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DNA methylation as a marker for prenatal smoke exposure in adults

DNA methylation as a marker for prenatal smoke exposure in adults Background: Prenatal smoke exposure is known to be robustly associated with DNA methylation among offspring in early life, but whether the association persists into adult- hood is unclear. This study aimed to investigate the long-term effect of maternal smoke exposure on DNA methylation in 754 women (mean age 30 years); to replicate findings in the same women 18 years later and in a cohort of 230 men (mean age 53 years); and to assess the extent to which a methylation score could predict prenatal smoke exposure. Methods: We first carried out an epigenome-wide association analysis for prenatal smoke exposure and performed replication analyses for the top CpG sites in the other samples. We derived a DNA methylation score based on previously identified CpG sites and generated receiver operating characteristic (ROC) curves to assess the performance of these scores as predictors of prenatal smoke exposure. Results: We observed associations at 15 CpG sites in 11 gene regions: MYO1G, FRMD4A, CYP1A1, CNTNAP2, ARL4C, AHRR, TIFAB, MDM4, AX748264, DRD1, FTO (false discovery rate<5%). Most of these associations were specific to exposure during pregnancy, were present 18 years later and were replicated in a cohort of men. A DNA methylation score could predict prenatal smoke exposure (30 years previously) with an area under the curve of 0.72 (95% confidence interval 0.69, 0.76). Conclusions: The results of this study provide robust evidence that maternal smoking in pregnancy is associated with changes in DNA methylation that persist in the exposed off- spring for many years after prenatal exposure. Key words: prenatal smoking, ALSPAC, DNA methylation, epigenetics, long-term, prediction, epigenome-wide association study, longitudinal V The Author(s) 2018. Published by Oxford University Press on behalf of the International Epidemiological Association. 1120 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1121 Key Messages We investigated the long-term impact of maternal smoking in pregnancy on epigenetic changes in the offspring by assessing differences in DNA methylation levels in adulthood. We observed associations at 15 CpG sites in 11 gene regions; most of these associations were specific to exposure during pregnancy, were found to persist until at least 48 years and were present in both men and women. A prenatal smoking score, derived by combining methylation values, could adequately predict whether the mothers of the adults smoked during pregnancy. The results of this study provide robust evidence that maternal smoking in pregnancy is associated with changes in DNA methylation that persist in the exposed offspring for many years after their prenatal exposure. These findings could have useful applications in epidemiological studies, e.g. by using DNA methylation signatures as a biosocial archive for exposure when data on maternal smoking during pregnancy are absent. Introduction prenatal tobacco smoke exposure on genome-wide methyl- ation is warranted. Cigarette smoke exposure during pregnancy is an environ- In this study, we aimed to assess the long-term impact mental stressor that has a profound effect on DNA methyl- 1 of prenatal tobacco smoke on DNA methylation in the ation in the exposed offspring. Previous work has context of the Avon Longitudinal Study of Parents and determined genome-wide changes in DNA methylation in 18,19 2 Children (ALSPAC) —a prospective birth cohort with response to smoke exposure in utero, with a recent data on reported maternal smoke exposure in pregnancy epigenome-wide association study (EWAS) meta-analysis and genome-wide DNA methylation levels of offspring in of methylation in newborn cord blood identifying over adulthood. We first conducted an analysis to investigate 6000 differentially methylated CpG sites (of which 568 3 associations between reported prenatal smoke exposure CpG sites surpassed the strict Bonferroni threshold). and DNA methylation in peripheral blood among women It is of interest to investigate the persistence of methyla- in ALSPAC (mean age 30 years). We next attempted to rep- tion marks into later life, as this presents the opportunity licate prenatal smoking-associated DNA methylation dif- to use methylation as an archive of historical exposure, ferences in peripheral blood of the women 18 years later particularly if methylation patterns can be robustly mod- 4–6 (mean age 48 years) and in men from the same study (the elled over time. In addition, persistent changes in DNA partners of these women) (mean age 52 years). Finally, we methylation might mediate at least some of the associa- aimed to assess the extent to which a prenatal smoking tions between smoke exposure in pregnancy and later-life 7 score, based on methylation at CpG sites previously shown health outcomes. to be associated with prenatal smoke exposure, could pre- Several studies have identified prenatal smoke exposure dict whether the mothers of the ALSPAC women smoked associated changes in methylation in childhood and adoles- 8 8–10 during pregnancy. cence in global methylation, candidate gene and 3,4,6,11–13 EWAS. However, few studies have investigated 14–17 the persistence of methylation change into adulthood. Methods Three such studies have been conducted in the New York Cohort and selection of participants City birth cohort, where prospectively assessed maternal smoking during pregnancy was found to be positively asso- ALSPAC is a large, prospective cohort study based in the ciated with global methylation in leukocytes of individuals south-west of England. A total of 14 541 pregnant women at age 43 years assessed using a methyl acceptance assay, resident in Avon, UK, with expected dates of delivery 1 inversely associated with levels of Sat2 methylation and April 1991 to 31 December 1992 were recruited and de- most recently associated with methylation at 17 CpG sites tailed information has been collected on these women and 17 18,19 on the Illumina Infinium HumanMethylation450 array, their offspring at regular intervals. The study website which remained even after adjustment for adult smoking contains details of all the data that are available through a status of the offspring. However, previous studies were fully searchable data dictionary (http://www.bris.ac.uk/ limited by low power due to small sample sizes and a more alspac/researchers/data-access/data-dictionary/). Written comprehensive assessment of the long-term impact of informed consent has been obtained for all ALSPAC Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 1122 International Journal of Epidemiology, 2018, Vol. 47, No. 4 participants. Ethical approval for the study was obtained surrogate variables and cell count fractions were included from the ALSPAC Ethics and Law Committee and the as additional covariates to adjust for technical batch and Local Research Ethics Committees. cell-type mixture (see Supplementary Material, available as We examined offspring DNA methylation in relation to Supplementary data at IJE online, for further details), al- reported maternal smoking during pregnancy in ALSPAC though we did not find strong evidence for associations be- using methylation data from the Illumina Infinium tween prenatal smoke exposure and derived cell types HumanMethylation450 (HM450) BeadChip assay (Supplementary Table 1, available as Supplementary data (Illumina, San Diego, CA, USA). We used data from at IJE online). women enrolled ALSPAC (n¼ 754) in the main analysis In order to evaluate the potential influence of own and looked to replicate findings in the same women ap- smoking, which might explain the persistence in methyla- proximately 18 years later (n¼ 656) and in men enrolled in tion signatures associated with intrauterine exposure, in- ALSPAC (n¼ 230). formation on the ALSPAC women’s own smoking status was obtained from a questionnaire administered at 18 weeks’ gestation. Women were asked whether they had Prenatal exposure variables smoked regularly pre-pregnancy, from which a dichoto- After recruitment of pregnant women into the ALSPAC mous variable for any tobacco smoking before pregnancy study, information was collected on both the women and was derived. To assess the impact of passive smoke expo- their partners, including details of their mothers’ smoking sure, women were also asked whether their partners behaviour. If the men and women reported that their smoked at the same time point and whether their fathers mothers had smoked, they were asked whether their moth- (as well as their mothers) had smoked previously. ers had smoked when they were pregnant with them and For the replication analysis, information on the wom- were given the responses yes/no/don’t know from which to en’s smoking status was also gathered in a questionnaire select. These data were analysed assuming that, for all administered approximately 18 years later. Furthermore, those who said don’t know, their mothers did smoke dur- information on the partner’s smoking status was obtained ing pregnancy, as has been done previously. We carried from a questionnaire administered to the partners approxi- out further sensitivity analysis excluding those individuals mately 21 years after the pregnancy. At these later time who were unsure of their mothers’ smoking status during points, the men and women were asked whether they cur- pregnancy and compared findings. rently smoked or whether they had smoked every day when a smoker in the past. From these data, a dichoto- mous variable for any previous tobacco smoking was DNA methylation assessment derived. We examined offspring DNA methylation in peripheral blood in ALSPAC men and women. As part of the Statistical analysis Accessible Resource for Integrated Epigenomics Studies 21 22 (ARIES) project, the