to embryonic development. Follow-up of identified genes showed correlations between gestational age and DNA methylation levels in fetal brain and lung tissue, as well as correlation with expression levels.
Conclusions:
We identified numerous CpGs differentially methylated in relation to gestational age at birth that appear to reflect fetal developmental processes across tissues. These findings may contribute to understanding mechanisms linking gestational age to health effects.
... It is possible that the influences of grandmaternal BMI and smoking on epigenetic changes can persist into later life of the mothers and then be transferred to the third generation. A few studies have investigated the mediating role of DNA methylation and have found that differential DNA methylations mediated the association between maternal smoking during pregnancy and birth outcomes, including lower BW (37), small for gestational age (38), and preterm birth (39). Further studies are needed to corroborate the epigenetic mechanism across two generations and explore how it may play a role in transgenerational effects. ...
Objective:
The aims of this study are to examine the potential association between grandmaternal BMI and grandchild's birth weight (BW) and whether maternal BW and BMI mediate this association.
Methods:
Data of 209 grandmother-mother pairs and 355 grandchildren from the Isle of Wight birth cohort in the UK were analyzed using path analysis.
Results:
An indirect effect of grandmaternal BMI on increasing grandchild's BW was mediated by maternal BW and BMI at age 18 years (indirect effects: β = 2.3 g/unit increase in grandmaternal BMI via maternal BW and β = 4.4 g via maternal BMI; P = 0.04). These two mediating effects of maternal BW and BMI confounded one another. Grandmaternal smoking during pregnancy had an indirect effect on decreasing grandchild's BW, dependent on maternal smoking during pregnancy and BW (indirect effects: β = -36.1 g compared with nonsmoking grandmothers via maternal smoking during pregnancy and β = -27.2 g via maternal BW; P = 0.005). Neither direct effect between grandmaternal BMI and grandchild's BW nor that between grandmaternal smoking during pregnancy and grandchild's BW was statistically significant.
Conclusions:
Larger grandmaternal BMI indirectly increased grandchild's BW via maternal BW and BMI. Grandmaternal smoking during pregnancy indirectly reduced grandchild's BW via maternal smoking during pregnancy and BW.
... However, the molecular pathways responsible for changes in placental structure and function in association with pregnancy outcomes have not been widely studied yet 19 . More recent studies highlight the importance of epigenetic mechanisms, primarily DNA methylation, in placental performance during pregnancy [20][21][22] . ...
Being born small (SGA) or large for gestational age (LGA) is associated with adverse birth outcomes and metabolic diseases in later life of the offspring. It is known that aberrations in growth during gestation are related to altered placental function. Placental function is regulated by epigenetic mechanisms such as DNA methylation. Several studies in recent years have demonstrated associations between altered patterns of DNA methylation and adverse birth outcomes. However, larger studies that reliably investigated global DNA methylation are lacking. The aim of this study was to characterize global placental DNA methylation in relationship to size for gestational age. Global DNA methylation was assessed in 1023 placental samples by LC-MS/MS. LGA offspring displayed significantly higher global placental DNA methylation compared to appropriate for gestational age (AGA; p < 0.001). ANCOVA analyses adjusted for known factors impacting on DNA methylation demonstrated an independent association between placental global DNA methylation and LGA births (p < 0.001). Tertile stratification according to global placental DNA methylation levels revealed a significantly higher frequency of LGA births in the third tertile. Furthermore, a multiple logistic regression analysis corrected for known factors influencing birth weight highlighted an independent positive association between global placental DNA methylation and the frequency of LGA births (p = 0.001).
... This association has been strongly confirmed by the PACE Consortium that identified in newborns at least 6000 CpG sites which resulted differentially methylated in response to maternal smoking [152]. In particular, the RUNX3 gene associated with airway hyper-responsiveness and asthma, resulted differentially methylated at seven loci in the placentas of smoking women who delivered SGA newborns [153]. ...
Increasing evidence suggests that early-life events can predispose the newborn to a variety of health issues in later life. In adverse pre- and perinatal conditions, oxidative stress appears to play an important role in the development of future pathological outcomes. From a molecular point of view, oxidative stress can result in genome damage and changes in DNA methylation that can in turn prime pathogenic mechanisms. Interestingly, both alterations have been related to a reciprocal regulation of oxidative stress. The aim of this review is to give a brief overview of the complex relationship linking oxidative stress to DNA damage and methylation and to go through the different sources of exposure that a neonate can encounter in utero or shortly after birth. In this context, the setup of methodologies to monitor the extent of oxidative stress, genomic damage and instability or the presence of altered methylation patterns contributes to the understanding on how the complex events occurring in early life can lead to either a healthy status or a pathological condition.
... They also identified increased inter-individual variability in third trimester samples [12]. Other studies have alsofound varied methylation differences associated with gestational age comparing placentas in the third trimester, as well as a global increase in methylation with gestational age (28-40 weeks) [13][14][15]. In addition, the placenta has the highest overall variability in DNA methylation when compared to other tissues [16]. ...
Background:
Preterm birth is a significant clinical problem and an enormous burden on society, affecting one in eight pregnant women and their newborns. Despite decades of research, the molecular mechanism underlying its pathogenesis remains unclear. Many studies have shown that preterm birth is associated with health risks across the later life course. The "fetal origins" hypothesis postulates that adverse intrauterine exposures are associated with later disease susceptibility. Our recent studies have focused on the placental epigenome at term. We extended these studies to genome-wide placental DNA methylation across a wide range of gestational ages. We applied methylation dependent immunoprecipitation/DNA sequencing (MeDIP-seq) to 9 placentas with gestational age from 25 weeks to term to identify differentially methylated regions (DMRs).
Results:
Enrichment analysis revealed 427 DMRs with nominally significant differences in methylation between preterm and term placentas (p < 0.01) and 21 statistically significant DMRs after multiple comparison correction (FDR p < 0.05), of which 62% were hypo-methylated in preterm placentas vs term placentas. The majority of DMRs were in distal intergenic regions and introns. Significantly enriched pathways identified by Ingenuity Pathway Analysis (IPA) included Citrulline-Nitric Oxide Cycle and Fcy Receptor Mediated Phagocytosis in macrophages. The DMR gene set overlapped placental gene expression data, genes and pathways associated evolutionarily with preterm birth.
Conclusion:
These studies form the basis for future studies on the epigenetics of preterm birth, "fetal programming" and the impact of environment exposures on this important clinical challenge.
... 126 In contrast, some CpG loci of RUNX3 (a gene with a role in T cell lymphopoiesis, which is linked with asthma and upper airway hyperresponsiveness), are hypermethylated in the placenta of pregnant women who smoke. 127 CYP1A1 (Cytochrome P450, family 1, subfamily A, polypeptide 1) and AHRR (aryl-hydrocarbon receptor repressor) are two genes involved in the detoxification metabolism of tobacco compounds such as PAH. CYP1A1, a member of the P450 cytochrome family, catalyzes the conversion of PAH to harmful carcinogenic intermediaries capable of forming DNA adducts. ...
... Furthermore, maternal tobacco smoking induced hypermethylation of cg04757093 within the body of the RUNX3 gene, which has been associated with premature birth. Specifically, increased methylation by one logit, increases 10 times the risk of premature birth (Maccani et al., 2014). ...
Among epigenetic mechanisms, DNA methylation has been widely studied with respect to many environmental factors. Smoking is a common factor which affects both global and gene-specific DNA methylation. It is supported that smoking directly affects DNA methylation, and these effects contribute to the development and progression of various diseases, such as cancer, lung and cardiovascular diseases and male infertility. In addition, prenatal smoking influences the normal development of the fetus via DNA methylation changes. The DNA methylation profile and its smoking-induced alterations helps to distinguish current from former smokers and non-smokers and can be used to predict the risk for the development of a disease. This review summarizes the DNA methylation changes induced by smoking, their correlation with smoking behavior and their association with various diseases and fetus development.
... In the last few years, animal and human studies have linked lifestyle factors to epigenetic changes, identifying the timing of early-life exposures as the factors for the different health outcomes in the offspring. In this regard, pregnant women are inevitably exposed to environmental insults of heterogeneous nature: not only nutrition, but also physical activity, tobacco smoking, alcohol consumption, environmental pollutants, psychological stress, and shift-work, all of which have been identified to modify epigenetic patterns [118][119][120][121][122][123][124][125][126][127][128][129][130][131][132] (Figure 1). ...
Gestational Diabetes Mellitus (GDM) is the most common metabolic condition during pregnancy and may result in short- and long-term complications for both mother and offspring.
The complexity of phenotypic outcomes seems influenced by genetic susceptibility, nutrient-gene interactions and lifestyle interacting with clinical factors. There are strong evidences that not only the adverse genetic background but also the epigenetic modifications in response to nutritional and environmental factors could influence the maternal hyperglycemia in pregnancy and the foetal metabolic programming. In this view, the correlation between epigenetic modifications and their transgenerational effects represents a very interesting field of study. The present review gives insight into the role of gene variants and their interactions with nutrients in GDM. In addition, we provide an overview of the epigenetic changes and their role in the maternal-foetal transmission of chronic diseases. Overall, the knowledge of epigenetic modifications induced by an adverse intrauterine and perinatal environment could shed light on the potential pathophysiological mechanisms of long-term disease development in the offspring and provide useful tools for their prevention.
... The promoter region of CYTIP and LINC00114 were found hypomethylated in preterm maternal peripheral blood [22], but no significant DNA methylation changes in maternal blood were identified in Parets's study [23]. Moreover, DNA methylation changes of UCN [24], OXTR [24,25], RUNX3 [26] and VEGF [27] were observed in PTB placenta. Regarding cord blood, Schroeder et al. [28] found that DNA methylation variations of 39 genes (including AVP, OXT, and CRHBP) were associated with gestational age (GA). ...
Background:
The etiology and mechanism of spontaneous preterm birth (sPTB) are still unclear. Accumulating evidence has documented that various environmental exposure scenarios may cause maternal and fetal epigenetic changes, which initiates the focus on whether epigenetics can contribute to the occurrence of sPTB. Therefore, we conducted the current study to examine and compare the DNA methylation changes associated with sPTB in placenta and cord blood.
Methods:
This hospital-based case-control study was carried out at three Women and Children's hospitals in South China, where 32 spontaneous preterm births and 16 term births were recruited. Genome-wide DNA methylation profiles of the placenta and cord blood from these subjects were measured using the Illumina HumanMethylation EPIC BeadChip, and sPTB-associated differential methylated CpG sites were identified using limma regression model, after controlling for major maternal and infant confounders. Further Gene Ontology analysis was performed with PANTHER in order to assess different functional enrichment of the sPTB-associated genes in placenta and cord blood.
Results:
After controlling for potential confounding factors, one differentially methylated position (DMP) in placenta and 31 DMPs in cord blood were found significantly associated with sPTB (Bonferroni corrected p < 0.05). The sPTB-associated CpG sites in placenta were mapped to genes that showed higher enrichment on biological processes including biological regulation, multicellular organismal process, and especially response to stimulus, while those in cord blood were mapped to genes that had higher enrichment on biological processes concerning cellular process, localization, and particularly metabolic process.
Conclusion:
Findings of this study indicated that DNA methylation alteration in both placenta and cord blood are associated with sPTB, yet the DNA methylation modification patterns may appear differently in placenta and cord blood.
... There is increasing evidence that maternal smoking may affect a child's global and gene-specific DNA methylation (DNAm) profiles as early as in utero [10][11][12][13][14][15][16][17][18][19][20][21]. An increasing number of genes have been identified for which the DNAm levels at specific CpG sites in newborns were significantly associated with maternal smoking during pregnancy, some of whichsuch as those encoding Aryl-hydrocarbon receptors repressor (AHRR), growth factor independent 1 transcriptional repressor (GFI1), and cytochrome P450, family 1, member A1 (CYP1A1)have been reported by multiple studies [10][11][12][13][14][15][16][17][18][19]21]. ...
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 the male fetus may be more vulnerable to the impact of maternal smoking on DNAm than the female; 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.
