Maternal blood metal concentrations and whole blood DNA methylation during pregnancy in the Early Autism Risk Longitudinal Investigation (EARLI)

Background: During pregnancy, the maternal epigenome may be responsive to environmental exposures. We tested whether maternal exposure to cadmium (Cd) results in differential maternal whole blood DNA methylation (DNAm) in early pregnancy.Methods: Maternal blood samples were collected at the initial study visit (during trimesters one or two of pregnancy) from 232 participants in the Early Autism Risk Longitudinal Investigation (EARLI) pregnancy cohort. We measured maternal blood Cd (n=215) using inductively coupled plasma mass spectrometry, which reflects recent exposure (blood t1/2 ~75 days), and measured maternal blood DNAm (n=201) on the Illumina 450K array; 93 non-smoking women had both measures available for analysis. Linear regression was used to test for site-specific associations between blood Cd and DNAm, adjusting for cell type composition and confounding variables.Results: The distribution of blood Cd was right skewed and log-transformed for statistical analyses. The geometric mean of blood Cd was 0.2 μg/L (Interquartile range = 0.13 μg/L). An interquartile range difference in blood Cd was associated with a 13.1% increase in B-cell proportions (95% CI: 1.0 – 25.2). In multivariable regression, six CpG sites were associated (p-value<10-5) with log blood Cd concentrations. At five of these sites, increasing blood Cd was associated with hypermethylation, and three corresponded to the genes LYN, TESC, and ESD. The CpG site near LYN was closest to genome-wide significance (p-value = 1.9x10-6), and an interquartile range difference in blood Cd was associated with an 8.4% increase in percent methylation at this site (95% CI: 5.1 – 11.6).Conclusion: We report site-specific associations between DNAm and blood Cd in early pregnancy. Future work will consider the persistence of DNAm marks. Identified sites may be potential biomarkers of Cd exposure that can inform future epidemiological studies or implicate downstream gene pathways affected by Cd exposure.

BackgroundPrenatal vitamin use is recommended before and during pregnancies for normal fetal development. Prenatal vitamins do not have a standard formulation, but many contain calcium, folic acid, iodine, iron, omega-3 fatty acids, zinc, and vitamins A, B6, B12, and D, and usually they contain higher concentrations of folic acid and iron than regular multivitamins in the US Nutrient levels can impact epigenetic factors such as DNA methylation, but relationships between maternal prenatal vitamin use and DNA methylation have been relatively understudied. We examined use of prenatal vitamins in the first month of pregnancy in relation to cord blood and placenta DNA methylation in two prospective pregnancy cohorts: the Early Autism Risk Longitudinal Investigation (EARLI) and Markers of Autism Risk Learning Early Signs (MARBLES) studies.ResultsIn placenta, prenatal vitamin intake was marginally associated with −0.52% (95% CI −1.04, 0.01) lower mean array-wide DNA methylation in EARLI, and associated with −0.60% (−1.08, −0.13) lower mean array-wide DNA methylation in MARBLES. There was little consistency in the associations between prenatal vitamin intake and single DNA methylation site effect estimates across cohorts and tissues, with only a few overlapping sites with correlated effect estimates. However, the single DNA methylation sites with p-value < 0.01 (EARLI cord nCpGs = 4068, EARLI placenta nCpGs = 3647, MARBLES cord nCpGs = 4068, MARBLES placenta nCpGs = 9563) were consistently enriched in neuronal developmental pathways.ConclusionsTogether, our findings suggest that prenatal vitamin intake in the first month of pregnancy may be related to lower placental global DNA methylation and related to DNA methylation in brain-related pathways in both placenta and cord blood.

