Environment‐induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children
2016; Springer Nature; Volume: 12; Issue: 3 Linguagem: Inglês
10.15252/msb.20156520
ISSN1744-4292
AutoresTobias Bauer, Saskia Trump, Naveed Ishaque, Loreen Thürmann, Lei Gu, Mario Bauer, Matthias Bieg, Zuguang Gu, Dieter Weichenhan, Jan‐Philipp Mallm, Stefan Röder, Gunda Herberth, Eiko Takada, Oliver Mücke, Marcus Winter, Kristin M. Junge, Konrad Grützmann, Ulrike Rolle‐Kampczyk, Qi Wang, Christian Lawerenz, Michael Borte, Tobias Polte, Matthias Schlesner, Michaela Schanné, Stefan Wiemann, Christina Geörg, Hendrik G. Stunnenberg, Christoph Plass, Karsten Rippe, Junichiro Mizuguchi, Carl Herrmann, Roland Eils, Irina Lehmann,
Tópico(s)Neonatal Respiratory Health Research
ResumoArticle24 March 2016Open Access Transparent process Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children Tobias Bauer Tobias Bauer orcid.org/0000-0002-4961-3639 Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Saskia Trump Saskia Trump Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Naveed Ishaque Naveed Ishaque Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Center for Personalized Oncology, DKFZ-HIPO, DKFZ, Heidelberg, Germany Search for more papers by this author Loreen Thürmann Loreen Thürmann Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Lei Gu Lei Gu Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Mario Bauer Mario Bauer Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Matthias Bieg Matthias Bieg Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Center for Personalized Oncology, DKFZ-HIPO, DKFZ, Heidelberg, Germany Search for more papers by this author Zuguang Gu Zuguang Gu Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Center for Personalized Oncology, DKFZ-HIPO, DKFZ, Heidelberg, Germany Search for more papers by this author Dieter Weichenhan Dieter Weichenhan Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Jan-Philipp Mallm Jan-Philipp Mallm Research Group Genome Organization & Function, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany Search for more papers by this author Stefan Röder Stefan Röder Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Gunda Herberth Gunda Herberth Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Eiko Takada Eiko Takada Department of Immunology, Tokyo Medical University, Tokyo, Japan Search for more papers by this author Oliver Mücke Oliver Mücke Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Marcus Winter Marcus Winter Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Kristin M Junge Kristin M Junge Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Konrad Grützmann Konrad Grützmann Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Ulrike Rolle-Kampczyk Ulrike Rolle-Kampczyk Department Metabolomics, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Qi Wang Qi Wang Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Christian Lawerenz Christian Lawerenz Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Michael Borte Michael Borte Municipal Hospital "St Georg" Children's Hospital, Leipzig, Germany Search for more papers by this author Tobias Polte Tobias Polte Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Department of Dermatology, Venerology and Allerology, Leipzig University Medical Center, Leipzig, Germany Search for more papers by this author Matthias Schlesner Matthias Schlesner Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Michaela Schanne Michaela Schanne Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Stefan Wiemann Stefan Wiemann Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Christina Geörg Christina Geörg Sample Processing Lab, National Center for Tumor Disease and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Hendrik G Stunnenberg Hendrik G Stunnenberg Department of Molecular Biology, Faculty of Science, Radboud University, Nijmegen, The Netherlands Search for more papers by this author Christoph Plass Christoph Plass Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Karsten Rippe Karsten Rippe Research Group Genome Organization & Function, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany Search for more papers by this author Junichiro Mizuguchi Junichiro Mizuguchi Department of Immunology, Tokyo Medical University, Tokyo, Japan Search for more papers by this author Carl Herrmann Carl Herrmann Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Institute of Pharmacy and Molecular Biotechnology and Bioquant Center, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Roland Eils Corresponding Author Roland Eils orcid.org/0000-0002-0034-4036 Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Center for Personalized Oncology, DKFZ-HIPO, DKFZ, Heidelberg, Germany Institute of Pharmacy and Molecular Biotechnology