... correspond to genes previously linked to PTB [66][67][68][69]. Ingenuity Pathway Analysis further identified Esr1 as one of the top upstream regulators impacted in the placental tissues following paternal developmental TCDD exposure (Table 1). ...
Preterm birth (PTB), parturition prior to 37 weeks gestation, is the leading cause of neonatal mortality. The causes of spontaneous PTB are poorly understood; however, recent studies suggest this condition may arise as a consequence of the parental fetal environment. Specifically, we previously demonstrated that developmental exposure of male mice (F1 animals) to the environmental endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was associated with reduced sperm quantity/quality in adulthood and control female partners frequently delivered preterm. Reproductive defects persisted in the F2 and F3 descendants and spontaneous PTB was common. Reproductive changes in the F3 males, the first generation without direct TCDD exposure, suggest the occurrence of epigenetic alterations in the sperm, which have the potential to impact placental development. Herein, we conducted an epigenetic microarray analysis of control and F1 male derived placentae, which identified 2171 differentially methylated regions (DMRs), including the progesterone receptor (Pgr) and insulin-like growth factor (Igf2). To assess if Pgr and Igf2 DNA methylation changes were present in sperm and persist in future generations, we assessed methylation and expression of these genes in F1/F3 sperm and F3-derived placentae. Although alterations in methylation and gene expression were observed, in most tissues, only Pgr reached statistical significance. Despite the modest gene expression changes in Igf2, offspring of F1 and F3 males consistently exhibited IUGR. Taken together, our data indicate that paternal developmental TCDD exposure is associated with transgenerational placental dysfunction, suggesting epigenetic modifications within the sperm have occurred. An evaluation of additional genes and alternative epigenetic mechanisms is warranted.
... Several genes were found to be differentially methylated in preterm placentas of which RUNX3, a tumor suppressor gene responsible for maintenance of normal immune system, was significantly associated with maternal smoking during pregnancy and reduced gestational age. 118 Khot et al showed that alterations in micronutrients such as vitamin B12 and folic acid and their metabolism may affect the enzymes involved in the production of methyl donors S-Adenosyl methionine (SAM), thereby affecting global DNA methylation in the plasma of women experiencing pPTB which in turn may have increased the global DNA methylation levels in the PTB placentas suggesting that methylation defects in the PT placentas may have serious consequences on the epigenome of the growing fetus. 119 Another study performed array-based genome-wide DNA methylome study and identified genes that were found to be differentially methylated in PTB placentas compared to term placentas. ...
Placenta, the first organ to be formed during gestation, plays a crucial role in intrauterine regulation of fetal growth and is involved in several functions during fetal development such as exchange of nutrients, wastes, and gases; protection against maternal immune rejection; and various metabolic and endocrine functions. Several studies have shown the regulation of epigenetic factors and the phenomenon of genomic imprinting in placentation and embryogenesis. Any gain or loss of imprint marks in the placenta has been shown to associate with severe placental defects which in turn affect both the mother and the growing fetus and can have long-term effects during adulthood. Using candidate and genome-wide high throughput approaches, several studies have shown association between aberrant epigenetic factors in the form of DNA methylation, histone modifications, and non-coding RNAs and placental defects in both human and animal models as well as using in vitro studies. In the current review, we discuss several placenta-related pathophysiologies and their association with various aberrant epigenetic factors and gene expression patterns in both in vivo and in vitro systems. This review will help the researchers gain insight into the recent evidences in the area of placentation and epigenetics and to design novel strategies to study and prevent the defects in this underestimated organ.
... Importantly, research in both humans (the focus of this review) and animal models (see Table 1) continues to characterize the influence of environmental exposures on epigenetic changes. Despite the complexity in humans, this work is significant because epigenetic mechanisms, such as DNA methylation profiles, may have utility not only as diagnostic biomarkers capable of predicting increased risk for exposure, behavioral deficits, and disease, but also as therapeutic targets (Ladd-Acosta, 2015; Maccani et al., 2013;Pajer et al., 2012). ...
The period of in utero development is one of the most critical windows during which adverse conditions and exposures may influence the growth and development of the fetus as well as its future postnatal health and behavior. Maternal substance use during pregnancy remains a relatively common but nonetheless hazardous in utero exposure. For example, previous epidemiological studies have associated prenatal substance exposure with reduced birth weight, poor developmental and psychological outcomes, and increased risk for diseases and behavioral disorders (e.g., externalizing behaviors like ADHD, conduct disorder, and substance use) later in life. Researchers are now learning that many of the mechanisms whereby adverse in utero exposures may affect key pathways crucial for proper fetal growth and development are epigenetic in nature, with the majority of work in humans considering DNA methylation specifically. This review will explore the research to date on epigenetic alterations tied to maternal substance use during pregnancy and will also discuss the possible role of DNA methylation in the robust relationship between maternal substance use and later behavioral and developmental sequelae in offspring.
... Additional targets include cancer, cell cycle, and metabolism-related genes (37, 59, 90,115,148,167,203). Prenatal exposure to tobacco smoke has been associated with alterations of genes related to fetal growth restriction (86,118), development (94,123,171), cancer and cell growth (160), and other processes (26,27,181,182,191). Among these genes, contactin-associated protein-like 2 (CNTNAP2), cytochrome P450, family 1, member A1 (CYP1A1), and myosin IG (MYO1G) are common across numerous studies (125). ...
DNA methylation is the most well studied of the epigenetic regulators in relation to environmental exposures. To date, numerous studies have detailed the manner by which DNA methylation is influenced by the environment, resulting in altered global and gene-specific DNA methylation. These studies have focused on prenatal, early-life, and adult exposure scenarios. The present review summarizes currently available literature that demonstrates a relationship between DNA methylation and environmental exposures. It includes studies on aflatoxin B1, air pollution, arsenic, bisphenol A, cadmium, chromium, lead, mercury, polycyclic aromatic hydrocarbons, persistent organic pollutants, tobacco smoke, and nutritional factors. It also addresses gaps in the literature and future directions for research. These gaps include studies of mixtures, sexual dimorphisms with respect to environmentally associated methylation changes, tissue specificity, and temporal stability of the methylation marks. Expected final online publication date for the Annual Review of Public Health Volume 39 is April 1, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Methylation at 1918 CpG sites was associated with MSDP (Maccani et al., 2013). Of those, APBA2, ATP10A, PTPRO, CASP8, CHFR, KLK10, L3MBTL, MLH1 and RB1 genes contained three or more CpG sites whose methylation was associated with MSDP. ...
The placenta is the first human organ to reach full development during pregnancy. It serves as a barrier but also as an interchange surface. Epigenetic changes observed in placental tissue may reflect intrauterine insults while also pointing to physiological pathways altered under exposure to such environmental threats. By means of a systematic search of the literature, 39 papers assessing human placental epigenetic signatures in association with either (i) psychosocial stress, (ii) maternal psychopathology, (iii) maternal smoking during pregnancy, and (iv) exposure to environmental pollutants, were identified. Their findings revealed placental tissue as a unique source of epigenetic variability that does not correlate with epigenetic patterns observed in maternal or newborn blood, tissues which are typically analyzed regarding prenatal stress. Studies regarding prenatal stress and psychopathology during pregnancy were scarce and exploratory in nature revealing inconsistent findings. Of note, there was a marked tendency towards placental hypomethylation in studies assessing either tobacco use during pregnancy or exposure to environmental pollutants suggesting the interaction between contaminant-derived metabolites and epigenetic machinery. This review highlights the need for further prospective longitudinal studies assessing long-term health effects of placental epigenetic signatures derived from exposure to several prenatal stressors.
... We have attempted to exclude multiple substance abusers from this study, but common factors such as smoking and antidepressants (Supplementary Table SI) can also affect embryonic development. Previous genomewide studies have shown that smoking affects the methylation level in placenta, but changes at the IGF2/H19 locus have not been reported (Suter et al. 2011;Maccani et al. 2013). Furthermore, we did not observe an association between smoking and specific changes in DNA methylation in this study. ...
Study Question
Does prenatal alcohol exposure (PAE) affect regulation of the insulin-like growth factor 2 (IGF2)/H19 locus in placenta and the growth-restricted phenotype of newborns?
Summary Answer
PAE results in genotype-specific trends in both placental DNA methylation at the IGF2/H19 locus and head circumference (HC) of newborns.
What is Known Already
PAE can disturb development of the nervous system and lead to restricted growth of the head, even microcephaly. To clarify the etiology of alcohol-induced growth restriction, we focused on the imprinted IGF2/H19 locus known to be important for normal placental and embryonic growth. The expression of IGF2 and a negative growth controller H19 are regulated by the H19 imprinting control region (H19 ICR) with seven-binding sites for the methylation-sensitive zinc-finger regulatory protein CTCF. A single nucleotide polymorphism rs10732516 G/A in the sixth-binding site has shown to associate with genotype-specific DNA methylation profiles at the H19 ICR.
Study Design, Size, Duration
By grouping 39 alcohol-exposed and 100 control samples according to rs10732516 polymorphism we explored alcohol-induced, genotype-specific changes in DNA methylation at the H19 ICR and the promoter region of H19 (H19 differentially methylated region). Also, IGF2 and H19 mRNA expression level in placenta as well as the phenotypes of newborns were examined.
Participants/Materials, Setting, Methods
We explored alcohol-induced, genotype-specific changes in placental DNA methylation by MassARRAY EpiTYPER and allele-specific changes by bisulphite sequencing. IGF2 and H19 expression in placenta were analyzed by quantitative PCR and the HC, birthweight and birth length of newborns were examined using national growth charts.
Main Results and the Role of Chance
We observed a consistent trend in genotype-specific changes in DNA methylation at H19 ICR in alcohol-exposed placentas. DNA methylation level in the normally highly methylated paternal allele of rs10732516 paternal A/maternal G genotype was decreased in alcohol-exposed placentas. In addition to decreased IGF2 mRNA expression in alcohol-exposed placentas of this specific genotype (P = 0.03), we observed significantly increased expression of H19 in relation to IGF2 when comparing all alcohol-exposed placentas to unexposed controls (P = 0.006). Furthermore, phenotypic examination showed a significant genotype-specific association between the alcohol exposure and HC of newborns (P = 0.001).
Limitations Reasons for Caution
Owing to the exceptional character of the alcohol-exposed human samples collected in this study, the sample size is restricted. An increased sample size and functional studies are needed to confirm these data and clarify the biological significance or causality of the observed associations.
Wider Implications of the Findings
Our results suggest that the rs10732516 polymorphism associates with the alcohol-induced alterations in DNA methylation profiles and head growth in a parent-of-origin manner. We also introduce a novel genotype-specific approach for exploring environmental effects on the IGF2/H19 locus and ultimately on embryonic growth.
Study Funding/Competing Interest(s)
This work was supported by the Academy of Finland (258304), The Finnish Foundation for Alcohol Studies, Finnish Cultural Foundation, Juho Vainio Foundation, Yrjö Jahnsson Foundation and Arvo and Lea Ylppö Foundation. No competing interests are declared.
... Human studies found epigenetic changes in placenta of women smoking during pregnancy throughout the genome [122]. Gene specific [123] alteration of DNA methylation, as well as reduced miRNA expression [124] have also been reported. Another study in buccal cell samples from prenatal tobacco-exposed children found lower methylation levels in the transposable element, Alu Yb8. ...
The epigenetics of an individual is in part built through an intrinsic program, such as an inherited template, and in part built by inputs of the environment. The latter improvises or in some cases permanently changes, the performance of genes from their normal course leads to structural or functional alterations. The influence of the environment is not limited to the contemporary stages; many may be traced before birth or transgenerational to their parental or ancestral time. This chapter reviews the environment, which have been found to influence the epigenetics to cast their actions. The exerting environment is divided here into mental or physiological environment, including enriched environment, learning experience, stress, etc., and the hazardous or chemical environment, including heavy metals, prescription drugs, and addictive substances. This review focuses on the links between the environment and its effect on various elements of epigenetics, and their influence on gene transcription.
