Effects of smoking on genome-wide DNA methylation profiles: A study of discordant and concordant monozygotic twin pairs
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eLife assessment
This study presents valuable findings from a study of identical twin pairs discordant and concordant for smoking to assess whether smoking has a direct effect on DNA methylation. The results are a valuable contribution as the study confirms the reported association between smoking and epigenetic profile is indeed due to the direct effects of constituents of tobacco smoke. The study design and methods applied by the authors are solid and provide a starting point for larger studies with rigorous laboratory approaches, as well as for assessing clinical impact. The work will be of broad interest to addiction researchers, genetic epidemiologists, and environmental scientists.
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Abstract
Smoking-associated DNA methylation levels identified through epigenome-wide association studies (EWASs) are generally ascribed to smoking-reactive mechanisms, but the contribution of a shared genetic predisposition to smoking and DNA methylation levels is typically not accounted for.
Methods:
We exploited a strong within-family design, that is, the discordant monozygotic twin design, to study reactiveness of DNA methylation in blood cells to smoking and reversibility of methylation patterns upon quitting smoking. Illumina HumanMethylation450 BeadChip data were available for 769 monozygotic twin pairs (mean age = 36 years, range = 18–78, 70% female), including pairs discordant or concordant for current or former smoking.
Results:
In pairs discordant for current smoking, 13 differentially methylated CpGs were found between current smoking twins and their genetically identical co-twin who never smoked. Top sites include multiple CpGs in CACNA1D and GNG12 , which encode subunits of a calcium voltage-gated channel and G protein, respectively. These proteins interact with the nicotinic acetylcholine receptor, suggesting that methylation levels at these CpGs might be reactive to nicotine exposure. All 13 CpGs have been previously associated with smoking in unrelated individuals and data from monozygotic pairs discordant for former smoking indicated that methylation patterns are to a large extent reversible upon smoking cessation. We further showed that differences in smoking level exposure for monozygotic twins who are both current smokers but differ in the number of cigarettes they smoke are reflected in their DNA methylation profiles.
Conclusions:
In conclusion, by analysing data from monozygotic twins, we robustly demonstrate that DNA methylation level in human blood cells is reactive to cigarette smoking.
Funding:
We acknowledge funding from the National Institute on Drug Abuse grant DA049867, the Netherlands Organization for Scientific Research (NWO): Biobanking and Biomolecular Research Infrastructure (BBMRI-NL, NWO 184.033.111) and the BBRMI-NL-financed BIOS Consortium (NWO 184.021.007), NWO Large Scale infrastructures X-Omics (184.034.019), Genotype/phenotype database for behaviour genetic and genetic epidemiological studies (ZonMw Middelgroot 911-09-032); Netherlands Twin Registry Repository: researching the interplay between genome and environment (NWO-Groot 480-15-001/674); the Avera Institute, Sioux Falls (USA), and the National Institutes of Health (NIH R01 HD042157-01A1, MH081802, Grand Opportunity grants 1RC2 MH089951 and 1RC2 MH089995); epigenetic data were generated at the Human Genomics Facility (HuGe-F) at ErasmusMC Rotterdam. Cotinine assaying was sponsored by the Neuroscience Campus Amsterdam. DIB acknowledges the Royal Netherlands Academy of Science Professor Award (PAH/6635).
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eLife assessment
This study presents valuable findings from a study of identical twin pairs discordant and concordant for smoking to assess whether smoking has a direct effect on DNA methylation. The results are a valuable contribution as the study confirms the reported association between smoking and epigenetic profile is indeed due to the direct effects of constituents of tobacco smoke. The study design and methods applied by the authors are solid and provide a starting point for larger studies with rigorous laboratory approaches, as well as for assessing clinical impact. The work will be of broad interest to addiction researchers, genetic epidemiologists, and environmental scientists.
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Reviewer #1 (Public Review):
Van Dongen et al. investigated the methylation signature of smoking found in the blood among monozygotic twins ascertained from the Netherlands Twin Register. With their unique study design (which by design controls for the influence of age and sex), the authors shed light on DNA methylation levels that vary with smoking status, as well as with smoking cessation. The authors novel study design examined of twin pairs concordant or discordant for smoking status (current, former, never). The authors performed an epigenome-wide association study (EWAS) and identified 13 genome-wide significant CpGs that were differentially methylated between the discordant twin current-never smoking pairs. Another EWAS conducted by the authors found 5 additional genome-wide significant CpGs among current-former smoking …
Reviewer #1 (Public Review):
Van Dongen et al. investigated the methylation signature of smoking found in the blood among monozygotic twins ascertained from the Netherlands Twin Register. With their unique study design (which by design controls for the influence of age and sex), the authors shed light on DNA methylation levels that vary with smoking status, as well as with smoking cessation. The authors novel study design examined of twin pairs concordant or discordant for smoking status (current, former, never). The authors performed an epigenome-wide association study (EWAS) and identified 13 genome-wide significant CpGs that were differentially methylated between the discordant twin current-never smoking pairs. Another EWAS conducted by the authors found 5 additional genome-wide significant CpGs among current-former smoking discordant pairs. Each of the 13 identified CpG sites between current-former twins have been previously identified as associated with smoking. The authors found that 3 of these 13 CpGs are located within 1Mb of a single nucleotide polymorphism (SNP) previously associated with smoking initiation, suggesting a role for the SNP in both genetic susceptibility of smoking as well as methylation. The authors tested for enrichment of the 13 CpGs within traits and pathways and found enrichment among smoking related traits, as well as the dopaminergic synapse pathway. Interestingly, the authors found that twin pairs discordant for former smoking (former smoking-never pair) had methylation levels that nearly returned to baseline (never smoking) after smoking cessation. These data broaden our understanding of methylation signatures in the blood using a concordant/discordant smoking and twin study design. The authors evaluated within-twin pair methylation differences for the 13 significant CpGs and found twins concordant for smoking status had very little difference between their methylation levels, yet those discordant for smoking status had larger differences with the current-never smoking twins having the largest differences. Importantly, using a dataset with both methylation and RNA sequencing data, the authors found higher methylation at three CpGs was associated with lower gene expression providing functional context for their findings. The authors correctly acknowledge the limitations of only having blood to evaluate methylation signatures and using a methylation array rather than bisulfite sequencing.