Background: Pregnancy measures of DNA methylation, an epigenetic mark, may be associated with autism spectrum disorder (ASD) development in children. Few ASD studies have considered prospective designs with DNA methylation measured in multiple tissues and tested overlap with ASD genetic risk loci. Objectives: To estimate associations between DNA methylation in maternal blood, cord blood, and placenta and later diagnosis of ASD, and to evaluate enrichment of ASD-associated DNA methylation for known ASD-associated genes. Methods: In the Early Autism Risk Longitudinal Investigation (EARLI), an ASD-enriched risk birth cohort, genome-scale maternal blood (early n=140 and late n=75 pregnancy), infant cord blood (n=133), and placenta (maternal n=106 and fetal n=107 compartments) DNA methylation was assessed on the Illumina 450k HumanMethylation array and compared to ASD diagnosis at 36 months of age. Differences in site-specific and global methylation were tested with ASD, as well as enrichment of single site associations for ASD risk genes (n=881) from the Simons Foundation Autism Research Initiative (SFARI) database. Results: No individual DNA methylation site was associated with ASD at genome-wide significance, however individual DNA methylation sites nominally associated with ASD (P<0.05) in each tissue were highly enriched for SFARI genes (cord blood P = 7.9x10-29, maternal blood early pregnancy P = 6.1x10-27, maternal blood late pregnancy P = 2.8x10-16, maternal placenta P= 5.6x10-15, fetal placenta P = 1.3x10-20). DNA methylation sites nominally associated with ASD across all five tissues overlapped at 144 (29.5%) SFARI genes. Conclusion: DNA methylation sites nominally associated with later ASD diagnosis in multiple tissues were enriched for ASD risk genes. Our multi-tissue study demonstrates the utility of examining DNA methylation prior to ASD diagnosis.

Background: Pregnancy measures of DNA methylation, an epigenetic mark, may be associated with autism spectrum disorder (ASD) development in children. Few ASD studies have considered prospective designs with DNA methylation measured in multiple tissues and tested overlap with ASD genetic risk loci. Objectives: To estimate associations between DNA methylation in maternal blood, cord blood, and placenta and later diagnosis of ASD, and to evaluate enrichment of ASD-associated DNA methylation for known ASD-associated genes. Methods: In the Early Autism Risk Longitudinal Investigation (EARLI), an ASD-enriched risk birth cohort, genome-scale maternal blood (early n=140 and late n=75 pregnancy), infant cord blood (n=133), and placenta (maternal n=106 and fetal n=107 compartments) DNA methylation was assessed on the Illumina 450k HumanMethylation array and compared to ASD diagnosis at 36 months of age. Differences in site-specific and global methylation were tested with ASD, as well as enrichment of single site associations for ASD risk genes (n=881) from the Simons Foundation Autism Research Initiative (SFARI) database. Results: No individual DNA methylation site was associated with ASD at genome-wide significance, however individual DNA methylation sites nominally associated with ASD (P<0.05) in each tissue were highly enriched for SFARI genes (cord blood P = 7.9x10-29, maternal blood early pregnancy P = 6.1x10-27, maternal blood late pregnancy P = 2.8x10-16, maternal placenta P= 5.6x10-15, fetal placenta P = 1.3x10-20). DNA methylation sites nominally associated with ASD across all five tissues overlapped at 144 (29.5%) SFARI genes. Conclusion: DNA methylation sites nominally associated with later ASD diagnosis in multiple tissues were enriched for ASD risk genes. Our multi-tissue study demonstrates the utility of examining DNA methylation prior to ASD diagnosis.

Background: Pregnancy measures of DNA methylation, an epigenetic mark, may be associated with autism spectrum disorder (ASD) development in children. Few ASD studies have considered prospective designs with DNA methylation measured in multiple tissues and tested overlap with ASD genetic risk loci. Objectives: To estimate associations between DNA methylation in maternal blood, cord blood, and placenta and later diagnosis of ASD, and to evaluate enrichment of ASD-associated DNA methylation for known ASD-associated genes. Methods: In the Early Autism Risk Longitudinal Investigation (EARLI), an ASD-enriched risk birth cohort, genome-scale maternal blood (early n=140 and late n=75 pregnancy), infant cord blood (n=133), and placenta (maternal n=106 and fetal n=107 compartments) DNA methylation was assessed on the Illumina 450k HumanMethylation array and compared to ASD diagnosis at 36 months of age. Differences in site-specific and global methylation were tested with ASD, as well as enrichment of single site associations for ASD risk genes (n=881) from the Simons Foundation Autism Research Initiative (SFARI) database. Results: No individual DNA methylation site was associated with ASD at genome-wide significance, however individual DNA methylation sites nominally associated with ASD (P<0.05) in each tissue were highly enriched for SFARI genes (cord blood P = 7.9x10-29, maternal blood early pregnancy P = 6.1x10-27, maternal blood late pregnancy P = 2.8x10-16, maternal placenta P= 5.6x10-15, fetal placenta P = 1.3x10-20). DNA methylation sites nominally associated with ASD across all five tissues overlapped at 144 (29.5%) SFARI genes. Conclusion: DNA methylation sites nominally associated with later ASD diagnosis in multiple tissues were enriched for ASD risk genes. Our multi-tissue study demonstrates the utility of examining DNA methylation prior to ASD diagnosis.