HM450 BeadChip has been used We first performed an epigenome-wide association analysis to generate epigenetic data on 1018 mother–offspring pairs in our largest sample: the ALSPAC women with methyla- in the ALSPAC cohort. A web portal has been constructed tion measured in peripheral blood taken at the time of en- to allow openly accessible browsing of aggregate ARIES rolment in the study (n¼ 754). CpG level methylation DNA methylation data (ARIES-Explorer) (http://www.arie [untransformed b-values, which is the ratio of the methyl- sepigenomics.org.uk/). Additional HM450 data have been ated probe intensity and the overall intensity and ranges generated on ALSPAC men (the partners of the women en- from 0 (no cytosine methylation) to 1 (complete cytosine rolled in ARIES) (n¼ 312). Details of sample handling and methylation)] was regressed against prenatal smoke expo- DNA methylation profiling are outlined in the sure (any maternal smoking during pregnancy) with adjust- Supplementary Material, available as Supplementary data ment for covariates (maternal age, parental social class, at IJE online. offspring age, top 10 SVs in the main model). We then assessed whether associations were robust to adjustment for own smoking status (including own smoking status as a Covariates further covariate and also running the EWAS stratified by Maternal age at birth and head of household social class own smoking status) and passive smoke exposure were included as covariates in these analyses, as they were (reported partner smoking). found to be most strongly associated with smoking status To evaluate the specificity of the intrauterine effect and during pregnancy in a previous study. In addition, 10 potential bias in our findings due to the role of passive Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1123 smoke exposure out of pregnancy or residual confounding, associated with both prenatal smoking and own smoking 23–25 we conducted two negative control tests : (i) perform- (Supplementary Figure 1, available as Supplementary data ing a comparison of the associations between parental at IJE online), we also assessed the performance of the pre- smoking (any paternal smoking and any maternal smok- natal smoking score adjusted for the own smoking score. ing) and methylation levels at the top CpG sites and (ii) We compared the AUC of pairs of ROC curves using the comparing associations between maternal smoking outside Delong test for difference computed by the roc.test func- of pregnancy and methylation levels with maternal smok- tion as part of the pROC package. ing during pregnancy and methylation levels at the same We also investigated the extent to which the prenatal sites. smoking scores could predict maternal smoking in preg- We next performed replication analyses for the top nancy independently of own smoking status. This was CpG sites [false discovery rate (FDR)< 0.05] in the other done by comparing methylation scores between four differ- samples, by conducting EWAS of maternal smoking in the ent groups of participants, determined based on their own ALSPAC women 18 years later and ALSPAC men, adjusted smoking status and that of their mothers during pregnancy: for the same covariates as in the main model for the non-smokers whose mothers never smoked in pregnancy, ALSPAC women at enrolment, and performing a look-up smokers whose mothers never smoked in pregnancy, non- of the CpGs identified in the main analysis. We also smokers whose mothers smoked in pregnancy and smokers assessed the association between prenatal smoke exposure whose mothers smoked in pregnancy. Pairwise compari- and methylation at the identified sites in cord blood in the sons between the groups were performed using two-tailed 3,4 ALSPAC cohort that has been investigated previously t-tests and a p-value for difference in methylation scores (Supplementary Table 2, available as Supplementary data obtained. In addition, a p-value for trend was obtained at IJE online). We next performed Pearson’s correlation from the linear regression of the methylation score on analysis in order to compare consistency in effect estimates smoking status, where the four groups were included in an between the main analysis and the replication analysis. ordered categorical variable. We also investigated the association between prenatal Analysis was performed using Stata (version 14) and R exposure to smoking and DNA methylation for the 568 (version 3.3.1). CpG sites previously found to be robustly associated with prenatal smoke in a cord blood meta-analysis at 3 Results Bonferroni significance (n¼ 6685), in each of the adult cohorts. We assessed the degree of inflation of association The cohort-specific summary statistics for this analysis are signal (lambda value) for these CpG sites compared with presented in Table 1. The ALSPAC women in our main that seen genome-wide across the samples and performed a analysis had a mean age of 30 years, whereas their mean Wilcoxon rank sum test to assess enrichment. age at follow-up was 48 years and the ALSPAC men had a Furthermore, we generated a DNA methylation score mean age of 53 years. Maternal ages at birth were similar for prenatal smoking based on these independently identi- between the ALSPAC men and women, as were parental fied 568 CpG sites and compared its ability to predict social class and rates of prenatal smoke exposure. Rates of whether the mothers of the ALSPAC adults had smoked own smoking varied quite substantially, from 16.9% in the during pregnancy with a score based on 19 CpG sites that ALSPAC women at the first time point to 33.8% in the reached Bonferroni significance in an EWAS of prenatal ALSPAC men. There was limited evidence for an associa- smoking conducted in peripheral blood of older children tion between prenatal smoke exposure and own smoking (n¼ 3187) and a score for own smoking, consisting of in the three study groups (Supplementary Table 3, avail- 2623 CpG sites that reached Bonferroni significance in the able as Supplementary data at IJE online). largest EWAS of own smoking to date (n¼ 9389 current vs We observed associations between 15 CpG sites and never smokers). Details of these studies and how the prenatal smoking exposure in women at age 30 at scores were generated are outlined in Supplementary Table FDR< 5% and 9 CpG sites that surpassed Bonferroni cor- 2, available as Supplementary data at IJE online, and the rection (Table 2). These sites were located in 11 gene Supplementary Material, available as Supplementary data regions and all but 2 have been previously identified in at IJE online. EWAS for maternal smoking at birth and into later 3,4,6,10–12,17 We generated receiver operating characteristic (ROC) life and all agree on direction of effect curves for the prenatal and own smoking methylation (Supplementary Table 4, available as Supplementary data scores and calculated the area under the curve (AUC) in or- at IJE online). der to assess the performance of these predictors using the Associations at CpG sites located near ARL4C pROC package in R. Given the overlap of CpG sites (cg05204104 and cg15016771), MDM4 (cg08241939) Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 1124 International Journal of Epidemiology, 2018, Vol. 47, No. 4 Table 1. Descriptive characteristics of participant groups in this study ALSPAC adult females ALSPAC adult females ALSPAC (Time Point 1) (Time Point 2) adult males N 754 656 230 Maternal age at birth (years) (SD) 27.6 (5.7) 27.8 (5.7) 28.2 (5.7) Social class (manual) (N, %) 347 (46.0) 301 (45.9) 92 (40.0) Prenatal smoke exposure (yes) (N, %) 216 (28.7) 179 (27.3) 73 (31.7) Age at follow-up (years) (SD) 30.3 (4.3) 48.1 (4.2) 53.4 (5.1) Sample type(s) Whole blood/white cells White cells/PBLs White cells/PBLs Own smoking (N,%) 127 (16.9) 144 (28.9) 75 (33.8) The sample size of individuals who reported own smoking was slightly smaller with N¼ 752, N¼ 498 and N¼ 222 for ALSPAC adult females (Time Point 1), ALSPAC adult females (Time Point 2) and ALSPAC adult males (Time Point 3), respectively. Table 2. DNA methylation changes associated with prenatal smoke exposure in ALSPAC women (Time Point 1) a b CpG site Chromosome Gene Position Basic model (N¼ 754 ) Adjusted model (N¼ 752) region Effect Standard P-value FDR Effect Standard P-value FDR size error size error cg22132788 7 MYO1G 45002486 0.062 0.008 1.70E-13 8.26E-08 0.058 0.008 9.77E-13 4.75E-07 cg12803068 7 MYO1G 45002919 0.111 0.015 9.82E-13 2.38E-07 0.097 0.014 1.71E-11 4.16E-06 cg11813497 10 FRMD4A 14372879 0.038 0.006 1.25E-10 2.02E-05 0.036 0.006 4.85E-10 7.85E-05 cg04180046 7 MYO1G 45002736 0.041 0.006 2.53E-10 3.07E-05 0.037 0.006 3.43E-09 3.33E-04 cg05549655 15 CYP1A1 75019143 0.007 0.001 1.21E-08 0.001 0.007 0.001 8.82E-10 1.07E-04 cg25949550 7 CNTNAP2 145814306 –0.005 0.001 1.48E-08 0.001 –0.004 0.001 2.41E-07 0.015 cg19089201 7 MYO1G 45002287 0.038 0.007 1.75E-08 0.001 0.034 0.007 1.88E-07 0.013 cg05204104 2 ARL4C 235403141 0.023 0.004 2.30E-08 0.001 0.023 0.004 4.12E-08 0.003 cg17924476 5 AHRR 323794 0.045 0.008 6.24E-08 0.003 0.040 0.008 5.33E-07 0.029 cg11429111 5 TIFAB 134813329 0.022 0.004 2.62E-07 0.013 0.019 0.004 2.90E-06 0.108 cg08241939 1 MDM4 204700816 0.022 0.004 5.04E-07 0.022 0.019 0.004 3.40E-06 0.110 cg11641006 2 AX748264 235213874 0.040 0.008 5.62E-07 0.023 0.029 0.006 1.27E-06 0.051 cg15016771 2 ARL4C 235403218 0.008 0.002 1.01E-06 0.036 0.007 0.002 3.35E-06 0.110 cg22807681 5 DRD1 174622933 0.023 0.005 1.04E-06 0.036 0.023 0.005 1.02E-06 0.045 cg26681628 16 FTO 54210550 0.037 0.008 1.27E-06 0.041 0.029 0.006 8.94E-06 0.193 Effect size¼ difference in methylation level (beta) between adult offspring of smokers and non-smokers in pregnancy. Entries in bold represent sites that surpassed the Bonferroni threshold. N¼ 216 smoked during pregnancy, N¼ 538 no smoking during pregnancy. Model includes adjustment for own smoking. and DRD1 (cg22807681) appear to be novel, although