Effects of smoking on genome-wide DNA methylation profiles: A study of discordant and concordant monozygotic twin pairs
Abstract
Background: Smoking-associated DNA methylation levels identified through epigenome-wide association studies (EWAS) 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, i.e., 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 behavior 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).
Data availability
The HumanMethylation450 BeadChip data from the NTR are available as part of the Biobank-based Integrative Omics Studies (BIOS) Consortium in the European Genome phenome Archive (EGA), under the accession code EGAD00010000887. They are also available upon request via the BBMRI-NL BIOS consortium (https://www.bbmri.nl/acquisition-use-analyze/bios). All NTR data can be requested by bona fida researchers (https://ntr-data-request.psy.vu.nl/). Because of the consent given by study participants the data cannot be made publicly available.The pipeline for DNA methylation-array analysis developed by the Biobank-based Integrative Omics Study (BIOS) consortium is available here: https://molepi.github.io/DNAmArray_workflow/ (https://doi.org/10.5281/zenodo.3355292). The code for the EWAS analysis in monozygotic twin pairs is included in additional file 9
Article and author information
Author details
Funding
National Institute on Drug Abuse (DA049867)
- Michael C Neale
ZonMw (NWO-Groot 480-15-001/674)
- Gonneke Willemsen
- Eco JC de Geus
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Human subjects: Informed consent was obtained from all participants. The study was approved by the Central Ethics Committee on Research Involving Human Subjects of the VU University Medical Centre, Amsterdam, an Institutional Review Board certified by the U.S. Office of Human Research Protections (IRB number IRB00002991 under Federal-wide Assurance- FWA00017598; IRB/institute codes, NTR 03-180).
Copyright
© 2023, van Dongen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Funding: Supported by the Brazilian Ministry of Health and the Brazilian National Research Council.