Epigenetic footprints: Investigating placental DNA methylation in the context of prenatal exposure to phenols and phthalates.

BACKGROUND: Endocrine disrupting compounds (EDCs) such as phthalates and phenols can affect placental functioning and fetal health, potentially via epigenetic modifications. We investigated the associations between pregnancy exposure to synthetic phenols and phthalates estimated from repeated urine sampling and genome wide placental DNA methylation. METHODS: The study is based on 387 women with placental DNA methylation assessed with Infinium MethylationEPIC arrays and with 7 phenols, 13 phthalates, and two non-phthalate plasticizer metabolites measured in pools of urine samples collected twice during pregnancy. We conducted an exploratory analysis on individual CpGs (EWAS) and differentially methylated regions (DMRs) as well as a candidate analysis focusing on 20 previously identified CpGs. Sex-stratified analyses were also performed. RESULTS: In the exploratory analysis, when both sexes were studied together no association was observed in the EWAS. In the sex-stratified analysis, 114 individual CpGs (68 in males, 46 in females) were differentially methylated, encompassing 74 genes (36 for males and 38 for females). We additionally identified 28 DMRs in the entire cohort, 40 for females and 42 for males. Associations were mostly positive (for DMRs: 93% positive associations in the entire cohort, 60% in the sex-stratified analysis), with the exception of several associations for bisphenols and DINCH metabolites that were negative. Biomarkers associated with most DMRs were parabens, DEHP, and DiNP metabolite concentrations. Some DMRs encompassed imprinted genes including APC (associated with parabens and DiNP metabolites), GNAS (bisphenols), ZIM2;PEG3;MIMT1 (parabens, monoethyl phthalate), and SGCE;PEG10 (parabens, DINCH metabolites). Terms related to adiposity, lipid and glucose metabolism, and cardiovascular function were among the enriched phenotypes associated with differentially methylated CpGs. The candidate analysis identified one CpG mapping to imprinted LGALS8 gene, negatively associated with ethylparaben. CONCLUSIONS: By combining improved exposure assessment and extensive placental epigenome coverage, we identified several novel genes associated with the exposure, possibly in a sex-specific manner.

Placental DNA methylation signatures of prenatal air pollution exposure and potential effects on birth outcomes: an analysis of three prospective cohorts.

BACKGROUND: Pregnancy air pollution exposure (PAPE) has been linked to a wide range of adverse birth and childhood outcomes, but there is a paucity of data on its influence on the placental epigenome, which can regulate the programming of physiological functions and affect child development. This study aimed to investigate the association between prenatal air pollutant exposure concentrations and changes in placental DNA methylation patterns, and to explore the potential windows of susceptibility and sex-specific alterations. METHODS: This multi-site study used three prospective population-based mother-child cohorts: EDEN, PELAGIE, and SEPAGES, originating from four French geographical regions (Nancy, Poitiers, Brittany, and Grenoble). Pregnant women were included between 2003 and 2006 for EDEN and PELAGIE, and between 2014 and 2017 for SEPAGES. The main eligibility criteria were: being older than 18 years, having a singleton pregnancy, and living and planning to deliver in one of the maternity clinics in one of the study areas. A total of 1539 mother-child pairs were analysed, measuring placental DNA methylation using Illumina BeadChips. We used validated spatiotemporally resolved models to estimate PM2·5, PM10, and NO2 exposure over each trimester of pregnancy at the maternal residential address. We conducted a pooled adjusted epigenome-wide association study to identify differentially methylated 5'-C-phosphate-G-3' (CpG) sites and regions (assessed using the Infinium HumanMethylationEPIC BeadChip array, n=871), including sex-specific and sex-linked alterations, and independently validated our results (assessed using the Infinium HumanMethylation450 BeadChip array, n=668). FINDINGS: We identified four CpGs and 28 regions associated with PAPE in the total population, 469 CpGs and 87 regions in male infants, and 150 CpGs and 66 regions in female infants. We validated 35% of the CpGs available. More than 30% of the identified CpGs were related to one (or more) birth outcome and most significant alterations were enriched for neural development, immunity, and metabolism related genes. The 28 regions identified for both sexes overlapped with imprinted genes (four genes), and were associated with neurodevelopment (nine genes), immune system (seven genes), and metabolism (five genes). Most associations were observed for the third trimester for female infants (134 of 150 CpGs), and throughout pregnancy (281 of 469 CpGs) and the first trimester (237 of 469 CpGs) for male infants. INTERPRETATION: These findings highlight the molecular pathways through which PAPE might affect child health in a widespread and sex-specific manner, identifying the genes involved in the major physiological functions of a developing child. Further studies are needed to elucidate whether these epigenetic changes persist and affect health later in life. FUNDING: French Agency for National Research, Fondation pour la Recherche Médicale, Fondation de France, and the Plan Cancer.