the four sites at MYO1G (cg22132788, cg12803068, sites at ARL4C were present in the extended list of FDR cg04180046 and cg19089201) did survive multiple testing significant sites in the previous cord blood EWAS. In con- adjustment, with the MYO1G site cg22132788 having the –13 trast to findings in cord blood, where smoking during preg- strongest association (P¼ 1.7 10 ), which is consistent 4,6,12 nancy has been associated approximately equally with with findings in children and adolescents. hyper and hypomethylation, the majority (14 out of 15 Results were robust to the additional inclusion of de- CpGs) showed long-term hypermethylation in this rived cell counts as covariates, with 14 of the 15 CpG sites analysis. surpassing the FDR< 5% threshold and the other CpG –6 Whereas, in most EWAS for cord blood methylation, site at DRD1 (cg22807681) with p¼ 5.22 10 AHRR (cg05575921) is the CpG site most consistently as- (Supplementary Table 5, available as Supplementary data 3,4,28,29 sociated with prenatal smoke exposure, this site did at IJE online). In sensitivity analysis where those partici- not survive adjustment for multiple tests in our persistence pants who were unsure of their mothers’ smoking status analysis (p¼ 0.0005, FDR> 5%). Rather, associations at during pregnancy were removed (n¼ 651 remained in the Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1125 analysis, n¼ 103 were excluded), 12 CpG sites were found MDM4 or DRD1 in the largest EWAS of own smoking to to be associated with prenatal smoke exposure at date. Findings were similar when analyses were stratified FDR< 5% in this smaller dataset (Supplementary Table 6, by own smoking status, i.e. with consistent effect estimates available as Supplementary data at IJE online). Nine of even among those women who had not previously smoked these CpG sites were overlapping with those in the main regularly themselves (Supplementary Table 7, available as analysis, with the remaining three CpG sites located in sim- Supplementary data at IJE online), although there was ilar gene regions [AHRR (cg05575921), TIFAB some evidence for a difference in effect sizes between (cg01952185) and FRMD4A (cg25464840)]. smokers and non-smokers at ARL4C (cg05204104), where One concern related to the identification of these signals the effect was larger among smokers than among non- is that they might reflect non-specific smoke exposure of smokers (p for interaction¼ 0.001). Furthermore, results the offspring over the life course rather than a ‘critical pe- were consistent when reported partner smoking was in- riod’ effect of smoke exposure in utero. In particular, 11 cluded as a covariate as an indicator of passive smoke ex- out of the 15 CpGs have been identified in relation to cur- posure (Supplementary Table 8, available as rent vs never smoking status at FDR< 0.05 in a recent Supplementary data at IJE online). EWAS of own smoking (Supplementary Table 4, avail- For the negative control tests, the parental comparison able as Supplementary data at IJE online). To account for showed consistently larger effect estimates for maternal this, we first examined whether past cigarette smoking by smoking than for paternal smoking, although confidence the adult themselves influenced these associations by in- intervals overlapped for some of the sites [FRMD4A cluding own smoking as a covariate in the model. (cg25464840), CYP1A1 (cg05549655), ARL4C Adjustment for own smoking attenuated associations at (cg05204104), AHRR (cg17924476) and MYO1G five CpG sites [TIFAB (cg11429111), MDM4 (cg04180046)] (Supplementary Figure 2, available as (cg08241939), AX748264 (cg11641006), ARL4C Supplementary data at IJE online). For the comparison of (cg15016771) and FTO (cg26681628)] that no longer associations between the offspring of women who smoked reached the FDR cut-off for significance (Table 2 and during pregnancy and the offspring of women who smoked Figure 1), although, on the whole, the magnitude of effect outside of pregnancy, effect estimates were consistently was only slightly reduced with this adjustment. larger for those reporting maternal smoking during preg- Furthermore, whereas methylation of CpG sites at nancy, this time without overlapping confidence intervals MYO1G, CNTNAP2 and AHRR have been consistently (Supplementary Figure 3, available as Supplementary data identified in relation to own smoking, no such associations at IJE online). Whereas, for MYO1G (cg12803066) and have been found with CpG sites at FDRM4A, CYP1A1, MYO1G (cg22132766), there was some evidence of asso- ciation between maternal smoking outside of pregnancy with methylation levels, suggestive of either residual con- founding in the intrauterine associations or a postnatal smoking effect, these findings may also be explained by misreporting of maternal smoking during pregnancy that was based on retrospective reports by the offspring in adulthood. This latter explanation is more likely given that the paternal smoking estimates at these sites were consis- tent with the null (Supplementary Figure 2, available as Supplementary data at IJE online). Effects at the top CpGs surpassing FDR correction in the ALSPAC women were found to be consistent in direc- tion although slightly attenuated in the follow-up analysis approximately 18 years later (Pearson’s correlation coeffi- cient, r¼ 0.92) (Figure 2 and Supplementary Figure 4, available as Supplementary data at IJE online). Furthermore, there was remarkable consistency in the di- rection and magnitude of effects at these CpGs in blood samples of the ALSPAC men (r¼ 0.77), with the exception Figure 1. Manhattan plot for EWAS of prenatal smoke exposure in of DRD1, where effects were not as consistently replicated. ALSPAC women (Time Point 1)*. *N¼ 752, adjusted for own smoking. We also compared for reference the effect of any maternal Solid horizontal line represents Bonferroni threshold; dotted horizontal line represents FDR correction (p< 0.05) threshold. smoking on cord blood methylation in the ALSPAC birth Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 1126 International Journal of Epidemiology, 2018, Vol. 47, No. 4 Figure 2. Replication of CpG sites observed below FDR (p< 0.05) threshold in ALSPAC women at a later time point (Time Point 2) and in ALSPAC men,* and comparison with effect of prenatal smoking on cord blood methylation in ALSPAC children. *Adjusted for own smoking in adult samples. N¼ 860 ALSPAC cord blood (reference), N¼ 752 ALSPAC women (Time Point 1), N¼ 498 ALSPAC women (Time Point 2), N¼ 222 ALSPAC men. Figure 3. Receiver operating characteristic (ROC) curves of prenatal and own smoking methylation scores for discriminating maternal smoking in pregnancy. Total N¼ 922; NB: sample size in the main analysis is smaller due to inclusion of additional covariates into those models that were miss- ing for some of the participants. Prenatal smoking methylation score (older children)¼ score derived from 19 CpG sites associated with maternal smoking in older children in an independent study. Prenatal smoking methylation score (newborns)¼ score derived from 568 CpG sites associated with maternal smoking in newborns in an independent study. Own smoking methylation score¼ score derived from 2623 CpG sites associated with smoking status in adults in an independent study. Scores were applied to methylation data from ALSPAC adult females at Time Point 1. Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1127 cohort at these CpGs and, again, both magnitude and di- To determine the extent to which methylation associa- rection of effect were similar (r¼ 0.92) with the exception tions with prenatal smoking were different from own of sites at ARL4C (cg05204104), AHRR (cg17924476) smoking associations, we constructed a similar score de- and DRD4 (cg22807681). At these sites, the difference in rived from 2623 CpG sites previously associated with own methylation was greater in the ALSPAC women than in smoking in adulthood. Whereas being strongly predictive the newborns (Figure 2 and Supplementary Figure 4, avail- of own smoking status in the ALSPAC women (AUC 0.88, able as Supplementary data at IJE online). 95% CI 0.85, 0.91), this score was only weakly associated In addition, we found that, among women with a mean with prenatal smoking compared with the 19-CpG prena- age of 30 years (N¼ 754), there was a strong signal of as- tal smoking score (AUC 0.57, 95% CI 0.53, 0.61; P for dif- –11 sociation above that expected by chance at CpG sites previ- ference¼ 3.0 10 )(Figure 3). In addition, the 19-CpG ously associated with prenatal smoke exposure in prenatal smoking methylation score was able to predict newborns [lambda¼ 2.62 vs 1.14 for all CpG sites on the with the same accuracy prenatal smoke exposure when ad- Illumina Infinium HumanMethylation450 (HM450) justed for the offspring smoking methylation score (AUC –16 BeadChip; Wilcoxon rank sum test p-value< 2.2 10 ] 0.71, 95% CI 0.68, 0.75; P for difference¼ 0.06) (Supplementary Figure 5, available as Supplementary data (Figure 3). at IJE online). Similarly, inflation of signals for prenatal The prenatal smoking methylation scores were higher in smoke exposure was seen in these women 18 years later individuals (both smokers and non-smokers) exposed to –15 (lambda¼ 1.54, p-value¼ 5.6 10 ) and in the ALSPAC prenatal smoking compared with non-smokers who were –4 men (lambda¼ 1.19, p-value¼ 2.2 10 ), compared with not exposed prenatally (i.e. the ‘OS.MS’ and ‘ONS.MS’ all CpG sites on the HM450 BeadChip (Supplementary group vs the ‘ONS.MNS’ baseline group; p-value for –16 Figure 5, available as Supplementary data at IJE online). trend< 2.0 10 )(Figure 4). The prenatal smoking A prenatal smoking methylation score, derived by com- methylation score derived from 568 CpG sites identified in bining methylation values at 568 CpG sites associated with newborns was not able to distinguish between non- prenatal