... Furthermore, in response to prenatal cigarette exposure, two other genes, NR3C1, a glucocorticoid receptor [50], and HSD11B2, a corticosteroid dehydrogenase isozyme [51], are found to be epigenetically regulated with hypermethylation in the placenta, associated with poor neurodevelopment in the offspring [52]. There are several differential methylation patterns identified in genes such as RUNX3 [46], PURA, GTF2H2, HKR1 [49], GCA, and GPR13 [53] that are associated with in-utero exposure to nicotine. The functions of these genes are not yet well understood in the developing placenta, but they may play important roles in one or the other metabolic pathways essential for healthy fetal development. ...
It is well established that the regulation of epigenetic factors, including chromatic reorganization, histone modifications, DNA methylation, and miRNA regulation, is critical for the normal development and functioning of the human brain. There are a number of maternal factors influencing epigenetic pathways such as lifestyle, including diet, alcohol consumption, and smoking, as well as age and infections (viral or bacterial). Genetic and metabolic alterations such as obesity, gestational diabetes mellitus (GDM), and thyroidism alter epigenetic mechanisms, thereby contributing to neurodevelopmental disorders (NDs) such as embryonic neural tube defects (NTDs), autism, Down’s syndrome, Rett syndrome, and later onset of neuropsychological deficits. This review comprehensively describes the recent findings in the epigenetic landscape contributing to altered molecular profiles resulting in NDs. Furthermore, we will discuss potential avenues for future research to identify diagnostic markers and therapeutic epi-drugs to reverse these abnormalities in the brain as epigenetic marks are plastic and reversible in nature.
... 14,15 More recently, epigenome-wide studies have reported distinct loci-specific DNA methylation patterns by in utero exposure to cigarette smoke in neonates and children. [15][16][17][18][19][20][21][22] Other studies focused on DNA methylation in adults have also identified loci associated with exposure to an individual's own cigarette smoking (active smoking), including current and lifetime smoking behavior and cumulative amount of smoke exposure. [23][24][25][26][27][28][29][30][31] There is considerable overlap in differentially methylated genes by prenatal smoke exposure and adult smoke exposures, and in studies of adult health outcomes, these genes have been linked to smoking related diseases, such as certain cancers and chronic pulmonary and neurological diseases, and relevant biological processes, such as cell regulation and differentiation, and chemical metabolism and detoxification . ...
Maternal smoking in pregnancy (MSP) has been associated with DNA methylation in specific CpG sites in infants and children. We investigated whether MSP, independent of own personal active smoking, was associated with midlife DNA methylation in CpGs that were previously identified in studies of MSP-DNA methylation in children. We used data on MSP collected from pregnant mothers of 89 adult women born in 1959-1964 and measured DNA methylation measured in blood (granulocytes) collected in 2001-2007 (mean age: 43 years). Seventeen CpGs were differentially methylated in MSP-exposed women, with multiple CpGs mapping to CYP1A1, MYO1G, AHRR, and GFI1. These associations were consistent in direction with prior studies (e.g., MSP associated with more and less methylation in AHRR and CYP1A1, respectively) and, with the exception of AHRR CpGs, were not substantially altered by adjustment for active smoking. These preliminary results confirm prior reports that MSP influences the offspring DNA methylation, and suggest that these effects may persist into adulthood, independently of active smoking.
Gestation and early life are highly susceptible to epigenetic alterations, as the fetal epigenome is undergoing large‐scale remodeling, including DNA demethylation and remethylation, which are followed by tissue‐specific methylation as cells undergo fate decisions. If these epigenetic changes are disrupted, the disruption may potentiate the development of disease later in life. This chapter introduces the use of epigenetic measurements as biomarkers of maternal exposure and their links to later health outcomes in the offspring. Further, it goes into the current state of the research across diverse environmental exposures such as to air pollution, metals, persistent organic pollutants, and nutrition. All in all, understanding the epigenetic effects of prenatal exposures may allow us to better assess the risk of adverse outcomes during pregnancy and later childhood diseases, as well as to inform strategies designed to prevent or reduce the impact of environmental exposure on women and children.
Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.
Objective: Preterm birth (PTB) has become a major public health concern as the leading cause of neonatal death, but little is understood about its etiology. Children born preterm are also at increased risk of long-term consequences such as neurodevelopmental disorders, adulthood hypertension and diabetes. Recent studies have indicated that DNA methylation may be involved in the occurrence of PTB as well as related adverse outcomes. The latest Infinium EPIC BeadChip extends the coverage of the genome and provides a better tool to help investigate the involvement of DNA methylation in these conditions.
Methods: We conducted this case-control study in three Women and Children’s hospitals in South China, and enrolled 32 spontaneous preterm births and 16 term births. We assessed placental DNA methylation profiling of these participants with the Infinium EPIC BeadChip. We identified PTB and gestational age (GA)-associated CpG sites with limma regression model, and applied seqlm to identify PTB-associated regions. We performed gene ontology analysis to further interpret functional enrichment of the identified differentially methylated genes in PTB.
Results: We identified a total of 8 differentially methylated positions (DMPs) that were significantly associated with PTB (FDR < 0.1) and a total of 15 DMPs that were associated with GA (FDR < 0.1). In the regional analysis, one differentially methylated region in the SLC23A1 gene overlapped with PTB-associated CpG site. The differentially methylated CpG sites in PTB were mapped to the genes involving in biological processes mainly regarding neurodevelopment, regulation of inflammation and metabolism.
Conclusion: Our findings suggested that preterm placenta have distinct DNA methylation alterations, and these alteration patterns established at birth provide insight into the long-term consequences of preterm birth.
We examined associations between maternal smoking and autism spectrum disorder (ASD) in children in a statewide population-based cohort and sibling comparison design using California birth records (n=2,015,104) with information on maternal smoking, demographics and pregnancy (2007-2010). ASD cases (n=11,722) were identified through California Department of Developmental Services records with diagnoses based on the Diagnostic and Statistical Manual of Mental Disorders-IV-R. We estimated odds ratios (OR) for ASD with/without intellectual disability in the full cohort using logistic regression, and conditional logistic regression in a sibling comparison. In the full cohort, the adjusted OR for ASD and maternal smoking 3-months before/during pregnancy compared to non-smoking was 1.15 (95%CI: 1.04, 1.26), and was similar in cases with (OR=1.12, 95%CI: 0.84, 1.49) and without intellectual disability (OR=1.15, 95%CI: 1.04, 1.27); heavy prenatal smoking (20+ cigarettes/day in any trimester) was related to 58% risk increase (95%CI: 23%, 101%). In the sibling comparison, the OR for heavy smoking was similarly elevated but the CI was wide. Our findings are consistent with an increased risk for ASD in offspring of mothers who smoked 20+ cigarettes/day during pregnancy; associations with lighter smoking were weaker.
Preterm birth (PTB) is a major public health challenge, and novel, sensitive approaches to predict PTB are still evolving. Epigenomic markers are being explored as biomarkers of PTB because of their molecular stability compared to gene expression. This approach is also relatively new compared to gene-based diagnostics, which relies on mutations or single nucleotide polymorphisms. The fundamental principle of epigenome diagnostics is that epigenetic reprogramming in the target tissue (e.g. placental tissue) might be captured by more accessible surrogate tissue (e.g. blood) using biochemical epigenome assays on circulating DNA that incorporate methylation, histone modifications, nucleosome positioning, and/or chromatin accessibility. Epigenomic-based biomarkers may hold great potential for early identification of the majority of PTBs that are not associated with genetic variants or mutations. In this review, we discuss recent advances made in the development of epigenome assays focusing on its potential exploration for association and prediction of PTB. We also summarize population-level cohort studies conducted in the USA and globally that provide opportunities for genetic and epigenetic marker development for PTB. In addition, we summarize publicly available epigenome resources and published PTB studies. We particularly focus on ongoing genome-wide DNA methylation and epigenome-wide association studies. Finally, we review the limitations of current research, the importance of establishing a comprehensive biobank, and possible directions for future studies in identifying effective epigenome biomarkers to enhance health outcomes for pregnant women at risk of PTB and their infants.
Aim:
To evaluate the association between spontaneous preterm birth (SPTB) and DNA methyltransferase (DNMT)1, 3A, 3B, and 3L gene polymorphisms, and their contribution to the clinical characteristics of women with SPTB and their newborns.
Methods:
This case-control study, conducted in 2018, enrolled 162 women with SPTB and 162 women with term delivery. DNMT1 rs2228611, DNMT3A rs1550117, DNMT3B rs1569686, DNMT3B rs2424913, and DNMT3L rs2070565 single nucleotide polymorphisms were genotyped using polymerase chain reaction and restriction fragment length polymorphism methods. The clinical characteristics included in the analysis were family history of preterm birth, maternal smoking, maternal age, gestational week at delivery, and fetal birth weight.
Results:
DNMT gene polymorphisms were not significantly associated with SPTB. DNMT3B rs1569686 and rs2424913 minor alleles (T) were significantly more frequent in women with familial PTB than in women with non-familial PTB, increasing the odds for familial PTB 3.30 and 3.54 times under dominant genetic models. They were also significantly more frequent in women with SPTB who smoked before pregnancy, reaching the most significant association under additive genetic models (odds ratio 6.86, 95% confidence interval 2.25-20.86, P<0.001; odds ratio 3.77, 95% confidence interval 1.36-10.52, P=0.011, respectively).
Conclusions:
DNMT3B rs1569686 and rs2424913 gene polymorphisms might be associated with positive family history of PTB and smoking status.
Cytochrome P450 17A1 (CYP17A1; EC: 1.14.14.19) is a critically important bifunctional enzyme with nicotinamide adenine dinucleotide phosphate (NADPH) as its cofactor that catalyzes the formation of all endogenous androgens. Its hydroxylase activity catalyzes the 17α-hydroxylation of pregnenolone (PREG)/progesterone (P4) to 17α-OH-pregnenolone/17α-OH-progesterone, and its 17,20-lyase activity converts 17α-OH-pregnenolone/17α-OH-progesterone to dehydroepiandrosterone/androstenedione. Androgens are required for male reproductive development, so androgen deficiency resulting from CYP17A1 inhibition may lead to reproductive disorders. There has been some advances on the study of environmental chemicals inhibiting mammalian CYP17A1 expression but no related review was available so we think it now necessary to review their characteristics and inhibiting properties.
Chronic exposure to aflatoxin B1 (AFB1) is linked to the development of hepatocellular carcinoma (HCC). To identify differentially methylated genes involved in AFB1-induced cell transformation, we analyzed DNA methylation patterns in immortal human hepatocyte L02 cells expressing an oncogenic H-Ras allele (L02R cells) and AFB1-transformed L02R (L02RT-AFB1) cells by performing genome-wide methylation profiling. We treated L02R cells with 0.3 μM AFB1 weekly and observed a transformed phenotype at the 17th week post-treatment. The transformed cells (L02RT-AFB1) could grow in an anchorage independent fashion and form tumors in immunodeficient mice. qRT-PCR was performed to examine whether gene methylation led to a reduction in gene expression of methylated candidate genes. As a result, the expression of the following seven genes including JUNB, RUNX3, NAV1, CXCR4, RARRES1, INTS1, and POLL was down-regulated in transformed L02RT-AFB1 cells. The reduction of gene expression of these genes could be reversed by treatment of 5-azadeoxycytidine. The methylated CpG sites of RUNX3 genes were verified using bisulfite sequencing PCR (BSP) assay. Furthermore, a dynamic change in RUNX3 methylation was observed over the course of AFB1-induced cell transformation, which was corresponded to the alteration of gene expression and the extent of DNA damage. In vitro study showed that methylation of RUNX3 tended to abate in L02R cells treated with AFB1 for a short-term period of time. Notably, hypermethylation of RUNX3 appeared in 70% (14/20) of human hepatocellular carcinomas. Moreover, LINE-1 hypomethylation and dynamic changes of DNMTs, TETs and MeCP2 expression were also observed during AFB1-induced transformation. Taken together, these observations suggest that aberrant methylation of RUNX3 and LINE-1 might be involved in AFB1-induced carcinogenesis.