BackgroundThe mother–child inheritance of DNA methylation (DNAm) variations could contribute to the inheritance of disease susceptibility across generations. However, no study has investigated patterns of mother–child associations in DNAm at the genome-wide scale. It remains unknown whether there are sex differences in mother–child DNAm associations.ResultsUsing genome-wide DNAm profiling data (721,331 DNAm sites, including 704,552 on autosomes and 16,779 on the X chromosome) of 396 mother–newborn pairs (54.5% male) from the Boston Birth Cohort, we found significant sex differences in mother–newborn correlations in genome-wide DNAm patterns (Spearman’s rho = 0.91–0.98; p = 4.0 × 10–8), with female newborns having stronger correlations. Sex differences in correlations were attenuated but remained significant after excluding X-chromosomal DNAm sites (Spearman’s rho = 0.91–0.98; p = 0.035). Moreover, 89,267 DNAm sites (12.4% of all analyzed, including 88,051 [12.5% of analyzed] autosomal and 1,216 [7.2% of analyzed] X-chromosomal sites) showed significant mother–newborn associations in methylation levels, and the top autosomal DNAm sites had high heritability than the genome-wide background (e.g., the top 100 autosomal DNAm sites had a medium h2 of 0.92). Additionally, significant interactions between newborn sex and methylation levels were observed for 11 X-chromosomal and 4 autosomal DNAm sites that were mapped to genes that have been associated with sex-specific disease/traits or early development (e.g., EFHC2, NXY, ADCYAP1R1, and BMP4). Finally, 18,769 DNAm sites (14,482 [77.2%] on the X chromosome) showed mother–newborn differences in methylation levels that were significantly associated with newborn sex, and the top autosomal DNAm sites had relatively small heritability (e.g., the top 100 autosomal DNAm sites had a medium h2 of 0.23). These DNAm sites were mapped to 2,532 autosomal genes and 978 X-chromosomal genes with significant enrichment in pathways involved in neurodegenerative and psychological diseases, development, neurophysiological process, immune response, and sex-specific cancers. Replication analysis in the Isle of Wight birth cohort yielded consistent results.ConclusionIn two independent birth cohorts, we demonstrated strong mother–newborn correlations in whole blood DNAm on both autosomes and ChrX, and such correlations vary substantially by sex. Future studies are needed to examine to what extent our findings contribute to developmental origins of pediatric and adult diseases with well-observed sex differences.

Background:Corticotropin-releasing hormone (CRH) plays a central role in regulating the secretion of cortisol which controls a wide range of biological processes. Fetuses overexposed to cortisol have increased risks of disease in later life. DNA methylation may be the underlying association between prenatal cortisol exposure and health effects. We investigated associations between maternal CRH levels and epigenome-wide DNA methylation of cord blood in offsprings and evaluated whether these associations persisted into mid-childhood.Methods:We investigated mother-child pairs enrolled in the prospective Project Viva pre-birth cohort. We measured DNA methylation in 257 umbilical cord blood samples using the HumanMethylation450 Bead Chip. We tested associations of maternal CRH concentration with cord blood cells DNA methylation, adjusting the model for maternal age at enrollment, education, maternal race/ethnicity, pre-pregnancy body mass index, parity, gestational age at delivery, child sex, and cell-type composition in cord blood. We further examined the persistence of associations between maternal CRH levels and DNA methylation in children’s blood cells collected at mid-childhood (N = 239, age: 6.7–10.3 years) additionally adjusting for the children’s age at blood drawn.Results:Maternal CRH levels are associated with DNA methylation variability in cord blood cells at 96 individual CpG sites (False Discovery Rate < 0.05). Among the 96 CpG sites, we identified 3 CpGs located near the LEP gene. Regional analyses confirmed the association between maternal CRH and DNA methylation near LEP. Moreover, higher maternal CRH levels were associated with higher blood-cell DNA methylation of the promoter region of LEP in mid-childhood (P < 0.05, β = 0.64, SE = 0.30).Conclusion:In our cohort, maternal CRH was associated with DNA methylation levels in newborns at multiple loci, notably in the LEP gene promoter. The association between maternal CRH and LEP DNA methylation levels persisted into mid-childhood.