smoke exposure in cord blood of newborns in a smokers whose mothers smoked in pregnancy compared previous study, could predict whether the mothers of the with smokers whose mothers did not smoke during preg- ALSPAC women smoked during pregnancy with an AUC nancy (difference in score between the ‘ONS.MS’ group 0.69 [95% confidence interval (CI) 0.67, 0.73]. This was and the ‘OS.MNS’ group¼ –0.07, p¼ 0.61) (Figure 4a). comparable with a score derived from 19 CpG sites previ- However, the score derived from 19 CpG sites which were ously associated with prenatal smoking in peripheral blood shown to persist in relation to prenatal smoke exposure 3 3 of older children, which had an AUC 0.72 (95% CI 0.69, based on an EWAS in older children was higher in non- 0.76; P for difference¼ 0.97) (Figure 3). smokers whose mothers smoked in pregnancy compared Figure 4. Box plots to assess differences in prenatal smoking methylation scores. Prenatal smoking methylation score (newborns)¼ score derived from 568 CpG sites associated with maternal smoking in newborns in an independent study. Prenatal smoking methylation score (older children)¼ score derived from 19 CpG sites associated with maternal smoking in older children in an independent study. ONS, MNS¼ offspring non-smoker, mother never smoked in pregnancy (N¼ 522); OS, MNS¼ offspring smoker, mother never smoked in pregnancy (N¼ 112); ONS, MS¼ offspring non-smoker, mother smoked in pregnancy (N¼ 222); OS, MS¼ offspring smoker, mother smoked in pregnancy (N¼ 66). NB: sample size in the main analysis is smaller due to inclusion of additional covariates into those models that were missing for some of the participants. Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 1128 International Journal of Epidemiology, 2018, Vol. 47, No. 4 with smokers whose mothers did not smoke in pregnancy Strengths of our study include the large sample size of (difference in score between the ‘ONS.MS’ group and the women with reported maternal smoking in pregnancy for ‘OS.MNS’ group¼ 0.01, p¼ 0.001) (Figure 4b). performing our initial EWAS analysis, the ability to adjust for own smoking status, the longitudinal assessment of dif- ferential methylation in a follow-up sample of these Discussion women and the replication analysis in men from the same In a large longitudinal cohort with genome-wide methyla- study. tion data, we identified 15 CpG sites that were differen- Although there was evidence of persistence for methyla- tially methylated in the peripheral blood of women over tion differences even after adjusting for own smoking sta- 30 years after exposure to prenatal smoking. Most of these tus in the adult offspring, there are limitations to signals remained in sensitivity analyses adjusted for own performing this type of adjustment analysis. As smoking and passive smoke exposure, and showed stron- parental smoking is strongly associated with their off- ger associations in relation to maternal smoke exposure in spring’s smoking initiation, own smoking serves as a pos- pregnancy compared with smoke exposure outside of preg- sible mediator on the path between prenatal smoking and nancy (by both mothers and fathers) indicating specificity offspring DNA methylation. This method of adjusting for of the intrauterine effect. Furthermore, we observed a per- a potential mediator in standard regression models to esti- sistent methylation signal related to prenatal smoke expo- mate the direct effect of an exposure may produce spurious 34,35 sure in peripheral blood 18 years later (i.e. at around the conclusions. Whereas an alternative method of using age of 48 years) and replicated in peripheral blood among life-course models previously provided more evidence for men in the ALSPAC cohort. the hypothesis that maternal smoking in pregnancy is the Many of these CpG sites have been previously identified ‘critical period’ for influencing persistent offspring methyl- in relation to prenatal smoke exposure in the offspring at ation profiles, this method could not be applied here given birth and the majority showed long-term hypermethyla- the limited amount of information on maternal smoking tion among the offspring of smokers. Findings are also reported by the ALSPAC men and women. consistent with a recent report that highlighted persistence A further limitation relates to cell-type heterogeneity, of DNA methylation levels related to prenatal smoke expo- given that the ALSPAC samples were obtained from a vari- sure into adulthood, which identified associations the ety of sources [white cells, whole blood and peripheral same CpG sites located in MYO1G and CYP1A1, and blood lymphocytes (PBLs)]. To account for this, we incor- other CpG sites in FTO and AHRR. porated surrogate variables into our models to account to For all of the study samples, there was also a strong sig- adjust for technical batch and cell-type mixture in order to nal of association above that expected by chance at 568 harmonize cellular variability of the samples and carried CpG sites previously associated with prenatal smoke expo- out sensitivity analysis that also adjusted for derived cell 3 37,38 sure in newborns from an independent study. In addition, counts. we found that a prenatal smoking score, derived by com- In addition, information on prenatal smoke exposure in bining methylation values at these CpG sites, could ade- the ALSPAC men and women was recorded retrospectively quately predict whether the mothers of the adults in by the adult offspring, rather than by prospective assess- ALSPAC smoked during pregnancy with an AUC 0.69 ment, and so may be subject to more misreporting. (95% CI 0.67, 0.73). A recent study identified a much Furthermore, in the ALSPAC men and women, rates of stronger predictive ability of a prenatal smoking methyla- maternal smoking in pregnancy were reported to be high tion score with AUC of 0.90 in a test set of cord blood in comparison with contemporary populations. This draws obtained from newborns in the MoBa cohort. The differ- to question the relevance of identified associations. ence in predictive ability is therefore likely attributed to the However, we have shown that many of the signals identi- 30-year difference in time since exposure and the genera- fied in adults were also present in cord blood of offspring tion of this methylation score using CpG sites identified in measured prospectively in a more contemporary cohort cord blood rather than adult peripheral blood. We also de- with lower rates of maternal smoking in pregnancy. rived a methylation score based on CpGs that showed evi- Overall, the results of this study provide robust evidence dence of a persistent difference in methylation in that maternal smoking in pregnancy is associated with peripheral blood of older offspring exposed to prenatal changes in DNA methylation that persist in the exposed smoking who had a marginally higher AUC of 0.72 (95% offspring for many years after their prenatal exposure. CI 0.67, 0.73) and was also able to distinguish non- Furthermore, these associations largely remain after adjust- smokers whose mothers smoked in pregnancy from smok- ing for the previous smoking history of the adults them- ers whose mothers did not smoke during pregnancy. selves and are in accordance with earlier studies Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1129 2. Richmond RC, Joubert BR. Contrasting the effects of intra- investigating prospectively assessed maternal smoking dur- uterine smoking and one-carbon micronutrient exposures on off- ing pregnancy in relation to global DNA methylation 14,15 spring DNA methylation. Epigenomics 2017;9:351–67. levels. 3. Joubert BR, Felix JF, Yousefi P, Bakulski KM, Just AC, Breton C. DNA methylation in newborns and maternal smoking in pregnancy: genome-wide consortium meta-analysis. Am J Hum Conclusion Genet 2016;98:680–96. 4. Richmond RC, Simpkin AJ, Woodward G et al. Prenatal expo- These findings could have useful applications in epidemio- sure to maternal smoking and offspring DNA methylation across logical studies, e.g. by using DNA methylation signatures the lifecourse: findings from the Avon Longitudinal Study of as a biosocial archive for historical exposure. Parents and Children (ALSPAC). Hum Mol Genet 2015;24: Furthermore, persistent changes in DNA methylation 2201–17. might mediate at least some of the associations between 5. Relton CL, Hartwig FP, Davey Smith G. From stem cells to the smoke exposure in pregnancy and later-life health out- law courts: DNA methylation, the forensic epigenome and the pos- comes. However, distinguishing mediation from other sibility of a biosocial archive. Int J Epidemiol 2015;44:1083–93. 6. Lee KWK, Richmond R, Hu PZ et al. Prenatal exposure to ma- association-driving mechanisms warrants further evalua- ternal cigarette smoking and DNA methylation: epigenome-wide tion with the integration of analytical techniques such as association in a discovery sample of adolescents and replication 41,42 two-step Mendelian randomization, transcriptomic in an independent cohort at birth through 17 years of age. analysis and the profiling of target tissues. Environ Health Persp 2015;123:193–99. 7. Nielsen CH, Larsen A, Nielsen AL. DNA methylation alterations in response to prenatal exposure of maternal cigarette smoking: Supplementary data a persistent epigenetic impact on health from maternal lifestyle? Arch Toxicol 2016;90:231–45. Supplementary data are available at IJE online. 