Associations of Maternal Cigarette Smoking in Pregnancy with Early Smoking Initiation and Asthma Development in the Offspring, Testing the Intermediate Role of DNA Methylation and the Role of the Glutathione S-Transferase (GST) Gene Single Nucleotide Polymorphisms (SNPs)

Maternal cigarette smoking during pregnancy (MSP) is a global health concern, with approximately half of smoking women continuing this habit throughout pregnancy. Epidemiological studies link in-utero exposure to cigarette smoke to detrimental outcomes, including an increased risk of asthma and smoking initiation in the offspring. While epidemiological research has shown the negative impacts of MSP, the underlying mechanisms remain elusive. Epigenetic modifications, specifically DNA methylation (DNAm), have been suggested as a mediator of the association between MSP and adverse health outcomes in the offspring. MSP has been associated with alterations in offspring DNAm. There is, however, limited understanding of individual variations in susceptibility to MSP. This dissertation comprises three studies, each addressing a different aspect of exposure to MSP. In the first study, we investigated whether polymorphisms in Glutathione S-transferase (GST) genes, known for their involvement in protecting against oxidative stress from cigarette smoke, influence the impact of MSP on offspring DNAm. Using data from the Isle of Wight Birth Cohort, we focused on individual susceptibilities to MSP based on GST gene polymorphisms (rs506008, rs574344, rs12736389, rs3768490, rs1537234, and rs1695). We found evidence that polymorphisms in GST genes modify the association of MSP and offspring DNAm in males and females. In the second study, we examined the association between MSP and the initiation of smoking in offspring. Furthermore, we explored the potential mediating role of DNAm at birth in this association. Offspring exposed to MSP showed earlier age of smoking initiation. In males 10 CpGs (cytosines followed by guanine where DNAm primarily occurs) and in females eight CpGs were identified in association with persistent smoking initiation. Furthermore, we identified seven CpGs in males and eight CpGs in females in association with smoking initiation. We found no evidence for a mediating role of DNAm in male offspring. However, in female offspring, DNAm at cg16774292 (LOC101928398) was identified as a significant mediator in the association between MSP and the initiation of smoking. In the third study, we examined the association between MSP and the asthma status of offspring at ages 1, 4, 10, and 18 years with a focus on investigating the potential mediating role of DNAm in this association. Overall, we showed an increased odds of asthma in those exposed to MSP. In an epigenome-wide approach, 52 CpGs in males and 36 CpGs in females were found to be associated with asthma repeated measures. However, regarding DNAm, associations reached statistical significance only in female offspring. We showed that in females, DNAm levels at cg00308823 (LAIR1) partially mediated the association of exposure to MSP and asthma status. Based on our current data, we found no such mediating CpG in males. Hence, DNAm is suggested to mediate the effect of exposure to MSP on offspring asthma in female subjects. Given that a potentially mediating role of DNAm allows to modify the odds of developing asthma even after intra-uterine exposure by changing DNA methylation, there is a need to also identify such DNAm sites in males

DNA methylation at birth is associated with lung function development until age 26 years

Little is known about whether DNA methylation (DNAm) of cytosine-phosphate-guanine (CpG) sites at birth predicts patterns of lung function development. We used heel prick DNAm from the F1-generation of Isle of Wight birth cohort (IOWBC-F1) for discovery of CpGs associated with lung function trajectories (forced expiratory volume in 1 s, forced vital capacity, their ratio, and forced expiratory flow at 25-75% of forced vital capacity) over the first 26 years, stratified by sex. We replicated the findings in the Avon Longitudinal Study of Parents and Children (ALSPAC) using cord blood DNAm. Epigenome-wide screening was applied to identify CpGs associated with lung function trajectories in 396 boys and 390 girls of IOWBC-F1. Replication in ALSPAC focussed on lung function at ages 8, 15 and 24 years. Statistically significantly replicated CpGs were investigated for consistency in direction of association between cohorts, stability of DNAm over time in IOWBC-F1, relevant biological processes and for association with gene expression (n=161) in IOWBC F2-generation (IOWBC-F2). Differential DNAm of eight CpGs on genes GLUL, MYCN, HLX, LHX1, COBL, COL18A1, STRA6, and WNT11 involved in developmental processes, were significantly associated with lung function in the same direction in IOWBC-F1 and ALSPAC, and showed stable patterns at birth, aged 10 and 18 years between high and low lung function trajectories in IOWBC-F1. CpGs on LHX1 and COL18A1 were linked to gene expression in IOWBC-F2. In two large cohorts, novel DNAm at birth were associated with patterns of lung function in adolescence and early adulthood providing possible targets for preventative interventions against adverse pulmonary function development.</p