There are a couple of aspects that would be useful to help with interpretation of their findings, such as whether presentation of a formal test for trend shows a linear relationship between overall DNA methylation and smoking pack-years and smoking quit time. It would help the reader if the authors could put their findings into context with what has been previously identified in studies such as the Framingham Heart Study or the prior twin study with concordant/discordant twins. While the findings are interesting, the moderate sample size and use of a methylation array rather than sequencing may ultimately lead this work to have only moderate impact on the field.
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Reviewer #2 (Public Review):
The authors aimed to test the effects of smoking on the methylome while controlling for genetics to test for evidence of whether previous studies on genetically-unrelated individuals were confounded by genetics.
The strengths of this study of genetics-independent associations between smoking exposure and DNA methylation using an epigenome-scale approach are (1) its moderate sample size for a twin study (50-100 ) to detect some of the larger effects sizes (10-15%) found in this study; (2) the thorough EWAS methodology including adjusting for cellular heterogeneity and the use of Bonferroni correction; (3) the use of a within identical twin pair design; (4) the strong overlap between the results and those of previous similar studies in genetically unrelated individuals. Weaknesses include the use of …
Reviewer #2 (Public Review):
The authors aimed to test the effects of smoking on the methylome while controlling for genetics to test for evidence of whether previous studies on genetically-unrelated individuals were confounded by genetics.
The strengths of this study of genetics-independent associations between smoking exposure and DNA methylation using an epigenome-scale approach are (1) its moderate sample size for a twin study (50-100 ) to detect some of the larger effects sizes (10-15%) found in this study; (2) the thorough EWAS methodology including adjusting for cellular heterogeneity and the use of Bonferroni correction; (3) the use of a within identical twin pair design; (4) the strong overlap between the results and those of previous similar studies in genetically unrelated individuals. Weaknesses include the use of methylation arrays that although targeted to putative regulatory regions, cover only around 2.5% of genomic CpGs, and the use of only a single tissue (blood). Both are acknowledged by the authors.
The authors achieved their aims and were able to test all their hypotheses. In general, the authors' claims were supported by their data, but they could empirically test for an association between methylation and expression at all top CpGs rather than just stating that a subset significantly associated.
This is an important set of findings for the field because genetic confounding has been levelled as a criticism of epigenomewide association studies. It therefore strengthens the evidence that environment (smoking) can change the methylome, assuming that the methylomes of each pair were similar prior to exposure.
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Reviewer #3 (Public Review):
In order to address their study question of a potential shared genetic predisposition to both smoking and DNA methylation level, they indicate that a MZ discordant pair analysis would be very powerful.
The authors draw on the well-characterized and very large prospective study of twins and family members from the Netherlands, the Netherlands Twin Register (NTR). Over 3000 cohort members have DNA methylation assessed by arrays (450k and Epic). Monozygotic twin pairs discordant and concordant for smoking are included in epigenome-wide analyses, and followed-up using enrichment and gene expression studies.
The results demonstrate that the strongest associations that have been seen in unrelated individuals (such as for AHRR) are seen in the discordant pairs but do not have the statistical power to confirm or …
Reviewer #3 (Public Review):
In order to address their study question of a potential shared genetic predisposition to both smoking and DNA methylation level, they indicate that a MZ discordant pair analysis would be very powerful.
The authors draw on the well-characterized and very large prospective study of twins and family members from the Netherlands, the Netherlands Twin Register (NTR). Over 3000 cohort members have DNA methylation assessed by arrays (450k and Epic). Monozygotic twin pairs discordant and concordant for smoking are included in epigenome-wide analyses, and followed-up using enrichment and gene expression studies.
The results demonstrate that the strongest associations that have been seen in unrelated individuals (such as for AHRR) are seen in the discordant pairs but do not have the statistical power to confirm or reject weaker (yet consistently seen) associations
Some mention of the effect of second-hand smoke (SHS) could be made as it is an exposure to smoke not due to one's own active smoking. As twin pairs often reside together or are in frequent contact/visiting - MZs more than DZ and females more than males, SHS may be attenuating differences between current and non-current smokers in discordant pairs rather than shared genetics. Likewise twin pairs often have the same or related occupation, and if smoking is common at their typical workplace (even if they work at different companies/employers), the non-smoking twin may be exposed to more tobacco than an unrelated never-smoker.
The study sample should be better described, especially with regard to how smoking behavior was assessed, and whether the twins in pairs discordant for smoking differ in characteristics that can affect DNA methylation. These details would be essential for understanding to what degree the observed findings are attributable to smoking.
The study provides important information on the smoking methylation relationship and supports the generally held view that smoking has a direct effect on methylation. Hence, methylation changes are a useful biomarker of current and past smoking. The current results indicate that confounding due to shared genetics is unlikely to be a major factor but some role cannot be excluded.
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