8. Breton CV, Byun HM, Wenten M, Pan F, Yang A, Gilliland FD. Prenatal tobacco smoke exposure affects global and gene-specific DNA methylation. Am J Respir Crit Care Med 2009;180:462–67. Funding 9. Toledo-Rodriguez M, Lotfipour S, Leonard G et al. Maternal This work was supported by the Integrative Epidemiology Unit smoking during pregnancy is associated with epigenetic modifi- which receives funding from the UK Medical Research Council and cations of the brain-derived neurotrophic factor-6 exon in ado- the University of Bristol (MC_UU_12013_1 and lescent offspring. Am J Med Genet B Neuropsychiatr Genet MC_UU_12013_2). This work was also supported by CRUK (grant 2010;153B:1350–54. number C18281/A19169) and the ESRC (grant number ES/ 10. Ladd-Acosta C, Shu C, Lee BK et al. Presence of an epigenetic N000498/1). The UK Medical Research Council and the Wellcome signature of prenatal cigarette smoke exposure in childhood. Trust (Grant ref: 102215/2/13/2) and the University of Bristol pro- Environ Res 2016;144:139–48. vide core support for ALSPAC. The Accessible Resource for 11. Breton CV, Siegmund KD, Joubert BR et al. Prenatal tobacco Integrated Epigenomics Studies (ARIES) was funded by the UK smoke exposure is associated with childhood DNA CpG methyl- Biotechnology and Biological Sciences Research Council (BB/ ation. PLoS One 2014;9:e99716. I025751/1 and BB/I025263/1). The methylation data generated on 12. Rzehak P, Saffery R, Reischl E et al. Maternal smoking during ALSPAC men (the partners of the women enrolled in ARIES) were pregnancy and DNA-methylation in children at age 5.5 years: funded by the Medical Research Council and the University of epigenome-wide-analysis in the European Childhood Obesity Bristol (MC_UU_12013_2). The funders had no role in study design, Project (CHOP)-Study. PLoS One 2016;11:e0155554. data collection and analysis, decision to publish or preparation of 13. Bauer T, Trump S, Ishaque N et al. Environment-induced epige- the manuscript. netic reprogramming in genomic regulatory elements in smoking mothers and their children. Mol Syst Biol 2016;12:861. Acknowledgements 14. Terry MB, Ferris JS, Pilsner R et al. Genomic DNA methylation among women in a multiethnic New York City birth cohort. 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DNA methylation as a marker for prenatal smoke exposure in adults

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Oxford University Press
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Copyright © 2022 International Epidemiological Association
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0300-5771
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1464-3685
DOI
10.1093/ije/dyy091
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Abstract

Background: Prenatal smoke exposure is known to be robustly associated with DNA methylation among offspring in early life, but whether the association persists into adult- hood is unclear. This study aimed to investigate the long-term effect of maternal smoke exposure on DNA methylation in 754 women (mean age 30 years); to replicate findings in the same women 18 years later and in a cohort of 230 men (mean age 53 years); and to assess the extent to which a methylation score could predict prenatal smoke exposure. Methods: We first carried out an epigenome-wide association analysis for prenatal smoke exposure and performed replication analyses for the top CpG sites in the other samples. We derived a DNA methylation score based on previously identified CpG sites and generated receiver operating characteristic (ROC) curves to assess the performance of these scores as predictors of prenatal smoke exposure. Results: We observed associations at 15 CpG sites in 11 gene regions: MYO1G, FRMD4A, CYP1A1, CNTNAP2, ARL4C, AHRR, TIFAB, MDM4, AX748264, DRD1, FTO (false discovery rate<5%). Most of these associations were specific to exposure during pregnancy, were present 18 years later and were replicated in a cohort of men. A DNA methylation score could predict prenatal smoke exposure (30 years previously) with an area under the curve of 0.72 (95% confidence interval 0.69, 0.76). Conclusions: The results of this study provide robust evidence that maternal smoking in pregnancy is associated with changes in DNA methylation that persist in the exposed off- spring for many years after prenatal exposure. Key words: prenatal smoking, ALSPAC, DNA methylation, epigenetics, long-term, prediction, epigenome-wide association study, longitudinal V The Author(s) 2018. Published by Oxford University Press on behalf of the International Epidemiological Association. 1120 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1121 Key Messages We investigated the long-term impact of maternal smoking in pregnancy on epigenetic changes in the offspring by assessing differences in DNA methylation levels in adulthood. We observed associations at 15 CpG sites in 11 gene regions; most of these associations were specific to exposure during pregnancy, were found to persist until at least 48 years and were present in both men and women. A prenatal smoking score, derived by combining methylation values, could adequately predict whether the mothers of the adults smoked during pregnancy. The results of this study provide robust evidence that maternal smoking in pregnancy is associated with changes in DNA methylation that persist in the exposed offspring for many years after their prenatal exposure. These findings could have useful applications in epidemiological studies, e.g. by using DNA methylation signatures as a biosocial archive for exposure when data on maternal smoking during pregnancy are absent. Introduction prenatal tobacco smoke exposure on genome-wide methyl- ation is warranted. Cigarette smoke exposure during pregnancy is an environ- In this study, we aimed to assess the long-term impact mental stressor that has a profound effect on DNA methyl- 1 of prenatal tobacco smoke on DNA methylation in the ation in the exposed offspring. Previous work has context of the Avon Longitudinal Study of Parents and determined genome-wide changes in DNA methylation in 18,19 2 Children (ALSPAC) —a prospective birth cohort with response to smoke exposure in utero, with a recent data on reported maternal smoke exposure in pregnancy epigenome-wide association study (EWAS) meta-analysis and genome-wide DNA methylation levels of offspring in of methylation in newborn cord blood identifying over adulthood. We first conducted an analysis to investigate 6000 differentially methylated CpG sites (of which 568 3 associations between reported prenatal smoke exposure CpG sites surpassed the strict Bonferroni threshold). and DNA methylation in peripheral blood among women It is of interest to investigate the persistence of methyla- in ALSPAC (mean age 30 years). We next attempted to rep- tion marks into later life, as this presents the opportunity licate prenatal smoking-associated DNA methylation dif- to use methylation as an archive of historical exposure, ferences in peripheral blood of the women 18 years later particularly if methylation patterns can be robustly mod- 4–6 (mean age 48 years) and in men from the same study (the elled over time. In addition, persistent changes in DNA partners of these women) (mean age 52 years). Finally, we methylation might mediate at least some of the associa- aimed to assess the extent to which a prenatal smoking tions between smoke exposure in pregnancy and later-life 7 score, based on methylation at CpG sites previously shown health outcomes. to be associated with prenatal smoke exposure, could pre- Several studies have identified prenatal smoke exposure dict whether the mothers of the ALSPAC women smoked associated changes in methylation in childhood and adoles- 8 8–10 during pregnancy. cence in global methylation, candidate gene and 3,4,6,11–13 EWAS. However, few studies have investigated 14–17 the persistence of methylation change into adulthood. Methods Three such studies have been conducted in the New York Cohort and selection of participants City birth cohort, where prospectively assessed maternal smoking during pregnancy was found to be positively asso- ALSPAC is a large, prospective cohort study based in the ciated with global methylation in leukocytes of individuals south-west of England. A total of 14 541 pregnant women at age 43 years assessed using a methyl acceptance assay, resident in Avon, UK, with expected dates of delivery 1 inversely associated with levels of Sat2 methylation and April 1991 to 31 December 1992 were recruited and de- most recently associated with methylation at 17 CpG sites tailed information has been collected on these women and 17 18,19 on the Illumina Infinium HumanMethylation450 array, their offspring at regular intervals. The study website which remained even after adjustment for adult smoking contains details of all the data that are available through a status of the offspring. However, previous studies were fully searchable data dictionary (http://www.bris.ac.uk/ limited by low power due to small sample sizes and a more alspac/researchers/data-access/data-dictionary/). Written comprehensive assessment of the long-term impact of informed consent has been obtained for all ALSPAC Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 1122 International Journal of Epidemiology, 2018, Vol. 47, No. 4 participants. Ethical approval for the study was obtained surrogate variables and cell count fractions were included from the ALSPAC Ethics and Law Committee and the as additional covariates to adjust for technical batch and Local Research Ethics Committees. cell-type mixture (see Supplementary Material, available as We examined offspring DNA methylation in relation to Supplementary data at IJE online, for further details), al- reported maternal smoking during pregnancy in ALSPAC though we did not find strong evidence for associations be- using methylation data from the Illumina Infinium tween prenatal smoke exposure and derived cell types HumanMethylation450 (HM450) BeadChip assay (Supplementary Table 1, available as Supplementary data (Illumina, San Diego, CA, USA). We used data from at IJE online). women enrolled ALSPAC (n¼ 754) in the main analysis In order to evaluate the potential influence of own and looked to replicate findings in the same women ap- smoking, which might explain the persistence in methyla- proximately 18 years later (n¼ 656) and in men enrolled in tion signatures associated with intrauterine exposure, in- ALSPAC (n¼ 230). formation on the ALSPAC women’s own smoking status was obtained from a questionnaire administered at 18 weeks’ gestation. Women were asked whether they had Prenatal exposure variables smoked regularly pre-pregnancy, from which a dichoto- After recruitment of pregnant women into the ALSPAC mous variable for any tobacco smoking before pregnancy study, information was collected on both the women and was derived. To assess the impact of passive smoke expo- their partners, including details of their mothers’ smoking sure, women were also asked whether their partners behaviour. If the men and women reported that their smoked at the same time point and whether their fathers mothers had smoked, they were asked whether their moth- (as well as their mothers) had smoked previously. ers had smoked when they were pregnant with them and For the replication analysis, information on the wom- were given the responses yes/no/don’t know from which to en’s smoking status was also gathered in a questionnaire select. These data were analysed assuming that, for all administered approximately 18 years later. Furthermore, those who said don’t know, their mothers did smoke dur- information on the partner’s smoking status was obtained ing pregnancy, as has been done previously. We carried from a questionnaire administered to the partners approxi- out further sensitivity analysis excluding those individuals mately 21 years after the pregnancy. At these later time who were unsure of their mothers’ smoking status during points, the men and women were asked whether they cur- pregnancy and compared findings. rently smoked or whether they had smoked every day when a smoker in the past. From these data, a dichoto- mous variable for any previous tobacco smoking was DNA methylation assessment derived. We examined offspring DNA methylation in peripheral blood in ALSPAC men and women. As part of the Statistical analysis Accessible Resource for Integrated Epigenomics Studies 21 22 (ARIES) project, the HM450 BeadChip has been used We first performed an epigenome-wide association analysis to generate epigenetic data on 1018 mother–offspring pairs in our largest sample: the ALSPAC women with methyla- in the ALSPAC cohort. A web portal has been constructed tion measured in peripheral blood taken at the time of en- to allow openly accessible browsing of aggregate ARIES rolment in the study (n¼ 754). CpG level methylation DNA methylation data (ARIES-Explorer) (http://www.arie [untransformed b-values, which is the ratio of the methyl- sepigenomics.org.uk/). Additional HM450 data have been ated probe intensity and the overall intensity and ranges generated on ALSPAC men (the partners of the women en- from 0 (no cytosine methylation) to 1 (complete cytosine rolled in ARIES) (n¼ 312). Details of sample handling and methylation)] was regressed against prenatal smoke expo- DNA methylation profiling are outlined in the sure (any maternal smoking during pregnancy) with adjust- Supplementary Material, available as Supplementary data ment for covariates (maternal age, parental social class, at IJE online. offspring age, top 10 SVs in the main model). We then assessed whether associations were robust to adjustment for own smoking status (including own smoking status as a Covariates further covariate and also running the EWAS stratified by Maternal age at birth and head of household social class own smoking status) and passive smoke exposure were included as covariates in these analyses, as they were (reported partner smoking). found to be most strongly associated with smoking status To evaluate the specificity of the intrauterine effect and during pregnancy in a previous study. In addition, 10 potential bias in our findings due to the role of passive Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1123 smoke exposure out of pregnancy or residual confounding, associated with both prenatal smoking and own smoking 23–25 we conducted two negative control tests : (i) perform- (Supplementary Figure 1, available as Supplementary data ing a comparison of the associations between parental at IJE online), we also assessed the performance of the pre- smoking (any paternal smoking and any maternal smok- natal smoking score adjusted for the own smoking score. ing) and methylation levels at the top CpG sites and (ii) We compared the AUC of pairs of ROC curves using the comparing associations between maternal smoking outside Delong test for difference computed by the roc.test func- of pregnancy and methylation levels with maternal smok- tion as part of the pROC package. ing during pregnancy and methylation levels at the same We also investigated the extent to which the prenatal sites. smoking scores could predict maternal smoking in preg- We next performed replication analyses for the top nancy independently of own smoking status. This was CpG sites [false discovery rate (FDR)< 0.05] in the other done by comparing methylation scores between four differ- samples, by conducting EWAS of maternal smoking in the ent groups of participants, determined based on their own ALSPAC women 18 years later and ALSPAC men, adjusted smoking status and that of their mothers during pregnancy: for the same covariates as in the main model for the non-smokers whose mothers never smoked in pregnancy, ALSPAC women at enrolment, and performing a look-up smokers whose mothers never smoked in pregnancy, non- of the CpGs identified in the main analysis. We also smokers whose mothers smoked in pregnancy and smokers assessed the association between prenatal smoke exposure whose mothers smoked in pregnancy. Pairwise compari- and methylation at the identified sites in cord blood in the sons between the groups were performed using two-tailed 3,4 ALSPAC cohort that has been investigated previously t-tests and a p-value for difference in methylation scores (Supplementary Table 2, available as Supplementary data obtained. In addition, a p-value for trend was obtained at IJE online). We next performed Pearson’s correlation from the linear regression of the methylation score on analysis in order to compare consistency in effect estimates smoking status, where the four groups were included in an between the main analysis and the replication analysis. ordered categorical variable. We also investigated the association between prenatal Analysis was performed using Stata (version 14) and R exposure to smoking and DNA methylation for the 568 (version 3.3.1). CpG sites previously found to be robustly associated with prenatal smoke in a cord blood meta-analysis at 3 Results Bonferroni significance (n¼ 6685), in each of the adult cohorts. We assessed the degree of inflation of association The cohort-specific summary statistics for this analysis are signal (lambda value) for these CpG sites compared with presented in Table 1. The ALSPAC women in our main that seen genome-wide across the samples and performed a analysis had a mean age of 30 years, whereas their mean Wilcoxon rank sum test to assess enrichment. age at follow-up was 48 years and the ALSPAC men had a Furthermore, we generated a DNA methylation score mean age of 53 years. Maternal ages at birth were similar for prenatal smoking based on these independently identi- between the ALSPAC men and women, as were parental fied 568 CpG sites and compared its ability to predict social class and rates of prenatal smoke exposure. Rates of whether the mothers of the ALSPAC adults had smoked own smoking varied quite substantially, from 16.9% in the during pregnancy with a score based on 19 CpG sites that ALSPAC women at the first time point to 33.8% in the reached Bonferroni significance in an EWAS of prenatal ALSPAC men. There was limited evidence for an associa- smoking conducted in peripheral blood of older children tion between prenatal smoke exposure and own smoking (n¼ 3187) and a score for own smoking, consisting of in the three study groups (Supplementary Table 3, avail- 2623 CpG sites that reached Bonferroni significance in the able as Supplementary data at IJE online). largest EWAS of own smoking to date (n¼ 9389 current vs We observed associations between 15 CpG sites and never smokers). Details of these studies and how the prenatal smoking exposure in women at age 30 at scores were generated are outlined in Supplementary Table FDR< 5% and 9 CpG sites that surpassed Bonferroni cor- 2, available as Supplementary data at IJE online, and the rection (Table 2). These sites were located in 11 gene Supplementary Material, available as Supplementary data regions and all but 2 have been previously identified in at IJE online. EWAS for maternal smoking at birth and into later 3,4,6,10–12,17 We generated receiver operating characteristic (ROC) life and all agree on direction of effect curves for the prenatal and own smoking methylation (Supplementary Table 4, available as Supplementary data scores and calculated the area under the curve (AUC) in or- at IJE online). der to assess the performance of these predictors using the Associations at CpG sites located near ARL4C pROC package in R. Given the overlap of CpG sites (cg05204104 and cg15016771), MDM4 (cg08241939) Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 1124 International Journal of Epidemiology, 2018, Vol. 47, No. 4 Table 1. Descriptive characteristics of participant groups in this study ALSPAC adult females ALSPAC adult females ALSPAC (Time Point 1) (Time Point 2) adult males N 754 656 230 Maternal age at birth (years) (SD) 27.6 (5.7) 27.8 (5.7) 28.2 (5.7) Social class (manual) (N, %) 347 (46.0) 301 (45.9) 92 (40.0) Prenatal smoke exposure (yes) (N, %) 216 (28.7) 179 (27.3) 73 (31.7) Age at follow-up (years) (SD) 30.3 (4.3) 48.1 (4.2) 53.4 (5.1) Sample type(s) Whole blood/white cells White cells/PBLs White cells/PBLs Own smoking (N,%) 127 (16.9) 144 (28.9) 75 (33.8) The sample size of