Prenatal exposure to triclosan assessed in multiple urine samples and placental DNA methylation.

A previous study reported positive associations of maternal urinary concentrations of triclosan, a synthetic phenol with widespread exposure in the general population, with placental DNA methylation of male fetuses. Given the high number of comparisons performed in -omic research, further studies were needed to validate and extend on these findings. Using a cohort of male and female fetuses with repeated maternal urine samples to assess exposure, we studied the associations between triclosan and placental DNA methylation. We assessed triclosan concentrations in two pools of 21 urine samples collected among 395 women from the SEPAGES cohort. We used Infinium Methylation EPIC arrays to measure DNA methylation in placental biopsies collected at delivery. We performed a candidate study restricted to a set of candidate CpGs (n = 500) identified in a previous work as well as an exploratory epigenome-wide association study to investigate the associations between triclosan and differentially methylated probes and regions. Analyses were conducted on the whole population and stratified by child's sex. Mediation analysis was performed to test whether heterogeneity of placental tissue may mediate the observed associations. In the candidate approach, we confirmed 18 triclosan-associated genes when both sexes were considered. After stratification for child's sex, triclosan was associated with 72 genes in females and three in males. Most of the associations were positive and several CpGs mapped to imprinted genes: FBRSL1, KCNQ1, RHOBTB3, and SMOC1. A mediation effect by placental tissue heterogeneity was identified for most of the observed associations. In the exploratory analysis, we identified a few isolated associations in the sex-stratified analysis. In line with a previous study on male placentas, our approach revealed several positive associations between triclosan exposure and placental DNA methylation. Several identified loci mapped to imprinted genes.

Epigenome-Wide Associations of Placental DNA Methylation and Behavioral and Emotional Difficulties in Children at 3 Years of Age.

The placenta is a key organ for fetal and brain development. Its epigenome can be regarded as a biochemical record of the prenatal environment and a potential mechanism of its association with the future health of the fetus. We investigated associations between placental DNA methylation levels and child behavioral and emotional difficulties, assessed at 3 years of age using the Strengths and Difficulties Questionnaire (SDQ) in 441 mother-child dyads from the EDEN cohort. Hypothesis-driven and exploratory analyses (on differentially methylated probes (EWAS) and regions (DMR)) were adjusted for confounders, technical factors, and cell composition estimates, corrected for multiple comparisons, and stratified by child sex. Hypothesis-driven analyses showed an association of cg26703534 (AHRR) with emotional symptoms, and exploratory analyses identified two probes, cg09126090 (intergenic region) and cg10305789 (PPP1R16B), as negatively associated with peer relationship problems, as well as 33 DMRs, mostly positively associated with at least one of the SDQ subscales. Among girls, most associations were seen with emotional difficulties, whereas in boys, DMRs were as much associated with emotional than behavioral difficulties. This study provides the first evidence of associations between placental DNA methylation and child behavioral and emotional difficulties. Our results suggest sex-specific associations and might provide new insights into the mechanisms of neurodevelopment.

Maternal blood pressure associates with placental DNA methylation both directly and through alterations in cell-type composition.

