Validation of differential DNA methylation in newborns exposed to tobacco smoke during gestation using bisulfite pyrosequencing.
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ABSTRACT:
SUBMITTER: Crute C
PROVIDER: S-EPMC9015814 | biostudies-literature | 2022
REPOSITORIES: biostudies-literature
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Validation of differential DNA methylation in newborns exposed to tobacco smoke during gestation using bisulfite pyrosequencing.
Crute Christine C Liao Yihan Y Son Esther E Grenier Carole C Huang Zhiqing Z Hoyo Cathrine C Murphy Susan K SK
microPublication biology 20220107
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Project description:BackgroundMaternal smoking during pregnancy is a major risk factor for adverse health outcomes. The main objective of the study was to assess the impact of in utero tobacco exposure on DNA methylation in children born at term with appropriate weight at birth.MethodsTwenty mother-newborn dyads, after uncomplicated pregnancies, in the absence of perinatal illness were included. All mothers were healthy with no cardiovascular risk factors, except for the associated risks among those mothers who smoked. Umbilical cord blood and maternal peripheral venous blood were collected and an epigenome-wide association study was performed using a 450 K epigenome-wide scan (Illumina Infinium HumanMethylation 450BeadChip) with adjustment to normalize the DNA methylation for data cell variability in whole blood.ResultsThe maternal plasmatic cotinine levels ranged from 10.70-115.40 ng/ml in the exposed group to 0-0.59 ng/ml in the non-exposed group. After adjusting for multiple comparisons in 427102 probes, statistically significant differences for 31 CpG sites, associated to 25 genes were observed. There was a greater than expected proportion of statistically-significant loci located in CpG islands (Fisher's exact test, p = 0.029) and of those CpG islands, 90.3% exhibit higher methylation levels in the exposed group. The most striking and significant CpG site, cg05727225, is located in the chromosome 11p15.4, within the adrenomedullin gene.ConclusionsIn utero tobacco exposure, even in the absence of fetal growth restriction, may alter the epigenome, contributing to global DNA hypomethylation. Therefore, DNA status can be used as a biomarker of prenatal insults. Considering the possibility to reverse epigenetic modifications, a window of opportunity exists to change the programmed chronic disease.
| S-EPMC4312439 | biostudies-literature
Project description:Different methodological approaches are available to assess DNA methylation biomarkers. In this study, we evaluated two sodium bisulfite conversion-dependent methods, namely pyrosequencing and methylation-specific qPCR (MS-qPCR), with the aim of measuring the closeness of agreement of methylation values between these two methods and its effect when setting a cut-off. Methylation of tumor suppressor gene p16/INK4A was evaluated in 80 lung cancer patients from which cytological lymph node samples were obtained. Cluster analyses were used to establish methylated and unmethylated groups for each method. Agreement and concordance between pyrosequencing and MS-qPCR was evaluated with Pearson's correlation, Bland-Altman, Cohen's kappa index and ROC curve analyses. Based on these analyses, cut-offs were derived for MS-qPCR. An acceptable correlation (Pearson's R2 = 0.738) was found between pyrosequencing (PYRmean) and MS-qPCR (NMP; normalized methylation percentage), providing similar clinical results when categorizing data as binary using cluster analysis. Compared to pyrosequencing, MS-qPCR tended to underestimate methylation for values between 0 and 15%, while for methylation >30% overestimation was observed. The estimated cut-off for MS-qPCR data based on cluster analysis, kappa-index agreement and ROC curve analysis were much lower than that derived from pyrosequencing. In conclusion, our results indicate that independently of the approach used for estimating the cut-off, the methylation percentage obtained through MS-qPCR is lower than that calculated for pyrosequencing. These differences in data and therefore in the cut-off should be examined when using methylation biomarkers in the clinical practice.