Association of Maternal DNA Methylation and Offspring Birthweight

This study aims to evaluate the association of maternal DNA methylation (DNAm) during pregnancy and offspring birthweight. One hundred twenty-two newborn-mother dyads from the Isle of Wight (IOW) cohort were studied to identify differentially methylated cytosine-phosphate-guanine sites (CpGs) in maternal blood associated with offspring birthweight. Peripheral blood samples were drawn from mothers at 22-38 weeks of pregnancy for epigenome-wide DNAm assessment using the Illumina Infinium HumanMethylation450K array. Candidate CpGs were identified using a course of 100 repetitions of a training and testing process with robust regressions. CpGs were considered informative if they showed statistical significance in at least 80% of training and testing samples. Linear mixed models adjusting for covariates were applied to further assess the selected CpGs. The Swedish Born Into Life cohort was used to replicate our findings (n = 33). Eight candidate CpGs corresponding to the genesLMF1,KIF9,KLHL18,DAB1,VAX2,CD207,SCT,SCYL2,DEPDC4,NECAP1, andSFRS3in mothers were identified as statistically significantly associated with their children's birthweight in the IOW cohort and confirmed by linear mixed models after adjusting for covariates. Of these, in the replication cohort, three CpGs (cg01816814, cg23153661, and cg17722033 withpvalues = 0.06, 0.175, and 0.166, respectively) associated with four genes (LMF1,VAX2,CD207, andNECAP1) were marginally significant. Biological pathway analyses of three of the genes revealed cellular processes such as endocytosis (possibly sustaining an adequate maternal-fetal interface) and metabolic processes such as regulation of lipoprotein lipase activity (involved in providing substrates for the developing fetus). Our results contribute to an epigenetic understanding of maternal involvement in offspring birthweight. Measuring DNAm levels of maternal CpGs may in the future serve as a diagnostic tool recognizing mothers at risk for pregnancies ending with altered birthweights.Peer reviewe

Association of asthma and rhinitis with epigenetics of coronavirus related genes

Introduction: susceptibility factors for coronavirus disease 2019 (COVID-19) include sex and medical conditions such as asthma and rhinitis. DNA methylation (DNAm) is associated with asthma, rhinitis, and several viruses. We examined associations of asthma/rhinitis with DNAm at CpGs located on coronavirus related genes, and if these associations were sex-specific.Methods: in total, n = 242 subjects aged 26 years from the Isle of Wight Birth Cohort were included in the study. Linear regressions were used to examine sex specific and non-specific associations of DNAm at CpGs on coronavirus related genes with asthma/rhinitis status. Associations of DNAm with gene expression in blood were assessed for functional relevance of identified CpGs.Results: statistically significant interaction effects of asthma or rhinitis with sex were identified at 40 CpGs for asthma and 27 CpGs for rhinitis. At 21 CpGs, DNAm was associated with asthma, and at 45 CpGs with rhinitis, regardless of sex. Assessment of functional relevance of the identified CpGs indicated a potential of epigenetic regulatory functionality on gene activity at 14 CpGs for asthma and 17 CpGs for rhinitis, and of those 6 CpGs for asthma and 7 CpGs for rhinitis were likely to be sex-specific.Conclusion: subjects with asthma/rhinitis may have altered susceptibility to COVID-19 due to changes in their DNAm associated with these conditions. Sex specificity on association of asthma/rhinitis with DNAm at certain CpGs, and on the association of DNAm at asthma/rhinitis-linked CpGs with gene expression have the potential to explain the reported sex-specificity in COVID-19 morbidity and mortality.</p

Methylation of host genes associated with coronavirus infection from birth of 26 years

DNA methylation (DNAm) patterns over time at 1146 CpGs on coronavirus-related genes were assessed to understand whether the varying differences in susceptibility, symptoms, and the outcomes of the SARS-CoV-2 infection in children and young adults could be explained through epigenetic alterations in a host cell’s transcriptional apparatus to coronaviruses. DNAm data from the Isle of Wight birth cohort (IOWBC) at birth, 10, 18, and 26 years of age were included. Linear mixed models with repeated measurements stratified by sex were used to examine temporal patterns, and cluster analysis was performed to identify CpGs following similar patterns. CpGs on autosomes and sex chromosomes were analyzed separately. The association of identified CpGs and expression of their genes were evaluated. Pathway enrichment analyses of the genes was conducted at FDR = 0.05. DNAm at 635 of the 1146 CpGs on autosomes showed statistically significant time effects (FDR = 0.05). The 635 CpGs were classified into five clusters with each representing a unique temporal pattern of DNAm. Of the 29 CpGs on sex chromosomes, DNAm at seven CpGs in males and eight CpGs in females showed time effects (FDR = 0.05). Sex-specific and non-specific associations of DNAm with gene expression were found at 24 and 93 CpGs, respectively. Genes which mapped the 643 CpGs represent 460 biological processes. We suggest that the observed variability in DNAm with advancing age may partially explain differing susceptibility, disease severity, and mortality of coronavirus infections among different age groups