individuals who reported own smoking was slightly smaller with N¼ 752, N¼ 498 and N¼ 222 for ALSPAC adult females (Time Point 1), ALSPAC adult females (Time Point 2) and ALSPAC adult males (Time Point 3), respectively. Table 2. DNA methylation changes associated with prenatal smoke exposure in ALSPAC women (Time Point 1) a b CpG site Chromosome Gene Position Basic model (N¼ 754 ) Adjusted model (N¼ 752) region Effect Standard P-value FDR Effect Standard P-value FDR size error size error cg22132788 7 MYO1G 45002486 0.062 0.008 1.70E-13 8.26E-08 0.058 0.008 9.77E-13 4.75E-07 cg12803068 7 MYO1G 45002919 0.111 0.015 9.82E-13 2.38E-07 0.097 0.014 1.71E-11 4.16E-06 cg11813497 10 FRMD4A 14372879 0.038 0.006 1.25E-10 2.02E-05 0.036 0.006 4.85E-10 7.85E-05 cg04180046 7 MYO1G 45002736 0.041 0.006 2.53E-10 3.07E-05 0.037 0.006 3.43E-09 3.33E-04 cg05549655 15 CYP1A1 75019143 0.007 0.001 1.21E-08 0.001 0.007 0.001 8.82E-10 1.07E-04 cg25949550 7 CNTNAP2 145814306 –0.005 0.001 1.48E-08 0.001 –0.004 0.001 2.41E-07 0.015 cg19089201 7 MYO1G 45002287 0.038 0.007 1.75E-08 0.001 0.034 0.007 1.88E-07 0.013 cg05204104 2 ARL4C 235403141 0.023 0.004 2.30E-08 0.001 0.023 0.004 4.12E-08 0.003 cg17924476 5 AHRR 323794 0.045 0.008 6.24E-08 0.003 0.040 0.008 5.33E-07 0.029 cg11429111 5 TIFAB 134813329 0.022 0.004 2.62E-07 0.013 0.019 0.004 2.90E-06 0.108 cg08241939 1 MDM4 204700816 0.022 0.004 5.04E-07 0.022 0.019 0.004 3.40E-06 0.110 cg11641006 2 AX748264 235213874 0.040 0.008 5.62E-07 0.023 0.029 0.006 1.27E-06 0.051 cg15016771 2 ARL4C 235403218 0.008 0.002 1.01E-06 0.036 0.007 0.002 3.35E-06 0.110 cg22807681 5 DRD1 174622933 0.023 0.005 1.04E-06 0.036 0.023 0.005 1.02E-06 0.045 cg26681628 16 FTO 54210550 0.037 0.008 1.27E-06 0.041 0.029 0.006 8.94E-06 0.193 Effect size¼ difference in methylation level (beta) between adult offspring of smokers and non-smokers in pregnancy. Entries in bold represent sites that surpassed the Bonferroni threshold. N¼ 216 smoked during pregnancy, N¼ 538 no smoking during pregnancy. Model includes adjustment for own smoking. and DRD1 (cg22807681) appear to be novel, although the four sites at MYO1G (cg22132788, cg12803068, sites at ARL4C were present in the extended list of FDR cg04180046 and cg19089201) did survive multiple testing significant sites in the previous cord blood EWAS. In con- adjustment, with the MYO1G site cg22132788 having the –13 trast to findings in cord blood, where smoking during preg- strongest association (P¼ 1.7 10 ), which is consistent 4,6,12 nancy has been associated approximately equally with with findings in children and adolescents. hyper and hypomethylation, the majority (14 out of 15 Results were robust to the additional inclusion of de- CpGs) showed long-term hypermethylation in this rived cell counts as covariates, with 14 of the 15 CpG sites analysis. surpassing the FDR< 5% threshold and the other CpG –6 Whereas, in most EWAS for cord blood methylation, site at DRD1 (cg22807681) with p¼ 5.22 10 AHRR (cg05575921) is the CpG site most consistently as- (Supplementary Table 5, available as Supplementary data 3,4,28,29 sociated with prenatal smoke exposure, this site did at IJE online). In sensitivity analysis where those partici- not survive adjustment for multiple tests in our persistence pants who were unsure of their mothers’ smoking status analysis (p¼ 0.0005, FDR> 5%). Rather, associations at during pregnancy were removed (n¼ 651 remained in the Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1125 analysis, n¼ 103 were excluded), 12 CpG sites were found MDM4 or DRD1 in the largest EWAS of own smoking to to be associated with prenatal smoke exposure at date. Findings were similar when analyses were stratified FDR< 5% in this smaller dataset (Supplementary Table 6, by own smoking status, i.e. with consistent effect estimates available as Supplementary data at IJE online). Nine of even among those women who had not previously smoked these CpG sites were overlapping with those in the main regularly themselves (Supplementary Table 7, available as analysis, with the remaining three CpG sites located in sim- Supplementary data at IJE online), although there was ilar gene regions [AHRR (cg05575921), TIFAB some evidence for a difference in effect sizes between (cg01952185) and FRMD4A (cg25464840)]. smokers and non-smokers at ARL4C (cg05204104), where One concern related to the identification of these signals the effect was larger among smokers than among non- is that they might reflect non-specific smoke exposure of smokers (p for interaction¼ 0.001). Furthermore, results the offspring over the life course rather than a ‘critical pe- were consistent when reported partner smoking was in- riod’ effect of smoke exposure in utero. In particular, 11 cluded as a covariate as an indicator of passive smoke ex- out of the 15 CpGs have been identified in relation to cur- posure (Supplementary Table 8, available as rent vs never smoking status at FDR< 0.05 in a recent Supplementary data at IJE online). EWAS of own smoking (Supplementary Table 4, avail- For the negative control tests, the parental comparison able as Supplementary data at IJE online). To account for showed consistently larger effect estimates for maternal this, we first examined whether past cigarette smoking by smoking than for paternal smoking, although confidence the adult themselves influenced these associations by in- intervals overlapped for some of the sites [FRMD4A cluding own smoking as a covariate in the model. (cg25464840), CYP1A1 (cg05549655), ARL4C Adjustment for own smoking attenuated associations at (cg05204104), AHRR (cg17924476) and MYO1G five CpG sites [TIFAB (cg11429111), MDM4 (cg04180046)] (Supplementary Figure 2, available as (cg08241939), AX748264 (cg11641006), ARL4C Supplementary data at IJE online). For the comparison of (cg15016771) and FTO (cg26681628)] that no longer associations between the offspring of women who smoked reached the FDR cut-off for significance (Table 2 and during pregnancy and the offspring of women who smoked Figure 1), although, on the whole, the magnitude of effect outside of pregnancy, effect estimates were consistently was only slightly reduced with this adjustment. larger for those reporting maternal smoking during preg- Furthermore, whereas methylation of CpG sites at nancy, this time without overlapping confidence intervals MYO1G, CNTNAP2 and AHRR have been consistently (Supplementary Figure 3, available as Supplementary data identified in relation to own smoking, no such associations at IJE online). Whereas, for MYO1G (cg12803066) and have been found with CpG sites at FDRM4A, CYP1A1, MYO1G (cg22132766), there was some evidence of asso- ciation between maternal smoking outside of pregnancy with methylation levels, suggestive of either residual con- founding in the intrauterine associations or a postnatal smoking effect, these findings may also be explained by misreporting of maternal smoking during pregnancy that was based on retrospective reports by the offspring in adulthood. This latter explanation is more likely given that the paternal smoking estimates at these sites were consis- tent with the null (Supplementary Figure 2, available as Supplementary data at IJE online). Effects at the top CpGs surpassing FDR correction in the ALSPAC women were found to be consistent in direc- tion although slightly attenuated in the follow-up analysis approximately 18 years later (Pearson’s correlation coeffi- cient, r¼ 0.92) (Figure 2 and Supplementary Figure 4, available as Supplementary data at IJE online). Furthermore, there was remarkable consistency in the di- rection and magnitude of effects at these CpGs in blood samples of the ALSPAC men (r¼ 0.77), with the exception Figure 1. Manhattan plot for EWAS of prenatal smoke exposure in of DRD1, where effects were not as consistently replicated. ALSPAC women (Time Point 1)*. *N¼ 752, adjusted for own smoking. We also compared for reference the effect of any maternal Solid horizontal line represents Bonferroni threshold; dotted horizontal line represents FDR correction (p< 0.05) threshold. smoking on cord blood methylation in the ALSPAC birth Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 1126 International Journal of Epidemiology, 2018, Vol. 47, No. 4 Figure 2. Replication of CpG sites observed below FDR (p< 0.05) threshold in ALSPAC women at a later time point (Time Point 2) and in ALSPAC men,* and comparison with effect of prenatal smoking on cord blood methylation in ALSPAC children. *Adjusted for own smoking in adult samples. N¼ 860 ALSPAC cord blood (reference), N¼ 752 ALSPAC women (Time Point 1), N¼ 498 ALSPAC women (Time Point 2), N¼ 222 ALSPAC men. Figure 3. Receiver operating characteristic (ROC) curves of prenatal and own smoking methylation scores for discriminating maternal smoking in pregnancy. Total N¼ 922; NB: sample size in the main analysis is smaller due to inclusion of additional covariates into those models that were miss- ing for some of the participants. Prenatal smoking methylation score (older children)¼ score derived from 19 CpG sites associated with maternal smoking in older children in an independent study. Prenatal smoking methylation score (newborns)¼ score derived from 568 CpG sites associated with maternal smoking in newborns in an independent study. Own smoking methylation score¼ score derived from 2623 CpG sites associated with smoking status in adults in an independent study. Scores were applied to methylation data from ALSPAC adult females at Time Point 1. Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1127 cohort at these CpGs and, again, both magnitude and di- To determine the extent to which methylation associa- rection of effect were similar (r¼ 0.92) with the exception tions with prenatal smoking were different from own of sites at ARL4C (cg05204104), AHRR (cg17924476) smoking associations, we constructed a similar score de- and DRD4 (cg22807681). At these sites, the difference in rived from 2623 CpG sites previously associated with own methylation was greater in the ALSPAC women than in smoking in adulthood. Whereas being strongly predictive the newborns (Figure 2 and Supplementary Figure 4, avail- of own smoking status in the ALSPAC women (AUC 0.88, able as Supplementary data at IJE online). 