Up to 20% of women smoke during pregnancy and, although many fetuses are exposed to tobacco smoke in utero, not all experience similar adverse outcomes.1,8–12
This discrepancy cannot be accounted for by dose effect alone and, despite decades of research, the mechanisms leading to attenuation of birth weight and related adverse outcomes are still largely unknown—likely because they are complex, involving interaction between epidemiologic, genetic, epigenetic and socio-demographic factors.
Of note and with respect to both our current and prior work, evidence to date suggests that these factors converge on a limited number of metabolic pathways that convert the vast majority of over 4,000 compounds found in tobacco smoke to reactive, potentially harmful and, in some instances, excretable intermediates.1–4,8–14
Potentially harmful DNA adducts (metabolic products of polycyclic aromatic hydrocarbons; PAH) are known to cross or collect in the placenta of smokers.1
PAH compounds together with nitrosamines comprise likely carcinogenic species in tobacco smoke, and are metabolized in a sequential series of two-phase enzymatic metabolic reactions.1–4
Phase I enzymes (such as CYP1A1) metabolically activate PAH compounds into oxidized derivatives, resulting in reactive oxygen intermediates capable of covalently binding DNA to form adducts. In turn, these reactive electrophilic intermediates can be detoxified by phase II enzymes, such as the glutathione S
-transferase (GSTT1), via conjugation with endogenous species to form hydrophilic glutathione conjugates, which are then readily excretable. Thus, the coordinated expression of these enzymes and their relative balance may determine the extent of cellular DNA damage and related development of adverse outcomes.
In an effort to understand the relationship between epigenetic regulation and genetic susceptibility to in utero tobacco exposure from a systems biology approach, we previously characterized known metabolic functional candidate polymorphisms along well-described metabolic pathways using a targeted-genomic approach. We demonstrated that while deletion of fetal GSTT1
significantly modified birth weight in smokers, it did not fully account for growth restriction per se.8
However, further interrogations demonstrated that tobacco exposure significantly increases placental expression of a phase I metabolite gene (CYP1A1)
in association with differential promoter methylation at a critical XRE binding element.13
Importantly, the methylation status of this region was correlated with the expression level of CYP1A1
, irrespective of maternal smoke exposure. In this study, we sought to extend our previous analysis and set out to determine if site-specific CpG methylation changes are associated with maternal smoking on a genome-wide level and whether these changes correlated with gene transcription.
Our work presented herein is the first to undertake a rigorous genome-wide approach to relate site-specific alterations in the methylome with meaningful changes in gene expression revealing signature pathways that are associated with smoking-mediated fetal growth attenuation. Using the Illumina genome-wide methylation and gene expression platforms in a well-matched nested cohort, we have described our measured alterations in the placental transcriptome and methylome. Development of an analysis workbench enabled interrogation of significant correlation (r > 0.70) between a given gene's promoter or CGI methylation and its expression. We demonstrate that expression of 623 genes and methylation of 1,024 CpG dinucleotides are significantly altered among smokers, with only 38 CpGs differing by a methylation level of 10% or greater. When we further apply linear regression models, altered placental site-specific CpG methylation at as few as 6 sites is attributed to a significant reduction in infant birth weight among smokers.
Our findings are consistent with those of other investigators who similarly reported that maternal smoking is associated with modified placental gene expression.14,21
A study of five control placentas and five placentas from smoking mothers reported differential expression of 174 genes, including changes in the level of Phase I enzymes that metabolize polycyclic aromatic hydrocarbons from tobacco smoke.21
A similar study including 12 smokers and 64 non-smokers characterized differential expression of 241 genes including genes involved in xenobiotic processing and coagulation.14
At a functional level, we and other investigators have similarly demonstrated an association between maternal active and passive smoking and evidence of oxidative stress16,20,24,31
and hypoxia inducible factors (such as HIF1α).25
In this study, we expand upon these findings to correlate site-specific CpG methylation with meaningful alterations in oxidative stress and hypoxia pathways.
There are several methodological strengths in our study. We utilized the Illumina platform to run concomitant genome-wide gene expression and DNA methylation analysis, allowing us to determine differentially expressed genes and differentially methylated CpG sites, and correlating the two to discover meaningful alterations in placental signature pathways that occur in association with maternal smoking. Specifically, we calculated Pearson coefficients (significance at −0.7 < r < 0.7) to determine whether a significant correlation of gene expression and site-specific CpG methylation within the gene's promoter or enhancer exists. In the absence of stratification by virtue of maternal smoking, in toto analysis (n = 36) reveals that only 13 genes demonstrate a significant correlation (either inverse or directional) between expression and methylation (Table S1
). However, when stratified by maternal smoking distinct and impressively significant variance in correlative methylation and gene expression occurs with an observed 18-fold increase in correlative placental gene expression (25 versus 438 genes, p < 0.0001; and Table S1
). We conclude that exposure to maternal smoking is associated with neither a global nor indiscriminate change in placental DNA methylation but rather occurs at specific CpG dinucleotides, which deregulate a significant number of genes in the transcriptome.
An additional strength to our study arises from the employment of linear regression analysis to control for potential interactions. This was needed because, although maternal smoking is associated with a significant decrease in fetal birth weight and renders risk of SGA birth across maternal strata,8,12,20,28
large population-derived studies have also shown that female infants weigh on-average 120 to 200 grams less than males.30
After Bonferroni correction, the variation of methylation level at 6 CpG sites in female newborn samples could be explained by smoking status to infant weight interactions, revealing that as few as 6 essential sites in the placental methylome are modified in association with maternal smoking to significantly influence birth weight. Others29
have similarly employed integrated computational and multivariate analysis approaches.
As further evidence of our attempt to be rigorous in our analyses, we employed two independent pathway tools (i.e., IPA and DAVID). Ingenuity Pathway Analysis (IPA) of the 438 differentially methylated genes among smokers reveals that the top canonical pathways include oxidative phosphorylation, mitochondrial dysfunction and HIF1α signaling with molecular enrichment along cell death, morphology and cell signaling signatures; we have independently confirmed that these pathways are functionally disrupted at the level of cellular physiology employing immunohistochemistry and in situ analysis (In press).28
Our observed gene signature pathways are of likely biological and clinical significance. First, our data is consistent with our functional cellular analysis of placentas, demonstrating the significant increased presence of markers of oxidative damage among smokers, namely 8-OHdG and 4-HNE.16,24,28
Second, HIF1α is a transcription factor that senses hypoxia to ultimately regulate transcription of these same pathways.25
Given that chronic fetal hypoxia due to utero-placental insufficiency in tobacco-exposed fetuses has long been hypothesized as a potential underlying physiologic mechanism that plays a role in growth attenuation, our findings allow for the convergence of multiple lines of data.8–12,24–28
Employing these methodologies in a nested cohort design, we have completed a robust analysis and demonstrated that biologically relevant and statistically significant deregulation of placental methylation correlates with gene expression. Development of a comprehensive analysis workbench enabled integrated analysis of parallel Illumina-based tiling array-based placental methylome and transcriptome data to reveal that altered placental site-specific CpG methylation at as few as 6 sites along signature pathways may contribute to a significant reduction in infant birth weight among smokers. When considered in the context of the implications for the biology of the development and programming of disease, these studies suggest that a common perinatal exposure (such as maternal smoking) deregulates placental methylation, which correlates with meaningful alterations in gene expression. We speculate that our methodologies and observations will lay the groundwork for further interrogations into the role of epigenomic deregulation of common perinatal exposures, which in turn have the potential to profoundly impact the health across that same individual's lifespan.