Nicotine and Its downstream metabolites in maternal and cord sera: biomarkers of prenatal smoking exposure associated with offspring DNA methylation

Nicotine is a major constituent of cigarette smoke. Its primary metabolite in maternal and cord sera, cotinine, is considered a biomarker of prenatal smoking. Nicotine and cotinine half-lives are decreased in pregnancy due to their increased rate of metabolism and conversion to downstream metabolites such as norcotinine and 3-hydroxycotinine. Hence, downstream metabolites of nicotine may provide informative biomarkers of prenatal smoking. In this study of three generations (F0-mothers, F1-offspring who became mothers, and F2-offspring), we present a biochemical assessment of prenatal smoking exposure based on maternal and cord sera levels of nicotine, cotinine, norcotinine, and 3-hydroxycotinine. As potential markers of early effects of prenatal smoking, associations with differential DNA methylation (DNAm) in the F1- and F2-offspring were assessed. All metabolites in maternal and cord sera were associated with self-reported prenatal smoking, except for nicotine. We compared maternal self-report of smoking in pregnancy to biochemical evidence of prenatal smoking exposure. Self-report of F0-mothers of F1 in 1989–1990 had more accuracy identifying prenatal smoking related to maternal metabolites in maternal serum (sensitivity = 94.6%, specificity = 86.9%) compared to self-reports of F1-mothers of F2 (2010–2016) associated with cord serum markers (sensitivity = 66.7%, specificity = 78.8%). Nicotine levels in sera showed no significant association with any DNAm site previously linked to maternal smoking. Its downstream metabolites, however, were associated with DNAm sites located on the MYO1G, AHRR, and GFI1 genes. In conclusion, cotinine, norcotinine, and 3-hydroxycotinine in maternal and cord sera provide informative biomarkers and should be considered when assessing prenatal smoking. The observed association of offspring DNAm with metabolites, except for nicotine, may imply that the toxic effects of prenatal nicotine exposure are exerted by downstream metabolites, rather than nicotine. If differential DNA methylation on the MYO1G, AHRR, and GFI1 genes transmit adverse effects of prenatal nicotine exposure to the child, there is a need to investigate whether preventing changes in DNA methylation by reducing the metabolic rate of nicotine and conversion to harmful metabolites may protect exposed children

Polymorphisms in Glutathione S-Transferase (<i>GST</i>) Genes Modify the Effect of Exposure to Maternal Smoking Metabolites in Pregnancy and Offspring DNA Methylation

Maternal smoking in pregnancy (MSP) affects the offspring’s DNA methylation (DNAm). There is a lack of knowledge regarding individual differences in susceptibility to exposure to MSP. Glutathione S-transferase (GST) genes are involved in protection against harmful oxidants such as those found in cigarette smoke. This study aimed to test whether polymorphisms in GST genes influence the effect of MSP on offspring DNAm. Using data from the Isle of Wight birth cohort, we assessed the association of MSP and offspring DNAm in 493 mother-child dyads (251 male, 242 female) with the effect-modifying role of GST gene polymorphism (at rs506008, rs574344, rs12736389, rs3768490, rs1537234, and rs1695). MSP was assessed by levels of nicotine and its downstream metabolites (cotinine, norcotinine, and hydroxycotinine) in maternal sera. In males, associations of hydroxycotinine with DNAm at cg18473733, cg25949550, cg11647108, and cg01952185 and norcotinine with DNAm at cg09935388 were modified by GST gene polymorphisms (p-values GST gene polymorphisms (p-values GST genes in DNAm’s susceptibility to MSP

Assessing the effect of childbearing on blood DNA methylation through comparison of parous and nulliparous females

