Development and Validation of a Novel Placental DNA Methylation Biomarker of Maternal Smoking during Pregnancy in the ECHO Program

Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function (VCSIP): Rationale, design, and methods of a randomized, controlled trial of vitamin C supplementation in pregnancy for the primary prevention of effects of in utero tobacco smoke exposure on infant lung function and respiratory health

The field of DNA methylation research is rapidly evolving, focusing on disease and phenotype changes over time using methylation measurements from diverse tissue sources and multiple array platforms. Consequently, identifying the extent of longitudinal, inter-tissue, and inter-platform variation in DNA methylation is crucial for future advancement. DNA methylation was measured in 375 individuals, with 197 of those having 2 blood sample measurements ~10 years apart. Whole-blood samples were measured on Illumina Infinium 450K and EPIC methylation arrays, and buccal samples from a subset of 58 participants were measured on EPIC array. The data were analyzed with the aims to examine the correlation between methylation levels in longitudinal blood samples in 197 individuals, examine the correlation between methylation levels in the blood and buccal samples in 58 individuals, and examine the correlation between blood methylation profiles assessed on the EPIC and 450K arrays in 83 individuals. We identified 136,833, 7674, and 96,891 CpGs significantly and strongly correlated (>0.50) longitudinally, across blood and buccal samples as well as array platforms, respectively. A total of 3674 of these CpGs were shared across all three sets. Analysis of these shared CpGs identified previously found associations with aging, ancestry, and 7016 mQTLs as well.

DNA methylation analysis is used to identify novel genetic loci associated with circulating fibrinogen levels in blood

Exposure to particulate air pollution during early pregnancy is associated with placental DNA methylation

Gene-specific placental DNA methylation patterns differ between normal pregnancies and pregnancies complicated by hypertension. However, whether global placental DNA methylation is associated with maternal blood pressure remains controversial. Using multiple linear regression models, we analysed the association between maternal mean arterial pressure (MAP) at the third trimester of pregnancy and global DNA methylation in the placenta in 922 mothers using LC-ESI-MS/MS. To better characterize the contribution of genetic or epigenetic mechanisms, we performed isolated analyses in mothers with and without a family history of hypertension. Mean placental global DNA methylation was 3.00 ± 0.46%. A significant negative correlation between placental global DNA methylation and mean arterial blood pressure (MAP) in the third trimester could be observed (P = 0.023, r = -0.075). This association remained significant after adjusting for confounders. In placenta samples from mothers with a family history of hypertension, mean maternal MAP was higher (86.1 ± 8.1 vs. 84.6 ± 7.5, P < 0.01) and placental global DNA methylation was lower (2.94 ± 0.43 vs. 3.04 ± 0.47, P < 0.01) compared with samples without a family history of hypertension. Furthermore, the significant independent negative correlation between global placental DNA methylation and MAP was only found in mothers without a family history of hypertension. This study showed an independent negative correlation between placental global DNA methylation and maternal MAP in mothers without a family history of hypertension.

Modelling of the public health effects of smoking in pregnancy in Wales

Placental DNA methylation varies across gestation and is associated with obstetrical complications. Cell-free DNA (cfDNA) from maternal plasma could provide a noninvasive approach to study placental DNA methylation in ongoing pregnancies. However, research on maternal cfDNA methylation is limited and technologically challenging. Therefore, we aimed to investigate DNA methylation in maternal cfDNA and placental tissues across gestation using the innovative methylation DNA sequencing (MeD-seq) technology. Secondly, we explored the origins of methylation differences in maternal cfDNA across gestation, and aimed to identify gestational age-associated placental DNA methylation markers directly in cfDNA. We longitudinally collected maternal cfDNA in all three trimesters and at birth (n = 10), alongside placental tissues from first trimester, second trimester, and term pregnancies (all n = 10), and used previously collected maternal blood buffy coat samples (n = 20). Different placental cell types, including syncytiotrophoblasts/cytotrophoblasts (SCTs/CTBs) (n = 10), extravillous trophoblasts (n = 7), and syncytial knotting (n = 3), and maternal cell types including spiral arteries (n = 3) and endometrial epithelium (n = 3), were isolated using laser capture microdissection. Differentially methylated regions (DMRs) identified in cfDNA from pregnant compared to non-pregnant women (n = 6) ranged from 798 to 2163 in first and third trimesters, respectively. Gradual DNA methylation changes were observed across gestation in cfDNA, placental tissues, and trophoblasts. We showed an increase in DMRs in cfDNA, that overlap with DNA methylation in placental tissues and especially trophoblasts, and in DNA methylation of placenta-specific markers across gestation, reflecting an increased placental-originated cfDNA fraction. Among 110 DMRs between first trimester and term placental tissues, those related to NXPH4, EPS8L2, AMOTL1, and IRX2 had the strongest association with gestational age in cfDNA, for which comparable associations were found in SCTs/CTBs. These DMRs were all hypomethylated in maternal buffy coat samples. This study indicates the feasibility of identifying gestational age-dependent placental DNA methylation marks in maternal cfDNA and can serve as a reference for future studies.

