Prenatal tobacco smoke exposure was associated with small yet significant changes in global and CpG-specific DNA methylation. Exposed children had lower mean methylation for AluYb8 repetitive elements compared with unexposed children. Moreover, a preliminary scan of specific CpG loci suggested that eight genes were hypermethylated in exposed children, of which two genes were validated by pyrosequencing. Genetic susceptibility appeared to play a role in smoking-related effects based on differences in LINE1 methylation in children with the common GSTM1 null genotype and in CpG-specific methylation in children with a common GSTP1 haplotype. These findings provide some of the first evidence that the life-long effects of in utero exposures may be mediated through alterations in DNA methylation patterns.
Although changes in methylation have been evaluated extensively in relation to cancer, research into whether prenatal environmental exposures can alter methylation in humans is nascent. Nutritional differences during pregnancy affect methylation in humans and animals (16
). Aberrant promoter methylation in smoking-related lung tumors has been found for several genes (18
). Moreover, tobacco smoke generates oxidative stress, which can cause a wide range of DNA lesions. Such DNA lesions can interfere with the binding of DNA methyltransferases to DNA, resulting in global hypomethylation (20
Only one study in humans has documented an association between prenatal tobacco smoke exposure and global methylation (21
). Terry and colleagues reported that prenatal exposure resulted in higher levels of DNA methylation. In contrast, we found prenatal exposure results in decreased global methylation. These inconsistent findings may be related to differences in the assays and tissues used to measure methylation in the two studies. Terry and colleagues measured methylation in isolated mononuclear cells from whole blood, whereas in our study methylation was measured in buccal cells. The [3
H]-methyl acceptance assay used by Terry and colleagues is limited by high day-to-day variability and inaccurate measurements of DNA concentration (22
). In contrast, pyrosequencing is a highly sensitive, quantitative, and high-throughput technique for DNA methylation detection (24
). Moreover, the methyl acceptance assay infers global methylation by measuring the ability of DNA to accept radiolabeled methyl groups. It is unclear how much this assay should correlate with specific methylation of AluYb8, which has a high level of intrinsic methylation.
Two genes demonstrated consistent differential methylation dependent upon prenatal smoke exposure status. AXL, a receptor tyrosine kinase, promotes antiapoptosis, mitogenesis, invasion, and cell survival (25
). The CpG site in AXL was located in the promoter at −223 base pairs upstream of the transcription start site in part of the core promoter region located −556 to −182, which is a known Sp1/Sp3 transcription factor binding site (26
). PTPRO, a protein tyrosine phosphatase receptor, has been linked to differentiation and axonogenesis of central and peripheral nervous system neurons during gestation (27
). The CpG site in PTPRO was located in the promoter at −371 base pairs upstream of the transcription start site. Gene expression in the promoters of AXL and PTPRO has previously been correlated with DNA methylation levels (26
). Although we were not directly able to evaluate correlations between smoking-induced methylation changes and subsequent changes in gene expression in our population, future research in this area is warranted. Given that these genes are susceptible to changes in methylation, exposure to tobacco smoke during gestation could alter expression of these genes, the consequences of which are unknown.
Genes involved in metabolism of tobacco smoke, particularly GSTM1 and GSTP1, can alter risks of childhood diseases that are associated with tobacco smoke (7
). We hypothesized that functional variants or haplotypes in these genes alter the associations between tobacco smoke exposure and methylation levels. Consistent with this hypothesis, we observed that the effects of maternal smoking on LINE1 methylation varied among children with and without the common GSTM1 null genotype. Smoke-related hypermethylation of CpG loci in MET and SNCG was also increased more in children who had two copies of the GSTP1 “0100” haplotype compared with those with fewer copies. We previously found this haplotype to protect against asthma in the larger CHS population, and variants in GSTM1 and GSTP1 are involved in asthma susceptibly from exposure to maternal smoking (10
In this study, we observed that LINE1 was hypomethylated, whereas single CpG loci in eight genes were hypermethylated, in response to prenatal tobacco smoke exposure. Though not fully understood, this trend of global hypomethylation with region-specific hypermethylation has been observed previously in cancers (29
). Global hypomethylation is believed to result in chromosomal instability and increased mutation events, whereas promoter hypermethylation can affect gene expression. Global hypomethylation could result from smoke-related ROS DNA damage during gestation if it prevents maintenance methyltransferases from binding (20
). Tobacco smoke exposure could also affect de novo
methylation in specific gene promoters, perhaps by incomplete erasure during methylation reprogramming that occurs in the embryo after fertilization.
Although the results of this study show that differences in methylation can be detected in children exposed to prenatal tobacco smoke, the differences were small for the endpoints evaluated. Therefore, the potential for bias needs to be considered in interpreting our findings. The modest differences in methylation level may result from having investigated CpG methylation in only 807 genes from a cancer panel in which some genes may have been less pertinent to the exposure evaluated. Examining a larger number of genes, using a genome-wide approach, or using a custom-targeted panel might identify additional genes with larger differences in methylation levels.
An association between prenatal tobacco smoke exposure and global methylation was observed only for AluYb8 but not LINE1; however, these are different repetitive elements with different residence times in the human genome and are not expected to respond to environmental stimuli in the same manner. The two repetitive elements are controlled through different mechanisms and have different transcription patterns in response to cellular stressors (30
). Moreover, other modifying factors likely exist that we have failed to adequately capture, such as any interaction between genotypes and exposure on DNA methylation. Our analyses of GSTM1 suggest this may be the case.
The association with prenatal tobacco smoke may be explained by other factors not addressed in this study. Women who smoke during pregnancy may have a less healthy lifestyle in general than women who do not, which could include differences in diet, alcohol consumption, or the use of illegal drugs. Smoking has also been associated with reduced folate levels, and folate-deficient diets have been associated with global hypomethylation and gene-specific hypermethylation (31
Given the limitations of collecting airway cells in children, we chose to use buccal mucosa cells, an aero-digestive tract epithelium, as an easily measurable surrogate for respiratory tract epithelium. Although the ideal tissue to use for assessing epigenetic changes in studies of respiratory health has not been defined, previous studies have demonstrated that DNA methylation changes of the oral epithelium can serve as a surrogate tissue for measuring DNA methylation changes of the lung (32
Our data support the utility of Illumina as an epigenome-wide screening platform but also demonstrate the need to use more quantitative DNA methylation assays, such as pyrosequencing, to confirm gene-specific analysis. We used a two-step approach—an Illumina methylation panel followed by pyrosequencing—to identify CpG loci associated with prenatal tobacco smoke exposure. This approach may become the paradigm for how these studies are performed. The purpose of the Illumina screen was to identify the most promising candidates for further follow-up and replication, based first on the magnitude of associations detected and then by statistical significance as a way to further narrow the list. Therefore, we did not adjust for multiple comparisons.
In this study, we provide some of the first evidence that differences in methylation patterns occur in children exposed in utero to tobacco smoke and that these differences can be detected in DNA from buccal cells, an aerodigestive tissue cell type. Exposure to maternal smoking affected two types of DNA methylation that have been associated with disease phenotypes: global methylation and promoter CpG island methylation. The effects of smoke exposure differed among children with common variants in genes involved in the detoxification of tobacco smoke. Our findings support the urgent need for research to understand better the potential mechanisms for life-long or transgenerational effects after preventable environmental exposures.