and Bioquant Center, University of Heidelberg, Heidelberg, Germany Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany Search for more papers by this author Irina Lehmann Corresponding Author Irina Lehmann Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Tobias Bauer Tobias Bauer orcid.org/0000-0002-4961-3639 Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Saskia Trump Saskia Trump Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Naveed Ishaque Naveed Ishaque Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Center for Personalized Oncology, DKFZ-HIPO, DKFZ, Heidelberg, Germany Search for more papers by this author Loreen Thürmann Loreen Thürmann Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Lei Gu Lei Gu Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Mario Bauer Mario Bauer Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Matthias Bieg Matthias Bieg Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Center for Personalized Oncology, DKFZ-HIPO, DKFZ, Heidelberg, Germany Search for more papers by this author Zuguang Gu Zuguang Gu Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Center for Personalized Oncology, DKFZ-HIPO, DKFZ, Heidelberg, Germany Search for more papers by this author Dieter Weichenhan Dieter Weichenhan Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Jan-Philipp Mallm Jan-Philipp Mallm Research Group Genome Organization & Function, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany Search for more papers by this author Stefan Röder Stefan Röder Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Gunda Herberth Gunda Herberth Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Eiko Takada Eiko Takada Department of Immunology, Tokyo Medical University, Tokyo, Japan Search for more papers by this author Oliver Mücke Oliver Mücke Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Marcus Winter Marcus Winter Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Kristin M Junge Kristin M Junge Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Konrad Grützmann Konrad Grützmann Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Ulrike Rolle-Kampczyk Ulrike Rolle-Kampczyk Department Metabolomics, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Qi Wang Qi Wang Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Christian Lawerenz Christian Lawerenz Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Michael Borte Michael Borte Municipal Hospital "St Georg" Children's Hospital, Leipzig, Germany Search for more papers by this author Tobias Polte Tobias Polte Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Department of Dermatology, Venerology and Allerology, Leipzig University Medical Center, Leipzig, Germany Search for more papers by this author Matthias Schlesner Matthias Schlesner Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Michaela Schanne Michaela Schanne Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Stefan Wiemann Stefan Wiemann Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Christina Geörg Christina Geörg Sample Processing Lab, National Center for Tumor Disease and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Hendrik G Stunnenberg Hendrik G Stunnenberg Department of Molecular Biology, Faculty of Science, Radboud University, Nijmegen, The Netherlands Search for more papers by this author Christoph Plass Christoph Plass Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Karsten Rippe Karsten Rippe Research Group Genome Organization & Function, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany Search for more papers by this author Junichiro Mizuguchi Junichiro Mizuguchi Department of Immunology, Tokyo Medical University, Tokyo, Japan Search for more papers by this author Carl Herrmann Carl Herrmann Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Institute of Pharmacy and Molecular Biotechnology and Bioquant Center, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Roland Eils Corresponding Author Roland Eils orcid.org/0000-0002-0034-4036 Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Center for Personalized Oncology, DKFZ-HIPO, DKFZ, Heidelberg, Germany Institute of Pharmacy and Molecular Biotechnology and Bioquant Center, University of Heidelberg, Heidelberg, Germany Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany Search for more papers by this author Irina Lehmann Corresponding Author Irina Lehmann Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany Search for more papers by this author Author Information Tobias Bauer1,‡, Saskia Trump2,‡, Naveed Ishaque1,3,‡, Loreen Thürmann2,‡, Lei Gu1,15,‡, Mario Bauer2,‡, Matthias Bieg1,3, Zuguang Gu1,3, Dieter Weichenhan4, Jan-Philipp Mallm5, Stefan Röder2, Gunda Herberth2, Eiko Takada6, Oliver Mücke4, Marcus Winter2, Kristin M Junge2, Konrad