| S-EPMC7730915 | biostudies-literature
Project description:We generated genome-wide methylation levels, using whole-genome bisulfite sequencing, for sorted CD4+ cells from cordblood of newborns exposed vs. unexposed to tobacco smoke in utero
2019-03-13 | GSE109212 | GEO
Project description:Background: Maternal smoking during pregnancy is a major risk factor for adverse health outcomes. The main objective of the study was to assess the impact of in utero tobacco exposure on DNA methylation in children born at term with appropriate weight at birth. Methods: Twenty mother-newborn dyads, after uncomplicated pregnancies, in the absence of perinatal illness were included. All mothers were healthy with no cardiovascular risk factors, except for the associated risks among those mothers who smoked. Umbilical cord blood (for methylation arrays) and maternal peripheral venous blood (for cotine level measurement) were collected and an epigenome-wide association study was performed using a 450K epigenome-wide scan (Illumina Infinium HumanMethylation 450BeadChip) with adjustment to normalize the DNA methylation for data cell variability in whole blood. Results: The maternal plasmatic cotinine levels ranged from 10.70-115.40 ng/ml in the exposed group to 0-0.59 ng/ml in the non-exposed group. After adjusting for multiple comparisons in 427102 probes, statistically significant differences for 31 CpG sites, associated to 25 genes were observed. There was a greater than expected proportion of statistically-significant loci located in CpG islands (Fisher’s exact test, p=0.029) and of those CpG islands, 90.3% exhibit higher methylation levels in the exposed group. The most striking and significant CpG site, cg05727225, is located in the chromosome 11p15.4, within the adrenomedullin gene. Conclusions: In utero tobacco exposure, even in the absence of fetal growth restriction, may alter the epigenome, contributing to global DNA hypomethylation. Therefore, DNA status can be used as a biomarker of prenatal insults. Considering the possibility to reverse epigenetic modifications, a window of opportunity exists to change the programmed chronic disease.
2015-02-16 | GSE64316 | GEO
Project description:Background: Maternal smoking during pregnancy is a major risk factor for adverse health outcomes. The main objective of the study was to assess the impact of in utero tobacco exposure on DNA methylation in children born at term with appropriate weight at birth. Methods: Twenty mother-newborn dyads, after uncomplicated pregnancies, in the absence of perinatal illness were included. All mothers were healthy with no cardiovascular risk factors, except for the associated risks among those mothers who smoked. Umbilical cord blood (for methylation arrays) and maternal peripheral venous blood (for cotine level measurement) were collected and an epigenome-wide association study was performed using a 450K epigenome-wide scan (Illumina Infinium HumanMethylation 450BeadChip) with adjustment to normalize the DNA methylation for data cell variability in whole blood. Results: The maternal plasmatic cotinine levels ranged from 10.70-115.40 ng/ml in the exposed group to 0-0.59 ng/ml in the non-exposed group. After adjusting for multiple comparisons in 427102 probes, statistically significant differences for 31 CpG sites, associated to 25 genes were observed. There was a greater than expected proportion of statistically-significant loci located in CpG islands (FisherM-bM-^@M-^Ys exact test, p=0.029) and of those CpG islands, 90.3% exhibit higher methylation levels in the exposed group. The most striking and significant CpG site, cg05727225, is located in the chromosome 11p15.4, within the adrenomedullin gene. Conclusions: In utero tobacco exposure, even in the absence of fetal growth restriction, may alter the epigenome, contributing to global DNA hypomethylation. Therefore, DNA status can be used as a biomarker of prenatal insults. Considering the possibility to reverse epigenetic modifications, a window of opportunity exists to change the programmed chronic disease. Twenty mother-newborn dyads, after uncomplicated pregnancies, in the absence of perinatal illness were included. All mothers were healthy with no cardiovascular risk factors, except for the associated risks among those mothers who smoked. Umbilical cord blood were collected and an epigenome-wide association study was performed using a 450K epigenome-wide scan (Illumina Infinium HumanMethylation 450BeadChip) with adjustment to normalize the DNA methylation for data cell variability in whole blood.
2015-02-16 | E-GEOD-64316 | biostudies-arrayexpress
Project description:Bisulfite sequencing (BS-seq) technology measures DNA methylation at single nucleotide resolution. A key task in BS-seq data analysis is to identify differentially methylation (DM) under different conditions. Here we provide a tutorial for BS-seq DM analysis using Bioconductor package DSS. DSS uses a beta-binomial model to characterize the sequence counts from BS-seq, and implements rigorous statistical method for hypothesis testing. It provides flexible functionalities for a variety of DM analyses.
| S-EPMC8171293 | biostudies-literature
Project description:Resistance to chemotherapy is a major complication during treatment of cancer patients. Hypermethylation of the MGMT gene alters DNA repair and is associated with longer survival of glioblastoma patients treated with alkylating agents. Therefore, MGMT promoter methylation plays an important role as a predictive biomarker for chemotherapy resistance. To adopt this established correlation into a molecular diagnosis procedure, we compared and optimized three experimental techniques [combined bisulfite restriction analysis, a primer extension- and denaturing high-performance liquid chromatography-based method named SIRPH (SNuPE ion pair-reverse phase high-performance liquid chromatography), and pyrosequencing] with regard to their accuracy of detecting MGMT promoter methylation. Initially, bisulfite sequencing was used to obtain a comprehensive methylation profile of the MGMT promoter region in 22 glioblastoma samples and in three normal brain controls. Next, we statistically identified CpG sites that best discriminate between methylated and unmethylated MGMT promoters. These results were then used to design optimal combined bisulfite restriction analysis, SIRPH, and pyrosequencing assays for accurate and cost-efficient assessment of MGMT promoter methylation. We compared all three techniques with regard to their reliability and reproducibility on well-characterized tumor samples. The optimized pyrosequencing assay performed best and provides a sensitive, robust, and easy-to-use method for quantitative assessment of MGMT methylation, for both snap-frozen and paraffin-embedded specimens.