Importance: Women with a history of depression are less likely to breastfeed and experience more breastfeeding challenges. Potential biological mechanisms that link maternal depression with poor breastfeeding outcomes have not been investigated. Objective: Examine associations between maternal lifetime history of depression and maternal DNA methylation at the oxytocin receptor gene (OXTR), and associations between maternal OXTR DNA methylation and breastfeeding outcomes. Design: Longitudinal observational data from the Norwegian Mother, Father and Child Cohort Study (MoBa) were analysed. Setting: MoBa is a population-based nationwide pregnancy cohort study conducted by the Norwegian Institute of Public Health.Participants: N=3,607 participants selected from three subsets of the MoBa cohort for whom DNA methylation data was available. Exposures: Lifetime history of major depression was self-reported during pregnancy (week 15) using 6 items that closely correspond to the DSM-III criteria for lifetime major depression. Main outcomes and measures: Blood samples were collected at week 16-18 of pregnancy, and DNA methylation was measured using the Illumina Methylation EPIC BeadChip 850K array. This array includes 22 CpG sites on the OXTR gene, which were used in analyses. Mothers self-reported breastfeeding initiation (breastmilk given to child in first month post-birth), breastfeeding maintenance (breastmilk given to child for 6 months or more) and breastfeeding problems.Results: Maternal lifetime history of depression was not associated with DNA methylation at the OXTR gene. There was some evidence that maternal DNA methylation at the OXTR gene was associated with breastfeeding outcomes. The strongest evidence was for an association between the CpG site cg26455676 and the maintenance of breastfeeding to 6 months after birth (odds ratio=1.59, 95% confidence intervals=1.11-2.27, raw p=0.01, adjusted p=0.04). Higher levels of maternal DNA methylation at CpG cg26455676 was associated with a greater likelihood of maintaining breastfeeding to 6 months after birth. Conclusions and relevance: This highly novel study highlights the intriguing possibility that maternal DNA methylation at genes important for breastfeeding may be associated with breastfeeding experiences. Further understanding of why women have vastly differing breastfeeding experiences would assist with the targeting of efforts to support breastfeeding women, particularly those who are vulnerable, such as mothers who have depression.

915 Maternal occupation is associated with maternal global dna (hydroxy) methylation in the second trimester of pregnancy

Anxiety disorders (ANX) are a prevalent public health burden that significantly impair daily functioning and decrease quality of life. A growing body of research suggests that DNA methylation (DNAm), an epigenetic modification that can impact gene expression, may be altered in ANX. The current review used a systematic approach to identify and synthesize the literature regarding methylome-wide association studies (MWASs) of ANX in humans. We screened 804 articles returned by a search in PubMed in May 2025 and identified 12 studies for inclusion. All included studies examined ANX-associated DNAm in blood. In total, 2,023 DNAm sites corresponding to 985 genes were significantly associated with ANX. No DNAm sites significantly replicated across studies and four nominally replicated. This is likely a result of a lack of replication attempts, small sample sizes, and differences in data analysis choices. Findings suggest that ANX-associated DNAm may promote dysregulation of immune and inflammatory processes, some possibly sex-dependent. Collectively, the findings from studies included in this review provide preliminary evidence of ANX-related alterations to DNAm in whole blood and multiple blood cell-types. Future MWASs of ANX could benefit from larger sample sizes, a standardized analytic pipeline, longitudinal study designs, and the examination of DNAm in additional cell-types and tissues.