Abstract Background Pregnancy and childbirth have been connected to modified risk of a wide variety of conditions in later life, including neurodegenerative disorders and cancers. The presence, extent, and direction of the effect that childbearing status has on decreasing or increasing the risk of these conditions differs depending on the disease. The mechanisms by which pregnancy and childbirth modify the risk of diseases are still unknown. DNA methylation (DNAm) alterations that occur during pregnancy and persist after childbirth may help us understand this phenomenon. Results Blood DNAm was available from 89 women (28 parous; 61 nulliparous) at ages 18 and 26 years in the Isle of Wight birth cohort; no significant differences in the population characteristics were present between the analyzed population and the full cohort. We performed an epigenome-wide association study on 389,355 CpGs and identified 184 CpGs to be significantly differentially methylated between parous and nulliparous women after adjusting for confounders and multiple testing. Of these CpGs, 105 had regression coefficients in the same direction in an independent Mexico City based ELEMENT cohort, of which 13 were significant (replication P < 0.05). These 13 CpGs were associated with 16 unique genes. DNAm levels tracked with gene expression in 3 of the replicated genes, one of which (TM2D3) was differentially expressed in parous vs nulliparous women. Gene disease association analysis identified a network of parous-associated diseases. Conclusions Our results suggest that pregnancy and childbirth lead to DNAm changes in parous women and these changes persist at least 6 months and up to 8 years postpartum. Parous-related CpG sites may play a role in how childbearing status modifies risk of later life diseases in women. Further studies are needed to explore the linkage and mechanism

Association of prenatal acetaminophen use and acetaminophen metabolites with DNA methylation of newborns: analysis of two consecutive generations of the Isle of Wight birth cohort

Acetaminophen is used by nearly two-thirds of pregnant women. Although considered safe, studies have demonstrated associations between prenatal acetaminophen use and adverse health outcomes in offspring. Since DNA methylation (DNAm) at birth may act as an early indicator of later health, assessments on whether DNAm of newborns is associated with gestational acetaminophen use or its metabolites are needed. Using data from three consecutive generations of the Isle of Wight cohort (F0-grandmothers, F1-mothers, and F2-offspring) we investigated associations between acetaminophen metabolites in F0 serum at delivery with epigenome-wide DNAm in F1 (Guthrie cards) and between acetaminophen use of F1 and F2-cord-serum levels with F2 cord blood DNAm. In epigenome-wide screening, we eliminated non-informative DNAm sites followed by linear regression of informative sites. Based on repeated pregnancies, indication bias analyses tested whether acetaminophen indicated maternal diseases or has a risk in its own right. Considering that individuals with similar intake process acetaminophen differently, metabolites were clustered to distinguish metabolic exposures. Finally, metabolite clusters from F1-maternal and F2-cord sera were tested for their associations with newborn DNAm (F1 and F2). Twenty-one differential DNAm sites in cord blood were associated with reported maternal acetaminophen intake in the F2 generation. For 11 of these cytosine-phosphate-guanine (CpG) sites, an indication bias was excluded and five were replicated in F2 with metabolite clusters. In addition, metabolite clusters showed associations with 25 CpGs in the F0-F1 discovery analysis, of which five CpGs were replicated in the F2-generation. Our results suggest that prenatal acetaminophen use, measured as metabolites, may influence DNAm in newborns

Association of Maternal DNA Methylation and Offspring Birthweight

This study aims to evaluate the association of maternal DNA methylation (DNAm) during pregnancy and offspring birthweight. One hundred twenty-two newborn-mother dyads from the Isle of Wight (IOW) cohort were studied to identify differentially methylated cytosine-phosphate-guanine sites (CpGs) in maternal blood associated with offspring birthweight. Peripheral blood samples were drawn from mothers at 22-38 weeks of pregnancy for epigenome-wide DNAm assessment using the Illumina Infinium HumanMethylation450K array. Candidate CpGs were identified using a course of 100 repetitions of a training and testing process with robust regressions. CpGs were considered informative if they showed statistical significance in at least 80% of training and testing samples. Linear mixed models adjusting for covariates were applied to further assess the selected CpGs. The Swedish Born Into Life cohort was used to replicate our findings (n = 33). Eight candidate CpGs corresponding to the genesLMF1,KIF9,KLHL18,DAB1,VAX2,CD207,SCT,SCYL2,DEPDC4,NECAP1, andSFRS3in mothers were identified as statistically significantly associated with their children's birthweight in the IOW cohort and confirmed by linear mixed models after adjusting for covariates. Of these, in the replication cohort, three CpGs (cg01816814, cg23153661, and cg17722033 withpvalues = 0.06, 0.175, and 0.166, respectively) associated with four genes (LMF1,VAX2,CD207, andNECAP1) were marginally significant. Biological pathway analyses of three of the genes revealed cellular processes such as endocytosis (possibly sustaining an adequate maternal-fetal interface) and metabolic processes such as regulation of lipoprotein lipase activity (involved in providing substrates for the developing fetus). Our results contribute to an epigenetic understanding of maternal involvement in offspring birthweight. Measuring DNAm levels of maternal CpGs may in the future serve as a diagnostic tool recognizing mothers at risk for pregnancies ending with altered birthweights.Peer reviewe