Grützmann2, Ulrike Rolle-Kampczyk7, Qi Wang1, Christian Lawerenz1, Michael Borte8, Tobias Polte2,9, Matthias Schlesner1, Michaela Schanne10, Stefan Wiemann10, Christina Geörg11, Hendrik G Stunnenberg12, Christoph Plass4, Karsten Rippe5, Junichiro Mizuguchi6, Carl Herrmann1,13,‡, Roland Eils 1,3,13,14,‡ and Irina Lehmann 2,‡ 1Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany 2Department of Environmental Immunology, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany 3Heidelberg Center for Personalized Oncology, DKFZ-HIPO, DKFZ, Heidelberg, Germany 4Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany 5Research Group Genome Organization & Function, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany 6Department of Immunology, Tokyo Medical University, Tokyo, Japan 7Department Metabolomics, Helmholtz Centre for Environmental Research Leipzig - UFZ, Leipzig, Germany 8Municipal Hospital "St Georg" Children's Hospital, Leipzig, Germany 9Department of Dermatology, Venerology and Allerology, Leipzig University Medical Center, Leipzig, Germany 10Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany 11Sample Processing Lab, National Center for Tumor Disease and German Cancer Research Center (DKFZ), Heidelberg, Germany 12Department of Molecular Biology, Faculty of Science, Radboud University, Nijmegen, The Netherlands 13Institute of Pharmacy and Molecular Biotechnology and Bioquant Center, University of Heidelberg, Heidelberg, Germany 14Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany 15Present address: Division of Newborn Medicine, Children's Hospital Boston and, Department of Cell Biology, Harvard Medical School, Boston, USA ‡These authors contributed equally to this work *Corresponding author. E-mail: [email protected] *Corresponding author. E-mail: [email protected] Molecular Systems Biology (2016)12:861https://doi.org/10.15252/msb.20156520 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood. Synopsis Genome-wide epigenomic and transcriptomic data reveal that maternal smoking induces important changes in the methylome of mothers and their children that affect in particular enhancer regions. These changes are stably maintained for years in the epigenome of the child. Comprehensive genome-wide sequencing reveals global changes in DNA methylation, chromatin states, and gene expression in mothers and their children associated with smoking during pregnancy. Such DNA methylation changes are stably maintained for years in the child. Taking into account genotype effects on methylation reveals a core set of genotype-independent, environmental driven DMRs, which are massively enriched in enhancers. Epigenetic deregulation of JNK2 enhancer at time of birth is linked to impaired lung function later in children's life. Introduction Altered epigenetic patterns represent an attractive explanation for understanding the phenotypic changes associated with environmental exposure. By affecting DNA methylation, post-translational histone modification, or non-coding RNA (ncRNA) signaling, environmental factors may cause persistent perturbations of regulatory pathways and thus induce an altered susceptibility for disease (Aguilera et al, 2010). Many lines of evidence indicate that early life and in particular the prenatal period represent a window of high vulnerability to environmental impacts with consequences for disease risk later in children's life. The subsequent manifestation of a disease may occur with long latency periods as shown in the Dutch Hunger Winter study. This study revealed that starvation during pregnancy increased the risk for several diseases later in children's life including type II diabetes, cardiovascular diseases, or decreased cognitive function (Brown et al, 1995; Susser et al, 1996; Roseboom et al, 2001; Painter et al, 2005; Veenendaal et al, 2013). Experimental models suggest that the in utero nutritional environment resulting from starvation induces epigenetic modifications including altered DNA methylation (Radford et al, 2014) and the generation of small RNAs (Rechavi et al, 2014) that are inherited in the next generation. One of the most common hazardous prenatal exposures is maternal smoking. Prenatal exposure to tobacco smoke was described as a risk factor for a multitude of different diseases in the child, including lung diseases, obesity, and cancer (Hemminki & Chen, 2006; Oken et al, 2008; Neuman et al, 2012). Several studies have focused on DNA methylation in cord blood to elucidate the influence of smoking and other prenatal exposures on the newborn's epigenome by analyzing global DNA methylation changes (i.e., in repetitive elements) or methylation of a limited number of preselected CpG probes (i.e., 27k methylation or 450k arrays) (Breton et al, 2009; Joubert