95% CI 0.85, 0.91), this score was only weakly associated In addition, we found that, among women with a mean with prenatal smoking compared with the 19-CpG prena- age of 30 years (N¼ 754), there was a strong signal of as- tal smoking score (AUC 0.57, 95% CI 0.53, 0.61; P for dif- –11 sociation above that expected by chance at CpG sites previ- ference¼ 3.0 10 )(Figure 3). In addition, the 19-CpG ously associated with prenatal smoke exposure in prenatal smoking methylation score was able to predict newborns [lambda¼ 2.62 vs 1.14 for all CpG sites on the with the same accuracy prenatal smoke exposure when ad- Illumina Infinium HumanMethylation450 (HM450) justed for the offspring smoking methylation score (AUC –16 BeadChip; Wilcoxon rank sum test p-value< 2.2 10 ] 0.71, 95% CI 0.68, 0.75; P for difference¼ 0.06) (Supplementary Figure 5, available as Supplementary data (Figure 3). at IJE online). Similarly, inflation of signals for prenatal The prenatal smoking methylation scores were higher in smoke exposure was seen in these women 18 years later individuals (both smokers and non-smokers) exposed to –15 (lambda¼ 1.54, p-value¼ 5.6 10 ) and in the ALSPAC prenatal smoking compared with non-smokers who were –4 men (lambda¼ 1.19, p-value¼ 2.2 10 ), compared with not exposed prenatally (i.e. the ‘OS.MS’ and ‘ONS.MS’ all CpG sites on the HM450 BeadChip (Supplementary group vs the ‘ONS.MNS’ baseline group; p-value for –16 Figure 5, available as Supplementary data at IJE online). trend< 2.0 10 )(Figure 4). The prenatal smoking A prenatal smoking methylation score, derived by com- methylation score derived from 568 CpG sites identified in bining methylation values at 568 CpG sites associated with newborns was not able to distinguish between non- prenatal smoke exposure in cord blood of newborns in a smokers whose mothers smoked in pregnancy compared previous study, could predict whether the mothers of the with smokers whose mothers did not smoke during preg- ALSPAC women smoked during pregnancy with an AUC nancy (difference in score between the ‘ONS.MS’ group 0.69 [95% confidence interval (CI) 0.67, 0.73]. This was and the ‘OS.MNS’ group¼ –0.07, p¼ 0.61) (Figure 4a). comparable with a score derived from 19 CpG sites previ- However, the score derived from 19 CpG sites which were ously associated with prenatal smoking in peripheral blood shown to persist in relation to prenatal smoke exposure 3 3 of older children, which had an AUC 0.72 (95% CI 0.69, based on an EWAS in older children was higher in non- 0.76; P for difference¼ 0.97) (Figure 3). smokers whose mothers smoked in pregnancy compared Figure 4. Box plots to assess differences in prenatal smoking methylation scores. Prenatal smoking methylation score (newborns)¼ score derived from 568 CpG sites associated with maternal smoking in newborns in an independent study. Prenatal smoking methylation score (older children)¼ score derived from 19 CpG sites associated with maternal smoking in older children in an independent study. ONS, MNS¼ offspring non-smoker, mother never smoked in pregnancy (N¼ 522); OS, MNS¼ offspring smoker, mother never smoked in pregnancy (N¼ 112); ONS, MS¼ offspring non-smoker, mother smoked in pregnancy (N¼ 222); OS, MS¼ offspring smoker, mother smoked in pregnancy (N¼ 66). NB: sample size in the main analysis is smaller due to inclusion of additional covariates into those models that were missing for some of the participants. Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 1128 International Journal of Epidemiology, 2018, Vol. 47, No. 4 with smokers whose mothers did not smoke in pregnancy Strengths of our study include the large sample size of (difference in score between the ‘ONS.MS’ group and the women with reported maternal smoking in pregnancy for ‘OS.MNS’ group¼ 0.01, p¼ 0.001) (Figure 4b). performing our initial EWAS analysis, the ability to adjust for own smoking status, the longitudinal assessment of dif- ferential methylation in a follow-up sample of these Discussion women and the replication analysis in men from the same In a large longitudinal cohort with genome-wide methyla- study. tion data, we identified 15 CpG sites that were differen- Although there was evidence of persistence for methyla- tially methylated in the peripheral blood of women over tion differences even after adjusting for own smoking sta- 30 years after exposure to prenatal smoking. Most of these tus in the adult offspring, there are limitations to signals remained in sensitivity analyses adjusted for own performing this type of adjustment analysis. As smoking and passive smoke exposure, and showed stron- parental smoking is strongly associated with their off- ger associations in relation to maternal smoke exposure in spring’s smoking initiation, own smoking serves as a pos- pregnancy compared with smoke exposure outside of preg- sible mediator on the path between prenatal smoking and nancy (by both mothers and fathers) indicating specificity offspring DNA methylation. This method of adjusting for of the intrauterine effect. Furthermore, we observed a per- a potential mediator in standard regression models to esti- sistent methylation signal related to prenatal smoke expo- mate the direct effect of an exposure may produce spurious 34,35 sure in peripheral blood 18 years later (i.e. at around the conclusions. Whereas an alternative method of using age of 48 years) and replicated in peripheral blood among life-course models previously provided more evidence for men in the ALSPAC cohort. the hypothesis that maternal smoking in pregnancy is the Many of these CpG sites have been previously identified ‘critical period’ for influencing persistent offspring methyl- in relation to prenatal smoke exposure in the offspring at ation profiles, this method could not be applied here given birth and the majority showed long-term hypermethyla- the limited amount of information on maternal smoking tion among the offspring of smokers. Findings are also reported by the ALSPAC men and women. consistent with a recent report that highlighted persistence A further limitation relates to cell-type heterogeneity, of DNA methylation levels related to prenatal smoke expo- given that the ALSPAC samples were obtained from a vari- sure into adulthood, which identified associations the ety of sources [white cells, whole blood and peripheral same CpG sites located in MYO1G and CYP1A1, and blood lymphocytes (PBLs)]. To account for this, we incor- other CpG sites in FTO and AHRR. porated surrogate variables into our models to account to For all of the study samples, there was also a strong sig- adjust for technical batch and cell-type mixture in order to nal of association above that expected by chance at 568 harmonize cellular variability of the samples and carried CpG sites previously associated with prenatal smoke expo- out sensitivity analysis that also adjusted for derived cell 3 37,38 sure in newborns from an independent study. In addition, counts. we found that a prenatal smoking score, derived by com- In addition, information on prenatal smoke exposure in bining methylation values at these CpG sites, could ade- the ALSPAC men and women was recorded retrospectively quately predict whether the mothers of the adults in by the adult offspring, rather than by prospective assess- ALSPAC smoked during pregnancy with an AUC 0.69 ment, and so may be subject to more misreporting. (95% CI 0.67, 0.73). A recent study identified a much Furthermore, in the ALSPAC men and women, rates of stronger predictive ability of a prenatal smoking methyla- maternal smoking in pregnancy were reported to be high tion score with AUC of 0.90 in a test set of cord blood in comparison with contemporary populations. This draws obtained from newborns in the MoBa cohort. The differ- to question the relevance of identified associations. ence in predictive ability is therefore likely attributed to the However, we have shown that many of the signals identi- 30-year difference in time since exposure and the genera- fied in adults were also present in cord blood of offspring tion of this methylation score using CpG sites identified in measured prospectively in a more contemporary cohort cord blood rather than adult peripheral blood. We also de- with lower rates of maternal smoking in pregnancy. rived a methylation score based on CpGs that showed evi- Overall, the results of this study provide robust evidence dence of a persistent difference in methylation in that maternal smoking in pregnancy is associated with peripheral blood of older offspring exposed to prenatal changes in DNA methylation that persist in the exposed smoking who had a marginally higher AUC of 0.72 (95% offspring for many years after their prenatal exposure. CI 0.67, 0.73) and was also able to distinguish non- Furthermore, these associations largely remain after adjust- smokers whose mothers smoked in pregnancy from smok- ing for the previous smoking history of the adults them- ers whose mothers did not smoke during pregnancy. selves and are in accordance with earlier studies Downloaded from https://academic.oup.com/ije/article/47/4/1120/5026413 by DeepDyve user on 14 July 2022 International Journal of Epidemiology, 2018, Vol. 47, No. 4 1129 2. 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Journal

International Journal of Epidemiology – Oxford University Press

Published: Aug 1, 2018

Keywords: pregnancy; smoking; adult; dna methylation; methylation; smoke; prenatal care; alspac study; smoking in pregnancy; offspring; epigenome; prenatal exposure; mothers

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