Background:
Infants whose mothers smoked during pregnancy demonstrate lifelong decreases in pulmonary function. DNA methylation changes associated with maternal smoking during pregnancy have been described in placenta and cord blood at delivery, in fetal lung, and in buccal epithelium and blood during childhood. We recently demonstrated in a randomized clinical trial (NCT00632476) that vitamin C supplementation to pregnant smokers can lessen the impact of maternal smoking on offspring pulmonary function and decrease the incidence of wheeze at one year of age.
Objectives:
To determine if vitamin C supplementation reduces changes in offspring methylation in response to maternal smoking and whether methylation at specific CpGs is also associated with respiratory outcomes.
Methods:
Targeted bisulfite sequencing was performed using a subset of placentas, cord bloods and buccal samples collected during the NCT00632476 trial followed by independent validation of selected cord blood DMRs using bisulfite amplicon sequencing.
Results:
The majority (69.03%) of CpGs with ≥ 10% methylation difference between placebo and non-smoker groups were restored (by at least 50%) towards non-smoker levels with vitamin C treatment. A significant proportion of restored CpGs were associated with phenotypic outcome with greater enrichment among hypomethylated CpGs.
Conclusions:
We identified a pattern of normalization in DNA methylation by vitamin C supplementation across multiple loci. The consistency of this pattern across tissues and time suggests a systemic and persistent effect on offspring DNA methylation. Further work is necessary to determine how genome-wide changes in DNA methylation may mediate or reflect persistent effects of maternal smoking on lung function.
The effects of cigarette smoking and alcohol consumption on the length of gestation were examined in a prospective study of 30,596 pregnant women in northern California. Preterm births (<37 weeks' gestation) were 20% more common in women smoking at least one pack of cigarettes per day. This effect was strongest for births occurring before 33 weeks, where the excess was 60%. This excess was not accounted for by differences in maternal age, education, ethnicity, time prenatal care began, drinking during pregnancy, or eight other potential confounding factors. The results indicate a probable effect of smoking on the time of parturition, which is additional to its well-known effect on intrauterine growth retardation. The effect of alcohol consumption on preterm births was also examined, but no consistent trends were found.(JAMA 1986;255:82-84)
Purpose
In the UK the accepted standards for the management of HIV‐positive pregnant women are the British HIV Association Guidelines [ 1 ]. The guidelines were updated in 2012 and make reference to conflicting evidence regarding the risk of premature birth; some studies suggest an increased risk of preterm delivery (PTD) in women treated with protease inhibitors (PIs) but others do not. Several of these studies have been conducted in developing countries and not in a setting analogous to UK healthcare. The aim of this study was to examine data from HIV‐positive pregnant women attending a large UK teaching hospital, comparing the rates of PTD in women on PI‐based regimes to those on non‐PI based regimes. In addition, known and possible confounding risk factors for prematurity were considered.
Methods
We analysed retrospective data from the files of HIV‐positive women attending a UK maternity center with specialist HIV care between 2003 and 2012. Inclusion criteria were women who delivered live‐born infant(s) beyond 24 weeks gestation, and who were taking anti‐retroviral treatment at some point during the first 37 weeks of their pregnancy. Preterm delivery was defined as delivery at less than 37 weeks. Multi‐variable logistic regression adjusted for repeated measures was used to compare the rates of PTD in women on non‐PI‐based regimes with those on PI‐based regimes; adjusting for possible covariates, including the percentage increase in CD4 count during pregnancy, the timing of HAART initiation, and concurrent genital tract infections.
Results
A total of 208 pregnancies in 157 women were included in the study. The overall prematurity rate was 11.1% (PI=11.1%, non‐PI=10.8%), of which 5.8% were before 34 weeks. Analysis of the data demonstrated no statistically significant increased risk between PI and non‐PI based regimes, which persisted following adjustments for known and possible confounding factors.
Conclusions
In this study 11.1% of HIV‐positive women experienced PTD; in the general UK population 7.5% of pregnancies end in PTD [ 2 ]. The inclusion of a PI in the mother's HIV drug regime was not associated with an increased risk for PTD. An association between PTD and CD4 count increase in pregnancy of borderline significance was noted; p=0.053. This is particularly relevant for women commencing HAART during pregnancy in whom, although over 1.5 times as likely to have PTD as those on treatment at conception, this was not statistically significant.
The transcription factor Ets1 contributes to the differentiation of CD8 lineage cells in the thymus, but how it does so is not understood. In this study, we demonstrate that Ets1 is required for the proper termination of CD4 expression during the differentiation of major histocompatability class 1 (MHC I)-restricted thymocytes, but not for other events associated with their positive selection, including the initiation of cytotoxic gene expression, corticomedullary migration, or thymus exit. We further show that Ets1 promotes expression of Runx3, a transcription factor important for CD8 T cell differentiation and the cessation of Cd4 gene expression. Enforced Runx3 expression in Ets1-deficient MHC I-restricted thymocytes largely rescued their impaired Cd4 silencing, indicating that Ets1 is not required for Runx3 function. Finally, we document that Ets1 binds at least two evolutionarily conserved regions within the Runx3 gene in vivo, supporting the possibility that Ets1 directly contributes to Runx3 transcription. These findings identify Ets1 as a key player during CD8 lineage differentiation and indicate that it acts, at least in part, by promoting Runx3 expression.
Background: Epigenetic modifications, such as DNA methylation, due to in utero exposures may play a critical role in early programming for childhood and adult illness. Maternal smoking is a major risk factor for multiple adverse health outcomes in children, but the underlying mechanisms are unclear.
Objective: We investigated epigenome-wide methylation in cord blood of newborns in relation to maternal smoking during pregnancy.
Methods: We examined maternal plasma cotinine (an objective biomarker of smoking) measured during pregnancy in relation to DNA methylation at 473,844 CpG sites (CpGs) in 1,062 newborn cord blood samples from the Norwegian Mother and Child Cohort Study (MoBa) using the Infinium HumanMethylation450 BeadChip (450K).
Results: We found differential DNA methylation at epigenome-wide statistical significance (p-value < 1.06 × 10–7) for 26 CpGs mapped to 10 genes. We replicated findings for CpGs in AHRR, CYP1A1, and GFI1 at strict Bonferroni-corrected statistical significance in a U.S. birth cohort. AHRR and CYP1A1 play a key role in the aryl hydrocarbon receptor signaling pathway, which mediates the detoxification of the components of tobacco smoke. GFI1 is involved in diverse developmental processes but has not previously been implicated in responses to tobacco smoke.
Conclusions: We identified a set of genes with methylation changes present at birth in children whose mothers smoked during pregnancy. This is the first study of differential methylation across the genome in relation to maternal smoking during pregnancy using the 450K platform. Our findings implicate epigenetic mechanisms in the pathogenesis of the adverse health outcomes associated with this important in utero exposure.
Preterm birth affects over 12% of all infants born in the US yet the biology of early delivery remains unclear, including whether epigenetic mechanisms are involved. We examined associations of maternal and umbilical cord blood long interspersed nuclear element-1 (LINE-1) DNA methylation with length of gestation and odds of preterm birth in singleton pregnancies in Project Viva. In white blood cells from maternal blood during 1(st) trimester (n=914) and 2(nd) trimester (n=922), and from venous cord blood at delivery (n=557), we measured LINE-1 by pyrosequencing (expressed as %5 methyl cytosines within the LINE-1 region analyzed [%5mC]). We ran linear regression models to analyze differences in gestation length, and logistic models for odds of preterm birth (<37 v. ≥37 weeks gestation), across quartiles of LINE-1. Mean(SD) LINE-1 levels were 84.3(0.6), 84.5(0.4), and 84.6(0.7) %5mC for 1(st) trimester, 2(nd) trimester and cord blood, respectively. Mean(SD) gestational age was 39.5(1.8) weeks, and 6.5% of infants were born preterm. After adjustment for maternal age, race/ethnicity, BMI, education, smoking status, and fetal sex, women with the highest vs. lowest quartile of 1(st) trimester LINE-1 had longer gestations (0.45 weeks [95% CI 0.12, 0.78]) and lower odds of preterm birth (OR 0.40 [0.17, 0.94]), whereas associations with cord blood LINE-1 were in the opposite direction (-0.45 weeks, -0.83, -0.08) and (OR 4.55 [1.18, 17.5]). In conclusion, higher early pregnancy LINE-1 predicts lower risk of preterm birth. In contrast, preterm birth is associated with lower LINE-1 in cord blood.
The aim of this study was to investigate the promoter hypermethylation as diagnostic markers to detect malignant prostate cells and as prognostic markers to predict the clinical recurrence of prostate cancer.
DNA was isolated from prostate cancer and normal adjacent tissues. After bisulfite conversion, methylation of 14,495 genes was evaluated using the Methylation27 microarrays in 238 prostate tissues. We analyzed methylation profiles in four different groups: (i) tumor (n = 198) versus matched normal tissues (n = 40), (ii) recurrence (n = 123) versus nonrecurrence (n = 75), (iii) clinical recurrence (n = 80) versus biochemical recurrence (n = 43), and (iv) systemic recurrence (n = 36) versus local recurrence (n = 44). Group 1, 2, 3, and 4 genes signifying biomarkers for diagnosis, prediction of recurrence, clinical recurrence, and systemic progression were determined. Univariate and multivariate analyses were conducted to predict risk of recurrence. We validated the methylation of genes in 20 independent tissues representing each group by pyrosequencing.
Microarray analysis revealed significant methylation of genes in four different groups of prostate cancer tissues. The sensitivity and specificity of methylation for 25 genes from 1, 2, and 4 groups and 7 from group 3 were shown. Validation of genes by pyrosequencing from group 1 (GSTP1, HIF3A, HAAO, and RARβ), group 2 (CRIP1, FLNC, RASGRF2, RUNX3, and HS3ST2), group 3 (PHLDA3, RASGRF2, and TNFRSF10D), and group 4 (BCL11B, POU3F3, and RASGRF2) confirmed the microarray results.
Our study provides a global assessment of DNA methylation in prostate cancer and identifies the significance of genes as diagnostic and progression biomarkers of prostate cancer.
Here we presented that the expression of RUNX3 was significantly decreased in 75 cases of clear cell renal cell carcinoma (CCRCC) tissues (p<0.05). Enforced RUNX3 expression mediated 786-O cells to exhibit inhibition of growth, G1 cell-cycle arrest and metastasis in vitro, and to lost tumorigenicity in nude mouse model in vivo. RUNX3-induced growth suppression was found partially to regulate various proteins, including inhibition of cyclinD1, cyclinE, cdk2, cdk4 and p-Rb, but increase of p27(Kip1), Rb and TIMP-1. Therefore, RUNX3 had the function of inhibiting the proliferative and metastatic abilities of CCRCC cells by regulating cyclins and TIMP1.
The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses – the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferroni-type procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.
Altered methylation at Insulin-like Growth Factor 2 (IGF2) regulatory regions has previously been associated with obesity, and several malignancies including colon, esophageal, and prostate adenocarcinomas, presumably via changes in expression and/or loss of imprinting, but the functional significance of these DNA methylation marks have not been demonstrated in humans. We examined associations among DNA methylation at IGF2 differentially methylated regions (DMRs), circulating IGF2 protein concentrations in umbilical cord blood (UCB) and birth weight in newborns.
Questionnaire data were obtained from 300 pregnant women recruited between 2005 and 2009. UCB DNA methylation was measured by bisulfite pyrosequencing. UCB plasma concentrations of soluble IGF2 were measured by ELISA assays. Generalized linear regression models were used to examine the relationship between DMR methylation and IGF2 levels.
Lower IGF2 DMR methylation was associated with elevated plasma IGF2 protein concentrations (β = -9.87, p < 0.01); an association that was stronger in infants born to obese women (pre-pregnancy BMI > 30 kg/m(2), β = -20.21, p < 0.0001). Elevated IGF2 concentrations were associated with higher birth weight (p < 0.0001) after adjusting for maternal race/ethnicity, pre-pregnancy BMI, cigarette smoking, gestational diabetes, and infant sex. These patterns of association were not apparent at the H19 DMR.
Our data suggest that variation in IGF2 DMR methylation is an important mechanism by which circulating IGF2 concentrations, a putative risk factor for obesity and cancers of the colon, esophagus, and prostate, are modulated; associations that may depend on pre-pregnancy obesity.