et al, 2012; Murphy et al, 2012; Markunas et al, 2014; Kupers et al, 2015; Richmond et al, 2015). From these earlier epidemiological studies, information on global and site-specific methylation changes is available, but the insights derived from those studies remain very limited. Importantly, these earlier investigations, being based on 450k methylation array data, covered only a small fraction of the genome and, for example, lack information on enhancer regulatory elements located outside of promoters. Although changes of epigenetic modifications due to environmental cues early in life may persist over time, genome-wide data for studying longitudinal stability of epigenetic patterns in humans are still missing. Earlier studies have shown evidence for long-term stability of a limited number of methylation loci. However, to what extend this is a global trend, or only limited to some methylation loci is unclear, given the sparse coverage of the probes from the methylation array. In this study, we address the following questions: When and where in the genome are epigenetic marks set by environmental factors? What is the contribution of the genetic sequence variation to changes in DNA methylation? Do those changes that are associated with maternal smoking persist over years or do they appear only transiently? Furthermore, do DNA methylation changes contribute to early programming for disease? To address these questions, we performed a comprehensive epigenetic characterization within the LINA mother–child birth cohort (Herberth et al, 2006, 2011) to dissect the link between environmental exposure and epigenetic signals. We first studied the DNA methylome at single base pair resolution in both children and their mothers around time of birth and until 4 years after birth. To decipher the regulatory role of DNA methylation changes associated with smoking, we performed histone modification ChIP-seq of four histone modification marks to segment the genome into distinct regulatory elements and linked the environmentally associated differential DNA methylation to transcription as measured by RNA sequencing. Finally, we show that DNA methylation changes in conjunction with histone modifications are related to disease development later in children's life. Results Maternal smoking is associated with genome-wide DNA methylation changes that are different between mothers and their children A variety of studies has investigated the impact of maternal smoking on epigenetic changes in newborn children and assessed the stability of such epigenetic marks over time. Note that these studies focused on DNA methylation changes disregarding equally important changes on the chromatin level (Joubert et al, 2012; Kupers et al, 2015; Lee et al, 2015; Richmond et al, 2015). Further, these studies were performed on the basis of DNA methylation arrays covering as few as 5% of the entire set of CpG dinucleotides in the genome located primarily in promoter regions. To overcome the limitations of presently widely used DNA array methylation arrays offering only a narrow view on the DNA methylome, we here followed a radically different discovery and validation strategy (Fig 1). First, we set out to perform whole-genome bisulfite sequencing (WGBS) in a set of mothers alongside with their children and we went further to seek functional support for our findings. For that, we performed detailed analysis of chromatin configuration changes encompassing DNA methylation changes and further studied genome-wide changes in gene expression by RNA sequencing as a functional readout of the concerted action of DNA methylation and chromatin configuration changes. Finally, we set out to validate selected findings in the entire discovery cohort and in an independent replication cohort (Fig 1). Figure 1. Discovery and validation strategyTo overcome the limitations of presently widely used DNA array methylation arrays offering only a narrow view on the DNA methylome, we followed a different discovery and validation strategy. First, we performed whole-genome bisulfite sequencing (WGBS) in a set of mothers alongside with their children. WGBS was carried out in 16 mother–child pairs, a rather small sample number compared to the typical sizes of epigenetic studies performed on DNA methylation arrays. However, we compensated this potential drawback by a subsequent comprehensive functional validation. We performed a detailed analysis of chromatin configuration and DNA methylation changes—including longitudinal stability analyses—and further studied genome-wide gene expression by RNA sequencing as a functional readout of the concerted action of the observed epigenetic perturbations. In a targeted analysis, we finally validate selected findings in the entire discovery