BackgroundThe capacity of technologies measuring DNA methylation (DNAm) is rapidly evolving, as are the options for applicable bioinformatics methods. The most commonly used DNAm microarray, the Illumina Infinium HumanMethylation450 (450K array), has recently been replaced by the Illumina Infinium HumanMethylationEPIC (EPIC array), nearly doubling the number of targeted CpG sites. Given that a subset of 450K CpG sites is absent on the EPIC array and that several tools for both data normalization and analyses were developed on the 450K array, it is important to assess their utility when applied to EPIC array data. One of the most commonly used 450K tools is the pan-tissue epigenetic clock, a multivariate predictor of biological age based on DNAm at 353 CpG sites. Of these CpGs, 19 are missing from the EPIC array, thus raising the question of whether EPIC data can be used to accurately estimate DNAm age. We also investigated a 71-CpG epigenetic age predictor, referred to as the Hannum method, which lacks 6 probes on the EPIC array. To evaluate these epigenetic clocks in EPIC data properly, a prior assessment of the effects of data preprocessing methods on DNAm age is also required.MethodsDNAm was quantified, on both the 450K and EPIC platforms, from human primary monocytes derived from 172 individuals. We calculated DNAm age from raw, and three different preprocessed data forms to assess the effects of different processing methods on the DNAm age estimate. Using an additional cohort, we also investigated DNAm age of peripheral blood mononuclear cells, bronchoalveolar lavage, and bronchial brushing samples using the EPIC array.ResultsUsing monocyte-derived data from subjects on both the 450K and EPIC, we found that DNAm age was highly correlated across both raw and preprocessing methods (r > 0.91). Thus, the correlation between chronological age and the DNAm age estimate is largely unaffected by platform differences and normalization methods. However, we found that the choice of normalization method and measurement platform can lead to a systematic offset in the age estimate which in turn leads to an increase in the median error. Comparing the 450K and EPIC DNAm age estimates, we observed that the median absolute difference was 1.44–3.10 years across preprocessing methods.ConclusionsHere, we have provided evidence that the epigenetic clock is resistant to the lack of 19 CpG sites missing from the EPIC array as well as highlighted the importance of considering the technical variance of the epigenetic when interpreting group differences below the reported error. Furthermore, our study highlights the utility of epigenetic age acceleration measure, the residuals from a linear regression of DNAm age on chronological age, as the resulting values are robust with respect to normalization methods and measurement platforms.

Collection of RNA for analysis of noncoding RNA, protein for histone modifications and plasma for exosomes/ extracellular vesicles is the next frontier of understanding how the environment affects gene regulation.

BackgroundWe previously reported improved respiratory outcomes in babies born to pregnant smokers supplemented with vitamin C (500 mg/day) versus placebo in a randomized clinical trial. Improved respiratory outcomes persisted to 5 years of age and were associated with buccal DNA methylation (DNAm) measured using the InfiniumMethylationEPIC array. The objective of this study was to examine associations of vitamin C treatment and lung function with buccal DNAm using a custom-content Asthma&Allergy array enriched for asthma and allergy loci likely to have a functional impact on gene expression.ResultsWe profiled DNAm at 36,999 CpGs in loci previously associated with asthma or allergic diseases using custom-content Asthma&Allergy arrays in 137 subjects (65 placebo; 72 vitamin C) with pulmonary function testing (PFT) at the 5-year visit in the “Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function” (VCSIP) double-blind, placebo-controlled randomized clinical trial. We examined the association of buccal DNAm with (1) vitamin C treatment vs placebo, (2) forced expiratory flow between 25 and 75% of expired volume (FEF25-75) and (3) wheeze at 4–6 years of age. We identified 9 genome-wide differentially methylated CpGs (DMCs; FDR < 0.05) and 2 differentially methylated regions (DMRs) between vitamin C and placebo subjects and one CpG associated with FEF25-75 at FDR significance. DNAm at 5 CpGs mediated a significant proportion of the vitamin C treatment effect on lung function, including 2 CpGs annotated to the SLC25A37 gene involved in mitochondrial iron transport.ConclusionsOur study revealed association of in utero vitamin C supplementation and childhood lung function with DNAm at novel loci, providing additional insight toward potential mechanisms for the persistent effects of vitamin C supplementation to pregnant smokers.Clinical trial registration ClinicalTrials.gov, NCT01723696 (Registered on November 6, 2011) and NCT03203603 (Registered on March 28, 2017).Supplementary InformationThe online version contains supplementary material available at 10.1186/s13148-025-01965-2.