BACKGROUND: Maternal blood pressure levels reflect cardiovascular adaptation to pregnancy and proper maternal-fetal exchanges through the placenta and are very sensitive to numerous environmental stressors. Maternal hypertension during pregnancy has been associated with impaired placental functions and with an increased risk for children to suffer from cardiovascular and respiratory diseases later on. Investigating changes in placental DNA methylation levels and cell-type composition in association with maternal blood pressure could help elucidate its relationships with placental and fetal development. METHODS: Taking advantage of a large cohort of 666 participants, we investigated the association between epigenome-wide DNA methylation patterns in the placenta, measured using the Infinium HumanMethylation450 BeadChip, placental cell-type composition, estimated in silico, and repeated measurements of maternal steady and pulsatile blood pressure indicators during pregnancy. RESULTS: At the site-specific level, no significant association was found between maternal blood pressure and DNA methylation levels after correction for multiple testing (false discovery rate < 0.05), but 5 out of 24 previously found CpG associations were replicated (p-value < 0.05). At the regional level, our analyses highlighted 64 differentially methylated regions significantly associated with at least one blood pressure component, including 35 regions associated with mean arterial pressure levels during late pregnancy. These regions were found enriched for genes implicated in lung development and diseases. Further mediation analyses show that a significant part of the association between steady blood pressure-but not pulsatile pressure-and placental methylation can be explained by alterations in placental cell-type composition. In particular, elevated blood pressure levels are associated with a decrease in the ratio between mesenchymal stromal cells and syncytiotrophoblasts, even in the absence of preeclampsia. CONCLUSIONS: This study provides the first evidence that the association between maternal steady blood pressure during pregnancy and placental DNA methylation is both direct and partly explained by changes in cell-type composition. These results could hint at molecular mechanisms linking maternal hypertension to lung development and early origins of childhood respiratory problems and at the importance of controlling maternal blood pressure during pregnancy.

Pregnancy exposure to phthalates and DNA methylation in male placenta - An epigenome-wide association study.

BACKGROUND: Exposure to phthalates during pregnancy may alter DNA methylation in the placenta, a crucial organ for the growth and development of the fetus. OBJECTIVES: We studied associations between urinary concentrations of phthalate biomarkers during pregnancy and placental DNA methylation. METHODS: We measured concentrations of 11 phthalate metabolites in maternal spot urine samples collected between 22 and 29 gestational weeks in 202 pregnant women. We analyzed DNA methylation levels in placental tissue (fetal side) collected at delivery. We first investigated changes in global DNA methylation of repetitive elements Alu and LINE-1. We then performed an adjusted epigenome-wide association study using IlluminaHM450 BeadChips and identified differentially methylated regions (DMRs) associated with phthalate exposure. RESULTS: Monobenzyl phthalate concentration was inversely associated with placental methylation of Alu repeats. Moreover, all phthalate biomarkers except for monocarboxy-iso-octyl phthalate and mono(2-ethyl-5-hydroxyhexyl) phthalate were associated with at least one DMR. All but three DMRs showed increased DNA methylation with increased phthalate exposure. The largest identified DMR (22 CpGs) was positively associated with monocarboxy-iso-nonyl phthalate and encompassed heat shock proteins (HSPA1A, HSPA1L). The remaining DMRs encompassed transcription factors and nucleotide exchange factors, among other genes. CONCLUSIONS: This is the first description of genome-wide modifications of placental DNA methylation in association with pregnancy exposure to phthalates. Our results suggest epigenetic mechanisms by which exposure to these compounds could affect fetal development. Of interest, four identified DMRs had been previously associated with maternal smoking, which may suggest particular sensitivity of these genomic regions to the effect of environmental contaminants.

Intron retention and its impact on gene expression and protein diversity: A review and a practical guide.