cohort. Download figure Download PowerPoint To study the impact of maternal smoking on DNA methylation in both mothers and children, we performed WGBS of whole blood samples from 32 individuals (maternal blood at 36th week of gestation for eight smoking mothers and eight non-smoking mothers and cord blood from their respective child; Table EV1; Fig EV1) at up to three different time points (for three children from smoking and three from non-smoking mothers at year one and four, and their respective mothers at year one; Table EV1; Fig EV1; for overview of discovery/validation strategy, see Fig 1). Click here to expand this figure. Figure EV1. Cohort and sample overviewThe diagram visualizes the number of mother–child pairs enrolled in the study together with the number of available blood samples (left). For the different experimental approaches, the number of samples analyzed is given starting at time of birth/pregnancy up to year 4 after birth (right). Download figure Download PowerPoint Heterogeneity of whole blood samples may be a possible confounder in our analysis. Therefore, we excluded the possibility that differences in cell type composition in the smoking exposed/non-exposed groups would give rise to DNA methylation changes by assessing promoter methylation levels from seven lineage markers reflecting the blood cell type composition in each sample (see Appendix Supplementary Methods). The analysis showed that the variation in cellular composition in response to tobacco smoke exposure was in the range of 1–7% for main blood cell types in mothers and children with significant reduced granulocyte and increased B-cell numbers in newborn children from smoking mothers (Table EV2). To exclude differentially methylated regions (DMRs) that were solely caused by differences in cellular blood composition between exposed and non-exposed samples, we used a threshold of 10% for DMR calling (Fig EV2, Appendix Supplementary Methods). Click here to expand this figure. Figure EV2. FDR and number of DMRs at different methylation difference theresholds Median false discovery rate (FDR) estimated by permutation analysis at different cutoffs for mean methylation changes of DMRs (ΔMethylation). Number of DMRs after filtering called at different mean methylation changes. The 10% ΔMethylation cutoff is marked and indicates a fair balance between sensitivity and specificity while offering acceptable FDRs (9.7% in children, 11.4% in mothers). Download figure Download PowerPoint Previous studies assessing the impact of smoking on differential DNA methylation had reported changes for single CpGs in the range of 1–25% (Zeilinger et al, 2013), indicating that our requirement of a 10% change represents a conservative threshold. To ensure consistently different methylation for all samples between groups, we excluded DMRs by a moderated t-statistics (P > 0.1) and permutation analysis. We thus identified 9743 and 8409 significant (P < 0.1, ΔMethylation > 0.1) DMRs in mothers and children, respectively, in the comparison of the smoking and the non-smoking individuals at time of birth (Fig 2, Table EV3). Using a random shuffling procedure, we determined that the median false discovery rate (FDR) level was 12.4% for children and 11.2% for mothers (see Appendix Supplementary Methods). Note that less than 5% of these DMRs are covered by CpG probes from the 450k platform. We finally conducted a FDR analysis based on permutation analysis of DMRs with subsequent filtering with a range of thresholds for ∆Methylation (1–25%). The obtained results implied that the 10% cutoff offers an optimal balance between high sensitivity and medium specificity, which are required when comparing groups of healthy individuals (see Appendix Supplementary Methods, Fig EV2). Figure 2. Mothers and children harbor a large number of differentially methylated regions between smokers and non-smokers at time of birth Circular representation of DNA methylation levels for mothers (outer circle) and children (inner circle). The height of each bar indicates the methylation change between the smoking and non-smoking group (dark hue: hypermethylation, light hue: hypomethylation). Bar plots represent the number of hypo- versus hypermethylated DMRs for all DMRs and ngDMRs in children and mothers. Number of genes predicted to interact with DMRs and ngDMRs. Annotation of allDMRs/ngDMRs according to genomic categories (lower panel). Download figure Download PowerPoint Beyond environmental factors, the individual genotype also effects DNA methylation. A single nucleotide polymorphism (SNP) may destroy the CpG context and thus directly induce differential DNA methylation in cis by reducing the methylation to 0 (0.5) in case of a
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