Maternal smoking during pregnancy has been associated with adverse effects on respiratory health. Whereas the epidemiologic link is incontrovertible, the mechanisms responsible for this association are still poorly understood. Although cigarette smoke has many toxic constituents, nicotine, the major addictive component in cigarette smoke, may play a more significant role than previously realized. The objectives of this study were to determine whether exposure to nicotine prenatally leads to alterations in pulmonary function and airway geometry in offspring, and whether α7 nicotinic acetylcholine receptors (nAChRs) mediate these effects. In a murine model of in utero nicotine exposure, pulmonary function, airway size and number, methacholine response, and collagen deposition were examined. Exposure periods included Gestation Days 7-21, Gestation Day 14 to Postnatal Day 7, and Postnatal Days 3-15. Prenatal nicotine exposure decreases forced expiratory flows in offspring through α7 nAChR-mediated signals, and the critical period of nicotine exposure was between Prenatal Day 14 and Postnatal Day 7. These physiologic changes were associated with increased airway length and decreased diameter. In addition, adult mice exposed to prenatal nicotine exhibit an increased response to methacholine challenge, even in the absence of allergic sensitization. Collagen expression was increased between adjacent airways and vessels, which was absent in α7 nAChR knockout mice. These observations provide a unified mechanism of how maternal smoking during pregnancy may lead to lifelong alterations in offspring pulmonary function and increased risk of asthma, and suggest potential targets to counteract those effects.
The human placenta facilitates the exchange of nutrients, gas and waste between the fetal and maternal circulations. It also protects the fetus from the maternal immune response. Due to its role at the feto-maternal interface, the placenta is subject to many environmental exposures that can potentially alter its epigenetic profile. Previous studies have reported gene expression differences in placenta over gestation, as well as inter-individual variation in expression of some genes. However, the factors contributing to this variation in gene expression remain poorly understood.
In this study, we performed a genome-wide DNA methylation analysis of gene promoters in placenta tissue from three pregnancy trimesters. We identified large-scale differences in DNA methylation levels between first, second and third trimesters, with an overall progressive increase in average methylation from first to third trimester. The most differentially methylated genes included many immune regulators, reflecting the change in placental immuno-modulation as pregnancy progresses. We also detected increased inter-individual variation in the third trimester relative to first and second, supporting an accumulation of environmentally induced (or stochastic) changes in DNA methylation pattern. These highly variable genes were enriched for those involved in amino acid and other metabolic pathways, potentially reflecting the adaptation of the human placenta to different environments.
The identification of cellular pathways subject to drift in response to environmental influences provide a basis for future studies examining the role of specific environmental factors on DNA methylation pattern and placenta-associated adverse pregnancy outcomes.
The epidemic of allergic disease in early life is one of the clearest indicators that the developing immune system is vulnerable to modern environmental changes. A range of environmental exposures epidemiologically associated with allergic disease have been shown to have effects on the foetal immune function in pregnancy, including microbial burden, dietary changes and environmental pollutants. Preliminary studies now suggest that these early effects on immune development may be mediated epigenetically through a variety of processes that collectively modify gene expression and allergic susceptibility and that these effects are potentially heritable across generations. It is also possible that rising rates of maternal allergy, a recognised direct risk factor for infant allergic disease, may be further amplifying the effects of environmental changes. Whilst effective prevention strategies are the ultimate goal in reversing the allergy epidemic, the specific environmental drivers, target genes, and intracellular pathways and mechanisms of early life immune programming are still unclear. It is hoped that identifying genes that are differentially regulated in association with subsequent allergic disease will assist in identifying causal pathways and upstream contributing environmental factors. In this way, epigenetic paradigms are likely to provide valuable insights into how the early environment can be modified to more favourably drive immune development and reverse the allergic epidemic.
Several studies linking alterations in differential placental methylation with pregnancy disorders have implicated (de)regulation of the placental epigenome with fetal programming and later-in-life disease. We have previously demonstrated that maternal tobacco use is associated with alterations in promoter methylation of placental CYP1A1 and that these changes are correlated with CYP1A1 gene expression and fetal growth restriction. In this study we sought to expand our analysis of promoter methylation by correlating it to gene expression on a genome-wide scale. Employing side-by-side IlluminaHG-12 gene transcription with Infinium27K methylation arrays, we interrogated correlative changes in placental gene expression and DNA methylation associated with maternal tobacco smoke exposure at an epigenome-wide level and in consideration of signature gene pathways. We observed that the expression of 623 genes and the methylation of 1024 CpG dinucleotides are significantly altered among smokers, with only 38 CpGs showing significant differential methylation (differing by a methylation level of ≥10%). We identified a significant Pearson correlation (≥0.7 or ≤-0.7) between placental transcriptional regulation and differential CpG methylation in only 25 genes among non-smokers but in 438 genes among smokers (18-fold increase, p < 0.0001), with a dominant effect among oxidative stress pathways. Differential methylation at as few as 6 sites was attributed to maternal smoking-mediated birth weight reduction in linear regression models with Bonferroni correction (p < 1.8 × 10(-6)). These studies suggest that a common perinatal exposure (such as maternal smoking) deregulates placental methylation in a CpG site-specific manner that correlates with meaningful alterations in gene expression along signature pathways.
The placenta acts not only as a conduit of nutrient and waste exchange between mother and developing fetus, but also functions as a regulator of the intrauterine environment. Recent work has identified changes in the expression of candidate genes, often through epigenetic alteration, which alter the placenta's function and impact fetal growth. In this study, we used the Illumina Infinium HumanMethylation27 BeadChip array to examine genome-wide DNA methylation patterns in 206 term human placentas. Semi-supervised recursively partitioned mixture modeling was implemented to identify specific patterns of placental DNA methylation that could differentially classify intrauterine growth restriction (IUGR) and small for gestational age (SGA) placentas from appropriate for gestational age (AGA) placentas, and these associations were validated in a masked testing series of samples. Our work demonstrates that patterns of DNA methylation in human placenta are reliably and significantly associated with infant growth and serve as a proof of principle that methylation status in the human term placenta can function as a marker for the intrauterine environment, and could potentially play a critical functional role in fetal development.
Despite the success of genome-wide association studies (GWASs) in identifying loci associated with common diseases, a substantial proportion of the causality remains unexplained. Recent advances in genomic technologies have placed us in a position to initiate large-scale studies of human disease-associated epigenetic variation, specifically variation in DNA methylation. Such epigenome-wide association studies (EWASs) present novel opportunities but also create new challenges that are not encountered in GWASs. We discuss EWAS design, cohort and sample selections, statistical significance and power, confounding factors and follow-up studies. We also discuss how integration of EWASs with GWASs can help to dissect complex GWAS haplotypes for functional analysis.
The association between secondhand smoke (SHS) exposure and bladder cancer is inconclusive. Epigenetic alterations in bladder tumors have been linked to primary cigarette smoking and could add to the biological plausibility of an association between SHS exposure and bladder cancer.
SHS exposure is associated with DNA methylation in bladder tumors.
Using an array-based approach, we profiled DNA methylation from never smoking cases of incident bladder cancer. Analyses examined associations between individual loci's methylation with SHS variables (exposure in adulthood, childhood, occupationally, and total exposure). A canonical pathway analysis was used to find pathways significantly associated with each SHS exposure type.
There is a trend toward increased methylation of numerous CpG loci with increasing exposure to adulthood, occupational, and total SHS. Discrete associations between methylation extent of several CpG loci and SHS exposures demonstrated significantly increased methylation of these loci across all types of SHS exposure. CpGs with SHS-related methylation alterations were associated with genes in pathways involved in carcinogenesis, immune modulation, and immune signaling.
Exposures to SHS in adulthood, childhood, occupationally, and in total are each significantly associated with changes in DNA methylation of several CpG loci in bladder tumors, adding biological plausibility to SHS as a risk factor for bladder cancer.
Monocytes are versatile cells that can express different functional programs in response to microenvironmental signals. We show that primary blood monocytes secrete the CXCL12 chemokine, and express the CXCR4 and CXCR7 receptors, leading to an autocrine/paracrine loop that contribute to shape monocyte differentiation to a distinct type of macrophages, with an enhanced expression of CD4, CD14, and CD163, or dendritic cells, with a reduced functional ability to stimulate antigen-specific T-lymphocyte responses. The in vivo relevance of CXCL12 production by mononuclear phagocytes was studied in metastatic melanoma tissues by a thoroughly immunofluorescence phenotyping of CXCL12(high) expressing cells, which were CD45(+), coexpressed the macrophage antigens CD68, CD163, and CD209 and constituted the 60%-90% of tumor-associated macrophages. Microarray analysis of primary monocytes revealed that the vascular endothelial growth factor and the angiogenic chemokine CCL1 mRNA levels were up-regulated in response to CXCL12, leading to enhanced expression of both proteins. In addition, we found that CXCL12 autocrine/paracrine signaling down-regulates the expression of the transcription factor RUNX3 and contributes to maintain the long-term CD4 and CD14 expression in monocytes/macrophages. Together, these results suggest that autocrine CXCL12 production modulates differentiation of monocytes toward a distinct program with proangiogenic and immunosuppressive functions.
The Illumina BeadArray is a popular platform for profiling DNA methylation, an important epigenetic event associated with gene silencing and chromosomal instability. However, current approaches rely on an arbitrary detection P-value cutoff for excluding probes and samples from subsequent analysis as a quality control step, which results in missing observations and information loss. It is desirable to have an approach that incorporates the whole data, but accounts for the different quality of individual observations.
We first investigate and propose a statistical framework for removing the source of biases in Illumina Methylation BeadArray based on several positive control samples. We then introduce a weighted model-based clustering called LumiWCluster for Illumina BeadArray that weights each observation according to the detection P-values systematically and avoids discarding subsets of the data. LumiWCluster allows for discovery of distinct methylation patterns and automatic selection of informative CpG loci. We demonstrate the advantages of LumiWCluster on two publicly available Illumina GoldenGate Methylation datasets (ovarian cancer and hepatocellular carcinoma).
R package LumiWCluster can be downloaded from http://www.unc.edu/∼pfkuan/LumiWCluster.
Maternal cigarette smoking during pregnancy is associated with poor fetal outcome and aberrant miRNA expression is associated with adverse pregnancy outcomes. In 25 human placentas, we analyzed the expression of four candidate miRNA previously implicated in growth and developmental processes: miR-16, miR-21, miR-146a, and miR-182, and used three immortalized placental cell lines to identify if specific components of cigarette smoke were responsible for alterations to miRNA expression. miR-16, miR-21, and miR-146a were significantly downregulated in cigarette smoke-exposed placentas compared to controls. TCL-1 cells exposed to both nicotine and benzo(a)pyrene exhibited significant, dose-dependent downregulation of miR-146a. These results suggest that miR-146a is particularly responsive to exposures, and that smoking may elicit some of its downstream effects through alteration of miRNA expression.
Environmental exposures in-utero may alter the epigenome, thus impacting chromosomal stability and gene expression. We hypothesized that in utero exposures to maternal smoking and perfluoroalkyl compounds (PFCs) are associated with global DNA hypomethylation in umbilical cord serum. Our objective was to determine if global DNA methylation could be used as a biomarker of in utero exposures to maternal smoking and PFCs. Using an ELISA-based method, global DNA methylation was quantified in umbilical cord serum from 30 newborns with high (> 10 ng/ml, mean 123.8 ng/ml), low (range 1-10 ng/ml, mean 1.6 ng/ml) and very low (< 1 ng/ml, mean 0.06 ng/ml) cord serum cotinine levels. Y chromosome analysis was performed to rule out maternal DNA cross-contamination. Cord serum global DNA methylation showed an inverse dose response to serum cotinine levels (p< 0.001). Global DNA methylation levels in cord blood were the lowest among newborns with smoking mothers (mean=15.04%; 95% CI, 8.4, 21.7) when compared to babies of mothers who were second-hand smokers (21.1%; 95% CI, 16.6, 25.5) and non-smokers (mean=29.2%; 95% CI, 20.1, 38.1). Global DNA methylation was inversely correlated with serum PFOA (r= -0.72, p < 0.01) but not PFOS levels. Serum Y chromosome analyses did not detect maternal DNA cross-contamination. This study supports the use of global DNA methylation status as a biomarker of in utero exposure to cigarette smoke and PFCs.