Background: Pregnancy measures of DNA methylation, an epigenetic mark, may be associated with autism spectrum disorder (ASD) development in children. Few ASD studies have considered prospective designs with DNA methylation measured in multiple tissues and tested overlap with ASD genetic risk loci.Objectives: To estimate associations between DNA methylation in maternal blood, cord blood, and placenta and later diagnosis of ASD, and to evaluate enrichment of ASD-associated DNA methylation for known ASD-associated genes.Methods: In the Early Autism Risk Longitudinal Investigation (EARLI), an ASD-enriched risk birth cohort, genome-scale maternal blood (early n = 140 and late n = 75 pregnancy), infant cord blood (n = 133), and placenta (maternal n = 106 and fetal n = 107 compartments) DNA methylation was assessed on the Illumina 450k HumanMethylation array and compared to ASD diagnosis at 36 months of age. Differences in site-specific and global methylation were tested with ASD, as well as enrichment of single site associations for ASD risk genes (n = 881) from the Simons Foundation Autism Research Initiative (SFARI) database.Results: No individual DNA methylation site was associated with ASD at genome-wide significance, however, individual DNA methylation sites nominally associated with ASD (P < 0.05) in each tissue were highly enriched for SFARI genes (cord blood P = 7.9 × 10–29, maternal blood early pregnancy P = 6.1 × 10–27, maternal blood late pregnancy P = 2.8 × 10–16, maternal placenta P = 5.6 × 10–15, fetal placenta P = 1.3 × 10–20). DNA methylation sites nominally associated with ASD across all five tissues overlapped at 144 (29.5%) SFARI genes.Conclusion: DNA methylation sites nominally associated with later ASD diagnosis in multiple tissues were enriched for ASD risk genes. Our multi-tissue study demonstrates the utility of examining DNA methylation prior to ASD diagnosis.

OPS 39: Metals and biomarkers, Room 411, Floor 4, August 26, 2019, 1:30 PM - 3:00 PM Background/Aim: Metals exposures have important health effects in pregnancy, and blood measures reflect recent exposure (cadmium half-life (t1/2) =~75 days, manganese t1/2=~40 days, mercury t1/2=~70 days, selenium t1/2=~100 days), and lead t1/2=~30 days). The maternal epigenome may be responsive to these exposures. We tested whether metals are associated with concurrent differential maternal whole blood DNA methylation. Methods: In the Early Autism Risk Longitudinal Investigation (EARLI) cohort, we measured first or second trimester maternal blood metals concentrations (215 participants) using inductively coupled plasma mass spectrometry. Maternal blood DNA methylation was measured in the same specimens on the Illumina 450K array (201 participants); 97 non-smoking women had both measures available for analysis. Linear regression was used to test for site-specific associations between individual metals and DNA methylation, adjusting for cell type composition and confounding variables. In targeted enrichment analysis, genes with altered DNA methylation (p-value<0.05) were tested for overlap with innate immunity genes. Discovery gene ontology analysis was conducted on the top 1,000 sites associated with each metal to elucidate downstream pathways. Results: In multivariable regression, we observed DNA methylation sites associated (p-value<10-5) with cadmium (n=3), manganese (n=21), mercury (n=1), and lead (n=63). The top sites associated with cadmium, manganese, mercury, and lead, were near the genes ITPKB, ARID2, SPTBN2, and CYP24A1 respectively. Cadmium and manganese associated DNA methylation sites had the greatest enrichment for innate immunity genes (Fischer's test p-value<10-3). Cadmium was associated with cell adhesion (q-value<0.01), manganese was associated with cellular metabolism (q-value<0.01), mercury was associated with organ morphogenesis (q-value<0.01), and lead was associated with cell adhesion (q-value<0.001) and nervous system development (q-value<0.001). Conclusions: We report site-specific associations between DNA methylation and blood metals in early pregnancy. Sites associated with cadmium, manganese, mercury, and lead may be potential biomarkers of exposure or implicate downstream gene pathways.