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; Maternal smoking in pregnancy is associated with adverse health outcomes in children, including cancers; underlying mechanisms may include epigenetic modifications. Using Illumina's 450K array, we previously identified differential DNA methylation related to maternal smoking during pregnancy at 26 CpG sites (CpGs) in 10 genes in newborn cord bloods from the Norwegian Mother and Child Cohort Study (MoBa). Whether these methylation signals in newborns reflect &lt;i&gt;in utero&lt;/i&gt; exposure only or possibly epigenetic inheritance of smoking-related modifications is unclear.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Methods:&lt;/b&gt; We therefore evaluated the impact of the timing of mother's smoking (before or during pregnancy using cotinine measured at 18 weeks gestation), the father's smoking before conception, and the grandmother's smoking during her pregnancy with the mother on methylation at these 26 CpGs in 1,042 MoBa newborns. We used robust linear regression, adjusting for covariates, applying Bonferroni correction.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; The strongest and only statistically significant associations were observed for sustained smoking by the mother during pregnancy through at least gestational week 18 (&lt;i&gt;P&lt;/i&gt; &lt; 1.6 × 10&lt;sup&gt;−5&lt;/sup&gt; for all 26 CpGs). We observed no statistically significant differential methylation due to smoking by the mother before pregnancy or that ceased by week 18, father's smoking before conception, or grandmother's smoking while pregnant with the mother.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Differential methylation at these CpGs in newborns seems to reflect sustained &lt;i&gt;in utero&lt;/i&gt; exposure rather than epigenetic inheritance.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Impact:&lt;/b&gt; Smoking cessation in early pregnancy may negate effects on methylation. Analyses of maternal smoking during pregnancy and offspring health outcomes, including cancer, limited to ever smoking might miss true associations. &lt;i&gt;Cancer Epidemiol Biomarkers Prev; 23(6); 1007–17. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; Maternal smoking in pregnancy is associated with adverse health outcomes in children, including cancers; underlying mechanisms may include epigenetic modifications. Using Illumina's 450K array, we previously identified differential DNA methylation related to maternal smoking during pregnancy at 26 CpG sites (CpGs) in 10 genes in newborn cord bloods from the Norwegian Mother and Child Cohort Study (MoBa). Whether these methylation signals in newborns reflect &lt;i&gt;in utero&lt;/i&gt; exposure only or possibly epigenetic inheritance of smoking-related modifications is unclear.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Methods:&lt;/b&gt; We therefore evaluated the impact of the timing of mother's smoking (before or during pregnancy using cotinine measured at 18 weeks gestation), the father's smoking before conception, and the grandmother's smoking during her pregnancy with the mother on methylation at these 26 CpGs in 1,042 MoBa newborns. We used robust linear regression, adjusting for covariates, applying Bonferroni correction.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; The strongest and only statistically significant associations were observed for sustained smoking by the mother during pregnancy through at least gestational week 18 (&lt;i&gt;P&lt;/i&gt; &lt; 1.6 × 10&lt;sup&gt;−5&lt;/sup&gt; for all 26 CpGs). We observed no statistically significant differential methylation due to smoking by the mother before pregnancy or that ceased by week 18, father's smoking before conception, or grandmother's smoking while pregnant with the mother.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Differential methylation at these CpGs in newborns seems to reflect sustained &lt;i&gt;in utero&lt;/i&gt; exposure rather than epigenetic inheritance.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Impact:&lt;/b&gt; Smoking cessation in early pregnancy may negate effects on methylation. Analyses of maternal smoking during pregnancy and offspring health outcomes, including cancer, limited to ever smoking might miss true associations. &lt;i&gt;Cancer Epidemiol Biomarkers Prev; 23(6); 1007–17. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Maternal smoking in pregnancy is associated with adverse health outcomes in children, including cancers; underlying mechanisms may include epigenetic modifications. Using Illumina's 450K array, we previously identified differential DNA methylation related to maternal smoking during pregnancy at 26 CpG sites (CpGs) in 10 genes in newborn cord bloods from the Norwegian Mother and Child Cohort Study (MoBa). Whether these methylation signals in newborns reflect in utero exposure only or possibly epigenetic inheritance of smoking-related modifications is unclear. We therefore evaluated the impact of the timing of mother's smoking (before or during pregnancy using cotinine measured at 18 weeks gestation), the father's smoking before conception, and the grandmother's smoking during her pregnancy with the mother on methylation at these 26 CpGs in 1,042 MoBa newborns. We used robust linear regression, adjusting for covariates, applying Bonferroni correction. The strongest and only statistically significant associations were observed for sustained smoking by the mother during pregnancy through at least gestational week 18 (P < 1.6 × 10(-5) for all 26 CpGs). We observed no statistically significant differential methylation due to smoking by the mother before pregnancy or that ceased by week 18, father's smoking before conception, or grandmother's smoking while pregnant with the mother. Differential methylation at these CpGs in newborns seems to reflect sustained in utero exposure rather than epigenetic inheritance. Smoking cessation in early pregnancy may negate effects on methylation. Analyses of maternal smoking during pregnancy and offspring health outcomes, including cancer, limited to ever smoking might miss true associations. Cancer Epidemiol Biomarkers Prev; 23(6); 1007-17. ©2014 AACR.