Maternal smoking in pregnancy is associated with reduced birth weight and childhood lung function. This study determined when maternal smoking first influences fetal growth and how this relates to childhood respiratory outcomes.
A longitudinal cohort of 1924 pregnant women was recruited. Fetal ultrasound measurements at 11 weeks (crown-rump length, CRL) and at 20 weeks gestation (femur length, FL, and biparietal diameter, BPD) and birth measurements were recorded. Childhood respiratory symptoms and spirometry were ascertained.
Of the 1924 original study participants, fetal size was determined in 903 in the first trimester, 1544 in the second trimester and at term in 1737 infants. Maternal smoking when first pregnant was reported in 593 (31%) and was not associated with reduced CRL. There was an inverse exposure-response relationship between cigarette consumption and FL (mean reduction in lowest compared with highest tertile 0.91 cm, p=0.033). Birth weight and length of those born to mothers who did (n=331) and did not (n=56) reduce cigarette consumption were similar and reduced compared with 186 infants whose mothers quit during the first trimester (p < or = 0.020). Children of mothers who continued smoking had increased wheeze at age 2 years (OR 1.58, p=0.017) and GP visits with wheeze at age 5 years (OR 2.18, p=0.030) and mean reduction in forced expiratory volume in 1 s of 62 ml (p=0.014) compared with controls.
Maternal smoking is associated with reduced fetal measurements in the second and third trimesters but not in the first trimester. Mothers who do not quit smoking during the first trimester deliver smaller infants who go on to have adverse respiratory outcomes in childhood.
IgA is a specific isotype required for mucosal immunity and is the most abundant Ab produced in vivo. Recently, several inductive signals for IgA class switch recombination have been identified; however, the molecular details of the action of these signals and the specific factors acting in B cells remain elusive. In this study, we show that combination of retinoic acid (RA) and TGF-beta1 with other factors induced a much higher frequency of IgA-switched cells than reported previously. In addition, IgA production is severely impaired in Runx2-Runx3 double-deficient mice. In Runx2-Runx3-deficient B cells, both RA- and TGF-beta1-dependent inductions of alpha germline transcription are completely blocked. These data suggest that Runx proteins play an essential role in IgA class switching acting downstream of RA and TGF-beta1 signaling.
Forkhead box P3 (FOXP3)(+)CD4(+)CD25(+) inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-beta (TGF-beta) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4(+) T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-beta (CBFbeta) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4(+) T cells into Foxp3(+) iT reg cells was significantly decreased in adoptively transferred Cbfb(F/F) CD4-cre naive T cells into Rag2(-/-) mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and Cbfb(F/F) CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4(+) CD25(high) CD127(-) T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-beta mRNA compared with CD4(+)CD25(-) cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-beta-induced Foxp3 expression in iT reg cell differentiation and function.
Prenatal exposure to tobacco smoke increases the risk for diseases later in the child's life that may be mediated through alterations in DNA methylation.
To demonstrate that differences in DNA methylation patterns occur in children exposed to tobacco smoke and that variation in detoxification genes may alter these associations.
Methylation of DNA repetitive elements, LINE1 and AluYb8, was measured using bisulfite conversion and pyrosequencing in buccal cells of 348 children participating in the Children's Health Study. Gene-specific CpG methylation differences associated with smoke exposure were screened in 272 participants in the Children's Health Study children using an Illumina GoldenGate panel. CpG loci that demonstrated a statistically significant difference in methylation were validated by pyrosequencing. Estimates were standardized across loci using a Z score to enable cross-comparison of results.
DNA methylation patterns were associated with in utero exposure to maternal smoking. Exposed children had significantly lower methylation of AluYb8 (beta, -0.31; P = 0.03). Differences in smoking-related effects on LINE1 methylation were observed in children with the common GSTM1 null genotype. Differential methylation of CpG loci in eight genes was identified through the screen. Two genes, AXL and PTPRO, were validated by pyrosequencing and showed significant increases in methylation of 0.37 (P = 0.005) and 0.34 (P = 0.02) in exposed children. The associations with maternal smoking varied by a common GSTP1 haplotype.
Life-long effects of in utero exposures may be mediated through alterations in DNA methylation. Variants in detoxification genes may modulate the effects of in utero exposure through epigenetic mechanisms.
Problem: Smoking among nonpregnant women contributes to reduced fertility, and smoking during pregnancy is associated with delivery of preterm infants, low infant birthweight, and increased infant mortality. After delivery, exposure to secondhand smoke can increase an infant's risk for respiratory tract infections and for dying of sudden infant death syndrome. During 2000-2004, an estimated 174,000 women in the United States died annually from smoking-attributable causes, and an estimated 776 infants died annually from causes attributed to maternal smoking during pregnancy.
To investigate the influence of prospectively measured smoking during pregnancy on aspects of neonatal behavior in a large community sample.
Participants were mothers and infants from the Providence, Rhode Island, cohort of the National Collaborative Perinatal Project enrolled between 1960 and 1966. Mothers with pregnancy/medical complications and infants with medical complications and/or born premature or of low birth weight were excluded. The final sample included 962 mother-infant pairs, 23% of whom were black. Maternal smoking was measured prospectively at each prenatal visit. Neonatal behavior was assessed by using the Graham-Rosenblith Behavioral Examination of the Neonate. Items from the examination were reduced to 3 subscales: irritability, muscle tone, and response to respiratory challenge.
Sixty-two percent of the sample reported smoking during pregnancy, with 24% of smokers reporting smoking 1 pack per day or more. We found a significant influence of maternal smoking exposure (none, moderate/less than 1 pack per day, heavy/1 pack per day or more) on irritability and muscle tone in the neonate, with exposed infants showing greater irritability and hypertonicity. Effects remained significant after controlling for significant covariates: maternal socioeconomic status, age, and race and infant birth weight and age. Posthoc tests suggested particular effects of heavy smoking on increased infant irritability and both moderate and heavy smoking exposure on increased muscle tone.
In a large community sample, exposure to maternal smoking was associated with increased irritability and hypertonicity in neonates. Exposure to maternal smoking did not influence neonatal response to respiratory challenge. This study is the largest-scale investigation to date of the effects of maternal smoking (heavy and moderate) on examiner-assessed neonatal behavior. Given the associations between both maternal smoking and infant irritability and later behavioral dysregulation, results have important implications for early identification and intervention with at-risk offspring.
Asthma is a complex heritable disease that is increasing in prevalence and severity, particularly in developed countries such as the United States, where 11% of the population is affected. The contribution of environmental and genetic factors to this growing epidemic is currently not well understood. We developed the hypothesis, based on previous literature, that changes in DNA methylation resulting in aberrant gene transcription may enhance the risk of developing allergic airway disease. Our findings indicate that in mice, a maternal diet supplemented with methyl donors enhanced the severity of allergic airway disease that was inherited transgenerationally. Using a genomic approach, we discovered 82 gene-associated loci that were differentially methylated after in utero supplementation with a methyl-rich diet. These methylation changes were associated with decreased transcriptional activity and increased disease severity. Runt-related transcription factor 3 (Runx3), a gene known to negatively regulate allergic airway disease, was found to be excessively methylated, and Runx3 mRNA and protein levels were suppressed in progeny exposed in utero to a high-methylation diet. Moreover, treatment with a demethylating agent increased Runx3 gene transcription, further supporting our claim that a methyl-rich diet can affect methylation status and consequent transcriptional regulation. Our findings indicate that dietary factors can modify the heritable risk of allergic airway disease through epigenetic mechanisms during a vulnerable period of fetal development in mice.
This report, which is based upon data gathered from 7,499 patients, shows an incidence of premature births at private hospitals which is approximately twice as great for smoking mothers as it is for nonsmoking mothers. The prematurity rate increases with the number of cigarettes smoked per day. The highest prematurity rates are for heavy smokers and the lowest rates for nonsmokers.
The County Hospital represents a different population in which socioeconomic factors may affect the picture. The prematurity rate for nonsmokers is relatively higher at the County Hospital than at the private hospitals and there is less difference between the prematurity rate for smokers and non-smokers. Mexicans delivered at the County Hospital report less smoking than any other ethnic group and show the lowest prematurity rate.
The writer is grateful to Dr. William B. Michael for his expert guidance in tabulating and interpreting the data presented. To the following doctors who critically previewed this report goes a vote of thanks: M. E. Cosand, L. Corsa, Jr., C. H. Thienes, L. H. Lonergan, H. N. Mozar, B. W. Halstead, F. R. Lemon, and W. F. Norwood.
Much credit is due to the following supervisors and their assistants who were responsible for gathering the information for the questionnaires: Mary Hyatt, County Hospital Obstetrical Supervisor; Sister Mary Elaine, St. Bernardine’s Hospital Records Librarian; Ruth Wipperman, Loma Linda Sanitarium and Hospital Obstetrical Supervisor.
AIMS
To determine whether maternal smoking during pregnancy is a risk factor for reported wheeze in early childhood that is independent of postnatal environmental tobacco smoke (ETS) exposure and other known risk factors.
METHODS
A total of 8561 mothers and infants completed questions about smoking during pregnancy, ETS exposure, and the mother's recall of wheeze during early childhood.
RESULTS
A total of 1869 (21.8%) children had reported wheeze between 18 and 30 months of age, and 3496 (40.8%) had reported wheeze in one or more of the three study periods (birth to 6 months, 6–18 months, 18–30 months). The risk of wheeze between 18 and 30 months of age was higher if the mother smoked during pregnancy. This relation did not show a dose–response effect and became less obvious after adjustment for the effects of other factors. Average daily duration of ETS exposure reported at 6 months of age showed a dose–response effect and conferred a similar risk of reported wheeze. Factors associated with early childhood wheeze had the following adjusted odds ratios: maternal history of asthma 2.03 (1.74 to 2.37); preterm delivery 1.66 (1.30 to 2.13); male sex 1.42 (1.28 to 1.59); rented accommodation 1.29 (1.11 to 1.51); and each additional child in household 1.13 (1.04 to 1.24).
CONCLUSIONS
Maternal smoking during pregnancy may be a risk factor for reported wheeze during early childhood that is independent of postnatal ETS exposure. For wheeze between 18 and 30 months of age, light smoking during the third trimester of pregnancy appears to confer the same risk as heavier smoking.