| S-EPMC1899414 | biostudies-literature
Project description:Background: With advances in next-generation sequencing technologies, the bisulfite conversion of genomic DNA followed by sequencing has become the predominant technique for quantifying genome-wide DNA methylation at single-base resolution. A large number of computational approaches are available in literature for identifying differentially methylated regions in bisulfite sequencing data, and more are being developed continuously. Results: Here, we focused on a comprehensive evaluation of commonly used differential methylation analysis methods and describe the potential strengths and limitations of each method. We found that there are large differences among methods, and no single method consistently ranked first in all benchmarking. Moreover, smoothing seemed not to improve the performance greatly, and a small number of replicates created more difficulties in the computational analysis of BS-seq data than low sequencing depth. Conclusions: Data analysis and interpretation should be performed with great care, especially when the number of replicates or sequencing depth is limited.
| S-EPMC8345583 | biostudies-literature
Project description:Cytosine methylation is an important DNA epigenetic modification. In vertebrates, methylation occurs at CpG sites, which are dinucleotides where a cytosine is immediately followed by a guanine in the DNA sequence from 5' to 3'. When located in the promoter region of a gene, DNA methylation is often associated with transcriptional silencing of the gene. Aberrant DNA methylation is associated with the development of various diseases such as cancer. Bisulfite sequencing (BS-seq) is the current "gold-standard" technology for high-resolution profiling of DNA methylation. Reduced representation bisulfite sequencing (RRBS) is an efficient form of BS-seq that targets CpG-rich DNA regions in order to save sequencing costs. A typical bioinformatics aim is to identify CpGs that are differentially methylated (DM) between experimental conditions. This workflow demonstrates that differential methylation analysis of RRBS data can be conducted using software and methodology originally developed for RNA-seq data. The RNA-seq pipeline is adapted to methylation by adding extra columns to the design matrix to account for read coverage at each CpG, after which the RRBS and RNA-seq pipelines are almost identical. This approach is statistically natural and gives analysts access to a rich collection of analysis tools including generalized linear models, gene set testing and pathway analysis. The article presents a complete start to finish case study analysis of RRBS profiles of different cell populations from the mouse mammary gland using the Bioconductor package edgeR. We show that lineage-committed cells are typically hyper-methylated compared to progenitor cells and this is true on all the autosomes but not the sex chromosomes. We demonstrate a strong negative correlation between methylation of promoter regions and gene expression as measured by RNA-seq for the same cell types, showing that methylation is a regulatory mechanism involved in epithelial linear commitment.
| S-EPMC5747346 | biostudies-literature
Project description:BackgroundThe pathophysiological role of SERPINA1 in respiratory health may be more strongly determined by the regulation of its expression than by common genetic variants. A family based study of predominantly smoking adults found methylation at two Cytosine-phosphate-Guanine sites (CpGs) in SERPINA1 gene to be associated with chronic obstructive pulmonary disease risk. The objective of this study was to confirm the association of lung function with SERPINA1 methylation in general population samples by testing a comprehensive set of CpGs in the SERPINA gene cluster. We considered lung function level and decline in adult smokers from three European population-based cohorts and lung function level and growth in tobacco-smoke exposed children from a birth cohort.MethodsDNA methylation using Illumina Infinium Human Methylation 450 k and EPIC beadchips and lung function were measured at two time points in 1076 SAPALDIA, ECRHS and NFBC adult cohort participants and 259 ALSPAC children. Associations of methylation at 119 CpG sites in the SERPINA gene cluster (PP4R4-SERPINA13P) with lung functions and circulating alpha-1-antitripsin (AAT) were assessed using multivariable cross-sectional and longitudinal regression models.ResultsMethylation at cg08257009 in the SERPINA gene cluster, located 32 kb downstream of SERPINA1, not annotated to a gene, was associated with FEV1/FVC at the Bonferroni corrected level in adults, but not in children. None of the methylation signals in the SERPINA1 gene showed associations with lung function after correcting for multiple testing.ConclusionsThe results do not support a role of SERPINA1 gene methylation as determinant of lung function across the life course in the tobacco smoke exposed general population exposed.
| S-EPMC6103990 | biostudies-literature