ABSTRACTStudies have linked maternal pre-pregnancy obesity and hyperglycaemia with metabolic and neurodevelopmental complications in childhood. DNA methylation (DNAm) might enable foetal adaptations to environmental adversities through important gene loci. NEGR1 is involved in both energy balance and behaviour regulation. The aim of this study was to investigate associations between placental DNAm at the NEGR1 gene locus and childhood anthropometric and neurodevelopmental profiles in preschoolers. We analysed 276 mother-child dyads from Gen3G, a prospective birth cohort from Sherbrooke. At 3yo (40.4 ± 3.0 months), we measured body mass index (BMI) and the mothers reported on offspring neurobehavior using the Strengths and Difficulties Questionnaire (SDQ). We quantified DNAm levels at 30 CpGs at the NEGR1 locus using the MethylationEPIC Array in placental biopsies. DNAm at four CpGs located before NEGR1 second exon predicted child’s BMI z-score (cg26153364: β=−0.16 ± 0.04; p=0.008, cg23166710: β=0.14 ± 0.08; p=0.03) and SDQ total score (cg04932878: β=0.22 ± 1.0; p= 3.0x10−4, cg16525738: β=−0.14 ± 0.18; p=0.01, cg23166710: β=−0.13 ± 0.36; p= 0.04), explaining 4.2% (p=0.003) and 7.3% (p= 1.3 x 10−4) of BMI-z and SDQ variances. cg23166710 was associated with both childhood phenotypes and correlated with NEGR1 placental expression (r=−0.22, p=0.04), suggesting its possible functional role. Together, maternal metabolic characteristics during pregnancy with NEGR1 DNAm levels explained 7.4% (p=4.2 x 10−4) of BMI-z and 14.2% (p=2.8 x 10−7) of SDQ variance at 3yo. This longitudinal study suggests that placental NEGR1 DNAm is associated with adiposity and neurodevelopment in preschool children and highlights its potential role in their comorbidity.

ABSTRACTIllumina HumanMethylation450 BeadChip (450K) has been commonly used to investigate DNA methylation in human tissues. Recently, it has been replaced by Illumina HumanMethylationEPIC BeadChip (EPIC) covering over 850,000 CpGs distributed genome-wide. Many consortia have now datasets coming from both arrays and aspire to analyze the two together. The placenta shows a high number of intermediate methylation levels and is often investigated for obstetric/birth outcomes, and potentially for long-term programming in offspring. We performed a systematic comparison between the two arrays using 108 duplicate placental samples from Gen3G birth cohort. We find that placenta shows a high per-sample correlation between the arrays, and higher median correlations at individual CpGs than those reported for blood. We identify 26,340 probes with absolute difference in per cent methylation >10%. We conclude that EPIC and 450K placental data can be combined, and we provide two lists of CpGs that should be excluded to avoid misleading results.