Intron retention (IR) occurs when a complete and unspliced intron remains in mature mRNA. An increasing body of literature has demonstrated a major role for IR in numerous biological functions, including several that impact human health and disease. Although experimental technologies used to study other forms of mRNA splicing can also be used to investigate IR, a specialized downstream computational analysis is optimal for IR discovery and analysis. Here we provide a review of IR and its biological implications, as well as a practical guide for how to detect and analyze it. Several methods, including long read third generation direct RNA sequencing, are described. We have developed an R package, FakIR, to facilitate the execution of the bioinformatic tasks recommended in this review and a tutorial on how to fit them to users aims. Additionally, we provide guidelines and experimental protocols to validate IR discovery and to evaluate the potential impact of IR on gene expression and protein output. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Splicing Regulation/Alternative Splicing RNA Methods > RNA Analyses in vitro and In Silico.

TALC: Transcript-level Aware Long-read Correction.

MOTIVATION: Long-read sequencing technologies are invaluable for determining complex RNA transcript architectures but are error-prone. Numerous 'hybrid correction' algorithms have been developed for genomic data that correct long reads by exploiting the accuracy and depth of short reads sequenced from the same sample. These algorithms are not suited for correcting more complex transcriptome sequencing data. RESULTS: We have created a novel reference-free algorithm called Transcript-level Aware Long-Read Correction (TALC) which models changes in RNA expression and isoform representation in a weighted De Bruijn graph to correct long reads from transcriptome studies. We show that transcript-level aware correction by TALC improves the accuracy of the whole spectrum of downstream RNA-seq applications and is thus necessary for transcriptome analyses that use long read technology. AVAILABILITY AND IMPLEMENTATION: TALC is implemented in C++ and available at https://github.com/lbroseus/TALC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Challenges in detecting and quantifying intron retention from next generation sequencing data.

Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. An increasing body of literature has demonstrated a major role for IR in numerous biological functions and in disease. Here we give an overview of the different computational approaches for detecting IR events from sequencing data. We show that these are based on different biological and computational assumptions that may lead to dramatically different results. We describe the various approaches for mitigating errors in detecting intron retention and for discovering IR signatures between different conditions.

GECKO is a genetic algorithm to classify and explore high throughput sequencing data.

Comparative analysis of high throughput sequencing data between multiple conditions often involves mapping of sequencing reads to a reference and downstream bioinformatics analyses. Both of these steps may introduce heavy bias and potential data loss. This is especially true in studies where patient transcriptomes or genomes may vary from their references, such as in cancer. Here we describe a novel approach and associated software that makes use of advances in genetic algorithms and feature selection to comprehensively explore massive volumes of sequencing data to classify and discover new sequences of interest without a mapping step and without intensive use of specialized bioinformatics pipelines. We demonstrate that our approach called GECKO for GEnetic Classification using k-mer Optimization is effective at classifying and extracting meaningful sequences from multiple types of sequencing approaches including mRNA, microRNA, and DNA methylome data.

Rare RNF213 variants in the C-terminal region encompassing the RING-finger domain are associated with moyamoya angiopathy in Caucasians.

Moyamoya angiopathy (MMA) is a cerebral angiopathy affecting the terminal part of internal carotid arteries. Its prevalence is 10 times higher in Japan and Korea than in Europe. In East Asian countries, moyamoya is strongly associated to the R4810K variant in the RNF213 gene that encodes for a protein containing a RING-finger and two AAA+ domains. This variant has never been detected in Caucasian MMA patients, but several rare RNF213 variants have been reported in Caucasian cases. Using a collapsing test based on exome data from 68 European MMA probands and 573 ethnically matched controls, we showed a significant association between rare missense RNF213 variants and MMA in European patients (odds ratio (OR)=2.24, 95% confidence interval (CI)=(1.19-4.11), P=0.01). Variants specific to cases had higher pathogenicity predictive scores (median of 24.2 in cases versus 9.4 in controls, P=0.029) and preferentially clustered in a C-terminal hotspot encompassing the RING-finger domain of RNF213 (P<10-3). This association was even stronger when restricting the analysis to childhood-onset and familial cases (OR=4.54, 95% CI=(1.80-11.34), P=1.1 × 10-3). All clinically affected relatives who were genotyped were carriers. However, the need for additional factors to develop MMA is strongly suggested by the fact that only 25% of mutation carrier relatives were clinically affected.