BackgroundMaternal mood disorders and their treatment during pregnancy may have effects on the offspring epigenome. We aim to evaluate associations of maternal prenatal antidepressant use, anxiety, and depression with cord blood DNA methylation across the genome at birth and test for persistence of associations in early and mid-childhood blood DNA.MethodsA discovery phase was conducted in Project Viva, a prospective pre-birth cohort study with external replication in an independent cohort, the Generation R Study. In Project Viva, pregnant women were recruited between 1999 and 2002 in Eastern Massachusetts, USA. In the Generation R Study, pregnant women were recruited between 2002 and 2006 in Rotterdam, the Netherlands. In Project Viva, 479 infants had data on maternal antidepressant use, anxiety, depression, and cord blood DNA methylation, 120 children had DNA methylation measured in early childhood (~ 3 years), and 460 in mid-childhood (~ 7 years). In the Generation R Study, 999 infants had data on maternal antidepressants and cord blood DNA methylation. The prenatal antidepressant prescription was obtained from medical records. At-mid pregnancy, symptoms of anxiety and depression were assessed with the Pregnancy-Related Anxiety Scale and the Edinburgh Postnatal Depression Scale in Project Viva and with the Brief Symptom Inventory in the Generation R Study. Genome-wide DNA methylation was measured using the Infinium HumanMethylation450 BeadChip in both cohorts.ResultsIn Project Viva, 2.9% (14/479) pregnant women were prescribed antidepressants, 9.0% (40/445) experienced high pregnancy-related anxiety, and 8.2% (33/402) reported symptoms consistent with depression. Newborns exposed to antidepressants in pregnancy had 7.2% lower DNA methylation (95% CI, − 10.4, − 4.1; P = 1.03 × 10−8) at cg22159528 located in the gene body of ZNF575, and this association replicated in the Generation R Study (β = − 2.5%; 95% CI − 4.2, − 0.7; P = 0.006). In Project Viva, the association persisted in early (β = − 6.2%; 95% CI − 10.7, − 1.6) but not mid-childhood. We observed cohort-specific associations for maternal anxiety and depression in Project Viva that did not replicate.ConclusionsThe ZNF575 gene is involved in transcriptional regulation but specific functions are largely unknown. Given the widespread use of antidepressants in pregnancy, as well as the effects of exposure to anxiety and depression, implications of potential fetal epigenetic programming by these risk factors and their impacts on development merit further investigation.

Early patterning of DNA methylation (DNAm) may play an important role in later disease development. To better understand intergenerational epigenetic inheritance, we investigated the correlation between DNAm in blood in mother-newborn and in father-newborn pairs in the Isle of Wight (IoW) birth cohort. For parent-newborn pairs (n = 48), offspring DNAm was measured in cord blood and the parent’s DNAm in whole blood. Mothers’ DNAm was analyzed at birth (Guthrie card), age 18, early and late pregnancy respectively, and fathers’ DNAm was measured during the mother’s pregnancy. Linear regressions were applied to assess the intergenerational correlation of parental DNAm with that of offspring. Among various pairs of mother-newborn and father-newborn DNAm, the pairs where the mothers’ DNAm was measured at age 18 years exhibited the highest number of CpGs with significant intergenerational correlation in DNAm, with 1829 CpGs (0.54%) of the 338,526 CpGs studied (FDR < 0.05). Amongst these 1829 CpGs, 986 (54%) are known quantitative trait loci (QTL) for CpG methylation (methQTL). When the mother’s DNAm was assessed at early pregnancy, the number of CpGs showing intergenerational correlation was the smallest (384 CpGs, 0.11%). The second smallest number of such CpGs (559 CpGs, 0.17%) was found when investigating DNAm in offspring cord blood and father pairs. The low proportions of intergenerationally correlated CpGs suggest that epigenetic inheritance is limited.

One-carbon metabolism is essential for multiple cellular processes and can be assessed by the concentration of folate metabolites in the blood. One-carbon metabolites serve as methyl donors that are required for epigenetic regulation. Deficiencies in these metabolites are associated with a variety of poor health outcomes, including adverse pregnancy complications. DNA methylation is known to vary with one-carbon metabolite concentration, and therefore may modulate the risk of adverse pregnancy outcomes. This study addresses changes in one-carbon indices over pregnancy and the relationship between maternal and child DNA methylation and metabolite concentrations by leveraging data from 24 mother-infant dyads. Five of the 13 metabolites measured from maternal blood and methylation levels of 993 CpG sites changed over the course of pregnancy. In dyads, maternal and fetal one-carbon concentrations were highly correlated, both early in pregnancy and at delivery. The 993 CpG sites whose methylation levels changed over pregnancy in maternal blood were also investigated for associations with metabolite concentrations in infant blood at delivery, where five CpG sites were associated with the concentration of at least one metabolite. Identification of CpG sites that change over pregnancy may result in better characterization of genes and pathways involved in maintaining a healthy, term pregnancy.