Background: Hypermethylation of a CpG-rich promoter (CpG island) blocks expression of the corresponding gene. The CpG island methylator phenotype (CIMP), defined as a variable pattern of hypermethylation of CpG islands in tumor suppressor genes, may be associated with carcinogenesis. To determine whether CIMP is associated with the development of hepatocellular carcinoma (HCC) and with exposure to environmental agents, we examined the methylation status of CpG islands in HCCs from countries with various HCC risks. Methods: We examined the methylation status of 12 CpG islands (eight for known genes) in 85 HCC tumors from various geographic locations by use of bisulfite–polymerase chain reaction methylation assays and analyzed results with univariate and multivariable methods. All statistical tests were two-sided. Results: Eight CpG islands were hypermethylated. The frequency of hypermethylation in the 85 tumors was 62% for the estrogen receptor (ER), 42% for p16, 18% for cyclooxygenase-2, 21% for the T-type calcium channel gene, 38% for MINT31, 28% for MINT1, 15% for MINT27, and 11% for MINT2 (the latter four CpG islands are not yet associated with genes). Methylation levels of the eight frequently methylated CpG islands were positively correlated (from R =. 2 [P = .05] to R =. 6 [P<.001]), supporting the presence of CIMP. p16 methylation had statistically significant geographic variation (34.4% in tumors from China and Egypt versus 12.2% in tumors from the United States and Europe, difference = 22.2%; 95% confidence interval [CI] = 11.2% to 33.2%; P<.001). Similar geographic variations were observed for ER methylation and CIMP. This observation was partly related to higher methylation in tumors from patients with cirrhosis (33.6% for patients with cirrhosis versus 11.7% for those without it; difference = 21.9%; 95% CI = 10.9% to 32.8%; P<.001) or hepatitis (34.2% for patients with hepatitis versus 6.2% for those without it; difference = 28%; 95% CI = 18.3% to 37.6%; P<.001). Conclusion: Geographic variations in the methylation status of various CpG islands indicate that environmental factors may influence the frequent and concordant degree of hypermethylation in multiple genes in HCC tumors. [J Natl Cancer Inst 2002;94:755–61]
Angelman syndrome is a neurodevelopmental disorder characterized by mental retardation, severe speech disorder, facial dysmorphism, secondary microcephaly, ataxia, seizures, and abnormal behaviors such as easily provoked laughter. It is most frequently caused by a de novo maternal deletion of chromosome 15q11-q13 (about 70-90%), but can also be caused by paternal uniparental disomy of chromosome 15q11-q13 (3-7%), an imprinting defect (2-4%) or in mutations in the ubiquitin protein ligase E3A gene UBE3A mostly leading to frame shift mutation. In addition, for patients with overlapping clinical features (Angelman-like syndrome), mutations in methyl-CpG binding protein 2 gene MECP2 and cyclin-dependent kinase-like 5 gene CDKL5 as well as a microdeletion of 2q23.1 including the methyl-CpG binding domain protein 5 gene MBD5 have been described. Here, we describe a patient who carries a de novo 5Mb-deletion of chromosome 15q11.2-q13.1 known to be associated with Angelman syndrome and a further, maternally inherited deletion 2q21.3 (~364 kb) of unknown significance. In addition to classic features of Angelman syndrome, she presented with severe infections in the first year of life, a symptom that has not been described in patients with Angelman syndrome. The 15q11.2-q13.1 deletion contains genes critical for Prader-Willi syndrome, the Angelman syndrome causing genes UBE3A and ATP10A/C, and several non-imprinted genes: GABRB3 and GABRA5 (both encoding subunits of GABA A receptor), GOLGA6L2, HERC2 and OCA2 (associated with oculocutaneous albinism II). The deletion 2q21.3 includes exons of the genes RAB3GAP1 (associated with Warburg Micro syndrome) and ZRANB3 (not disease-associated). Despite the normal phenotype of the mother, the relevance of the 2q21.3 microdeletion for the phenotype of the patient cannot be excluded, and further case reports will need to address this point.
A prospective study was performed on the effect of smoking on pregnancy, using information from 6 363 pregnancies with known smoking habits. Of these, 2806 (44%) smoked during pregnancy, and 2731 (97%) of the 2 806 reported that they smoked during the whole pregnancy. The well-known effect on prematurity rate and mean birth weight was verified in this study. A 50% increase of prematurity rate was registered among smoking women compared with non-smoking women. The mean birth weight reduction was 170 g among live-born, non-malformed children. These effects were found to occur irrespective of other variables studied: maternal age, parity, and whether the pregnancy was wanted. The body length, head circumference, and shoulder circumference were found to be reduced in children born to smoking women. Placental weight was also reduced, but the ratio placental weight over body weight increased with smoking. No effect on the malformation rate was observed, but the series is too small to exclude a teratogenic effect of smoking. Children were followed to 1 year of age. The death risk for a child of a smoking woman was found to be increased to 1.6 times that for a child of a non-smoking woman. No significant difference in stillbirth rate was found, but an effect was found on neo-natal death both before and after the age of 1 week, although only among non-premature children. Among children dying before the age of 1 week, a significant increase in frequency of abruptio placentae was noted. No other specific cause of death was noted. An overall increased risk of spontaneous abortion among smoking women was verified; this was shown to be almost completely due to the association between the fact that the pregnancy was unwanted and smoking. An even higher incidence of smoking was seen among women with induced abortions. Spontaneous abortion is also associated with unwanted pregnancies, perhaps due to the presence of induced abortions among the group recorded as spontaneous abortions. Smoking was shown to be associated only with late spontaneous abortions. If there is an abortifacient effect of smoking, it must be very slight. The decreased rate of preeclamptic complications among smoking women, described by earlier authors, was verified. A similar decrease in frequency of morning sickness was also found. No effect of smoking on the mean Apgar score of surviving, non-malformed children was seen. The sex ratio among children born to smoking women differed slightly from that born to non-smoking women, but this difference was not statistically significant.
To investigate the role of Runx3 gene CpG island methylation in the development of human gastric carcinoma.
A total of 150 tumor specimens from patients with gastric carcinoma and 50 normal tissue specimens were selected. Methylation specific PCR (MSP) and pyrosequencing (PS) were used to detect the methylation status of Runx3 gene promoter.
Compared to normal tissue samples, a significant increase of CpG island methylation status of Runx3 gene was observed in gastric carcinomas (MSP: 67.3% vs. 40.0%, P = 0.002; PS: 76.0% vs. 30.0%, P < 0.01). Runx3 gene methylation was only related to tumor size (P < 0.05) based on MSP analysis. PS test however showed that the extent of methylation of Runx3 gene was related to the tumor size (P = 0.004), Lauren's classification (P = 0.043), depth of invasion (P < 0.01), lymph node metastasis (P = 0.021) and TNM staging (P = 0.045).
Methylation status of Runx3 gene detectable by PS is closely correlated with clinicopathological parameters of gastric carcinoma, including tumor size, Lauren's classification, depth of invasion, lymph node metastasis and TNM staging. PS is more sensitive than MSP in the detection of Runx3 gene methylation, which may serve as an important marker for early diagnosis and treatment of gastric carcinoma.
Helicobacter pylori has been recognized as a definite carcinogen for gastric cancer (GC); however, the pathogenesis of H. pylori infection remains unclear. Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene whose deficiency is causally related to GC. However, in H. pylori infection-associated GC, the role of RUNX3 has not been studied.
The authors used real-time methylation-specific polymerase chain reaction analysis to determine methylation status of the RUNX3 promoter in a spectrum of gastric lesions, including 220 samples of chronic atrophic gastritis, 196 samples of intestinal metaplasia, 134 samples of gastric adenoma, 102 samples of dysplasia, and 202 samples of GC with paired noncancerous mucosa tissues and corresponding blood specimens. The association of abnormal methylation with precancerous gastric lesions was evaluated along with the association between RUNX3 methylation and H. pylori infection, and the concordance of methylation levels was investigated between serum and tissues.
The results indicated that increasing RUNX3 promoter methylation was correlated with distinct stages of GC progression. GC tissues had the highest methylation proportion (75.2%) compared with precancerous gastric lesions, including chronic atrophic gastritis (15.9%), intestinal metaplasia (36.7%), gastric adenoma (41.8%), and dysplasia (54.9%). H. pylori infection, a major risk factor for GC, contributed to the inactivation of RUNX3 in gastric epithelial cells through promoter hypermethylation. The levels of RUNX3 methylation in serum were in significant concordance with the methylation levels observed in GC tissues (P = .887).
The current findings supported RUNX3 methylation as a risk factors for the carcinogenesis of chronic atrophic gastritis with H. pylori infection and indicated that circulating RUNX3 methylation is a valuable biomarker for the detection of early GC. Cancer 2012.
Emerging evidence indicates that RUNX3 is a tumor suppressor in breast cancer. RUNX3 is frequently inactivated in human breast cancer cell lines and cancer samples by hemizygous deletion of the Runx3 gene, hypermethylation of the Runx3 promoter, or cytoplasmic sequestration of RUNX3 protein. Inactivation of RUNX3 is associated with the initiation and progression of breast cancer. Female Runx3(+/-) mice spontaneously develop ductal carcinoma, and overexpression of RUNX3 inhibits the proliferation, tumorigenic potential, and invasiveness of breast cancer cells. This review is intended to summarize these findings and discuss the tumor suppressor function of RUNX3 in breast cancer.
Intrauterine smoke exposure (IUS) is a strong risk factor for development of airways responsiveness and asthma in childhood. Runt-related transcription factors (RUNX1-3) have critical roles in immune system development and function. We hypothesized that genetic variations in RUNX1 would be associated with airway responsiveness in asthmatic children and that this association would be modified by IUS. Family-based association testing analysis in the Childhood Asthma Management Program genome-wide genotype data showed that 17 of 100 RUNX1 single-nucleotide polymorphisms (SNPs) were significantly (P < 0.03-0.04) associated with methacholine responsiveness. The association between methacholine responsiveness and one of the SNPs was significantly modified by a history of IUS exposure. Quantitative PCR analysis of immature human lung tissue with and without IUS suggested that IUS increased RUNX1 expression at the pseudoglandular stage of lung development. We examined these associations by subjecting murine neonatal lung tissue with and without IUS to quantitative PCR (N = 4-14 per group). Our murine model showed that IUS decreased RUNX expression at postnatal days (P)3 and P5 (P < 0.05). We conclude that 1) SNPs in RUNX1 are associated with airway responsiveness in asthmatic children and these associations are modified by IUS exposure, 2) IUS tended to increase the expression of RUNX1 in early human development, and 3) a murine IUS model showed that the effects of developmental cigarette smoke exposure persisted for at least 2 wk after birth. We speculate that IUS exposure-altered expression of RUNX transcription factors increases the risk of asthma in children with IUS exposure.
Rabphilin-3A-like (RPH3AL) protein functions in the regulation of hormone exocytosis, and mutations in the RPHA3L gene have been associated with tumorigenesis in colorectal cancer (CRC). We evaluated the potential use of anti-RPH3AL autoantibodies as a marker for CRC detection.
Sera from 84 patients with CRC and 63 healthy controls were analysed for the presence of RPH3AL autoantibodies with a Western blotting assay.
The frequencies of RPH3AL autoantibodies in the early stage, advanced stage and all CRC patients were 64.7%, 78.0% and 72.6%, respectively. These values are significantly higher than the frequency of RPH3AL autoantibodies in healthy controls (15.9%, P<0.001). Although the presence of RPH3AL autoantibodies did not correlate with clinical parameters, RPH3AL autoantibodies were found in 69.4% (34/49) of CRC patients who were negative for carcinoembryonic antigen. The value of the area under the receiver operating characteristic curve of RPH3AL autoantibody was 0.84, which suggests that screening for these autoantibodies could potentially be used for CRC diagnosis.
Circulating RPH3AL autoantibodies are prevalent in patients with CRC, and detection of these autoantibodies might provide a novel non-invasive approach for CRC diagnosis.
The placenta is of utmost importance for intrauterine fetal development and growth. Deregulation of placentation can lead to adverse outcomes for both mother and fetus, e.g. gestational trophoblastic disease (GTD), pre-eclampsia and fetal growth retardation. A significant factor in placental development and function is epigenetic regulation.
This review summarizes the current knowledge in the field of epigenetics in relation to placental development and function. Relevant studies were identified by searching PubMed, Medline and reference sections of all relevant studies and reviews.
Epigenetic regulation of the placenta evolves during preimplantation development and further gestation. Epigenetic marks, like DNA methylation, histone modifications and non-coding RNAs, affect gene expression patterns. These expression patterns, including the important parent-of-origin-dependent gene expression resulting from genomic imprinting, play a pivotal role in proper fetal and placental development. Disturbed placental epigenetics has been demonstrated in cases of intrauterine growth retardation and small for gestational age, and also appears to be involved in the pathogenesis of pre-eclampsia and GTD. Several environmental effects have been investigated so far, e.g. ethanol, oxygen tension as well as the effect of several aspects of assisted reproduction technologies on placental epigenetics.
Studies in both animals and humans have made it increasingly clear that proper epigenetic regulation of both imprinted and non-imprinted genes is important in placental development. Its disturbance, which can be caused by various environmental factors, can lead to abnormal placental development and function with possible consequences for maternal morbidity, fetal development and disease susceptibility in later life.
DNA methylation is one of the most intensely studied epigenetic modifications in mammals. In normal cells, it assures the proper regulation of gene expression and stable gene silencing. DNA methylation is associated with histone modifications and the interplay of these epigenetic modifications is crucial to regulate the functioning of the genome by changing chromatin architecture. The covalent addition of a methyl group occurs generally in cytosine within CpG dinucleotides which are concentrated in large clusters called CpG islands. DNA methyltransferases are responsible for establishing and maintenance of methylation pattern. It is commonly known that inactivation of certain tumor-suppressor genes occurs as a consequence of hypermethylation within the promoter regions and a numerous studies have demonstrated a broad range of genes silenced by DNA methylation in different cancer types. On the other hand, global hypomethylation, inducing genomic instability, also contributes to cell transformation. Apart from DNA methylation alterations in promoter regions and repetitive DNA sequences, this phenomenon is associated also with regulation of expression of noncoding RNAs such as microRNAs that may play role in tumor suppression. DNA methylation seems to be promising in putative translational use in patients and hypermethylated promoters may serve as biomarkers. Moreover, unlike genetic alterations, DNA methylation is reversible what makes it extremely interesting for therapy approaches. The importance of DNA methylation alterations in tumorigenesis encourages us to decode the human epigenome. Different DNA methylome mapping techniques are indispensable to realize this project in the future.
RUNX proteins are heterodimeric factors that play crucial roles during development and differentiation of cells of the immune system. The RUNX3 transcription factor controls lineage decisions during thymopoiesis and T-cell differentiation, and modulates myeloid cell effector functions. We now report the characterization of the human RUNX3/p33 isoform, generated by splicing out a Runt DNA-binding domain-encoding exon, and whose transcriptional activities differ from those of the prototypic RUNX3/p44 molecule. Unlike RUNX3/p44, RUNX3/p33 is induced upon maturation of monocyte-derived dendritic cells (MDDC), and is unable to transactivate the regulatory regions of the CD11a, CD11c and CD49e integrin genes. Overexpression of RUNX3/p33 in myeloid cell lines led to diminished expression of genes involved in inflammatory responses. Moreover, overexpression of RUNX3/p33 down-modulated the basal level of IL-8 production from immature monocyte-derived dendritic cells (MDDC). Besides, siRNA-mediated knock-down of RUNX3 led to diminished levels of IL-8 RNA in immature MDDC, and modulated the neutrophil-recruiting capacity of myeloid cell line supernatants. Since IL-8 promotes neutrophil chemotaxis and degranulation during inflammatory responses, and exerts mitogenic and angiogenic actions within tumor microenvironment, our results imply that myeloid RUNX3 expression regulates the recruitment of leukocytes towards inflammatory foci and might also contribute to human cancer progression.
The aetiology of autism is still largely unknown despite analyses from family and twin studies demonstrating substantial genetic role in the aetiology of the disorder. Data from linkage studies and analyses of chromosomal abnormalities identified 15q11-q13 as a region of particular aetiopathogenesis interest. We screened a set of markers spanning two known imprinted, maternally expressed genes, UBE3A and ATP10A, harboured in this candidate region. We replicated evidence of linkage disequilibrium (LD) at marker D15S122, located at the 5' end of UBE3A and originally reported by Nurmi et al. (2001). The potential role of UBE3A in our family-based association study is further supported by the association of two haplotypes that include one of the alleles of D15S122 and by the transmission disequilibrium test (TDT) evidence of the same allele in a parent of origin effect analysis. In a secondary analysis, we provided the first evidence of a significant association between first word delay and psychomotor regression with the 15q11-q13 region. Our data support a potential role of UBE3A in the complex pathogenic mechanisms of autism.
The metabolic pathways used by higher-eukaryotic organisms to deal with potentially carcinogenic xenobiotic compounds from tobacco smoke have been well characterized. Carcinogenic compounds such as polycyclic aromatic hydrocarbons are metabolized sequentially in 2 phases: in phase I, CYP1A1 catalyzes conversion into harmful hydrophilic DNA adducts, whereas in phase II, GSTT1 enables excretion via conjugation into polar electrophiles. In an effort to understand susceptibility to in utero tobacco exposure, we previously characterized known metabolic functional polymorphisms and demonstrated that although deletion of fetal GSTT1 significantly modified birth weight in smokers, no polymorphism fully accounted for fetal growth restriction. Because smoking up-regulates CYP1A1 expression, we hypothesized that nonallelic (epigenetic) dysregulation of placental CYP1A1 expression via alterations in DNA methylation (meCpG) may further modify fetal growth. In the present article, we compared placental expression of multiple CYP family members among gravidae and observed significantly increased CYP1A1 expression among smokers relative to controls (4.4-fold, P < .05). To fully characterize CYP1A1 meCpG status, bisulfite modification and sequencing of the entire proximal 1-kilobase promoter (containing 59 CpG sites) were performed. CpG sites immediately proximal to the 5′-xenobiotic response element transcription factor binding element were significantly hypomethylated among smokers (55.6% vs 45.9% meCpG, P = .027), a finding that uniquely correlated with placental gene expression (r = 0.737, P = .007). Thus, in utero tobacco exposure significantly increases placental CYP1A1 expression in association with differential methylation at a critical xenobiotic response element.
There is considerable evidence for induction of differential risk of noncommunicable diseases in human by variation in the quality of the early life environment. Studies in animal models show that induction and
stability of induced changes in the phenotype of the offspring involve altered epigenetic regulation by DNAmethylation and covalentmodifications of histones. These findings indicate that such epigenetic changes are highly gene specific and function at the level of individualCpGdinucleotides.
Interventions using supplementation with folic acid ormethyl donors during pregnancy, or folic acid after weaning, alter the phenotype and epigenotype induced by maternal dietary constraint during gestation. This suggests a possible means for reducing risk of induced noncommunicable disease, although the design and conduct of such interventions may require caution. The purpose of this review is to discuss recent advances in understanding the mechanism that underlies the early life origins of disease and to place these studies in a broader life-course context.
We recently reported an autistic proband and affected sibling with maternally inherited microduplications within the 15q13.1 and 15q13.3 regions that contain a total of 4 genes. The amyloid precursor protein-binding protein A2 (APBA2) gene is located within the 15q13.1 duplication and encodes a neuronal adaptor protein essential to synaptic transmission that interacts directly with NRXN1 at the presynaptic membrane. We interpreted this as evidence for a putative role of APBA2 in autism as larger maternal duplications of 15q11-q13 are the most common known cause of autism. We therefore resequenced 512 subjects with autism spectrum disorder (ASD) and 463 controls, and identified 7 novel nonsynonymous coding variants in ASD subjects compared with 4 in controls. Five of the seven variants in the ASD group were predicted to affect protein function, alter residues conserved across 18 species, or both. All of the variants for which parental DNA was available were inherited. We also found two different nonsynonymous variants in two siblings with autism: (1) a paternally inherited heterozygous 6 bp deletion and (2) a maternally inherited heterozygous missense mutation, the latter also found in a single control. These results indicate compound heterozygous mutations of APBA2 in this autism sibship. The co-occurrence of two nonsynonymous mutations in both affected siblings in a single family, each transmitted from a different unaffected parent, suggest a role for APBA2 mutations in rare individuals with ASD.
Thymoma is a thymic epithelial neoplasm which induces T cell development. However, the frequency of mature CD4(+) T cells in thymomas is lower than in normal thymi. Recently, CD4/CD8 lineage commitment has been elucidated in animal model. The zinc finger transcription factor Th-POK is a critical factor to CD4(+) T cell development in CD4/CD8 lineage commitment, whereas CD8(+) T cell development requires the transcription factor Runx3. These factors antagonize in CD4/CD8 lineage commitment. In this study, we examined Th-POK and Runx3 mRNA expression in the T cell subsets of human normal thymus and thymoma. A quantitative reverse transcriptase-polymerase chain reaction examination revealed that Th-POK expression in normal thymi was higher in the CD4(+)CD8(-) subset than in the CD4(+)CD8(+) and CD4(-)CD8(+) subsets. In thymomas, Th-POK expression in the CD4(+)CD8(-) subset was significantly lower than that in normal thymi, and was significantly correlated with the proportion of CD3(+) cells in the CD4(+)CD8(-) subset. However, Th-POK expressions of the CD3(+)CD4(+)CD8(+) and CD3(+)CD4(+)CD8(-) subsets were not impaired in thymomas compared to normal thymi. These results suggest that thymoma neoplastic epithelial cells can induce Th-POK expression similarly to the normal thymic epithelial cells. In addition, there was no significant difference in Runx3 expression between normal thymi and thymomas. Therefore, CD4/CD8 lineage commitment dependent on Th-POK and Runx3 system seems to be working even in the neoplastic environment formed by human thymomas.
Pregnancy is arguably the most critical period of developmental programming. Here, we particularly focus on the emerging paradigm that disease propensity is epigenetically determined by maternal exposures that have the capacity to activate or silence fetal genes through alterations in DNA and histone methylation, histone acetylation, and chromatin structure.
The most notable recent candidate to emerge in this role has been dietary folate, a methyl donor clearly associated with changes in gene expression and disease susceptibility through gene hypermethylation. Animal studies also provide the first evidence that the allergy protective effects of microbial exposure in pregnancy may be mediated by changes in methylation of Th1 genes of the offspring. There is also emerging evidence that perinatal differences in immune function of allergy-prone newborns extend beyond previously recognized differences in effector T cell (Th1/Th2) function, to also include differences in neonatal regulatory T cell (Treg) and Th17 function, and moreover, that these pathways are also epigenetically regulated.
New studies reinforce the importance of in-utero exposures (including dietary nutrients, microbial products, cigarette smoking, and certain maternal mediations) in fetal immune development and in programming the susceptibility to asthma and allergic disease.
Epigenetics is focused on understanding the control of gene expression beyond what is encoded in the sequence of DNA. Central to growing interest in the field is the hope that more can be learned about the epigenetic regulatory mechanisms underlying processes of human development and disease. Researchers have begun to examine epigenetic alterations - such as changes in promoter DNA methylation, genomic imprinting, and expression of miRNA - to learn more about epigenetic regulation in the placenta, an organ whose proper development and function are crucial to the health, growth, and survival of the developing fetus. A number of studies are now making important links between alterations to appropriate epigenetic regulation in the placenta and diseases of gestation and early life. In addition, these studies are adding important insight into our understanding of trophoblast biology and differentiation as well as placental immunology. Examining epigenetic alterations in the placenta will prove especially important in the search for biomarkers of exposure, pathology, and disease risk and can provide critical insights into the biology of development and pathogenesis of disease. Thus, epigenetic alterations may aid in disease diagnosis and prognosis as well as in targeting new treatment and prevention strategies.
We have previously reported that irradiation of mice in utero significantly increased the tumor incidence in the offspring of irradiated mothers. The joint effects of irradiation and cigarette smoking (CS) on tumor incidence and on the process of carcinogenesis were investigated.
Pregnant C57Bl/6J female mice were irradiated with a single dose of gamma-ray (1 Gy or 3 Gy) and/or exposed to CS of IR3 non-filtered cigarettes before or during pregnancy, tumors were investigated both with histological and immunohistochemical methods.
Longer exposure (60 days) of the mice to CS before pregnancy and irradiation during pregnancy significantly increased the tumor incidence in the mothers and their offspring. Parallel activation of Caspase-8 and inactivation of Caspase-9 was found.
Joint exposure of mice to prolonged CS before pregnancy and irradiation during pregnancy significantly increased the tumor incidence both in the mothers and their offspring.
In intestinal epithelial cells, inactivation of APC, a key regulator of the Wnt pathway, activates beta-catenin to initiate tumorigenesis. However, other alterations may be involved in intestinal tumorigenesis. Here we found that RUNX3, a gastric tumor suppressor, forms a ternary complex with beta-catenin/TCF4 and attenuates Wnt signaling activity. A significant fraction of human sporadic colorectal adenomas and Runx3(+/-) mouse intestinal adenomas showed inactivation of RUNX3 without apparent beta-catenin accumulation, indicating that RUNX3 inactivation independently induces intestinal adenomas. In human colon cancers, RUNX3 is frequently inactivated with concomitant beta-catenin accumulation, suggesting that adenomas induced by inactivation of RUNX3 may progress to malignancy. Taken together, these data demonstrate that RUNX3 functions as a tumor suppressor by attenuating Wnt signaling.